Phosphate Buffer Solution Group Research Articles

[Effects of gelatin methacrylate anhydride hydrogel loaded with small extracellular vesicles derived from human umbilical cord mesenchymal stem cells in the treatment of full-thickness skin defect wounds in mice].

Objective: To investigate the effects of gelatin methacrylate anhydride (GelMA) hydrogel loaded with small extracellular vesicles derived from human umbilical cord mesenchymal stem cells (hUCMSCs-sEVs) in the treatment of full-thickness skin defect wounds in mice. Methods: This study was an experimental study. hUCMSCs-sEVs were extracted by ultracentrifugation, their morphology was observed through transmission electron microscope, and the expression of CD9, CD63, tumor susceptibility gene 101 (TSG101), and calnexin was detected by Western blotting. The human umbilical vein endothelial cells (HUVECs), the 3rd and 4th passages of human epidermal keratinocytes (HEKs) and human dermal fibroblasts (HDFs) were all divided into blank control group (routinely cultured) and hUCMSC-sEV group (cultured with the cell supernatant containing hUCMSCs-sEVs). The cell scratch test was performed and the cell migration rates at 6, 12, and 24 h after scratching were calculated, the cell Transwell assay was performed and the number of migration cells at 12 h after culture was calculated, and the proportion of proliferating cells was detected by 5-acetylidene-2'-deoxyuridine and Hoechst staining at 24 h after culture, with sample numbers being all 3. The simple GelMA hydrogel and the GelMA hydrogel loaded with hUCMSCs-sEVs (hereinafter referred to as hUCMSC-sEV/GelMA hydrogel) were prepared. Then the micromorphology of 2 kinds of hydrogels was observed under scanning electron microscope, the distribution of hUCMSCs-sEVs was observed by laser scanning confocal microscope, and the cumulative release rates of hUCMSCs-sEVs at 0 (immediately), 2, 4, 6, 8, 10, and 12 d after soaking hUCMSC-sEV/GelMA hydrogel in phosphate buffer solution (PBS) were measured and calculated by protein colorimetric quantification (n=3). Twenty-four 6-week-old male C57BL/6J mice were divided into PBS group, hUCMSC-sEV alone group, GelMA hydrogel alone group, and hUCMSC-sEV/GelMA hydrogel group according to the random number table, with 6 mice in each group, and after the full-thickness skin defect wounds on the back of mice in each group were produced, the wounds were performed with PBS injection, hUCMSC-sEV suspenson injection, simple GelMA coverage, and hUCMSC-sEV/GelMA hydrogel coverage, respectively. Wound healing was observed on post injury day (PID) 0 (immediately), 4, 8, and 12, and the wound healing rates on PID 4, 8, and 12 were calculated, and the wound tissue was collected on PID 12 for hematoxylin-eosin staining to observe the structure of new tissue, with sample numbers being both 6. Results: The extracted hUCMSCs-sEVs showed a cup-shaped structure and expressed CD9, CD63, and TSG101, but barely expressed calnexin. At 6, 12, and 24 h after scratching, the migration rates of HEKs (with t values of 25.94, 20.98, and 20.04, respectively), HDFs (with t values of 3.18, 5.68, and 4.28, respectively), and HUVECs (with t values of 4.32, 19.33, and 4.00, respectively) in hUCMSC-sEV group were significantly higher than those in blank control group (P<0.05). At 12 h after culture, the numbers of migrated HEKs, HDFs, and HUVECs in hUCMSC-sEV group were 550±23, 235±9, and 856±35, respectively, which were significantly higher than 188±14, 97±6, and 370±32 in blank control group (with t values of 22.95, 23.13, and 17.84, respectively, P<0.05). At 24 h after culture, the proportions of proliferating cells of HEKs, HDFs, and HUVECs in hUCMSC-sEV group were significantly higher than those in blank control group (with t values of 22.00, 13.82, and 32.32, respectively, P<0.05). The inside of simple GelMA hydrogel showed a loose and porous sponge-like structure, and hUCMSCs-sEVs was not observed in it. The hUCMSC-sEV/GelMA hydrogel had the same sponge-like structure, and hUCMSCs-sEVs were uniformly distributed in clumps. The cumulative release rate curve of hUCMSCs-sEVs from hUCMSC-sEV/GelMA hydrogel tended to plateau at 2 d after soaking, and the cumulative release rate of hUCMSCs-sEVs was (59.2±1.8)% at 12 d after soaking. From PID 0 to 12, the wound areas of mice in the 4 groups gradually decreased. On PID 4, 8, and 12, the wound healing rates of mice in hUCMSC-sEV/GelMA hydrogel group were significantly higher than those in the other 3 groups (P<0.05); the wound healing rates of mice in GelMA hydrogel alone group and hUCMSC-sEV alone group were significantly higher than those in PBS group (P<0.05). On PID 8 and 12, the wound healing rates of mice in hUCMSC-sEV alone group were significantly higher than those in GelMA hydrogel alone group (P<0.05). On PID 12, the wounds of mice in hUCMSC-sEV/GelMA hydrogel group showed the best wound epithelization, loose and orderly arrangement of dermal collagen, and the least number of inflammatory cells, while the dense arrangement of dermal collagen and varying degrees of inflammatory cell infiltration were observed in the wounds of mice in the other 3 groups. Conclusions: hUCMSCs-sEVs can promote the migration and proliferation of HEKs, HDFs, and HUVECs which are related to skin wound healing, and slowly release in GelMA hydrogel. The hUCMSC-sEV/GelMA hydrogel as a wound dressing can significantly improve the healing speed of full-thickness skin defect wounds in mice.

[Influences and mechanism of extracellular vesicles from dermal papilla cells of mice on human hypertrophic scar fibroblasts].

Objective: To investigate the influences and mechanism of extracellular vesicles from dermal papilla cells (DPC-EVs) of mice on human hypertrophic scar fibroblasts (HSFs). Methods: The study was an experimental research. The primary dermal papilla cells (DPCs) of whiskers were extracted from 10 6-week-old male C57BL/6J mice and identified successfully. The DPC-EVs were extracted from the 3rd to 5th passage DPCs by ultracentrifugation, and the morphology was observed through transmission electron microscope and the particle diameter was detected by nanoparticle tracking analyzer (n=3) at 24 h after culture. The 3rd passage of HSFs were divided into DPC-EV group and phosphate buffer solution (PBS) group, which were cultured with DPC-EVs and PBS, respectively. The cell scratch test was performed and cell migration rate at 24 h after scratching was calculated (n=5). The cell proliferation levels at 0 (after 12 h of starvation treatment and before adding DPC-EVs or PBS), 24, 48, 72, and 96 h after culture were detected by using cell counting kit 8 (n=4). The protein expressions of α-smooth muscle actin (α-SMA) and collagen typeⅠ (ColⅠ) in cells at 24 h after culture were detected by immunofluorescence method and Western blotting, and the protein expression of Krüppel-like factor 4 (KLF4) in cells at 24 h after culture was detected by Western blotting. After the 3rd passage of HSFs were cultured with DPC-EVs for 24 h, the cells were divided into blank control group, KLF4 knockdown group, and KLF4 overexpression group according to the random number table. The cells in blank control group were only routinely cultured for 48 h. The cells in KLF4 knockdown group and KLF4 overexpression group were incubated with KLF4 knockdown virus for 24 h, then the cells in KLF4 knockdown group were routinely cultured for 24 h while the cells in KLF4 overexpression group were incubated with KLF4 overexpression virus for 24 h. The protein expressions of KLF4, α-SMA, and ColⅠ in cells were detected by Western blotting at 48 h after culture. Results: At 24 h after culture, the extracted DPC-EVs showed vesicular structure with an average particle diameter of 108.8 nm. At 24 h after scratching, the migration rate of HSFs in PBS group was (54±10)%, which was significantly higher than (29±8)% in DPC-EV group (t=4.37, P<0.05). At 48, 72, and 96 h after culture, the proliferation levels of HSFs in DPC-EV group were significantly lower than those in PBS group (with t values of 4.06, 5.76, and 6.41, respectively, P<0.05). At 24 h after culture, the protein expressions of α-SMA and ColⅠ of HSFs in DPC-EV group were significantly lower than those in PBS group, while the protein expression of KLF4 was significantly higher than that in PBS group. At 48 h after culture, compared with those in blank control group, the protein expression of KLF4 of HSFs in KLF4 knockdown group was down-regulated, while the protein expressions of α-SMA and ColⅠ were both up-regulated; compared with those in KLF4 knockdown group, the protein expression of KLF4 of HSFs in KLF4 overexpression group was up-regulated, while the protein expressions of ColⅠ and α-SMA were down-regulated. Conclusions: The DPC-EVs of mice can inhibit the proliferation and migration of human HSFs and significantly inhibit the expressions of fibrosis markers α-SMA and ColⅠ in human HSFs by activating KLF4.

[Effects of cerium oxide nanoenzyme-gelatin methacrylate anhydride hydrogel in the repair of infected full-thickness skin defect wounds in mice].

Objective: To investigate the effects of cerium oxide nanoenzyme-gelatin methacrylate anhydride (GelMA) hydrogel (hereinafter referred to as composite hydrogel) in the repair of infected full-thickness skin defect wounds in mice. Methods: This study was an experimental study. Cerium oxide nanoenzyme with a particle size of (116±9) nm was prepared by hydrothermal method, and GelMA hydrogel with porous network structure and good gelling performance was also prepared. The 25 μg/mL cerium oxide nanoenzyme which could significantly promote the proliferation of human skin fibroblasts and had high superoxide dismutase activity was screened out. It was added to GelMA hydrogel to prepare composite hydrogel. The percentage of cerium oxide nanoenzyme released from the composite hydrogel was calculated after immersing it in phosphate buffer solution (PBS) for 3 and 7 d. The red blood cell suspension of mice was divided into PBS group, Triton X-100 group, cerium oxide nanoenzyme group, GelMA hydrogel group, and composite hydrogel group, which were treated with corresponding solution. The hemolysis of red blood cells was detected by microplate reader after 1 h of treatment. The bacterial concentrations of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli were determined after being cultured with PBS, cerium oxide nanoenzyme, GelMA hydrogel, and composite hydrogel for 2 h. The sample size in all above experiments was 3. Twenty-four 8-week-old male BALB/c mice were taken, and a full-thickness skin defect wound was prepared in the symmetrical position on the back and infected with MRSA. The mice were divided into control group without any drug intervention, and cerium oxide nanoenzyme group, GelMA hydrogel group, and composite hydrogel group applied with corresponding solution, with 6 mice in each group. The wound healing was observed on 3, 7, and 14 d after injury, and the remaining wound areas on 3 and 7 d after injury were measured (the sample size was 5). The concentration of MRSA in the wound exudation of mice on 3 d after injury was measured (the sample size was 3), and the blood flow perfusion in the wound of mice on 5 d after injury was observed using a laser speckle flow imaging system (the sample size was 6). On 14 d after injury, the wound tissue of mice was collected for hematoxylin-eosin staining to observe the newly formed epithelium and for Masson staining to observe the collagen situation (the sample size was both 3). Results: After immersion for 3 and 7 d, the release percentages of cerium oxide nanoenzyme in the composite hydrogel were about 39% and 75%, respectively. After 1 h of treatment, compared with that in Triton X-100 group, the hemolysis of red blood cells in PBS group, GelMA hydrogel group, cerium oxide nanoenzyme group, and composite hydrogel group was significantly decreased (P<0.05). Compared with that cultured with PBS, the concentrations of MRSA and Escherichia coli cultured with cerium oxide nanoenzyme, GelMA hydrogel, and composite hydrogel for 2 h were significantly decreased (P<0.05). The wounds of mice in the four groups were gradually healed from 3 to 14 d after injury, and the wounds of mice in composite hydrogel group were all healed on 14 d after injury. On 3 and 7 d after injury, the remaining wound areas of mice in composite hydrogel group were (29±3) and (13±5) mm2, respectively, which were significantly smaller than (56±12) and (46±10) mm2 in control group and (51±7) and (38±8) mm2 in cerium oxide nanoenzyme group (with P values all <0.05), but was similar to (41±5) and (24±9) mm2 in GelMA hydrogel group (with P values both >0.05). On 3 d after injury, the concentration of MRSA on the wound of mice in composite hydrogel group was significantly lower than that in control group, cerium oxide nanoenzyme group, and GelMA hydrogel group, respectively (with P values all <0.05). On 5 d after injury, the volume of blood perfusion in the wound of mice in composite hydrogel group was significantly higher than that in control group, cerium oxide nanoenzyme group, and GelMA hydrogel group, respectively (P<0.05). On 14 d after injury, the wound of mice in composite hydrogel group basically completed epithelization, and the epithelization was significantly better than that in the other three groups. Compared with that in the other three groups, the content of collagen in the wound of mice in composite hydrogel group was significantly increased, and the arrangement was also more orderly. Conclusions: The composite hydrogel has good biocompatibility and antibacterial effect in vivo and in vitro. It can continuously sustained release cerium oxide nanoenzyme, improve wound blood perfusion in the early stage, and promote wound re-epithelialization and collagen synthesis, therefore promoting the healing of infected full-thickness skin defect wounds in mice.

Establishment and Validation of a Mouse Bacterial Vaginosis Model.

Bacterial vaginosis (BV) is a common vaginal infection without a reliable animal model. To establish a novel mouse BV model, we evaluated multiple parameters of various identified bacteria-infected mice, including Staphylococcus aureus (SA), Escherichia coli (EC), Streptococcus agalactiae, β-Hemolytic streptococcus, and Gardnerella vaginalis (GV). Mature female KM mice were randomly allocated to a vehicle group (group A, without any treatment) and experimental groups. After vaginal secretions were harvested, experimental groups were divided into phosphate buffer solution group (PBS, group B), control group including SA, and EC with a 1:1 ratio (group C), SA, EC, and Streptococcus agalactiae with a 1:2:1 ratio group (group D), SA, EC, and β-Hemolytic streptococcus with a 1:2:1 ratio group (group E), and GV group (group F). The vaginal secretions of experimental mice were collected by flushing with 100 mL sterile PBS on days 2, 4, and 6. Vaginal secretions were examined by Gram staining, sialidase assay, ammonia test, and pH value measurement. IL-6 and IL-10 levels in mouse serum were detected by enzyme-linked immunosorbent assay. Hematoxylineosin staining and mouse cervicovaginal tissue histopathological scores were observed. The diagnostic test results were analyzed by logistic regression analysis and receiver operating characteristic curves. The Shapiro-Wilk analysis of variance, or rank-sum test, was used for normal distribution analysis. Pearson's correlation and chi-squared test determined the correlation and comparison data expressed as a percentage or frequency. There was less severe vaginal morphology in GV-infected mice compared to other bacteria-infected mice. The sialidase assay, the ammonia test, and the pH values of vaginal secretions showed significant differences between GV-infected and uninfected mice. Serum IL-6 and IL-10 levels and vaginal histological scoring increased in other bacteria-infected mice, but GV-infected mice showed only a mildly increasing trend of IL-10 levels and vaginal histological scoring compared to control mice. GV-infected mice showed clinical features similar to human BV infection, including vaginal anatomical and pathological indices, and biochemical and immune parameters. Serum IL-10 level has potential for use in BV diagnosis.

[Influence of family with sequence similarity 134, member B-mediated reticulophagy on lipopolysaccharide-induced apoptosis of mouse dendritic cells].

Objective: To investigate the influence of family with sequence similarity 134, member B (FAM134B)-mediated reticulophagy on lipopolysaccharide (LPS)-induced apoptosis of mouse dendritic cells (DCs), so as to provide a basis for improving the immune suppression of sepsis caused by wound infection and other factors. Methods: The experimental research methods were used. The DC line DC2.4 of the 3rd to 10th passage in the logarithmic growth stage was collected for experiments. DCs were divided into LPS stimulation 0 h (no stimulation) group, LPS stimulation 6 h group, LPS stimulation 12 h group, LPS stimulation 24 h group, and LPS stimulation 72 h group, which were cultured with 1 μg/mL LPS (the same concentration below) for the corresponding time. The protein expressions of FAM134B, microtubule-associated protein 1 light chain 3B (LC3B), and transporter protein SEC61B were determined by Western blotting, and the ratio of LC3B-Ⅱ/LC3B-Ⅰ was calculated (n=3). DCs were divided into phosphate buffer solution (PBS) group and LPS group for corresponding treatment. After 24 hours of culture, the expression of FAM134B and its co-localization with lysosomal probes and LC3B were detected using immunofluorescence method, while the number of autolysosomes in cells were observed through transmission electron microscope. DCs were divided into the FAM134B-knockdown group that were transfected with lentivirus containing small interfering RNA (siRNA) sequence of FAM134B gene and the empty vector group with empty lentivirus transfected. At post transfection hour 72, the fluorescence expression of cells was observed under the inverted fluorescence phase contrast microscope, meanwhile, the normally cultured DCs were set as blank control group, and the same observation was performed at the corresponding time point. DCs were divided into PBS alone group and LPS alone group, DCs successfully transfected with lentivirus containing siRNA sequence of FAM134B gene were divided into FAM134B-knockdown+PBS group and FAM134B-knockdown+LPS group, and DCs successfully transfected with empty lentivirus were divided into empty vector+PBS group and empty vector+LPS group. These cells were stimulated correspondingly and cultured for 24 hours. The protein expression of FAM134B was detected using Western blotting (n=3); the apoptotic rate of cells was determined by flow cytometry (n=3); the situation of apoptosis was observed by Hoechst staining, and the apoptotic rate was calculated (n=5); the protein expressions of cleaved cysteine aspartic acid specific protease-3 (caspase-3), B cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) were detected using Western blotting, and the ratio of Bax/Bcl-2 was calculated (n=5). Data were statistically analyzed with one-way analysis of variance (ANOVA), least significant difference test, and ANOVA for factorial design. Results: Compared with those in LPS stimulation 0 h group, the protein expressions of FAM134B of cells in LPS stimulation 12 h group and LPS stimulation 24 h group were significantly increased (P<0.05), the protein expressions of SEC61B of cells in LPS stimulation 6 h group, LPS stimulation 12 h group, LPS stimulation 24 h group, and LPS stimulation 72 h group were significantly decreased (P<0.05), and the ratios of LC3B-Ⅱ/LC3B-Ⅰ of cells in LPS stimulation 24 h group and LPS stimulation 72 h group were obviously increased (P<0.05). As the most significant changes of three proteins were seen in the cells of LPS stimulation 24 h group, 24 h was used as the duration of subsequent LPS stimulation. After 24 hours of culture, the expression of FAM134B and its co-localization with LC3B and lysosomal probes in the cells of LPS group were all significantly enhanced, with a significant increase in the number of autolysosomes in comparison with those in PBS group. Both the empty vector group and the FAM134B-knockdown group showed high intensity fluorescence in the cells at post transfection hour 72, but the blank control group showed no fluorescence in the cells at the corresponding time point. After 24 hours of culture, the protein expression of FAM134B of cells in FAM134B-knockdown+PBS group was significantly lower than the expressions in PBS alone group and empty vector+PBS group (with P values all <0.05), the protein expression of FAM134B of cells in FAM134B-knockdown+LPS group was significantly lower than the expressions in LPS alone group and empty vector+LPS group (with P values all <0.05), the protein expression of FAM134B of cells in LPS alone group was significantly higher than that in PBS alone group (P<0.05), while the protein expression of FAM134B of cells in empty vector+LPS group was significantly higher than that in empty vector+PBS group (P<0.05). After 24 hours of culture, flow cytometry assay revealed that the apoptotic rate of cells in PBS alone group, LPS alone group, empty vector+PBS group, empty vector+LPS group, FAM134B-knockdown+PBS group, and FAM134B-knockdown+LPS group were (13.3±0.8)%, (32.6±4.3)%, (17.0±1.5)%, (51.7±3.3)%, (52.4±3.1)%, and (62.3±2.6)%, respectively. After 24 hours of culture, compared with those in LPS alone group and empty vector+LPS group, the protein expression of cleaved caspase-3, the ratio of Bax/Bcl-2, and the apoptotic rates of cells detected by flow cytometry and Hoechst staining were significantly increased in FAM134B-knockdown+LPS group (P<0.05); compared with those in the corresponding PBS treatment group, namely, PBS alone group, empty vector+PBS group, and FAM134B-knockdown+PBS group, the protein expression of cleaved caspase-3, the ratio of Bax/Bcl-2, and the apoptotic rates of cells detected by flow cytometry and Hoechst staining were significantly increased in LPS alone group, empty vector+LPS group, and FAM134B-knockdown+LPS group (P<0.05). Conclusions: The activation of reticulophagy mediated by FAM134B in mouse DCs is enhanced and peaked in 24 hours under LPS stimulation, and the activated reticulophagy has a significant inhibitory effect on cell apoptosis.

Adoption of Ultrasound-guided Polymer Nanocarriers in Tumor Chemoradiotherapy and Oxidation Treatment.

The research was aimed at discussing the effectiveness of ultrasound-guided polymer nanocarriers in the clinical treatment of tumors by chemoradiotherapy and oxidation treatment. Twenty female Balb/cAnN (BALB/C) mice were selected as the research objects in the experiment. These mice were set up as tumor-bearing mice, and then ultrasound-guided polymers with different doses, including polyethylene glycol-poly 2-bromoethyl methacrylate (PEG-PBEMA) (Micelle group), free small molecules called l-ascorbyl palmitate (PA) (PA group), PA-micelle micellar particles (PA-Micelle group) prepared in the research, and phosphate buffer solution (PBS) (PBS group) were adopted. Besides, the growth of mice was recorded and compared after each operation. Meanwhile, different concentrations of PA-Micelle micellar particles and free small molecules of PA were added to the breast cancer cells of mice, and the concentration changes of glutathione (GSH) were detected to test the oxidation treatment ability of this method. According to the results of the experiment, the tumor volume of mice in the PA-Micelle group prepared in the research was the smallest followed by the PA group, and the tumor volume of mice in the Micelle group was the third smallest. The mice in the PBS group had the largest tumors among mice in all four groups. In oxidation treatment, the GSH concentration of mice in the PA-Micelle group was the lowest, while the GSH concentration of mice in the PA group was almost unchanged. The results of this experiment proved that the therapeutic effect of polymer nanocarriers in tumor chemotherapy and oxidation treatment was more significant than in traditional drug treatment.

[Influence of autologous adipose stem cell matrix gel on wound healing and scar hyperplasia of full-thickness skin defects in rabbit ears].

Objective: To investigate the influence of autologous adipose stem cell matrix gel on wound healing and scar hyperplasia of full-thickness skin defects in rabbit ears, and to analyze the related mechanism. Methods: Experimental research methods were adopted. The complete fat pads on the back of 42 male New Zealand white rabbits aged 2 to 3 months were cut to prepare adipose stem cell matrix gel, and a full-thickness skin defect wound was established on the ventral side of each ear of each rabbit. The left ear wounds were included in adipose stem cell matrix gel group (hereinafter referred to as matrix gel group), and the right ear wounds were included in phosphate buffer solution (PBS) group, which were injected with autologous adipose stem cell matrix gel and PBS, respectively. The wound healing rate was calculated on post injury day (PID) 7, 14, and 21, and the Vancouver scar scale (VSS) scoring of scar tissue formed on the wound (hereinafter referred to as scar tissue) was performed in post wound healing month (PWHM) 1, 2, 3, and 4. Hematoxylin-eosin staining was performed to observe and measure the histopathological changes of wound on PID 7, 14, and 21 and the dermal thickness of scar tissue in PWHM 1, 2, 3, and 4. Masson staining was performed to observe the collagen distribution in wound tissue on PID 7, 14, and 21 and scar tissue in PWHM 1, 2, 3, and 4, and the collagen volume fraction (CVF) was calculated. The microvessel count (MVC) in wound tissue on PID 7, 14, and 21 and the expressions of transforming growth factor β1 (TGF-β1) and α smooth muscle actin (α-SMA) in scar tissue in PWHM 1, 2, 3, and 4 were detected by immunohistochemical method, and the correlation between the expression of α-SMA and that of TGF-β1 in scar tissue in matrix gel group was analyzed. The expressions of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) in wound tissue were detected by enzyme-linked immunosorbent assay on PID 7, 14, and 21. The number of samples at each time point in each group was 6. Data were statistically analyzed with analysis of variance for repeated measurement, analysis of variance for factorial design, paired sample t test, least significant difference test, and Pearson correlation analysis. Results: On PID 7, the wound healing rate in matrix gel group was (10.3±1.7)%, which was close to (8.5±2.1)% in PBS group (P>0.05). On PID 14 and 21, the wound healing rates in matrix gel group were (75.5±7.0)% and (98.7±0.8)%, respectively, which were significantly higher than (52.7±6.7)% and (90.5±1.7)% in PBS group (with t values of 5.79 and 10.37, respectively, P<0.05). In PWHM 1, 2, 3, and 4, the VSS score of scar tissue in matrix gel group was significantly lower than that in PBS group (with t values of -5.00, -2.86, -3.31, and -4.45, respectively, P<0.05). Compared with the previous time point within the group, the VSS score of scar tissue at each time point after wound healing in the two groups was significantly increased (P<0.05), except for PWHM 4 in matrix gel group (P>0.05). On PID 7, the granulation tissue regeneration and epithelialization degree of the wounds between the two groups were similar. On PID 14 and 21, the numbers of fibroblasts, capillaries, and epithelial cell layers in wound tissue of matrix gel group were significantly more than those in PBS group. In PWHM 1, 2, 3, and 4, the dermal thickness of scar tissue in matrix gel group was significantly thinner than that in PBS group (with t values of -4.08, -5.52, -6.18, and -6.30, respectively, P<0.05). Compared with the previous time point within the group, the dermal thickness of scar tissue in the two groups thickened significantly at each time point after wound healing (P<0.05). Compared with those in PBS group, the collagen distribution in wound tissue in matrix gel group was more regular and the CVF was significantly increased on PID 14 and 21 (with t values of 3.98 and 3.19, respectively, P<0.05), and the collagen distribution in scar tissue was also more regular in PWHM 1, 2, 3, and 4, but the CVF was significantly decreased (with t values of -7.38, -4.20, -4.10, and -4.65, respectively, P<0.05). Compared with the previous time point within the group, the CVFs in wound tissue at each time point after injury and scar tissue at each time point after wound healing in the two groups were significantly increased (P<0.05), except for PWHM 1 in matrix gel group (P>0.05). On PID 14 and 21, the MVC in wound tissue in matrix gel group was significantly higher than that in PBS group (with t values of 4.33 and 10.10, respectively, P<0.05). Compared with the previous time point within the group, the MVC of wound at each time point after injury in the two groups was increased significantly (P<0.05), except for PID 21 in PBS group (P>0.05). In PWHM 1, 2, 3, and 4, the expressions of TGF-β1 and α-SMA in scar tissue in matrix gel group were significantly lower than those in PBS group (with t values of -2.83, -5.46, -5.61, -8.63, -10.11, -5.79, -8.08, and -11.96, respectively, P<0.05). Compared with the previous time point within the group, the expressions of TGF-β1 and α-SMA in scar tissue in the two groups were increased significantly at each time point after wound healing (P<0.05), except for the α-SMA expression in matrix gel group in PWHM 4 (P>0.05). There was a significantly positive correlation between the expression of α-SMA and that of TGF-β1 in scar tissue in matrix gel group (r=0.92, P<0.05). On PID 14 and 21, the expressions of VEGF (with t values of 6.14 and 6.75, respectively, P<0.05) and EGF (with t values of 8.17 and 5.85, respectively, P<0.05) in wound tissue in matrix gel group were significantly higher than those in PBS group. Compared with the previous time point within the group, the expression of VEGF of wound at each time point after injury in the two groups was increased significantly (P<0.05), and the expression of EGF was decreased significantly (P<0.05). Conclusions: Adipose stem cell matrix gel may significantly promote the wound healing of full-thickness skin defects in rabbit ears by promoting collagen deposition and expressions of VEGF and EGF in wound tissue, and may further inhibit the scar hyperplasia after wound healing by inhibiting collagen deposition and expressions of TGF-β1 and α-SMA in scar tissue.

[Influence of reactive oxygen species responsive self-assembled nanomicelle loaded with pyroptosis inhibitor on full-thickness skin defects in diabetic rats].

Objective: To investigate the influence of reactive oxygen species (ROS) responsive self-assembled nanomicelle loaded with pyroptosis inhibitor on full-thickness skin defects in diabetic rats. Methods: Experimental research methods were employed. A nucleotide-binding oligomerization domain (NOD) 1/2 inhibitor (NOD-IN-1) was encapsulated with nanomicelle polyethylene glycol-block-polypropylene sulfide (PEG-b-PPS), and the resulting product was called PEPS@NOD-IN-1. The morphology and hydration particle size of PEG-b-PPS and PEPS@NOD-IN-1 were observed by transmission electron microscope and particle size analyzer, respectively, and the encapsulation rate and drug loading rate of PEPS@NOD-IN-1 to NOD-IN-1 and the cumulative release rate of NOD-IN-1 by PEPS@NOD-IN-1 in phosphate buffer solution (PBS) alone and hydrogen peroxide-containing PBS within 40 h were measured and calculated by microplate reader, and the sample number was 3. Twenty-four male Sprague-Dawley rats aged 6-7 weeks were injected with streptozotocin to induce type 1 diabetes mellitus. Six full-thickness skin defect wounds were made on the back of each rat. The injured rats were divided into PBS group, NOD-IN-1 group, PEG-b-PPS group, and PEPS@NOD-IN-1 group with corresponding treatment according to the random number table, with 6 rats in each group. The wound healing was observed on post injury day (PID) 3, 7, and 12, and the wound healing rate was calculated. The ROS levels in wound tissue were detected by immunofluorescence method on PID 3. On PID 7, the granulation tissue thickness in wound was assessed by hematoxylin-eosin staining, the mRNA expressions of NOD1 and NOD2 were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction, and the protein expressions of NOD1, NOD2, and GSDMD-N terminals were detected by Western blotting. Six wounds from different rats in each group were taken for detection of the above indicators. Wound tissue (3 samples per group) was taken from rats in PBS group and PEPS@NOD-IN-1 group on PID 7, and transcriptome sequencing was performed using high-throughput sequencing technology platform. Differentially expressed genes (DEGs) significantly down-regulated in PEPS@NOD-IN-1 group as compared with PBS group were screened, and the enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed. The DEG heatmap of the NOD-like receptor pathway, a pyroptosis-related pathway, was made. Protein-protein interaction (PPI) analysis of DEGs in heatmap was performed through the STRING database to screen key genes of PEPS@NOD-IN-1 regulating the NOD-like receptor pathway. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and Tukey test. Results: PEG-b-PPS and PEPS@NOD-IN-1 were in spherical structures of uniform size, with hydration particle sizes of (134.2±3.3) and (143.1±2.3) nm, respectively. The encapsulation rate of PEPS@NOD-IN-1 to NOD-IN-1 was (60±5)%, and the drug loading rate was (15±3)%. The release of NOD-IN-1 from PEPS@NOD-IN-1 in PBS alone was slow, and the cumulative release rate at 40 h was only (12.4±2.3)%. The release of NOD-IN-1 from PEPS@NOD-IN-1 in hydrogen peroxide-containing PBS within 10 h was very rapid, and the cumulative release rate at 10 h reached (90.1±3.6)%. On PID 3 and 7, the wounds of rats in the four groups were gradually healed, and the healing in PEPS@NOD-IN-1 group was better than that in the other three groups. On PID 12, the wound scab area in PBS group was large, the wound epithelialization in NOD-IN-1 group and PEG-b-PPS group was obvious, and the wound in PEPS@NOD-IN-1 group was close to complete epithelialization. Compared with those in PBS group, NOD-IN-1 group, and PEG-b-PPS group, the wound healing rates on PID 7 and 12 in PEPS@NOD-IN-1 group were significantly increased (P<0.05), the level of ROS in wound tissue on PID 3 was significantly decreased (P<0.05), the thickness of granulation tissue in wound on PID 7 was significantly thickened (P<0.05), and the mRNA expressions of NOD1 and NOD2 and the protein expressions of NOD1, NOD2, and GSDMD-N terminals in wound tissue on PID 7 were significantly decreased (P<0.05). KEGG pathway analysis showed that DEGs significantly down-regulated in PEPS@NOD-IN-1 group as compared with PBS group were significantly enriched in NOD-like receptors, hypoxia-inducible factors, mitogen-activated protein kinases, and tumor necrosis factor (TNF) pathways. In the DEG heatmap of NOD-like receptor pathway, the genes regulating pyroptosis mainly involved NOD1, NOD2, NOD-like receptor thermoprotein domain-related protein 3, Jun, signal transduction and transcriptional activator 1 (STAT1), TNF-α-induced protein 3. The PPI results showed that NOD1, NOD2, and STAT1 were the key genes of PEPS@NOD-IN-1 regulating the NOD-like receptor pathway. Conclusions: PEPS@NOD-IN-1 can down-regulate the level of local ROS in wounds and the expression of NOD1, NOD2, and GSDMD-N terminals, the key regulators of pyroptosis, thereby promoting the repair of full-thickness skin defect wounds in diabetic rats. PEPS@NOD-IN-1 can also significantly down-regulate the pyroptosis, inflammation, and hypoxia-related pathways of wounds, and regulate NOD-like receptor pathways by down-regulating key genes NOD1, NOD2, and STAT1.

[Effects and mechanism of human umbilical vein endothelial cells-derived exosomes on wound healing in diabetic rabbits].

Objective: The investigate the effects and mechanism of exosomes derived from human umbilical vein endothelial cells (HUVECs) on wound healing in diabetes rabbits. Methods: The experimental research methods were used. The primary vascular endothelial cells (VECs) and human skin fibroblasts (HSFs) were extracted from skin tissue around ulcer by surgical excision of two patients with diabetic ulcer (the male aged 49 years and the female aged 58 years) admitted to Xiangya Third Hospital of Central South University in June 2019. The cells were successfully identified through morphological observation and flow cytometry. The HUVEC exosomes were extracted by ultracentrifugation and identified successfully by morphological observation, particle size detection, and Western blotting detection. Twenty female 3-month-old New Zealand rabbits were taken to create one type 2 diabetic full-thickness skin defect wound respectively on both sides of the back. The wounds were divided into exosomes group and phosphate buffer solution (PBS) group and treated accordingly, with 20 wounds in each group, the time of complete tissue coverage of wound was recorded. On PID 14, hematoxylin-eosin staining or Masson staining was performed to observe angiogenesis or collagen fiber hyperplasia (n=20). The VECs and HSFs were co-cultured with HUVEC exosomes for 24 h to observe the uptake of HUVEC exosomes by the two kinds of cells. The VECs and HSFs were divided to exosome group treated with HUVEC exosomes and PBS group treated with PBS to detect the cell proliferation on 4 d of culture with cell count kit 8, to detect and calculate the cell migration rate at 24 and 48 h after scratch by scratch test, to detect the cell migration number at 24 h of culture with Transwell test, and to detect the mRNA expressions of nuclear factor-erythroid 2-related factor 2 (NRF2) and transcription activating factor 3 (ATF3) by real time fluorescence quantitative reverse transcription polymerase chain reaction. Besides, the number of vascular branches and vascular length were observed in the tube forming experiment after 12 h of culture of VECs (n=3). The VECs and HSFs were taken and divided into PBS group and exosome group treated as before, and NRF2 interference group, ATF3 interference group, and no-load interference group with corresponding gene interference. The proliferation and migration of the two kinds of cells, and angiogenesis of VECs were detected as before (n=3). Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, independent sample t test, and least significant difference test. Results: The time of complete tissue coverage of wound in exosome group was (17.9±1.9) d, which was significantly shorter than (25.2±2.3) d in PBS group (t=4.54, P<0.05). On PID14, the vascular density of wound in PBS group was significantly lower than that in exosome group (t=10.12, P<0.01), and the collagen fiber hyperplasia was less than that in exosome group. After 24 h of culture, HUVEC exosomes were successfully absorbed by VECs and HSFs. The proliferative activity of HSFs and VECs in exosome group was significantly higher than that in PBS group after 4 d of culture (with t values of 54.73 and 7.05, respectively, P<0.01). At 24 and 48 h after scratch, the migration rates of HSFs (with t values of 3.42 and 11.87, respectively, P<0.05 or P<0.01) and VECs (with t values of 21.42 and 5.49, respectively, P<0.05 or P<0.01) in exosome group were significantly higher than those in PBS group. After 24 h of culture, the migration numbers of VECs and HSFs in exosome group were significantly higher than those in PBS group (with t values of 12.31 and 16.78, respectively, P<0.01). After 12 h of culture, the mRNA expressions of NRF2 in HSFs and VECs in exosome group were significantly higher than those in PBS group (with t values of 7.52 and 5.78, respectively, P<0.05 or P<0.01), and the mRNA expressions of ATF3 were significantly lower than those in PBS group (with t values of 13.44 and 8.99, respectively, P<0.01). After 12 h of culture, the number of vascular branches of VECs in exosome group was significantly more than that in PBS group (t=17.60, P<0.01), and the vascular length was significantly longer than that in PBS group (t=77.30, P<0.01). After 4 d of culture, the proliferation activity of HSFs and VECs in NRF2 interference group was significantly lower than that in PBS group and exosome group (P<0.05 or P<0.01); the proliferation activity of HSFs and VECs in ATF3 interference group was significantly higher than that in PBS group (P<0.05 or P<0.01) and significantly lower than that in exosome group (P<0.05 or P<0.01). At 24 and 48 h after scratch, the migration rates of HSFs and VECs in ATF3 interference group were significantly higher than those in PBS group (P<0.05 or P<0.01) and significantly lower than those in exosome group (P<0.05 or P<0.01). At 24 and 48 h after scratch, the migration rates of HSFs and VECs in NRF2 interference group were significantly lower than those in PBS group and exosome group (P<0.05 or P<0.01). After 24 h of culture, the migration numbers of VECs and HSFs in ATF3 interference group were significantly more than those in PBS group (P<0.05) and significantly less than those in exosome group (P<0.05 or P<0.01); the migration numbers of VECs and HSFs in NRF2 interference group were significantly less than those in PBS group and exosome group (P<0.01). After 12 h of culture, the vascular length and number of branches of VECs in NRF2 interference group were significantly decreased compared with those in PBS group and exosome group (P<0.01); the vascular length and number of branches of VECs in ATF3 interference group were significantly increased compared with those in PBS group (P<0.01) and were significantly decreased compared with those in exosome group (P<0.01). Conclusions: HUVEC exosomes can promote the wound healing of diabetic rabbits by promoting the proliferation and migration of VECs and HSFs, and NRF2 and ATF3 are obviously affected by exosomes in this process, which are the possible targets of exosome action.

[Effects of exosomes from hepatocyte growth factor-modified human adipose mesenchymal stem cells on full-thickness skin defect in diabetic mice].

Objective: To investigate the effects and mechanism of exosomes from hepatocyte growth factor (HGF)-modified human adipose mesenchymal stem cells (ADSCs) on full-thickness skin defect wounds in diabetic mice. Methods: The experimental study method was adopted. Discarded adipose tissue of 3 healthy females (10-25 years old) who underwent abdominal surgery in the Department of Plastic Surgery of First Affiliated Hospital of Air Force Medical University from February to May 2021 was collected, and primary ADSCs were obtained by collagenase digestion method and cultured for 7 days. Cell morphology was observed by inverted phase contrast microscope. The ADSCs of third passage were transfected with HGF lentivirus and cultured for 5 days, and then the fluorescence of cells was observed by imaging system and the transfection rate was calculated. The exosomes of ADSCs of the third to sixth passages and the HGF transfected ADSCs of the third to sixth passages were extracted by density gradient centrifugation, respectively, and named, ADSC exosomes and HGF-ADSC exosomes. The microscopic morphology of exosomes was observed by transmission electron microscopy, and the positive expressions of CD9, CD63, and CD81 of exosomes were detected by flow cytometry, respectively. Twenty-four 6-week-old male Kunming mice were selected to make the diabetic models, and full-thickness skin defect wounds were made on the backs of mice. According to the random number table method, the mice were divided into phosphate buffer solution (PBS) group, HGF alone group, ADSC exosome alone group, and HGF-ADSC exosome group, with 6 mice in each group, and treated accordingly. On post injury day (PID) 3, 7, 10, and 14, the wounds were observed and the wound healing rate was calculated; the blood flow intensity of wound base was detected by Doppler flowmeter and the ratio of relative blood flow intensity on PID 10 was calculated. On PID 10, the number of Ki67 positive cells in wounds was detected by immunofluorescence method, and the number of new-vascularity of CD31 positive staining and tubular neovascularization in the wounds was detected by immunohistochemistry method; the protein expressions of protein endothelial nitric oxide synthase (eNOS), phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), protein kinase B (Akt) and phosphorylated Akt (p-Akt) in wounds were detected by Western blotting, and the ratios of p-PI3K to PI3K and p-Akt to Akt were calculated. On PID 14, the defect length and collagen regeneration of wound skin tissue were detected by hematoxylin and eosin staining and Masson staining, respectively, and the collagen volume fraction (CVF) was calculated. The number of samples is 3 in all cases. Data were statistically analyzed with repeated measurement analysis of variance, one-way analysis of variance, and Tukey test. Results: After 7 days of culture, the primary ADSCs were spindle shaped and arranged in vortex shape after dense growth. After 5 days of culture, HGF transfected ADSCs of the third passage carried green fluorescence, and the transfection rate was 85%. The ADSC exosomes and HGF-ADSC exosomes were similar in microscopic morphology, showing vesicular structures with an average particle size of 103 nm and 98 nm respectively, and both were CD9, CD63, and CD81 positive. On PID 3, the wounds of mice in the 4 groups were all red and swollen, with a small amount of exudate. On PID 7, the wounds of HGF-ADSC exosome group were gradually reduced, while the wounds of the other three groups were not significantly reduced. On PID 10, the wounds in the 4 groups were all reduced and scabbed. On PID 14, the wounds in HGF-ADSC exosome group were basically healed, while the residual wounds were found in the other three groups. On PID 3, the healing rates of wounds in the four groups were similar (P>0.05); On PID 7 and 10, the wound healing rates in HGF-ADSC exosome group were significantly higher than those in PBS group, HGF alone group, and ADSC exosome alone group, respectively (with q values of 13.11, 13.11, 11.89, 12.85, 11.28, and 7.74, respectively, all P<0.01); on PID 14, the wound healing rate in HGF-ADSC exosome group was significantly higher than that in PBS group, HGF alone group, and ADSC exosome alone group (with q values of 15.50, 11.64, and 6.36, respectively, all P<0.01). On PID 3, there was no obvious blood supply in wound base of mice in the 4 groups. On PID 7, microvessels began to form in the wound base of HGF-ADSC exosome group, while the wound base of the other three groups was only congested at the wound edge. On PID 10, microvessel formation in wound base was observed in the other 3 groups except in PBS group, which had no obvious blood supply. On PID 14, the blood flow intensity of wound base in HGF-ADSC exosome group was stronger than that in the other 3 groups, and the distribution was uniform. On PID 10, the ratio of wound base relative blood flow intensity in HGF-ADSC exosome group was significantly higher than that in PBS group, HGF alone group, and ADSC exosome alone group (with q values of 23.73, 19.32, and 9.48, respectively, all P<0.01); The numbers of Ki67-positive cells and new-vascularity of wounds in HGF-ADSC exosome group were significantly higher than those in PBS group, HGF alone group, and ADSC exosome alone group, respectively (with q values of 19.58, 18.20, 11.04, 20.68, 13.79, and 8.12, respectively, P<0.01). On PID 10, the protein expression level of eNOS of wounds in HGF-ADSC exosome group was higher than that in PBS group, HGF alone group, and ADSC exosome alone group (with q values of 53.23, 42.54, and 26.54, respectively, all P<0.01); the ratio of p-PI3K to PI3K and the ratio of p-Akt to Akt of wounds in HGF-ADSC exosome group were significantly higher than those in PBS group, HGF alone group, and ADSC exosome alone group, respectively (with q values of 16.11, 11.78, 6.08, 65.54, 31.63, and 37.86, respectively, P<0.01). On PID 14, the length of skin tissue defect in the wounds of HGF-ADSC exosome group was shorter than that in PBS group, HGF alone group, and ADSC exosome alone group (with q values of 20.51, 18.50, and 11.99, respectively, all P<0.01); the CVF of wounds in HGF-ADSC exosome group was significantly higher than that in PBS group, HGF alone group and ADSC exosome alone group (with q values of 31.31, 28.52, and 12.35, respectively, all P<0.01). Conclusions: Human HGF-ADSC exosomes can significantly promote wound healing in diabetic mice by increasing neovascularization in wound tissue, and the mechanism may be related to the increased expression of eNOS in wounds by activating PI3K/Akt signaling pathway.

[Effect of P62 on the migration and motility of human epidermal cell line HaCaT in high glucose microenvironment and its mechanism].

Objective: To investigate the effect of P62 on the migration and motility of human epidermal cell line HaCaT in high glucose microenvironment and its possible molecular mechanism, so as to explore the mechanism of refractory diabetic foot wound healing. Methods: The method of experimental research was used. HaCaT cells in logarithmic growth phase was taken for experiment. The cells were collected and divided into normal control group (culture solution containing glucose with final molarity of 5.5 mmol/L) and high glucose (culture solution containing glucose with final molarity of 30.0 mmol/L) 24 h group, high glucose 48 h group, and high glucose 72 h group according to the random number table (the same grouping method below). The cells in normal control group were routinely cultured for 72 h, cells in high glucose 72 h group were cultured with high glucose for 72 h, cells in high glucose 48 h group were routinely cultured for 24 h then cultured with high glucose for 48 h, cells in high glucose 24 h group were routinely cultured for 48 h then cultured with high glucose for 24 h. Then the protein expression of P62 was detected by Western blotting. The cells were collected and divided into normal control group and high glucose group. After being correspondingly cultured for 48 h as before, the protein expression of P62 was detected by immunofluorescence method (indicated as green fluorescence). The cells were collected and divided into negative control small interfering RNA (siRNA) group, P62-siRNA-1 group, P62-siRNA-2 group, and P62-siRNA-3 group, and transfected with the corresponding reagents. At post transfection hour (PTH) 72, the protein expression of P62 was detected by Western blotting. The cells were collected and divided into normal glucose+negative control siRNA group, normal glucose+P62-siRNA group, high glucose+negative control siRNA group, and high glucose+P62-siRNA group. After the corresponding treatment, the protein expression of P62 was detected by Western blotting at PTH 72 h, the cell migration rate was detected and calculated at 24 h after scratching by scratch test, with the number of samples being 9; and the range of cell movement was observed and the trajectory velocity was calculated within 3 h under the living cell workstation, with the number of samples being 76, 75, 80, and 79 in normal glucose+negative control siRNA group, normal glucose+P62-siRNA group, high glucose+negative control siRNA group, and high glucose+P62-siRNA group, respectively. The cells were collected and divided into normal glucose+phosphate buffered solution (PBS) group, high glucose+PBS group, and high glucose+N-acetylcysteine (NAC) group. After the corresponding treatment, the protein expression of P62 at 48 h of culture was detected by Western blotting and immunofluorescence method, respectively. Except for scratch test and cell motility experiment, the number of samples was all 3 in the rest experiments. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: Compared with the protein expression in normal control group, the protein expressions of P62 of cells in high glucose 24 h group, high glucose 48 h group, and high glucose 72 h group were significantly increased (P<0.01). At 48 h of culture, the green fluorescence of P62 of cells in high glucose group was stronger than that in normal control group. At PTH 72, compared with the protein expression in negative control siRNA group, the protein expressions of P62 of cells in P62-siRNA-1 group, P62-siRNA-2 group, and P62-siRNA-3 group were significantly decreased (P<0.01). At PTH 72, compared with the protein expression in normal glucose+negative control siRNA group, the protein expression of P62 of cells in normal glucose+P62-siRNA group was significantly decreased (P<0.01), while the protein expression of P62 of cells in high glucose+negative control siRNA group was significantly increased (P<0.01); compared with the protein expression in high glucose+negative control siRNA group, the protein expression of P62 of cells in high glucose+P62-siRNA group was significantly decreased (P<0.01). At 24 h after scratching, compared with (55±7)% in normal glucose+negative control siRNA group, the cell migration rate in normal glucose+P62-siRNA group was significantly increased ((72±14)%, P<0.01), while the cell migration rate in high glucose+negative control siRNA group was significantly decreased ((37±7)%, P<0.01); compared with that in high glucose+negative control siRNA group, the cell migration rate in high glucose+P62-siRNA group was significantly increased ((54±10)%, P<0.01). Within 3 h of observation, the cell movement range in high glucose+negative control siRNA group was smaller than that in normal glucose+negative control siRNA group, while the cell movement range in normal glucose+P62-siRNA group was larger than that in normal glucose+negative control siRNA group, and the cell movement range in high glucose+P62-siRNA group was larger than that in high glucose+negative control siRNA group. Compared with that in normal glucose+negative control siRNA group, the cell trajectory speed in normal glucose+P62-siRNA group was significantly increased (P<0.01), while the cell trajectory speed in high glucose+negative control siRNA group was significantly decreased (P<0.01); compared with that in high glucose+negative control siRNA group, the cell trajectory speed in high glucose+P62-siRNA group was significantly increased (P<0.01). At 48 h of culture, compared with that in normal glucose+PBS group, the protein expression of P62 of cells in high glucose+PBS group was significantly increased (P<0.01); compared with that in high glucose+PBS group, the protein expression of P62 of cells in high glucose+NAC group was significantly decreased (P<0.01). At 48 h of culture, the green fluorescence of P62 of cells in high glucose+PBS group was stronger than that in normal glucose+PBS group, while the green fluorescence of P62 of cells in high glucose+NAC group was weaker than that in high glucose+PBS group. Conclusions: In HaCaT cells, high glucose microenvironment can promote the protein expression of P62; knockdown of P62 protein can promote the migration and increase the mobility of HaCaT cells; and the increase of reactive oxygen species in high glucose microenvironment may be the underlying mechanism for the increase of P62 expression.

The 16S rRNA Gene Sequencing of Gut Microbiota in Chickens Infected with Different Virulent Newcastle Disease Virus Strains.

Simple SummaryNewcastle disease (ND), which is caused by virulent Newcastle disease virus (NDV), is one of the most important viral diseases for chickens and birds. However, the intestinal pathogenesis of NDV is still poorly understood. To preliminarily investigate its intestinal pathogenesis mechanisms from the aspect of gut microbiota, the 16S rRNA gene sequencing technology was used to evaluate the gut microbiota composition changes post different virulent NDV infection. Results showed that different virulent NDV infection resulted in a different alteration of the gut microbiota in chickens, including a loss of probiotic bacteria and an expansion of some pathogenic bacteria. The above results suggest that NDV strains with different virulence have different impacts on chicken gut microbiota.Newcastle disease virus (NDV) which is pathogenic to chickens is characterized by dyspnea, diarrhea, nervous disorder and hemorrhages. However, the influence of different virulent NDV strain infection on the host gut microbiota composition is still poorly understood. In this study, twenty 21-day-old specific pathogen free (SFP) chickens were inoculated with either the velogenic Herts33 NDV strain, lentogenic La Sota NDV strain or sterile phosphate buffer solution (PBS). Subsequently, the fecal samples of each group were collected for 16S rRNA sequencing. The results showed that the gut microbiota were mainly dominated by Firmicutes, Bacteroidetes and Proteobacteria in both healthy and NDV infected chickens. NDV infection altered the structure and composition of gut microbiota. As compared to the PBS group, phylum Firmicutes were remarkably reduced, whereas Proteobacteria was significantly increased in the velogenic NDV infected group; the gut community structure had no significant differences between the lentogenic NDV infected group and the PBS group at phylum level. At genus level, Escherichia-Shigella was significantly increased in both the velogenic and lentogenic NDV infected groups, but the lactobacillus was only remarkably decreased in the velogenic NDV infected group. Collectively, different virulent strain NDV infection resulted in a different alteration of the gut microbiota in chickens, including a loss of probiotic bacteria and an expansion of some pathogenic bacteria. These results indicated that NDV strains with different virulence have different impacts on chicken gut microbiota and may provide new insights into the intestinal pathogenesis of NDV.

[Effects of transient receptor potential vanilloid type 4-specific activator on human vascular endothelial cell functions and blood supply of rat perforator flap and its mechanism].

Objective: To analyze the effects of transient receptor potential vanilloid type 4 (TRPV4) activation on the function and endothelial-to-mesenchymal transition (EndMT) of human umbilical vein endothelial cells (HUVECs), as well as to explore the effects of TRPV4 activation on blood perfusion and survival of rat perforator flap and the mechanism. Methods: The experimental research methods were used. The 3rd to 6th passages of HUVECs were used for experiments and divided into 0.5 μmol/L 4α-phorbol 12, 13-didecanoate (4αPDD) group, 1.0 μmol/L 4αPDD group, 3.0 μmol/L 4αPDD group, 10.0 μmol/L 4αPDD group, and phosphate buffer solution (PBS) group, which were cultivated in corresponding final molarity of 4αPDD and PBS, respectively. The cell proliferation activity at 6 and 12 h of culture was detected using cell counting kit-8 (CCK-8). Another batch of cells was acquired and divided into PBS group, 1 μmol/L 4αPDD group, and 3 μmol/L 4αPDD group, which were treated similarly as described before and then detected for cell proliferation activity at 6, 12, 24, and 48 h of culture. The residual scratch area of cells at post scratch hour (PSH) 12, 24, and 48 was detected by scratch test, and the percentage of the residual scratch area was calculated. The number of migrated cells at 24 and 48 h of culture was detected by Transwell experiment. The tube-formation assay was used to measure the number of tubular structures at 4 and 8 h of culture. The protein expressions of E-cadherin, N-cadherin, Slug, and Snail at 24 h of culture were detected by Western blotting. All the sample numbers in each group at each time point in vitro experiments were 3. A total of 36 male Sprague-Dawley rats aged 8 to 10 weeks were divided into delayed flap group, 4αPDD group, and normal saline group according to the random number table, with 12 rats in each group, and iliolumbar artery perforator flap models on the back were constructed. The flap surgical delay procedure was only performed in the rats in delayed flap group one week before the flap transfer surgery. Neither rats in 4αPDD group nor normal saline group had flap surgical delay; instead, they were intraperitoneally injected with 4αPDD and an equivalent mass of normal saline, respectively, at 10 min before, 24 h after, and 48 h after the surgery. The general state of flap was observed on post surgery day (PSD) 0 (immediately), 1, 4, and 7. The flap survival rates were assessed on PSD 7. The flap blood perfusion was detected by laser speckle contrast imaging technique on PSD 1, 4, and 7. The microvascular density in the flap's choke vessel zone was detected by immunohistochemical staining. All the sample numbers in each group at each time point in vivo experiments were 12. Data were statistically analyzed with analysis of variance for factorial design, analysis of variance for repeated measurement, one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: At 6 and 12 h of culture, there were no statistically significant differences in cell proliferation activity in the overall comparison among PBS group, 0.5 μmol/L 4αPDD group, 1.0 μmol/L 4αPDD group, 3.0 μmol/L 4αPDD group, and 10.0 μmol/L 4αPDD group (P>0.05). At 6, 12, 24, and 48 h of culture, there were no statistically significant differences in cell proliferation activity in the overall comparison among PBS group, 1 μmol/L 4αPDD group, and 3 μmol/L 4αPDD group (P>0.05). At PSH 12, the percentages of the residual scratch area of cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were close to that in PBS group (P>0.05). At PSH 24 and 48, compared with those in PBS group, the percentages of the residual scratch area of cells in 3 μmol/L 4αPDD group were significantly decreased (with t values of 2.83 and 2.79, respectively, P<0.05), while the percentages of the residual scratch area of cells in 1 μmol/L 4αPDD group showed no significant differences (P>0.05). At 24 h of culture, the number of migrated cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were close to that in PBS group (P>0.05). At 48 h of culture, the number of migrated cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD groups were significantly greater than that in PBS group (with t values of 6.20 and 9.59, respectively, P<0.01). At 4 h of culture, the numbers of tubular structures of cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were significantly greater than that in PBS group (with t values of 4.68 and 4.95, respectively, P<0.05 or <0.01). At 8 h of culture, the numbers of tubular structures of cells in 1 μmol/L 4αPDD and 3 μmol/L 4αPDD groups were similar to that in PBS group (P>0.05). At 24 h of culture, compared with those in PBS group, the protein expression level of E-cadherin of cells in 3 μmol/L 4αPDD group was significantly decreased (t=5.13, P<0.01), whereas there was no statistically significant difference in the protein expression level of E-cadherin of cells in 1 μmol/L 4αPDD group (P>0.05); the protein expression level of N-cadherin of cells in 3 μmol/L 4αPDD group was significantly increased (t=4.93, P<0.01), whereas there was no statistically significant difference in the protein expression level of N-cadherin of cells in 1 μmol/L 4αPDD group (P>0.05); the protein expression levels of Slug of cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were significantly increased (with t values of 3.85 and 6.52, respectively, P<0.05 or P<0.01); and the protein expression level of Snail of cells in 3 μmol/L 4αPDD group was significantly increased (t=4.08, P<0.05), whereas there was no statistically significant difference in the protein expression level of Snail of cells in 1 μmol/L 4αPDD group (P>0.05). There were no statistically significant differences in the protein expression levels of E-cadherin, N-cadherin, Slug, or Snail of cells between 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group (P>0.05). The general condition of flaps of rats in the three groups was good on PSD 0. On PSD 1, the flaps of rats in the three groups were basically similar, with bruising and swelling at the distal end. On PSD 4, the swelling of flaps of rats in the three groups subsided, and the distal end turned dark brown and necrosis occurred, with the area of necrosis in flaps of rats in normal saline group being larger than the areas in 4αPDD group and delayed flap group. On PSD 7, the necrotic areas of flaps of rats in the 3 groups were fairly stable, with the area of necrosis at the distal end of flap of rats in delayed flap group being the smallest. On PSD 7, the flap survival rates of rats in 4αPDD group ((80±13)%) and delayed flap group ((87±9)%) were similar (P>0.05), and both were significantly higher than (70±11)% in normal saline group (with t values of 2.24 and 3.65, respectively, P<0.05 or P<0.01). On PSD 1, the overall blood perfusion signals of rats in the 3 groups were basically the same, and the blood perfusion signals in the choke vessel zone were relatively strong, with a certain degree of underperfusion at the distal end. On PSD 4, the boundary between the surviving and necrotic areas of flaps of rats in the 3 groups became evident, and the blood perfusion signals in the choke vessel zone were improved, with the normal saline group's distal hypoperfused area of flap being larger than the areas in delayed flap group and 4αPDD group. On PSD 7, the blood perfusion signals of overall flap of rats had generally stabilized in the 3 groups, with the intensity of blood perfusion signal in the choke vessel zone and overall flap of rats in delayed flap group and 4αPDD group being significantly greater than that in normal saline group. On PSD 7, the microvascular density in the choke vessel zone of flap of rats in 4αPDD group and delayed flap group were similar (P>0.05), and both were significantly higher than that in normal saline group (with t values of 4.11 and 5.38, respectively, P<0.01). Conclusions: After activation, TRPV4 may promote the migration and tubular formation of human vascular endothelial cells via the EndMT pathway, leading to the enhanced blood perfusion of perforator flap and microvascular density in the choke vessel zone, and therefore increase the flap survival rate.

[Effects of exosomes from human adipose-derived mesenchymal stem cells on inflammatory response of mouse RAW264.7 cells and wound healing of full-thickness skin defects in mice].

Objective: To investigate the effects of exosomes from human adipose-derived mesenchymal stem cells (ADSCs) on inflammatory response of mouse RAW264.7 cells and wound healing of full-thickness skin defects in mice. Methods: The experimental research methods were adopted. The discarded adipose tissue was collected from 3 female patients (aged 10-25 years) who underwent abdominal surgery in the First Affiliated Hospital of Air Force Medical University. ADSCs were extracted from the adipose tissue by collagenase Ⅰ digestion and identified with flow cytometry. Exosomes were extracted from the human ADSCs by differential ultracentrifugation, the morphology of the exosomes was observed by transmission electron microscopy, the particle diameter of the exosomes was detected by nanoparticle tracking analyzer, and the protein expressions of CD9, CD63, tumor susceptibility gene 101 (TSG101), and β-actin were detected by Western blotting. The human ADSCs exosomes (ADSCs-Exos) and RAW264.7 cells were co-cultured for 12 h, and the uptake of RAW264.7 cells for human ADSCs-Exos was observed. The RAW264.7 cells were divided into phosphate buffer solution (PBS) group stimulated with PBS for suitable time, endotoxin/lipopolysaccharide (LPS) stimulation 2 h group, LPS stimulation 4 h group, LPS stimulation 6 h group, LPS stimulation 12 h group, and LPS stimulation 24 h group stimulated with LPS for corresponding time, with 3 wells in each group, and the mRNA expressions of interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), IL-6, and IL-10 were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) method. The RAW264.7 cells were divided into PBS group, LPS alone group, and LPS+ADSCs-Exos group, with 3 wells in each group, which were dealt correspondingly for the time screened out in the previous experiment, the mRNA expressions of IL-1β, TNF-α, IL-6, IL-10, trasforming growth factor β (TGF-β,) and vascular endothelial growth factor (VEGF) were detected by real time fluorescence quantitative RT-PCR method, and the protein expressions of inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1) were detected by Western blotting. Twenty-four 8-week-old male BALB/c mice were divided into PBS group and ADSCs-Exos group according to the random number table, with 12 mice in each group, and a full-thickness skin defect wound with area of 1 cm×1 cm was inflicted on the back of each mouse. Immediately after injury, the wounds of mice in the two groups were dealt correspondingly. On post injury day (PID) 1, the concentration of IL-1β and TNF-α in serum were detected by enzyme-linked immunosorbent assay, and the mRNA expressions of IL-1β, TNF-α, and IL-6 were detected by real time fluorescence quantitative RT-PCR method. On PID 3, 6, 9, 12, and 15, the wound healing was observed and the wound non-healing rate was calculated. On PID 15, the defect length of skin accessory and collagen volume fraction (CVF) were detected by hematoxylin eosin staining and Masson staining, respectively, the CD31 expression and neovascularization were detected by immunohistochemistry, and the ratio of Ki67 positive cells, the ratio of iNOS and Arg1 double positive cells, and the ratio of iNOS positive cells to Arg1 positive cells and their fluorescence intensities were detected by immunofluorescence method. The number of samples in animal experiments was 6. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and independent sample t test. Results: At 12 h of culture, the cells exhibited a typical spindle shape, which were verified as ADSCs with flow cytometry. The exosomes with a vesicular structure and particle diameters of 29-178 nm, were positively expressed CD9, CD63, and TSG101 and negatively expressed β-actin. After 12 h of co-culture, the human ADSCs-Exos were endocytosed into the cytoplasm by RAW264.7 cells. The mRNA expressions of IL-1β, TNF-α, IL-6, and IL-10 of RAW264.7 cells in LPS stimulation 2 h group, LPS stimulation 4 h group, LPS stimulation 6 h group, LPS stimulation 12 h group, and LPS stimulation 24 h group were significantly higher than those in PBS group (with t) values of 39.10, 14.55, 28.80, 4.74, 48.80, 22.97, 13.25, 36.34, 23.12, 18.71, 29.19, 41.08, 11.68, 18.06, 8.54, 43.45, 62.31, 22.52, 21.51, and 37.13, respectively, P<0.01). The stimulation 12 h with significant expressions of all the inflammatory factors was selected as the time point in the following experiment. After stimulation of 12 h, the mRNA expressions of IL-1β, TNF-α, IL-6, and IL-10 of RAW264.7 cells in LPS alone group were significantly higher than those in PBS group (with t values of 44.20, 51.26, 14.71, and 8.54, respectively, P<0.01); the mRNA expressions of IL-1β, TNF-α, and IL-6 of RAW264.7 cells in LPS+ADSCs-Exos group were significantly lower than those in LPS alone group (with t values of 22.89, 25.51, and 8.03, respectively, P<0.01), while the mRNA expressions of IL-10, TGF-β, and VEGF were significantly higher than those in LPS alone group (with t values of 9.89, 13.12, and 7.14, respectively, P<0.01). After stimulation of 12 h, the protein expression of iNOS of RAW264.7 cells in LPS alone group was significantly higher than that in PBS group and LPS+ADSCs-Exos group, respectively (with t values of 11.20 and 5.06, respectively, P<0.05 or P<0.01), and the protein expression of Arg1 was significantly lower than that in LPS+ADSCs-Exos group (t=15.01, P<0.01). On PID 1, the serum concentrations of IL-1β and TNF-α and the mRNA expressions of IL-1β, TNF-α, and IL-6 in wound tissue of mice in ADSCs-Exos group were significantly those in lower than PBS group (with t values of 15.44, 12.24, 9.24, 7.12, and 10.62, respectively, P<0.01). On PID 3, 6, 9, 12, and 15 d, the wound non-healing rates of mice in ADSCs-Exos group were (73.2±4.1)%, (53.8±3.8)%, (42.1±5.1)%, (24.1±2.8)%, and 0, which were significantly lower than (82.5±3.8)%, (71.2±4.6)%, (52.9±4.1)%, (41.5±3.6)%, and (14.8±2.5)% in PBS group, respectively (with t values of 4.77, 8.93, 5.54, 7.63, and 7.59, respectively, P<0.01). On PID 15, the defect length of skin accessory in wounds of mice in PBS group was significantly longer than that in ADSCs-Exos group (t=9.50, P<0.01), and the CVF was significantly lower than that in ADSCs-Exos group (t=9.15, P<0.01). On PID 15, the CD31 expression and the number of new blood vessels (t=12.99, P<0.01), in wound tissue of mice in ADSCs-Exos group were significantly more than those in PBS group, and the ratio of Ki67 positive cells was significantly higher than that in PBS group (t=7.52, P<0.01). On PID 15, the ratio of iNOS and Arg1 double positive cells in wound tissue of mice in PBS group was (12.33±1.97)%, which was significantly higher than (1.78±0.29)% in ADSCs-Exos group (t=13.04, P<0.01), the ratio of iNOS positive cells and the fluorescence intensity of iNOS were obviously higher than those of ADSCs-Exos group, and the ratio of Arg1 positive cells and the fluorescence intensity of Arg1 were obviously lower than those of ADSCs-Exos group. Conclusions: The human ADSCs-Exos can alleviate inflammatory response of mouse RAW264.7 cells, decrease macrophage infiltration and secretion of the pro-inflammatory cytokines, increase the secretion of anti-inflammatory cytokines to promote neovascularization and cell proliferation in full-thickness skin defect wounds of mice, hence accelerating wound healing.

The effect and mechanism of exosomes derived from human amniotic epithelial cells on the proliferation and migration of HaCaT in high glucose environment

Objective: To investigate the effect and mechanism of exosomes derived from human amniotic epithelial cells (hAEC-Exos) on the proliferation and migration of HaCaT in high glucose environment. Methods: The experimental research method was adopted. The amniotic membrane tissue was collected from 10 healthy pregnant women at full term delivery in the Department of Obstetrics and Gynecology of Fujian Medical University Union Hospital from January to June 2019, and the primary human amniotic epithelial cells (hAECs) were isolated. The growth status and morphological changes of the primary hAECs on the 2nd, 4th, and 7th day of culture were observed, and the expressions of the cells surface markers of CD73, CD90, CD29, CD34, and human leukocyte antigen DR (HLA-DR). The 2nd to 4th passages of hAECs were used in the following experiments. The hAEC-Exos were separated by ultracentrifugation method. The HaCaT and hAEC-Exos were co-cultured for 3 h, and the uptake of hAEC-Exos by HaCaT was observed by inverted fluorescence microscopy. The HaCaT were divided into phosphate buffer solution (PBS) group and hAEC-Exos group or dimethyl sulfoxide (DMSO)+PBS group, DMSO+hAEC-Exos group, and LY294002+hAEC-Exos group, which were dealt correspondingly, with 3 wells in each group. Cell counting kit 8 (CCK-8) method was used to detect cell proliferation activity after 0 (immediately), 12, 24, 36, 48, and 60 h of culture. The scratch test was conducted to detect the scratch healing at 0, 24, 48, and 72 h after the scratch, and the scratch healing rate was calculated, respectively. The Transwell experiment was conducted to detect the number of transmembrane cells after 48 h of culture. The Western blotting was used to detect the protein expressions of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), protein kinase B (Akt), and phosphorylated Akt (p-Akt) related to phosphatidylinositol 3-kinase-Akt-mTOR (PI3K-Akt-mTOR) pathway after 24 h of culture. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and independent sample t test. Results: Most of the primary hAECs were oval and uniform in size on the 2nd day of culture. The hAECs were arranged in a typical cobblestone-like monolayer on the 4th and 7th day of culture. The primary hAECs highly expressed CD73, CD90, and CD29 of mesenchymal stem cell related surface markers, and were with no or low expressions of CD34 and HLA-DR of hematopoietic stem cell related surface markers. After 3 h of culture, hAEC-Exos were successfully endocytosed by HaCaT into the cytoplasm and gathered around the nucleus. After 12, 24, 36, 48, and 60 h of culture, the proliferation activity of HaCaT in hAEC-Exos group was significantly higher than that in PBS group (t=3.691, 10.861, 12.121, 10.531, 14.931, P<0.01). At 24, 48, and 72 h after scratch, the scratch healing rates of HaCaT in PBS group were significantly lower than those in hAEC-Exos group (t=3.342, 6.427, 5.485, P<0.05 or P<0.01). After 48 h of culture, the number of transmembrane HaCaT in hAEC-Exos group was significantly more than that in PBS group (t=5.385, P<0.01). After 24 h of culture, the protein expressions of p-mTOR and p-Akt in HaCaT of hAEC-Exos group were significantly higher than those in PBS group (t=4.240, 5.586, P<0.01), while the protein expressions of mTOR and Akt in HaCaT of the two groups were similar (P>0.05). After 24 h of culture, the protein expressions of p-mTOR and p-Akt in HaCaT of DMSO+hAEC-Exos group were significantly higher than those in DMSO+PBS group (t=6.155, 8.338, P<0.01) and LY294002+hAEC-Exos group (t=5.030, 3.960, P<0.01), while the protein expressions of mTOR and Akt in HaCaT of the three groups were similar (P>0.05). The proliferation activity of HaCaT in DMSO+hAEC-Exos group at 12, 24, 36, 48, and 60 h of culture was 0.78±0.05, 1.23±0.07, 1.60±0.09, 1.86±0.09, and 2.03±0.08, which was significantly higher than 0.46±0.04, 0.69±0.07, 0.98±0.08, 1.16±0.08, and 1.26±0.11 in DMSO+PBS group (t=4.376, 7.398, 8.488, 9.766, 10.730, P<0.01). The proliferation activity of HaCaT in DMSO+hAEC-Exos group at 24, 36, 48, and 60 h was significantly higher than 0.96±0.09, 1.20±0.08, 1.39±0.08, and 1.55±0.10 in LY294002+hAEC-Exos group (t=3.639, 5.447, 6.605, 6.693, P<0.05 or P<0.01). The scratch healing rates of HaCaT in DMSO+hAEC-Exos group at 24, 48, and 72 h after scratch were significantly higher than those in DMSO+PBS group (t=4.003, 6.349, 7.714, P<0.01) and LY294002+hAEC-Exos group (t=3.805, 4.676, 4.067, P<0.05 or P<0.01). After 48 h of culture, the number of transmembrane HaCaT in DMSO+hAEC-Exos group was significantly more than that in DMSO+PBS group and LY294002+hAEC-Exos group, respectively (t=7.464, 1.232, P<0.01). Conclusions: PI3K-Akt-mTOR pathway can promote the proliferation and migration of HaCaT in high glucose environment by mediating hAEC-Exos.

[Effects and mechanism of hepatocyte growth factor-modified human adipose mesenchymal stem cells on wound healing of full-thickness skin defects in diabetic rats].

Objective: To investigate the effects and mechanism of hepatocyte growth factor (HGF)-modified human adipose mesenchymal stem cells (ADSCs) on the wound healing of full-thickness skin defects in diabetic rats. Methods: The experimental research method was adopted. The discarded abdominal adipose tissue was collected from a 35-year-old healthy female who underwent abdominal liposuction in the Department of Plastic Surgery of the First Affiliated Hospital of Air Force Medical University in December 2019. The long spindle-shaped primary ADSCs were obtained by collagenase digestion, and the third passage of cells were identified by flow cytometry to positively express ADSCs surface markers CD29 and CD90 and negatively express CD34 and CD45. The third passage of ADSCs were used for the subsequent experiments. ADSCs were transfected with lentivirus-mediated HGF for 4 h (obtaining HGF modified ADSCs) and then routinely cultured for 24 h. The cell morphology was observed under an inverted phase contrast microscope, and the transfection rate was calculated. Eighty-one male Sprague-Dawley rats aged 4 weeks were induced into diabetic rat model by high glucose and high fat diet combined with streptozotocin injection. A full-thickness skin defect wound of 1.5 cm×1.5 cm was made on the back of each rat. The injured rats were divided into phosphate buffer solution (PBS) group, ADSCs alone group, and HGF-modified ADSCs group according to the random number table, with 27 rats in each group. The rats were injected with the same volume of corresponding substances around the wound on post injury day (PID) 1, 3, and 7, respectively. Nine rats in each group were selected according to the random number table, the wound area of whom was measured on PID 0 (immediately), 3, 7, 10, and 14 (after injection on injection day), and the wound healing rates on PID 3, 7, 10, and 14 were calculated. Nine remaining rats in each group were sacrificed after injection on PID 3 and 7, respectively, and the skin tissue around the wound were collected. The mRNA expressions of inflammatory factors such as tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-10 on PID 3 and collagen type Ⅰ and Ⅲ on PID 7 were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The expression level of vascular endothelial growth factor (VEGF) was detected by enzyme-linked immunosorbent assay on PID 7. The protein expression of nuclear factor κb-p65 on PID 3 and phosphorylation level of protein kinase B (Akt) on PID 7 were detected by Western blotting. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: After 24 h of culture, the HGF-transfected human ADSCs showed good morphology, which was not different with the non-transfected ADSCs, and the transfection rate reached 90%. On PID 3, 7, 10, and 14, the wound healing rates of rats in HGF-modified ADSCs group were (31.5±1.0)%, (75.2±2.0)%, (92.2±1.3)%, and (99.1±1.8)%, respectively, being significantly higher than (21.4±1.3)%, (61.4±1.5)%, (80.1±2.1)%, and (92.4±1.8)% in PBS group and (25.1±2.1)%, (67.2±1.3)%, (89.3±1.4)%, and (95.1±2.1)% in ADSCs alone group (t=1.452, 0.393, 0.436, 0.211, 4.982, 3.011, 4.211, 7.503, P<0.05 or P<0.01). On PID 3, compared with those in PBS group and ADSCs alone group, the mRNA expressions of TNF-α and IL-1β and protein expression of nuclear factor κb-p65 in the skin tissue around the wound of rats in HGF-modified ADSCs group were significantly decreased (t=7.281, 17.700, 9.447, 6.231, 13.083, 7.783, P<0.01), and the mRNA expression of IL-10 in the skin tissue around the wound of rats in HGF-modified ADSCs group was significantly increased (t=-6.644, -6.381, P<0.01). On PID 7, compared with those in PBS group and ADSCs alone group, the mRNA expressions of collagen type Ⅰ and Ⅲ, the expression level of VEGF, and the phosphorylation level of Akt in the skin tissue around the wound of rats in HGF-modified ADSCs group were significantly increased (t=-5.126, -4.347, -5.058, -3.367, -10.694, -19.876, -4.890, -6.819, P<0.05 or P<0.01). Conclusions: HGF-modified human ADSCs can significantly promote the wound healing of full-thickness skin defects in diabetic rats. The mechanism may be related to the inhibition of TNF-α and IL-1β expression, the promotion of IL-10, collagen type Ⅰ and Ⅲ, and VEGF expression, which could be related to the inhibition of nuclear factor κB signaling pathway, and the promotion of Akt signaling pathway.

[Effects and mechanism of norepinephrine on the migration of bone marrow mesenchymal stem cells in mice].

Objective: To investigate the effects and mechanism of sympathetic neurotransmitter norepinephrine (NE) on the migration of bone marrow mesenchymal stem cells (BMSCs) in mice. Methods: (1) Twenty 3-week-old male C57BL/6 mice were sacrificed for isolating, culturing, and identifying BMSCs from the femur and tibia. Cells of the second or third passages were divided into phosphate buffer solution (PBS) group, 1 μmol/L NE group, 10 μmol/L NE group, and 100 μmol/L NE group, with 8 wells in each group. Cells in 1 μmol/L NE group, 10 μmol/L NE group, and 100 μmol/L NE group were cultured in low-sugar Dulbecco's modified eagle medium containing 1% volume fraction of fetal bovine serum (hereinafter referred to as low-serum medium) added with NE in final molarity of 1 μmol/L, 10 μmol/L, and 100 μmol/L, respectively. Cells in PBS group were cultured in low-serum medium added with the same volume of PBS. Before stimulation (0 d) and on stimulation day 1, 3, 5, cell counting kit 8 method was used to detect cell proliferation activity (expressed as the absorbance value). (2) In cell scratch test 1, cells were divided into PBS group and simple NE group. After the scratch test, cells in simple NE group were cultured with low-serum medium+ NE in final molarity of 10 μmol/L, and cells in PBS group were cultured with low-serum medium+ the same volume of PBS. In cell scratch test 2, cells were divided into PBS group, propranolol+ NE group, and phentolamine+ NE group. After the scratch test, cells in propranolol+ NE group were pretreated with low-serum medium+ propranolol in final molarity of 1 μmol/L for 30 minutes each day, cells in phentolamine+ NE group were pretreated with low-serum medium+ phentolamine in final molarity of 10 μmol/L for 30 minutes each day, and then they were cultured with low-serum medium+ NE in final molarity of 10 μmol/L. Cells in PBS group were cultured with low-serum medium+ the same volume of PBS. In cell scratch test 3, cells were divided into simple NE group, simple (2E, 6E)-2, 6-bis (4-pyridylmethylene) cyclohexanone (SC-66) group, and SC-66+ NE group. After the scratch test, cells in simple NE group was cultured with low-serum medium+ NE in final molarity of 10 μmol/L, cells in simple SC-66 group were cultured with low-serum medium after being pretreated with SC-66 in final molarity of 30 mmol/L for 30 minutes every day, cells in SC-66+ NE group were cultured with low-serum medium+ NE in final molarity of 10 μmol/L after being pretreated with SC-66 in final molarity of 30 mmol/L for 30 minutes every day. In the above 3 cell scratch tests, the sample numbers in each group were all 6, and the scratch healing rates at post scratch hour (PSH) 24, 48, and 72 were all calculated. (3) Cells were divided into PBS group, simple NE group, propranolol+ NE group, and phentolamine+ NE group, with 3 wells in each group. The lower chamber treatment methods of PBS group and simple NE group were the same as those of the same groups in cell scratch test 1. The lower chamber treatment of propranolol+ NE group and phentolamine+ NE group were the same as those of the same groups in cell scratch test 2. After the Transwell experiment was performed and the cells were routinely cultured for 24 hours, the migrated cells were counted. (4) Cells were divided into PBS group, simple NE group, propranolol+ NE group, and phentolamine+ NE group, with 2 dishes in each group. The cell treatment of PBS group and simple NE group were the same as those of the same groups in cell scratch test 1. The cell treatment of propranolol+ NE group and phentolamine+ NE group were the same as those of the same groups in cell scratch test 2. After 24 hours of routine culture, the phosphorylation level of protein kinase B (Akt) of cells was detected by Western blotting. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, independent sample t test, least significant difference t test, and Bonferroni correction. Results: (1) After 1 day of stimulation, the absorbance value of cells in 100 μmol/L NE group was significantly lower than that in PBS group (t=2.986, P<0.05). After 5 days of stimulation, the absorbance value of cells in 10 μmol/L NE group was significantly higher than that in PBS group (t=3.547, P<0.01). (2) In cell scratch test 1, at PSH 24, 48, and 72, the scratch healing rates of cells in simple NE group were (34.4±3.4)%, (52.5±4.7)%, and (70.0±3.8)%, which were significantly lower than (44.1±4.2)%, (80.0±3.6)%, and (95.9±2.2)% in PBS group (t=19.320, 128.319, 221.575, P<0.01). In cell scratch test 2, at PSH 24, 48, and 72, the scratch healing rates of cells in propranolol+ NE group were significantly lower than those in PBS group (t=4.073, 9.618, 15.272, P<0.01). In cell scratch test 3, at PSH 72, the scratch healing rates of cells in NE group was significantly lower than that in simple SC-66 group (t=8.862, P<0.01). At PSH 24, 48, and 72, the scratch healing rates of cells in SC-66+ NE group were significantly lower than those in simple SC-66 group (t=3.862, 4.290, 10.357, P<0.01). (3) The Transwell experiment showed that after 24 hours of culture, the numbers of migrated cells in simple NE group, propranolol+ NE group, and phentolamine+ NE group were significantly less than the number in PBS group (t=11.895, 10.196, 3.222, P<0.01). (4) After 24 hours of culture, the phosphorylation levels of Akt of cells in simple NE group and propranolol+ NE group were significantly higher than the level in PBS group (t=8.186, 5.996, P<0.01). Conclusions: NE can inhibit the migration of BMSCs in mice, a process in which the signal pathway of Akt is involved in its regulation.

[Effects of Astragaloside Ⅳ on exosome secretion and its microRNA-126 expression in human endothelial progenitor cells].

Objective: To investigate the effects of Astragaloside Ⅳ on the secretion of exosomes in human endothelial progenitor cells (EPCs) and the expression of microRNA (miRNA)-126 in exosomes. Methods: The umbilical cord blood from one healthy full-term newborn from the Department of Obstetrics and Gynecology of the First Affiliated Hospital of Hunan University of Traditional Chinese Medicine in 2019 was harvested for isolating mononuclear cells by density gradient centrifugation and cultured for 7 days. Morphological observation was performed during this period. Cells of the third passage were collected for identification by CD31 immunomagnetic bead sorting and double fluorescence staining. According to the random number table, the identified EPCs were divided into Astragaloside Ⅳ group and phosphate buffer solution (PBS) group. The cells in Astragaloside Ⅳ group were cultured with Astragaloside Ⅳ in final mass concentration of 100 mg/L for 24 hours, and the cells in PBS group were cultured with the same volume of PBS for 24 hours. After culture, the exosomes from the cell culture supernatant of the two groups were collected, and the expressions of characteristic markers of exosomes CD9, CD63, and CD81 were detected by Western blotting, the morphology of EPC exosomes (EPC-Exos) was observed under transmission electron microscope, and the particle size of EPC-Exos was detected by nanoparticle tracking analysis technique. The concentration of EPC-Exos was determined by dioctyl butyric acid method (the sample number was 3), and the expressions of miRNA-126-3p and miRNA-126-5p related to angiogenesis in EPC-Exos were determined by reverse transcription polymerase chain reaction (the sample number was 3). Data were statistically analyzed with independent sample t test. Results: (1) On the 4th day of culture, the cells began to adhere to the wall, and the multi-forms such as circle, fusiform, and strip appeared at the same time. On the 7th day of culture, the edge of the cells was clear and arranged like a paving stone, the central cells were round, and the surrounding cells were fusiform. (2) CD31 immunomagnetic beads sorting method identification showed that the membrane was stained with green fluorescence and the nucleus was stained with blue fluorescence. Double fluorescence staining method showed that the cells were orange-yellow. The cells were identified as EPCs. (3) After 24 hours of culture, the expressions of CD9, CD63, and CD81 in EPC-Exos were all positive, confirming that EPC-Exos were extracted successfully in this experiment. (4) After 24 hours of culture, the EPC-Exos of the two groups showed round membrane vesicles, and there was no significant difference in morphology. (5) After 24 hours of culture, the particle size of 98.7% EPC-Exos in Astragaloside Ⅳ group was 84.7 to 143.1 nm, and that of 98.0% EPC-Exos in PBS group was 88.7 to 123.5 nm. (6) After 24 hours of culture, the mass concentration of EPC-Exos in Astragaloside Ⅳ group was (310±5) μg/mL, which was significantly higher than (257±5) μg/mL in PBS group, t=13.369, P<0.01. (7) After 24 hours of culture, there were more miRNA-126-3p (t=16.062, P<0.01) and miRNA-126-5p (t=3.252, P<0.05) in EPC-Exos of Astragaloside Ⅳ group than in PBS group. Conclusions: Astragaloside Ⅳ can improve the function of human EPC secretory exosomes, and the secreted exosomes are loaded with miRNA-126.

[Mechanism study of dendritic epidermal T lymphocytes in promoting healing of full-thickness skin defects wound on mice by regulating the proliferation and differentiation of epidermal stem cells in mice].

Objective: To explore the mechanism of dendritic epidermal T lymphocytes (DETCs) in promoting healing of full-thickness skin defect wound on mice by regulating the proliferation and differentiation of epidermal stem cells (ESCs) in mice. Methods: (1) Ten 8-week-old wild type (WT) male C57BL/6 mice (the same sex and kind below) were sacrificed to collect the skin of back for extracting DETCs to culture. Five WT and five 8-week-old T cell receptor (TCR) δ(-)/(-) mice were selected and enrolled in WT control group and TCR δ(-)/(-) control group, respectively. A full-thickness skin defect wound with diameter of 6 mm was made on both sides of spinal line on the back of mice without any treatment after injury. Another fifteen 8-week-old TCR δ(-)/(-) mice were selected and divided into phosphate buffer solution (PBS), DETC, and insulin-like growth factor-Ⅰ(IGF-Ⅰ) groups according to the random number table (the same grouping method below), with 5 mice in each group, and the same full-thickness skin defect wound was made on each mouse. Immediately after injury, mice in PBS, DETC, and IGF-Ⅰ groups were injected subcutaneously around each wound with 10 μL sterile PBS , DETCs (cell concentration of 1×10(6)/mL), and 5 mg/mL recombinant mice IGF-Ⅰ, respectively. The percentage of the residual wound area was calculated on post injury day (PID) 2, 4, 6, and 8. (2) Three 8-week-old WT mice were enrolled in WT control group and nine 8-week-old TCR δ(-)/(-) mice were divided into TCR δ(-)/(-) control group, PBS group, and DETC group, with 3 mice in each group. The full-thickness skin defect wound was made as in experiment (1) . On PID 3, the protein expression of IGF-Ⅰ in the epidermis tissue of wound margin was detected by chemiluminescence imaging analyzer. (3) Three 8-week-old WT mice were enrolled in WT control group and six 8-week-old TCR δ(-)/(-) mice were divided into PBS and DETC groups, with 3 mice in each group, and the full-thickness skin defect wound was made as in experiment (1). On PID3, DETCs were extracted from the wound margin epidermis tissue to detect the percentage of DETCs expressing IGF-Ⅰ by flow cytometer. (4) The mice were taken as in experiment (2) and divided into WT control, PBS, DETC, and IGF-Ⅰ groups. A straight full-thickness skin defect incision with length of 3 cm was made in the direction of one inner ear. Mice in WT control group didn't have any other treatment after injury, and immediately after injury, mice in PBS, DETC, and IGF-Ⅰ groups were injected subcutaneously around each wound with 10 μL sterile PBS, DETCs (cell concentration of 1×10(6)/mL), and 5 mg/mL recombinant mice IGF-Ⅰ, respectively. On PID 12, epidermis tissue of wound margin was collected, and immunofluorescence staining was performed to observe the number of keratin 15 positive cells. (5) The same mice were collected, grouped, and treated as in experiment (4). On PID12, the epidermis tissue of wound margin was collected and immunofluorescence staining was performed to observe the number of keratin 10 positive cells. (6) Twenty 3-day-old WT mice (the same below) were sacrificed to collect the whole skin, which was used to extract ESCs, with 5 mice detecting one index. The ESCs were divided into DETC co-culture group and control group, which were added with 1 mL DETCs (cell concentration of 1.25×10(6)/mL) and DETC medium, respectively. The percentage of 5-ethynyl-2'-deoxyuridine (EdU) positive cell on culture day (CD) 3, the percentages of CD49f(+) CD71(-) and keratin 14 positive cells on CD 5, and the percentage of keratin 10 positive cell on CD 10 in 2 groups were detected by flow cytometer. (7) Twenty mice were taken to extract ESCs, with 5 mice detecting one index. The ESCs were divided into control group and IGF-Ⅰ group, which were added with 1 mL sterile PBS and 10 ng/mL recombinant mice IGF-Ⅰ, respectively. The percentages of EdU positive cell, CD49f(+) CD71(-) cell, keratin10 positive cell, and keratin 14 positive cell were detected as in experiment (6). The sample in each group of experiments (6) and (7) was three. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and t test. Results: (1) On PID 4, 6, and 8, the percentage of residual wound area in TCR δ(-)/(-) control group was significantly higher than that in WT control group (t=2.78, 3.39, 3.66, P<0.05 or P<0.01). The percentage of residual wound area in DETC group and IGF-Ⅰgroup on PID 4, 6, and 8 was apparently lower than that in PBS group (t=2.61, 3.21, 3.88, 2.84, 2.91, 2.49, P<0.05 or P<0.01). (2) On PID 3, the protein expression of IGF-Ⅰ in the epidermis tissue of wound margin of mice in TCR δ(-)/(-) control group was significantly lower than that in WT control group (t=17.34, P<0.01). The protein expression of IGF-Ⅰ in the epidermis tissue of wound margin of mice in DETC group was significantly higher than that in PBS group (t=11.71, P<0.01). (3) On PID 3, the percentage of DETCs expressing IGF-Ⅰ in the epidermis tissue of wound margin of mice in PBS group was significantly lower than that in WT control group and DETC group (t=24.95, 27.23, P<0.01). (4) On PID 12, the number of keratin 15 positive cells in the epidermis tissue of wound margin of mice in PBS group was significantly lower than that in WT control group, DETC group, and IGF-Ⅰ group (t=17.97, 11.95, 7.63, P<0.01). (5) The number of keratin 10 positive cells in the epidermis tissue of wound margin of mice in PBS group was significantly higher than that in WT control group, DETC group, and IGF-Ⅰ group (t=11.59, 9.51, 3.48, P<0.05 or P<0.01). (6) The percentages of EdU positive cells on CD 3, CD49f(+) CD71(-) cells on CD 5, and keratin 14 positive cells on CD 5 in DETC co-culture group were respectively (43.5±0.6)%, (66.5±0.5)%, (69.3±1.7)%, apparently higher than (32.3±1.3)%, (56.4±0.3)%, (54.9±1.3)% in control group (t=7.97, 17.10, 6.66, P<0.01). The percentage of keratin 10 positive cells on CD 10 in DETC co-culture group was (55.7±0.7)%, significantly lower than (67.1±1.2)% in control group (t=8.34, P<0.01). (7) The percentages of EdU positive cells on CD 3, CD49f(+) CD71(-) cells on CD 5, and keratin 14 positive cells on CD 5 in IGF-Ⅰ group were respectively (42.1±0.9)%, (81.1±1.3)%, (66.8±1.0)%, apparently higher than (32.4±0.7)%, (74.9±0.7)%, (52.0±1.9)% in control group (t=8.39, 4.24, 7.25, P<0.05 or P<0.01). The percentage of keratin 10 positive cells on CD 10 in IGF-Ⅰ group was (53.5±1.1)% , significantly lower than (58.2±0.3)% in control group (t=3.99, P<0.05). Conclusions: DETCs can promote the proliferation and anti-apoptotic potential of ESCs and inhibit their differentiation into end-stage by secreting IGF-Ⅰ, thus promoting wound healing of full-thickness skin defects in mice.

[Effects of hypoxia-pretreated rat adipose-derived mesenchymal stem cells conditioned medium on wound healing of rats with full-thickness defects].

Objective: To investigate the effects of hypoxia-pretreated rat adipose-derived mesenchymal stem cells (ADSCs) conditioned medium on wound healing of rats with full-thickness defects. Methods: (1) A 6-week-old male Sprague-Dawley rat was sacrificed by cervical dislocation, the bilateral inguinal adipose tissue was collected, the third generation ADSCs were isolated by collagenase digestion method, and the cells morphology was observed. The cells were harvested and divided into adipogenic induction group and osteogenic induction group according to the random number table (the same grouping method below), with 6 wells in each group. The cells in adipogenic induction group were cultured for 14 days to observe adipogenesis, and cells in osteogenic induction group were cultured for 28 days to observe osteogenesis. (2) The third generation ADSCs were collected and divided into normoxic group and hypoxic group. Cells in normoxic group was incubated in normal oxygen incubator with oxygen volume fraction of 21%, and cells in hypoxic group was incubated in low oxygen incubator with oxygen volume fraction of 2% respectively, with 3 samples in each group for each time point. Three samples in normoxic group on 3 h of culture and in hypoxic group on 3, 6, 12, 24, and 48 h of culture were collected for detecting the following indexes. The mRNA expressions of hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and peroxisome proliferator-activated receptor γ (PPAR-γ) were detected by real time fluorescent quantitative reverse transcription polymerase chain reaction. The cell culture supernatant in the two groups was collected, centrifuged, and filtered to obtain normoxic conditioned medium (normo-CM ) and hypoxic conditioned medium (hypo-CM). Enzyme linked immunosorbent assay was used to detect content of VEGF, transforming growth factor β (TGF-β), epidermal growth factor (EGF), and insulin-like growth factor (IGF) in conditioned medium. (3) Twenty-seven male Sprague-Dawley rats aged 6-8 weeks were collected and divided into phosphate buffer solution (PBS) group, normo-CM group, and hypo-CM group, with 9 rats in each group. A circular full-thickness skin defect wound with diameter of 1 cm was made on the back of each rat, and the wounds of rats in PBS, normo-CM, and hypo-CM groups were respectively dropped with 50 μL PBS, normo-CM, and hypo-CM. On post injury day (PID) 0, 3, 5, 7, 9, and 11, the gross condition of wound was observed, wound area was measured, and the non-healing rate of wound was calculated. The wound tissue was collected for hematoxylin eosin staining to observe inflammatory reaction of wound on PID 3, 9, and 11 and re-epithelialization of wound on PID 9. Masson staining was used to observe the collagen deposition and analyze collagen volume fraction of wound on PID 11. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, t test, and Bonferroni correction. Results: (1) The isolated cells showed a fusiform, in adherent growth and close arrangement when in low fusion degree. On 14 d of culture, the red lipid droplets stained with oil red O were observed in cells in adipogenic induction group, and on 28 d of culture, the red nodules stained with alizarin red S were observed in cells in osteogenic induction group. The cells were identified as ADSCs. (2) Compared with that in normoxic group, the mRNA expression of HIF-1α was significantly increased at 12 and 24 h of culture (t=5.43, 5.11, P<0.05), the mRNA expression of VEGF was significantly increased at 6 and 12 h of culture (t=3.29, 2.33, P<0.05 or P<0.01), the mRNA expression of bFGF was significantly increased at 12 h of culture (t=12.59, P<0.01) and significantly reduced at 48 h of culture (t=9.34, P<0.01), and the mRNA expression of PPAR-γ was significantly reduced at 3, 12, and 24 h of culture in hypoxic group (t=5.14, 6.56, 4.97, P<0.05). (3) Compared with that in normoxic group, the VEGF content was significantly increased at 3, 6, 12, 24, and 48 h of culture (t=5.74, 12.37, 14.80, 15.70, 34.63, P<0.05 or P<0.01), and the IGF content was significantly increased at 6, 12, 24, and 48 h of culture (t=5.65, 8.06, 20.12, 22.99, P<0.05 or P<0.01), and the content of TGF-β and EGF showed no obvious change at 3, 6, 12, 24, and 48 h of culture in hypoxic group. (4) From PID 0 to 11, the wound of rats in the three groups shrank to varying degrees, with no obvious infection or exudate. On PID 11, the wound area of rats in PBS group was still large, which was larger than that in normo-CM group, and the wound area of rats in hypo-CM group was basically healed. On PID 0, 3, and 5, the non-healing rates of wound of rats in the three groups were similar. On PID 7, the non-healing rates of wound of rats in normo-CM and hypo-CM groups were significantly lower than that in PBS group (t=10.26, 16.03, P<0.05). On PID 9, the non-healing rate of wound of rats in hypo-CM group was significantly lower than that of PBS group and normo-CM group, respectively (t=17.25, 6.89, P<0.05 or P<0.01), and the non-healing rate of wound of rats in normo-CM group was significantly lower than that in PBS group (t=8.81, P<0.05). On PID 11, the non-healing rate of wound of rats in hypo-CM group was (2.4±1.5)%, which was significantly lower than (20.0±5.0)% in PBS group and (7.7±1.7)% in normo-CM group (t= 30.15, 84.80, P<0.05). (5) On PID 3, the infiltration of inflammatory cells in the wound of rats in hypo-CM group was obviously more than those in the other two groups. On PID 9, the infiltration of inflammatory cells in the wound of rats in hypo-CM and normo-CM groups was obviously less than that in PBS group. On PID 11, the infiltration of inflammatory cells in the wound of rats in hypo-CM group was obviously less than those in PBS and normo-CM groups. On PID 9, the length of " epidermal migration tongue" on the wound of rats in hypo-CM group was longer than those of the other two groups, and the epidermis thickness was close to normal skin. On PID 11, compared with those in PBS and normo-CM groups, a large number of collagen deposits with dense structure, neat arrangement, and higher maturity were seen in the wound of rats in hypo-CM group. The wound collagen volume fraction of rats in PBS group was (22.90±1.25)%, which was significantly lower than (31.96±0.14)% in normo-CM group and (56.10±1.50)% in hypo-CM group (t=12.48, 29.43, P<0.05), and the wound collagen volume fraction of rats in normo-CM group was significantly lower than that in hypo-CM group (t=27.73, P<0.05). Conclusions: Hypoxia-pretreated can significantly enhance paracrine effect of rat ADSCs. Hypoxia-pretreated rat ADSC conditioned medium can accelerate the healing of full-thickness skin defect wound in rats by regulating inflammatory cell infiltration, promoting re-epithelialization and collagen deposition in the wound.