Phosphate-buffered Saline Group Research Articles

Normalization of Oxygen‐Induced Retinopathy in the Mouse Supplementation with Myo/Nog Cells

IntroductionRetinopathy of Prematurity (ROP) is the leading cause of blindness in children, affecting 50% of infants born prior to gestational week 32. ROP is the result of postpartum supplemental oxygen administered to compensate for underdeveloped lungs. However, hyperoxia inhibits proper development supplying the retina. When the infants are returned to ambient air, an imbalance is present between the oxygen concentration and metabolic demands of retinal neurons. This leads to a rapid induction of angiogenesis, leaky blood vessels, neovascular tuft formation, and cell death. Myo/Nog cells that express MyoD, Noggin, and brain‐specific angiogenesis inhibitor 1 (BAI1) exhibit neuroprotective properties in the eye and brain and are proposed to play a role in regulating angiogenesis in ROP.MethodsAn Oxygen‐induced retinopathy (OIR) protocol was implemented in neonatal C57BL/6J mice by exposing them on postnatal days 7‐12 (P7‐P12) to hyperoxia (75% oxygen) and then returning them to normal air (21% oxygen) from P12 until P21. On P15, groups receiving treatments were intravitreally injected with 1 µL of phosphate‐buffered saline (PBS) containing 2000 Myo/Nog cells isolated from the brain with the BAI1 antibody (BAI1+ group), 2000 unsorted brain cells (BAI1‐ group), or PBS alone (PBS group). Electroretinography (ERG) was conducted on P21 to assess retinal function. Eyes were extracted and either dissected for flat‐mount observation of vasculature and tufts, or processed for histology. The thickness of the retina and number of TUNEL+ apoptotic cells were quantified in tissue sections of eyes processed for histology. Adobe Photoshop (Adobe, San Jose, CA) was used to splice together 9 images that made up each whole retina from flat‐mounted eyes. The blood vessels were selected using the “Lasso tool” on photoshop, quantified by number of pixels, and then compared to the total number of pixels in the whole retina.ResultsOIR induced neovascular tuft formation. Exogenously added Myo/Nog cells normalized the retinopathy induced by the OIR model, reducing the number of tufts and improving the quality of the vasculature. The vasculature was most developed in retinas injected with brain derived Myo/Nog cells. ERG data shows a trend towards improvement of visual function with Myo/Nog cell injection.ConclusionMyo/Nog cell regulation of retinal vascularization may be related to the production of BAI1. Improved visual function seen with injection of Myo/Nog cells may be related to both a reduction of neovascular tufts and direct effects on retinal neurons. Therefore Myo/Nog cells have therapeutic potential in the prevention of ROP by normalizing the vasculature and preventing neuronal cell death.

Resolvin E1's Antimicrobial Potential Against Aggregatibacter Actinomycetemcomitans.

BackgroundMicroorganisms along with host response play a key role in the development of periodontal and peri-implant infections. Advanced periodontal and peri-implant diseases are most likely associated with bacterial plaques that trigger host immune response and eventually lead to the destruction of the attachment apparatus and bone loss around a tooth or a dental implant. A recent systematic review and meta-analysis revealed that Aggregatibacter actinomycetemcomitans had the highest association with peri-implantitis. Resolvin E1 (RvE1) is part of the specialized pro-resolving lipid mediator family biosynthesized from omega-3, polyunsaturated fatty acids (PUFAs), and eicosapentaenoic acid (EPA). Although RvE1 is an established anti-inflammatory agent, it was found that its application as a treatment or as a preventive drug had an indirect effect on the subgingival microbiota of both rats and rabbits with experimental periodontitis.AimThe aim of this study is to evaluate the direct antimicrobial effect of RvE1 on Aggregatibacter actinomycetemcomitans bacteria.Materials and MethodsThe study comprised three groups that underwent minimum inhibitory concentration (MIC) against Aggregatibacter actinomycetemcomitans. The first group was tested with the RvE1 working concentration of 5 ug/ml, the second group was tested with ethanol (EtOH), 10% as the working concentration, and the final group was diluted in phosphate-buffered saline (PBS) as the positive control. Optical density (OD600) was used for the comparison of bacterial growth among the tested groups. The experiment was conducted in three biological replicates. Data were analyzed using SPSS, and results were analyzed by using one-way analysis of variance (ANOVA) followed by post-hoc Bonferroni using a minimum level of significance (P-value) of 0.05.ResultsMinimum inhibitory concentration was 1.25 μg/ml and 5% for RvE1 and EtOH, respectively. RvE1's mean optical density (OD600) was 0.156 ± 0.021 and was significantly lower compared with all the other groups (P-value < 0.01). The EtOH group (mean OD600 0.178 ± 0.013) and the PBS group (mean OD600 0.1855 ± 0.022) did not reveal a significant difference (P-value = 0.185).ConclusionRvE1 demonstrated significant antimicrobial activity against A. actinomycetemcomitans with an MIC of 1.25 μg/ml. The RvE1 group showed significantly lower bacterial growth compared to the EtOH and PBS groups.

Implantation of Hypoxia-Induced Mesenchymal Stem Cell Advances Therapeutic Angiogenesis.

Hypoxia preconditioning enhances the paracrine abilities of mesenchymal stem cells (MSCs) for vascular regeneration and tissue healing. Implantation of hypoxia-induced mesenchymal stem cells (hi-MSCs) may further improve limb perfusion in a murine model of hindlimb ischemia. This study is aimed at determining whether implantation of hi-MSCs is an effective modality for improving outcomes of treatment of ischemic artery diseases. We evaluated the effects of human bone marrow-derived MSC implantation on limb blood flow in an ischemic hindlimb model. hi-MSCs were prepared by cell culture under 1% oxygen for 24 hours prior to implantation. A total of 1 × 105 MSCs and hi-MSCs and phosphate-buffered saline (PBS) were intramuscularly implanted into ischemic muscles at 36 hours after surgery. Restoration of blood flow and muscle perfusion was evaluated by laser Doppler perfusion imaging. Blood perfusion recovery, enhanced vessel densities, and improvement of function of the ischemia limb were significantly greater in the hi-MSC group than in the MSC or PBS group. Immunochemistry revealed that hi-MSCs had higher expression levels of hypoxia-inducible factor-1 alpha and vascular endothelial growth factor A than those in MSCs. In addition, an endothelial cell-inducing medium showed high expression levels of vascular endothelial growth factor, platelet endothelial cell adhesion molecule-1, and von Willebrand factor in hi-MSCs compared to those in MSCs. These findings suggest that pretreatment of MSCs with a hypoxia condition and implantation of hi-MSCs advances neovascularization capability with enhanced therapeutic angiogenic effects in a murine hindlimb ischemia model.

Association between anti-fibrillin-2 protein induced retinal degeneration via intravitreous delivery and activated TGF-β signalling in mice.

Fibrillin-2 (FBN2) is a major component of tissue microfibrils, and the decrease of FBN2 perturbs the signalling events mediated by transforming growth factor-β (TGF-β), thereby playing a role in macular degeneration. However, the association between the retinal degeneration resulting from the abnormality of FBN2 and the activation of TGF-β signalling has not been fully addressed. In the present study, the mice were divided into a normal control group (NC group), a phosphate-buffered saline (PBS) injection group (PBS group), and an anti-FBN2 protein injection group (anti-FBN2group), and the mice in PBS and anti-FBN2groups received the relevant treatment via the intravitreal injection once a week for three consecutive weeks. One week later after injection, the retinal morphology and visual function of the fundus were detected. Further, the expression of FBN2, TGF-β1, TGF-β2 and TGF-β3 in retina was measured using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. As a result, fundus examination suggests that after intravitreous injection of anti-FBN2 protein, there were a large patchy yellow white degeneration region and numerous pigmentations in the retina in anti-FBN2-treated mice; by contrast, there was no apparent change in mice from the NC and PBS groups. The retina suffered markedly damage, and the thickness of whole retina and outer nuclear layer markedly thinned. The expression of FBN2 was decreased whereas the levels of TGF-β1, TGF-β2 and TGF-β3 were upregulated. Together, our findings indicate that the intravitreous delivery of anti-FBN2 protein could induce retina degeneration in mice, accompanied by the higher activated TGF-β. The retinal degeneration mouse model established will provide a platform for the investigation of the retinal diseases.

Ginsenoside Rg1 Attenuates Premature Ovarian Failure of D-gal Induced POF Mice Through Downregulating p16INK4a and Upregulating SIRT1 Expression.

Premature ovarian failure (POF) refers to pathological amenorrhea before 40 years. To explore the regulatory effect of Rg1 on POF and clarify associated mechanisms. POF mice were induced by injecting with D-galactose (D-gal, 200 mg/kg/- day). Mice were divided into phosphate buffered saline (PBS), D-gal (POF mice), D-gal/Rg1 group (POF mice administrating D-gal/Rg1). Weight growth rate and ovarian weight coefficient were measured. Serum estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), superoxide dismutase (SOD), catalase (CAT) levels were examined using ELISA. The status of follicle and corpus luteum was determined using hematoxylin-eosin (HE) staining. P16INK4a and silent- mating type information regulation-2 homolog-1 (SIRT1) were determined using western blotting and RT-PCR. Weight growth rate and ovarian weight coefficient of mice in D-gal group were significantly decreased than PBS group (p<0.05). Serum E2, LH, SOD, CAT levels were significantly decreased, FSH levels were remarkably increased in D-gal group than PBS group (p<0.05). Rg1 (D-- gal/Rg1 group) significantly increased weight growth rate and ovarian weight coefficient, enhanced E2, LH, SOD, CAT levels and decreased FSH levels than D-gal group (p<0.05). HE staining demonstrated normal follicle morphology/structure of mice in PBS group and decreased the number of follicles, obvious vacuolation of corpus luteum and increased atretic follicles of mice in D-gal group. Compared with D-gal group, the number of follicles was increased, luteal follicles were decreased in mice in D-gal/Rg1 group (p<0.05). Rg1 significantly (D-gal/Rg1) downregulated p16INK4a and upregulated SIRT1 expression in ovarian tissues of mice compared to the D-gal group (p<0.05). Rg1 could delay premature ovarian failure in D-gal induced POF mouse model through downregulating p16INK4a and upregulating SIRT1 expression.

Tumor-Selective Cascade-Amplified Dual-Prodrugs Activation for Synergistic Oxidation-Chemotherapy

Tumor-Selective Cascade-Amplified Dual-Prodrugs Activation for Synergistic Oxidation-Chemotherapy

Effect of Bacillus subtilis, Enterococcus faecium, and Enterococcus faecalis supernatants on serotonin transporter expression in cells and tissues.

BACKGROUND Bacillus subtilis (B. subtilis), Enterococcus faecium (E. faecium), and Enterococcus faecalis (E. faecalis) are probiotics that are widely used in the clinical treatment of irritable bowel syndrome (IBS). Whether the supernatants of these three probiotics can improve gastrointestinal sensation and movement by regulating the serotonin transporter (SERT) expression needs to be clarified.AIMTo investigate whether B. subtilis, E. faecium, and E. faecalis supernatants can upregulate SERT expression in vitro and in vivo. METHODSCaco-2 and HT-29 cells were stimulated with probiotic culture supernatants for 12 and 24 h, respectively. A male Sprague-Dawley rat model of post-infectious irritable bowel syndrome (PI-IBS) was established and the rats were treated with phosphate-buffered saline (group A) and three probiotics culture supernatants (groups B, C, and D) for 4 wk. The levels of SERT were detected by quantitative PCR and western blotting. RESULTSThe levels of SERT at post-treatment 12 and 24 h were significantly elevated in Caco-2 cells treated with B. subtilis supernatant compared with those in the control group (aP < 0.05). Those levels were markedly upregulated in Caco-2 cells stimulated with E. faecium and E. faecalis supernatants at 24 h (aP < 0.05). In addition, SERT expression in groups B, C, and D was significantly higher than that in group A in the 2nd wk (aP < 0.05). Increased SERT expression was only found in group D in the 3rd wk (aP < 0.05). However, there was no significant difference in SERT expression between the groups in the last week (P > 0.05).CONCLUSIONThe supernatants of B. subtilis, E. faecium, and E. faecalis can upregulate SERT expression in intestinal epithelial cells and the intestinal tissues in the rat model of PI-IBS.

Oral Administration of Water Extract from Euglena gracilis Alters the Intestinal Microbiota and Prevents Lung Carcinoma Growth in Mice.

The antitumor effects of a partially purified water extract from Euglena gracilis (EWE) and EWE treated by boiling (bEWE) were evaluated using orthotopic lung cancer syngeneic mouse models with Lewis lung carcinoma (LLC) cells. Daily oral administration of either EWE or bEWE started three weeks prior to the inoculation of LLC cells significantly attenuated tumor growth as compared to the phosphate buffered saline (PBS) control, and the attenuation was further enhanced by bEWE. The intestinal microbiota compositions in both extract-treated groups were more diverse than that in the PBS group. Particularly, a decrease in the ratio of Firmicutes to Bacteroidetes and significant increases in Akkermansia and Muribaculum were observed in two types of EWE-treated groups. Fecal microbiota transplantation (FMT) using bEWE-treated mouse feces attenuated tumor growth to an extent equivalent to bEWE treatment, while tumor growth attenuation by bEWE was abolished by treatment with an antibiotic cocktail. These studies strongly suggest that daily oral administration of partially purified water extracts from Euglena gracilis attenuates lung carcinoma growth via the alteration of the intestinal microbiota.

Repair Effects of Bone Marrow Mesenchymal Stem Cells on Demyelination of Trigeminal Ganglion in Rats with Trigeminal Neuralgia.

ObjectiveThe current study investigated the effects of bone marrow mesenchymal stem cells (BMSCs) on pain behavior in rats with trigeminal neuralgia induced by infraorbital nerve chronic constriction injury (ION-CCI), and the repair effects of BMSCs on pathological changes in trigeminal ganglion demyelination.MethodsBMSCs or phosphate-buffered saline (PBS) alone were injected around trigeminal ganglion in ION-CCI rats via a rat brain stereotaxic apparatus. Mechanical pain threshold (von Frey test) and face grooming behavior were measured in each group. Recovery of demyelination of trigeminal ganglion was observed via electron microscopy 2 weeks later, and BMSC differentiation was observed via immunofluorescence.ResultsRats in the BMSC group exhibited significant improvements in mechanical pain threshold and face grooming behavior compared with the PBS group. BMSCs could repair demyelinating changes in trigeminal ganglion in ION-CCI rats. Only cells expressing GFAP, S-100, and p75 were observed via immunofluorescence, and no PKH67-labeled BMSCs were observed in the trigeminal ganglion. No BMSC differentiation was observed in the trigeminal ganglion.ConclusionInjection of BMSCs around the trigeminal ganglion could relieve trigeminal neuralgia effectively and repair trigeminal ganglion demyelination. No differentiation of BMSCs injected around the trigeminal ganglion into Schwann cells was observed. The mechanism of trigeminal neuralgia demyelination repair requires further investigation.

MicroRNA-499 improved septic myocardial dysfunction by regulating myosin heavy chain gene axis

To investigate the role and mechanism of microRNA-499 (miR-499) regulating α-myosin heavy chain (α-MHC) and β-myosin heavy chain (β-MHC) gene axis in septic myocardial dysfunction (SMD) and its significance. Sixty healthy adult male Sprague-Dawley (SD) rats were divided into phosphate buffered saline (PBS) control group (PBS group), lipopolysaccharide (LPS) induced SMD model group (LPS group), miR-499 agonist pretreatment group (agomir+LPS group), and miR-499 inhibitor pretreatment group (antagomir+LPS group) by random number table, with 15 rats in each group. SMD rat model was reproduced by intraperitoneal injection of LPS 10 mg/kg. The PBS group was intraperitoneally injected with the same amount of PBS. The two pretreatment groups were injected with agomir 30 mg/kg or antagomir 80 mg/kg through the caudal vein for 3 days, once a day. PBS group and LPS group were not pretreated. Echocardiography was detected 5 hours after LPS injection, and relevant indexes were recorded. The expression of miR-499 in plasma and myocardial tissue was detected by real-time quantitative polymerase chain reaction (qPCR). Western blotting was used to detect the protein expressions of α-MHC and β-MHC in myocardial tissue. Plasma N-terminal pro-brain natriuretic peptide (NT-proBNP), a marker of heart failure, was measured by electrochemiluminescence. Compared with the PBS group, the rats in LPS group were depressed. Additionally, LPS down-regulated the level of miR-499 in plasma and myocardial tissue, decreased α-MHC expression in myocardial tissue and up-regulated the expression of β-MHC. Echocardiography showed that left ventricular ejection fraction (LVEF), left ventricle fractional shortening (LVFS), cardiac output (CO), stroke volume (SV) and heart rate (HR) decreased by 49.1%, 59.2%, 48.8%, 39.4% and 15.9%, respectively, and the level of plasma NT-proBNP increased significantly in LPS group, indicating that LPS could induce cardiac dysfunction in rats. Compared with the LPS group, after pretreatment with agomir to overexpress the miR-499, LVEF and LVFS were significantly increased [LVEF: 0.662±0.020 vs. 0.323±0.024, LVFS: (36.16±1.43)% vs. (20.20±1.32)%, both P < 0.01], which suggested that the cardiac function of rats was improved in agomir+LPS group. At the same time, pretreatment with agomir significantly down-regulated the β-MHC protein expression (β-MHC/GAPDH: 0.74±0.04 vs. 2.97±0.34, P < 0.01), significantly up-regulated α-MHC protein expression (α-MHC/GAPDH: 1.59±0.05 vs. 0.74±0.14, P < 0.01), and significantly decreased the plasma NT-proBNP level (ng/L: 114.49±6.85 vs. 334.13±4.36, P < 0.01). After pretreatment with antagomir to inhibit the expression of miR-499, echocardiography showed that LVEF and LVFS were significantly lower than those in the LPS group [LVEF: 0.297±0.021 vs. 0.323±0.024, LVFS: (19.38±1.52)% vs. (21.20±1.32)%, both P < 0.01], which suggested that the cardiac function of rats was significantly inhibited. At the same time, pretreatment with antagomir significantly down-regulated α-MHC protein expression in myocardial tissue (α-MHC/GAPDH: 0.63±0.03 vs. 0.74±0.14, P < 0.01), significantly up-regulated β-MHC protein expression (β-MHC/GAPDH: 3.03±0.47 vs. 2.97±0.34, P < 0.01), and significantly increased the level of plasma NT-proBNP (ng/L: 373.91±4.23 vs. 334.13±4.36, P < 0.05). miR-499 could regulate the expression of α-MHC and β-MHC which improved cardiac dysfunction caused by sepsis. Targeted regulation of miR-499 expression may be an effective way to treat SMD.

Dental Pulp Stem Cells Transplantation Improves Passive Avoidance Memory and Neuroinflammation in Trimethyltin-Induced Alzheimer's Disease Rat Model

Background: According to the increasing incidence of Alzheimer's disease (AD), this study aimed to investigate the effect of dental pulp stem cells (DPSCs) transplantation on passive avoidance memory and neuroinflammation in trimethyltin (TMT)-induced AD rat model. Materials and Methods: In this experimental study, 18 male Wistar rats were randomly divided into three groups: the control rats, TMT+phosphate-buffered saline (PBS) group that received 8 mg/kg TMT plus 0.5 ml PBS, and TMT+DPSCs (TMT+1×106 cells/ml DPSC in 0.5 ml PBS). Then, after one month, a passive avoidance test was performed. Also, nuclear factor kappa-β (NF-Kβ) serum level and the percentage of damaged neurons in the hippocampus were determined. Results: DPSCs transplantation showed significantly increased step-through latency to the dark compartment compared to control and TMT+PBS groups 24 hours aftershock. Also, time spent in the dark compartment of the TMT+DPSCs group significantly decreased compared to control and TMT+PBS groups in 24 and 48 hours after shock (P&lt;0.05). Furthermore, DPSCs transplantation significantly decreased the NF-Kβ serum level and percentage of damaged pyramidal neurons of CA1 compared to the TMT+PBS group (P&lt;0.05). Conclusion: DPSCs transplantation improved memory and learning, regulated NF-Kβ serum level, and decreased damaged neurons of CA1 hippocampus in the TMT-induced AD rat model.

Enhancing Intersystem Crossing by Intermolecular Dimer-Stacking of Cyanine as Photosensitizer for Cancer Therapy

Enhancing Intersystem Crossing by Intermolecular Dimer-Stacking of Cyanine as Photosensitizer for Cancer Therapy

Effect of Src tyrosine kinase on a rat model of asthma

Src tyrosine kinase is a protein encoded by the Src gene. The present study aimed to determine the role of Src protein kinase in asthma using small interfering RNA (siRNA) technology. Several Src siRNAs were designed and the most effective siRNA pair was selected. A rat model of asthma was established using ovalbumin, and the rats were treated with Src siRNA, empty vector or phosphate-buffered saline (PBS). A non-asthmatic control group was also established. The rats were clinically observed and Src mRNA and protein levels were measured by reverse transcription-quantitative PCR and western blot analysis, respectively. Pathological observation of the lung tissue, counting of white blood cells (WBCs) and eosinophils (EOSs) and analysis of the concentrations of IL-5, IL-33 and IFN-γ in the bronchoalveolar lavage fluid were performed. The expression levels of Src mRNA in the control, PBS, empty vector and siRNA groups were 110±30.7x103, 253±55.4x103, 254±41.3x103 and 180±50.9x103, respectively. Histochemical analysis of the lung tissue of rats in the siRNA group exhibited a relatively complete lung structure and little damage to the alveolar cavity. Src protein expression and IL-5, IL-33 levels, WBC and EOS levels were positively correlated with Src mRNA expression, while the IFN-γ concentration was negatively correlated with Src mRNA expression. These results indicate that Src knockdown inhibits the release of tracheal inflammatory factors and significantly alleviates asthma in rats. In conclusion, the present study utilized a gene transfer technique to interfere with the expression of Src in rats, which decreased the levels of IL-5, IL-33, WBCs and EOSs and increased the level of IFN-γ; these changes effectively ameliorated the condition of the trachea in asthmatic rats.

Effects of N-trimethyl chitosan-recombinant tissue factor pathway inhibitor complex on avulsion flap with roll compaction in rat

Effects of N-trimethyl chitosan-recombinant tissue factor pathway inhibitor complex on avulsion flap with roll compaction in rat

Hydrogen peroxide potentiates antimicrobial photodynamic therapy in eliminating Candida albicans and Streptococcus mutans dual-species biofilm from denture base

BackgroundCandida albicans (C.albicans) is the primary pathogen of denture biofilm. Moreover, it could establish a cross-kingdom relationship with bacteria to enhance its virulence and resistance to antifungal drugs. This study aimed to investigate the efficacy of antimicrobial photodynamic therapy (aPDT) in combination with hydrogen peroxide (H2O2) against C.albicans and Streptococcus mutans (S.mutans) dual-species biofilm formed on polymethyl methacrylate (PMMA) disk, and explore its involved mechanisms. MethodsC.albicans and S.mutans were grown on PMMA disk for 48 h to form biofilm and received different treatments. The treatments included:1) phosphate-buffered saline (PBS) group,2) 100 mM H2O2 group,3) aPDT group,4) aPDT+ H2O2 and 5) H2O2+aPDT group. Colony forming units (CFU), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and scanning electron microscope (SEM) were used to evaluate the antimicrobial effects. Extracellular polysaccharide substance (EPS) production and observation, cell permeability of biofilm, and uptake of toluidine blue O (TBO) by biofilm were assessed to investigate the involved mechanism. ResultsThere was no significant difference between PBS group and H2O2 group in viable microorganisms and metabolic activity of biofilm. The treatment protocols containing aPDT group reduced microorganism numbers and metabolic activity when compared to PBS group or H2O2 group (P<0.05). H2O2+aPDT treatment showed the highest antimicrobial efficacy in comparison with other treatments (P<0.05). Pretreatment with H2O2 could decrease EPS production and enhance cell permeability, leading to increased TBO uptake in biofilm. ConclusionPretreatment with H2O2 improved aPDT efficiency in eliminating dual-species biofilm from PMMA disk by reducing EPS amount, enhancing cell permeability, and increasing TBO uptake.

Effects and mechanisms of allogeneic epidermal stem cells on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds

Objective: To investigate the effects and mechanisms of allogeneic epidermal stem cells (ESCs) on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds. Methods: Experimental research methods were applied. Primary ESCs that appeared paving stone-like after being cultured for 7 d were obtained by enzymatic digestion method from one 4-week-old male BALB/c-NU nude mouse (the same strain, age, and sex below). The cells of third passage were identified by flow cytometry to positively express ESC marker CD44 and negatively express CD45, meanwhile, the positive expression of ESC markers of p63 and integrin 6α, and negative expression of CD71 were identified by immunofluorescence method. The ESCs of third passage in the logarithmic growth phase were used for the following experiments. Twenty-six nude mice were equally divided into phosphate buffered saline (PBS) group and ESCs group according to the random number table. A full-thickness skin defect wound was made on the back of each nude mouse, and then the wounds of the two groups were sprayed with equal volumes of PBS and ESCs, respectively. The wounds were transplanted with full-thickness skin grafts cut from the backs of 4 other nude mice. Each ten nude mice from the two groups were selected, the wound healing and skin survival on post surgery day (PSD) 0 (immediately), 3, 7, 14, and 21 were observed, and the survival ratio and shrinkage rate of skin grafts on PSD 3, 7, 14, and 21 were calculated (the number of sample was the number of surviving skin grafts at each time point); the blood perfusion in the skin grafts on PSD 3, 7, and 14 was detected by the laser speckle blood flow imager, and the blood flow ratio of nude mice skin grafts in ESCs group to PBS group at each time point was calculated (the number of sample was the pair number of surviving skin grafts in group pairing at each time point). The skin graft tissue of each 3 nude mice remained in the two groups were collected on PSD 7, and the mRNA expressions and protein expressions of tumor necrosis factor α (TNF-α), interleukin 8 (IL-8), IL-10, type Ⅰ collagen, type Ⅲ collagen, and matrix metalloproteinase 9 (MMP-9) in the tissue were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Data were statistically analyzed with Log-rank test, analysis of variance for repeated measurement, one-way analysis of variance, independent sample t test, and Bonferroni correction. Results: Taking the condition on PSD 0 as a reference, the wounds of nude mice in the two groups healed gradually on PSD 3, 7, 14, and 21, and the shrinkage of skin grafts was gradually obvious. Among them, the shrinkage healing of wound of nude mice in PBS group was more significant than that in ESCs group. On PSD 3, the skin graft of 1 nude mouse failed in ESCs group, while the skin graft of 3 nude mice failed in PBS group. On PSD 7, the skin graft of another nude mouse failed in PBS group. The survival ratio of skin grafts of nude mice in the two groups was similar on PSD 3, 7, 14, and 21 (P>0.05). On PSD 3, 7, 14, and 21, the shrinkage rates of skin grafts of nude mice in ESCs group were (9.2±0.4)%, (19.7±1.2)%, (53.6±3.5)%, and (62.2±5.1)%, respectively, which was significantly lower than (11.0±0.9)%, (47.8±2.8)%, (86.1±7.1)%, and (89.7±9.0)% in PBS group (t=5.719, 26.650, 11.940, 7.617, P<0.01). On PSD 3, 7, and 14, blood perfusion signals were observed in the skin grafts of nude mice in the two groups. The average blood perfusion ratios of the skin grafts of nude mice in ESCs group to PBS group were greater than 1, and there was no statistically significant difference in the overall comparison of 3 time points (P>0.05). On PSD 7, compared with those of PBS group, the mRNA and protein expressions of TNF-α, IL-8, type Ⅰ collagen, and type Ⅲ collagen in the skin graft tissue of nude mice in ESCs group were significantly reduced, while the mRNA and protein expressions of IL-10 and MMP-9 in the skin graft tissue of nude mice in ESCs group were significantly increased (in mRNA comparison, t=2.823, 2.934, 2.845, 2.860, 3.877, 2.916, P<0.05). Conclusions: Allogeneic ESCs can reduce the shrinkage of allogeneic full-thickness skin grafts transplanted on full-thickness skin defect wounds in nude mice, promote the formation of new blood vessels between the skin graft and the wound, reduce inflammation and collagen protein expression, and promote the expression of MMP-9, thus improving the survival quality of skin grafts.

Preparation and roles of sliver-loaded viscous hydrogel in healing of full-thickness skin defect wounds with bacterial colonization in mice

Preparation and roles of sliver-loaded viscous hydrogel in healing of full-thickness skin defect wounds with bacterial colonization in mice

Nattokinase crude extract enhances oral mucositis healing

BackgroundNattokinase (NK) is a promising alternative in the prevention and treatment of cardiovascular diseases due to its potent fibrinolytic activity. In this study, we investigated the effect of crude nattokinase extract on the healing of acetic acid-induced oral mucositis in mice.MethodsBacillus subtilis culture media (BSCM) was isolated into the supernatant, named nattokinase crude extract (NCE), and the pellet was named Bacillus subtilis mass (BSM). An oral mucositis model was established in mice by applying 50% glacial acetic acid to the buccal mucosa. According to the treatment conditions, the mice were divided into BSCM, NCE, BSM and phosphate buffered saline (PBS) groups. The weight of the mice, oral mucositis healing score and histopathological examination were used to evaluate the treatment.ResultsFibrinolytic activities of BSCM, NCE and BSM were approximately 8069, 10,800 and 80 U/ml, respectively. The weight gain of mice in the NCE group was significantly different from the PBS group after three days’ treatment (p < 0.05). The oral mucositis score of NCE group was significantly higher than other groups (p < 0.05). The differences in histopathology scores between the NCE and other groups were statistically significant (p < 0.01).ConclusionsNCE could possess remarkable potential to reduce pain and promote oral mucositis healing with minimal safety concerns. In this study, we first report that NCE from the supernatant of Bacillus subtilis can promote the healing of oral mucositis, which extends the application scope of NK.

Anti-infective activity of Cratylia argentea lectin (CFL) against experimental infection with virulent Listeria monocytogenes in Swiss mice

BackgroundThe lectin from Cratylia argentea (CFL) is able to modulate the immune system response and is thus a potential phytotherapeutic substance. Hypothesis/PurposeIn this study, we investigated the role of CFL on control of bacterial infection caused by Listeria monocytogenes, the causative agent of human listeriosis. Study designSwiss mice were infected with L. monocytogenes and then treated with CFL. MethodsAdult Swiss mice weighing with 30–40 g were infected intraperitoneally with a bacterial suspension (0.2 ml; 1 × 107 CFU/ml). After 30 min, the mice were treated with CFL intravenously at concentrations of 0.1 or 10 mg/kg. Control mice received phosphate-buffered saline (PBS). The animals were euthanized 24 h after infection. ResultsWe observed that i.v. administration of CFL to Swiss mice did not cause acute toxicity, and reduced the leukocyte counts in the bloodstream 24 h after infection with virulent L. monocytogenes. There was a reduction in the bacterial burden within peritoneal macrophages after infection in CFL-treated mice. Accordingly, the bacterial counts in the bloodstream, spleen and liver also decreased in comparison with the PBS group. Histological damage in the spleen and liver was lower in mice that received CFL treatment. In vitro antimicrobial assays demonstrated that CFL does not inhibit the growth of L. monocytogenes. The mRNA expression of the anti-inflammatory cytokine IL-10 was enhanced with CFL treatment after infection. ConclusionThe lectin from C. argentea (CFL) has immunomodulatory and anti-infective properties of pharmacological interest for control of infectious diseases.

Effect and mechanism of TNF-α and etanercept on the invasion ability of extravillous trophoblast cell in URSA patients

Objective: To investigate the effect and mechanism of tumor necrosis factor α (TNF-α) and its inhibitor etanercept (ETA) on the invasion ability of extravillous trophoblast in patients with unexplained recurrent spontaneous abortion (URSA). Methods: (1) Patients were collected from March to June in 2019. They were divided into the URSA group (n=15) and the normal control group (n=15), according to whether diagnosed with URSA or not. The mRNA expression levels of TNF-α in villi tissue of patients in the two groups were detected by quantitative real-time PCR (qRT-PCR). (2) The mRNA and protein expression levels of matrix metalloproteinase-2 (MMP-2), Slug and CXC chemokine rceptor 4 (CXCR4) in HTR-8/SVneo cells were detected by qRT-PCR or western blot after being stimulated by exogenous TNF-α (0.2, 2, 20 ng/ml) alone or TNF-α along with ETA, or phosphate buffered saline (PBS) as control. (3) The invasion ability of HTR-8/SVneo cells was investigated by transwell test after stimulating by TNF-α alone or TNF-α along with ETA. (4) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells, which were stimulated by TNF-α (2 ng/ml) alone after nuclear factor-κB (NF-κB) inhibitor, BAY 11-7028, preconditioning, were detected by qRT-PCR or western blot. Results: (1) The mRNA expression level of TNF-α in villi tissue of URSA group (4.10±0.49) was 4.1 times as much as the normal control group (t=10.51, P<0.05). (2) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells of TNF-α group were significantly lower than those in PBS control group (P<0.05) and those in TNF-α along with ETA group (P<0.05). (3) The invasion ability of HTR-8/SVneo cells in TNF-α group was significantly decreased than PBS group and TNF-α along with ETA group (78±14 vs 373±26 vs 227±44, P<0.05). (4) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells with BAY 11-7028 preconditioning (mRNA: 1.03±0.10, 1.03±0.06, 1.09±0.08; protein: 1.09±0.03, 1.49±0.03, 1.12±0.03) were significantly higher than without preconditioning after being stimulated by TNF-α (all P<0.05). Conclusions: The expression of TNF-α in the villi of URSA patients is much higher than normal early pregnant women. TNF-α could decrease the capacity of invasion by suppressing the expression of MMP-2, Slug and CXCR4 through NF-κB signaling pathway in extravillous trophoblast cells. While ETA could improve the invasiveness capability of extravillous trophoblast cells through inhibiting the negative effect of TNF-α.