Number Of Inflammatory Cells Infiltration Research Articles

Effect of salvia miltiorrhiza combined with roxadustat on wound healing of full-thickness skin defects in diabetic rats and its mechanism

Effect of salvia miltiorrhiza combined with roxadustat on wound healing of full-thickness skin defects in diabetic rats and its mechanism

Isolation and characterization of a novel parvovirus from a red-crowned crane, China, 2021

BackgroundParvoviruses are icosahedral, nonenveloped viruses with single-stranded DNA genomes of approximately 5 kb in length. In recent years, parvoviruses have frequently mutated and expanded their host range to cause disease in many wild animals by altering their tissue tropism. Animal infection mainly results in acute enteritis and inflammation of other organs. In this study, we used a viral metagenomic method to detect a novel parvovirus species in a red-crowned crane that died due to severe diarrhea in China.ResultsThe presence of the viral genome in the kidney, lung, heart, liver, and intestine were confirmed by PCR. Histopathological examination of the intestine showed a large number of infiltrated inflammatory cells. The JL21/10 strain of the red-crowned crane parvovirus was first isolated from the intestine. Whole-genome sequence analysis showed that JL21/10 shared high identity with the red-crowned crane Parvovirinae strains yc-8 at the nucleotide level (96.61%). Phylogenetic analysis of the complete genome and NS1 gene revealed that the JL21/10 strain clustered with strains in chicken and revealed a close genetic relationship with the red-crowned crane parvovirus strains.The complete of VP2 gene analysis showed that JL21/10 shared identity with the red-crowned crane yc-8 strains (97.7%), chicken (55.4%),ducks(31.0%) and geese(30.1%) at the amino acid level. The result showed that red-crowned crane parvovirus may be cross-species transmission to chicken. However, There is little possibility of transmission to ducks and geese.ConclusionThis is the first isolation and identification of a parvovirus in red-crowned crane that was associated with severe diarrhea.

Ameliorating Osteoarthritis in Mice Using Silver Nanoparticles.

Knee osteoarthritis (KOA) is one of the most commonly encountered degenerative diseases of the joints in people over 45 years of age. Currently, there are not any effective therapeutics for KOA,and the only end-point strategy is total knee arthroplasty (TKA); therefore, KOA is associated with economic burdens and societal costs. The immune inflammatory response is involved in the occurrence and development of KOA. We previously established a mouse model of KOA using type II collagen. Hyperplasia of the synovial tissue was present in the model, alongside a large number of infiltrated inflammatory cells. Silver nanoparticles have substantial anti-inflammatory effects and have been widely used in tumor therapy and surgical drug delivery. Therefore, we evaluated the therapeutic effects of silver nanoparticles in a collagenase II-induced KOA model. The experimental results showed that silver nanoparticles significantly reduced synovial hyperplasia and the infiltration of neutrophils in the synovial tissue. Hence, this work demonstrates the identification of a novel strategy for OA and provides a theoretical basis for preventing the progress of KOA.

Natural β-carotene prevents acute lung injury induced by cyclophosphamide in mice.

IL-17 is associated with varied inflammatory and immune-related diseases. However, the biological function of IL-17 and its expression in acute lung damage are not entirely known. Thanks to the powerful antioxidant properties of β-carotene, we presumed that it would show a potent protecting effect against cyclophosphamide (CP) -induced acute lung injury (ALI) in mice. We studied the mechanisms underlying the effect of β-carotene supplementation against CP-induced ALI in mice. We isolated the β-carotene from Scenedesmus obliquus microalgae n-hexane extract and identified it by HPLC and 1H-NMR analysis. Within the experiments, 40 mice were assigned into five groups randomly: Group 1 (Control): Mice received saline. Group 2 (β-carotene control): Mice were administered β-carotene (40 mg/kg; orally) once daily for 10 sequent days without CP injection. Group 3 (CP): One i.p injection of 200 (mg/kg) of CP was given to mice. Group 4 and 5 (CP + β-carotene): Mice were administered β-carotene (20 and 40 mg/kg; orally) once a day for ten days following the CP injection. Lung samples were collected for lab analysis, after scarifying the animals at the experiment end. Administration of β-carotene orally reduced CP-induced ALI and inflammation. β-carotene significantly decreased wet-to-dry weight ratios (W/D), down-regulated IL-17, NF-κB, and IKBKB, decreased the contents of TNF-α, COX-2, and PKC, and increased the contents of SIRT1 and PPARγ in the lung tissues. β-carotene ameliorated the histopathological changes induced by CP and reduced the scoring number of inflammatory cell infiltration and emphysema when compared to CP. Consequently, we conclude natural β-carotene is a promising anti-inflammatory mediator for different inflammatory-related complications.

Effect of electroacupuncture of "Weizhong" (BL40) on iron metabolism disorder in rats with lumbar multifidus muscle injury

To observe the effect of electroacupuncture (EA) of "Weizhong" (BL40) on the disorder of iron metabolism and the level of oxidative stress after lumbar multifidus muscle injury (LMMI), so as to explore its mechanisms underlying promoting the repair of LMMI. Male SD rats were randomly divided into normal, model and EA groups (6 rats in each group). The LMMI model was established by injecting 0.5% bupivacaine (BPVC) solution (400 μL) into the lumbar multifidus muscle with the syringe-needle close to the spinous process (L4-L5). Twenty-four hours after successful establishment of the model, EA (2 Hz/15 Hz, 2 mA) was applied to bilateral BL40 for 30 min, once a day for 2 days. Histopathological changes of the multifid muscle were observed under microscope after H.E. staining, and the iron granules in the multifid muscle tissue observed after Prussian blue staining. The expression of glutathione synthase (GSS) was detected by Western blot, and the expressions of iron regulatory protein 1 (IRP1), ferroportin (Fpn), ferritin heavy chain 1 (FTH1, iron metabolism-related proteins) and gluta-thione peroxidase 4 (GPX4, functions in protecting cells against detrimental lipid peroxidation and governing a novel form of regulated necrotic cell death, called ferroptosis) mRNAs were detected by quantitative real-time PCR. The contents of glutathione (GSH) and malondialdehyde (MDA) were measured by biochemical methods. H.E. staining showed large areas of necrosis and breakage of muscle fibers, disordered arrangement of muscle fibers, widened muscle cell space, accompanying with a large number of inflammatory cell infiltration in the multifidus muscle tissue of the model group, which was relatively milder in the EA group. Outcomes of Prussian blue staining showed that compared with the normal group, there were more iron particles in the multifidus muscle tissue and enlarged muscle fiber gaps, which was also milder in the EA group. Compared with the normal group, the expression level of IRP1 mRNA and content of MDA were significantly increased (P<0.001), the expression levels of Fpn, FTH1 and GPX4 mRNAs and GSS protein, and the content of GSH were considerably decreased (P<0.001) in the model group. In comparison with the model group, the increase of IRP1 mRNA expression and MDA content, as well as the decrease of Fpn, FTH1 and GPX4 mRNAs expressions and GSH content were reversed in the EA group (P<0.001,P<0.05,P<0.01). EA of BL40 has a protect effect in BPVC-induced injury of lumbar multifidus muscle in rats, which may be related to its functions in improving iron metabolism to reduce oxidative damage by regulating expression of IRP1, Fpn and FTH1.

Effect of electroacupuncture on inflammatory signal expression in local tissues of rats with chronic pelvic pain syndrome based on purinergic 2X7 receptor/NOD-like receptor pyrin domain-containing 3 signal pathway.

To study the expression of inflammatory signal in local prostate tissue of chronic pelvic pain syndrome (CPPS) rats by electroacupuncture (EA) of Guanyuan (CV4), Zhongji (CV3), Huiyang (BL35) and Sanyinjiao (SP6), and to explore the possible mechanism of anti-inflammatory and analgesic effects of EA. METHODS：A total of 36 Sprague-Dawley male rats were randomly divided into three groups: control, model and EA (n=12 rats/group). The CPPS model was made by injection of CFA into ventral lobes of the prostate (0.1 mL). Electric acupuncture apparatus was applied to stimulate Guanyuan (CV4), Zhongji (CV3), bilateral Huiyang (BL35) and Sanyinjiao (SP6) acupoints in EA group. The general condition of rats was observed and the prostate index (PI) was calculated. The thermal pain threshold was collected after each therapeutic course. Histopathological changes of the prostate tissue were examined by hematoxylin-eosin staining method. The expression levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and prostaglandin E2 (PGE2) in prostatic homogenates were measured by enzyme linked immunosorbent assay (ELISA). Moreover, the expression levels of purinergic 2X7 receptor (P2X7R), NOD-like receptor pyrin domain-containing 3 (NLRP3), caspase-1 and interleukin-18 (IL-18) mRNA were quantified by quantitative real-time polymerase chain reaction. Compared with control group, the PI of rats increased, and the thermal pain threshold decreased significantly in model group. The morphological structure of prostate tissues of rats in model group was severely damaged with a large number of inflammatory cells infiltration. Additionally, the levels of TNF-α, IL-1β and PGE2 were higher, and the expressions of P2X7R, NLRP3, caspase-1 and IL-18 mRNA were higher than those in control group. After EA treatment, the PI was significantly decreased, the thermal pain threshold was significantly increased, and the tissue damage was significantly improved. The expressions of inflammatory cytokines were lower in EA group, and expression of P2X7R/NLRP3 pathway was down-regulated. The effect of EA at Guanyuan (CV4), Zhongji (CV3), Huiyang (BL35) and Sanyinjiao (SP6) can improve inflammation and pain symptoms of CPPS rats induced by Complete Freund's adjuvant (CFA). This suggests that EA at Guanyuan (CV4), Zhongji (CV3), Huiyang (BL35) and Sanyinjiao (SP6) can produce anti-inflammatory analgesia effect by preventing the activation of P2X7R/NLRP3 signal pathway, inhibit the release of inflammatory cytokines in CPPS rats, which may provide a putative novel target for the treatment of CPPS.

Effect and mechanism of Rho kinase inhibitor on intestinal injury in septic rats

To explore the effect of Rho kinase inhibitor on intestinal injury in septic rats and its possible mechanism. Thirty-two male Sprague-Dawley (SD) rats were randomly divided into sham operation group (Sham group), Rho kinase inhibitor Y-27632 control group (Y+Sham group), sepsis model group [cecal ligation and puncture (CLP) group] and Y-27632 pretreatment group (Y+CLP group), with 8 rats in each group. Rat sepsis model was reproduced by CLP. The rats in the Sham group and Y+Sham group were only separated and moved the cecum without ligation and perforation. The rats in the Y+Sham group and Y+CLP group were pretreated with intraperitoneal injection of Y-27632 solution 5 mg/kg 15 minutes before operation; the rats in the Sham group and CLP group were intraperitoneally injected with the same amount of phosphate buffered saline (PBS). Twenty-four hours after operation, the heart blood was collected and the serum diamine oxidase (DAO) content was determined by enzyme-linked immunosorbent assay (ELISA). Then the small intestine tissue was collected, the pathological changes of the intestinal tissue were observed under the light microscope after hematoxylin-eosin (HE) staining, and Chiu's score was performed. The positive expressions of Rho-related coiled-coil kinase 1 (ROCK1) and nuclear factor-κB (NF-κB) in intestinal tissue were detected by immunohistochemistry. ELISA was used to detect the levels of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in intestinal tissue homogenate. The intestinal tissue structure of the Sham group and Y+Sham group was intact and the mucosa was arranged neatly. Compared with the Sham group, the intestinal mucosa of the CLP group was arranged disorderly, with a large number of inflammatory cells infiltration, and the Chiu's score was significantly increased (3.83±0.27 vs. 0.12±0.11, P < 0.05), indicating that those rats suffered from septic intestinal injury. Compared with the CLP group, the degree of necrosis of intestinal epithelial cells in the Y+CLP group was reduced, a small amount of inflammatory cells infiltration was seen, and the Chiu's score was significantly decreased (2.85±0.21 vs. 3.83±0.27, P < 0.05), indicating that Y-27632 pretreatment could alleviate intestinal injury in septic rats. Compared with the Sham group, the positive expressions of intestinal tissue ROCK1 and NF-κB, the contents of serum DAO and intestinal homogenate TNF-α in the CLP group were significantly increased [ROCK1 expression (A value): 0.19 (0.18, 0.22) vs. 0.10 (0.09, 0.11), NF-κB expression (A value): 0.40±0.02 vs. 0.15±0.01, DAO (ng/L): 287.81±23.31 vs. 144.92±17.72, TNF-α (ng/L): 101.08±5.62 vs. 74.81±5.56, all P < 0.05], the level of intestinal homogenate IL-10 was significantly decreased (μg/L: 55.16±5.20 vs. 95.95±7.53, P < 0.05). Compared with the CLP group, the positive expressions of intestinal tissue ROCK1, NF-κB, the contents of serum DAO and intestinal homogenate TNF-α in the Y+CLP group were significantly decreased [ROCK1 expression (A value): 0.15 (0.13, 0.18) vs. 0.19 (0.18, 0.22), NF-κB expression (A value): 0.28±0.01 vs. 0.40±0.02, DAO (ng/L): 243.34±19.76 vs. 287.81±23.31, TNF-α (ng/L): 90.41±8.79 vs. 101.08±5.62, all P < 0.05], while the level of intestinal homogenate IL-10 was significantly increased (μg/L: 66.15±5.74 vs. 55.16±5.20, P < 0.05), indicating that the protective effect of Y-27632 pretreatment on sepsis intestinal injury rats might be related to the regulation of RhoA/ROCK1/NF-κB signaling pathway. Rho kinase inhibitors can reduce intestinal injury in septic rats, and the mechanism may be related to inhibiting RhoA/ROCK1/NF-κB signaling pathway and reducing intestinal inflammation in septic rats.

Protective effect of heat shock transcription factor 1 on acute lung injury in septic rats by regulating NOD-like receptor protein 3 inflammasome activation

To determine whether heat shock transcription factor 1 (HSF1) ameliorates sepsis induced acute lung injury (ALI) by regulating NOD-like receptor protein 3 (NLRP3) inflammasome activity in rat alveolar macrophages (AM). Twenty-four SPF Sprague-Dawley (SD) rats were divided into control group, lipopolysaccharide (LPS) group, overexpression empty vector+LPS group, and overexpression HSF1+LPS group by random number table method, with 6 rats in each group. The rat model of sepsis-induced ALI was reproduced by intraperitoneal injection of LPS (5 mg/kg); the rats in the control group were given the same volume of normal saline. The rats in the overexpression empty vector+LPS group and the overexpression HSF1+LPS group were instilled with 100 μL of overexpressed empty vector adenovirus or overexpressed HSF1 adenovirus through the trachea, respectively; the rats in the control group and LPS group were instilled with an equal volume of normal saline at the same time. At 6 hours after the model was reproduced, carotid blood was collected to determine the oxygenation index (PaO2/FiO2); lung tissue was obtained, and hematoxylin-eosin (HE) staining was performed to observe the pathological changes of lung tissue under a light microscope. Lung tissue wet/dry ratio (W/D) was determined. Immunohistochemistry was used to detect the positive expression of macrophage-specific marker antibody CD68. Western blotting was used to detect the protein expressions of HSF1 and NLRP3. Bronchoalveolar lavage fluid (BALF) was collected, and the levels of interleukins (IL-1β, IL-18) were detected by enzyme-linked immunosorbent assay (ELISA). In addition, BALF of normal rats was collected, and primary AM was isolated, cultured and divided into four groups. The AM in the blank control group was cultured normally without any treatment; the LPS group was treated with 1 mg/L LPS for 24 hours to reproduced the LPS stimulation model; AM in the overexpression empty vector+LPS group and the overexpression HSF1+LPS group were transfected with empty vector plasmid or overexpressed HSF1 plasmid, respectively, for 48 hours, and then the AM was treated with 1 mg/L LPS. The cell viability was detected by cell counting kit-8 (CCK-8). The protein expressions of HSF1 and NLRP3 in AM were detected by Western blotting. The levels of IL-1β and IL-18 in AM culture medium were determined by ELISA. Compared with the control group, the rat lung structure in the LPS group was severely injured, the alveolar cavity and pulmonary interstitium were congested and edema, the alveolar walls were significantly thickened and ruptured, accompanied by a large number of inflammatory cells infiltration. The lung W/D ratio increased, the infiltration degree of macrophages increased, PaO2/FiO2 decreased, HSF1 protein expression decreased and NLRP3 protein expression increased in lung tissue and AM, and IL-1β and IL-18 levels in BALF and AM culture medium increased. Compared with the LPS group, the degree of lung injury in the overexpression HSF1+LPS group was significantly improved, the lung W/D ratio was significantly reduced (4.76±0.16 vs. 6.93±0.33, P < 0.05), the macrophage infiltration was reduced, PaO2/FiO2 increased significantly [mmHg (1 mmHg ≈ 0.133 kPa): 397.62±19.46 vs. 280.12±37.42, P < 0.05], HSF1 protein expression was significantly up-regulated in lung tissue and AM (HSF1/GAPDH: 0.90±0.04 vs. 0.61±0.04 in lung tissue, 1.10±0.10 vs. 0.57±0.08 in AM, both P < 0.05), NLRP3 protein expression was significantly down-regulated (NLRP3/GAPDH: 0.75±0.14 vs. 1.05±0.11 in lung tissue, 0.81±0.09 vs. 1.14±0.17 in AM, both P < 0.05), and the contents of IL-1β and IL-18 in BALF and AM medium were significantly decreased [IL-1β (ng/L): 7.82±0.45 vs. 14.09±0.58 in BALF, 11.11±0.46 vs. 16.66±0.96 in AM; IL-18 (ng/L): 50.44±3.30 vs. 66.31±5.67 in BALF, 43.95±0.88 vs. 73.52±1.23 in AM, all P < 0.05]. There was no significant difference in the detection indicators between the overexpression empty vector+LPS group and the LPS group. HSF1 attenuates LPS-induced ALI in rats, and the mechanism may be related to the inhibition of NLRP3 inflammasome activation in AM.

Electroacupuncture ameliorates inflammatory response of mice with acute myocardial ischemia by regulating vascular endothelial growth factor C/vascular endothelial growth factor receptor 3 pathway

To observe the effect of electroacupuncture (EA) on the cardiac function, lymphatic markers, macrophage and inflammatory cytokines in acute myocardial ischemia (AMI) mice, so as to explore its mechanism in improving AMI. Male C57BL/6 mice were randomly divided into sham operation, model, EA, inhibitor and inhibitor+EA groups, with 10 mice in each group. AMI model was established by occlusion of left anterior descending coronary artery. For mice in the EA group and inhibitor+EA group, EA (1 mA, 2 Hz/15 Hz) was applied to bilateral "Shenmen"(HT7) and "Tongli"(HT5) for 30 min, once daily for consecutive 3 days. Mice in the inhibitor+EA group were given intraperitoneal injection of vascular endothelial growth factor receptor-3 (VEGFR-3) inhibitor SAR131675 30 min before the EA, while mice in the inhibitor group were given intraperitoneal injection of SAR131675 only. The electrocardiogram (ECG) of the neck-thoracic lead was recorded and analyzed by BL-420F biological function experiment system. Histopathologic changes of myocardial tissue were observed after H.E. staining. The contents of lactate dehydrogenase (LDH), cardiac troponin I (cTnI) in serum and interleukin-18 (IL-18) and interleukin-6 (IL-6) in ischemic myocardium were determined by ELISA. The expressions of hyaluronic acid receptor-1 (LYVE-1) and macrophage mar-ker CD68 (CD68) in the myocardial tissue were detected by immunofluorescence assay. The protein expression levels of vascular endothelial growth factor C (VEGF-C) and VEGFR-3 were detected by Western blot. Compared with the sham operation group, the ECG-ST level, the contents of serum LDH and cTnI, and the contents of IL-18 and IL-6 in the myocardial tissue were significantly increased (P<0.01), the expression of LYVE-1 and the protein expression levels of VEGF-C and VEGFR-3 in the myocardial tissue were significantly decreased (P<0.01), while the number of CD68 positive cells was significantly increased (P<0.01) in the model group. Compared with the model, inhibitor and inhibitor+EA groups, the ECG-ST level, the contents of serum LDH and cTnI, and the contents of IL-18 and IL-6 in the myocardial tissue were decreased (P<0.01), the expression of LYVE-1 and the expression level of VEGFR-3 protein were increased (P<0.01), while the number of CD68 positive cells was significantly decreased (P<0.01) in the EA group. Compared with the model and inhibitor groups, the expression level of VEGF-C was increased (P<0.01) in the EA group. Outcomes of H.E. staining showed that the myocardial fibers were disordered with a large number of inflammatory cell infiltration in the model group, which was milder in the EA group. Acupuncture can improve the inflammatory injury of AMI mice, which may be related to activate VEGF-C/VEGFR-3 pathway to promote lymphangiogenesis, reduce macrophage infiltration and inflammatory factors.

Effect of electroacupuncture combined with Zhuang-medicine-thread moxibustion on oxidative stress in gastric antrum of diabetic gastroparesis rats

To observe the effect of electroacupuncture (EA) combined with Zhuang-medicine-thread moxibustion on oxidative stress-related indicators in diabetic gastroparesis (DGP) rats, so as to explore its mechanism underlying improvement of DGP. Male SD rats were randomly divided into normal, model, medication, EA, Zhuang-medicine-thread moxibustion (moxibustion) and EA+Zhuang-medicine-thread moxibustion (combination) groups (15 rats in each group). The DGP model was established by intraperitoneal injection of streptozotocin (STZ). Rats of the medication group were treated by gavage of 0.15 mg/mL mosapride citrate suspension（10 mL/kg）. EA (10 Hz/50 Hz, 2 mA, 20 min) or Zhuang-medicine-thread moxibustion (3 cones) was applied to "Zhongwan" (CV12), bilateral "Neiguan" (PC6) and bilateral "Sanyinjiao" (SP6) of the related groups, once a day for 3 weeks. The body weight, blood glucose, gastric emptying rate and intestinal propulsion rate of rats were measured. The serum malondialdehyde (MDA) content was measured by thiobarbituric acid method, the serum supero-xide dismutase (SOD) activity was measured by xanthine oxidase method, and the serum reactive oxygen species (ROS) activity was detected by ELISA. HE staining was used to observe the pathological changes of gastric antrum. The expression of heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), nicotin-amide adenine dinucleotide phosphate oxidases (NOX4), peroxisome proliferators activated receptor-gamma coactivator 1α(PGC-1α) proteins and mRNAs in gastric antrum was detected by Western blot and real-time quantitative PCR, respectively. Compared with the normal group, the body weight, gastric emptying rate, intestinal propulsion rate, serum SOD activity, the expressions of HO-1, PGC-1α, total Nrf2 proteins and mRNAs, and Nrf2 nuclear translocation in gastric antrum were decreased (P<0.01), while the blood glucose, serum MDA content and ROS activity, NOX4 protein and mRNA expressions in gastric antrum were increased (P<0.01) in the model group. Compared with the model group, the blood glucose was decreased in the EA, moxibustion and combination groups (P<0.01); the body weight, gastric emptying rate, intestinal propulsion rate, and the expressions of HO-1 and PGC-1α mRNAs in gastric antrum were all increased in the four treatment groups (P<0.01, P<0.05), while the serum MDA content and ROS activity, NOX4 protein and mRNA expressions in gastric antrum were all decreased (P<0.01); the serum SOD activity and total Nrf2 protein expression in gastric antrum were increased in medication, moxibustion and combination groups (P<0.01, P<0.05); the expressions of HO-1 and PGC-1α proteins, and Nrf2 nuclear translocation in gastric antrum were increased in medication and combination groups (P<0.05, P<0.01); the expression of Nrf2 mRNA was increased in the medication, EA and combination groups (P<0.01, P<0.05). Compared with the combination group, the body weight, gastric emptying rate and intestine propulsion rate were decreased in the medication, EA and moxibustion groups(P<0.01, P<0.05), and the blood glucose increased (P<0.01); the serum MDA content and ROS activity, NOX4 protein and mRNA expressions in gastric antrum were increased (P<0.01, P<0.05), serum SOD activity, and the expressions of total Nrf2 protein, PGC-1α protein and mRNA, HO-1 mRNA in gastric antrum were decreased (P<0.01, P<0.05) in the EA and moxibustion groups; the expression of Nrf2 mRNA was decreased in the moxibustion group (P<0.05). HE staining showed a large number of inflammatory cell infiltration in the lamina propria and submucosa, and the gastric glands in the lamina propria were significantly expanded, the submucosa was severely edematous in the model group, which were relative milder in the four treatment groups. EA combined with Zhuang-medicine-thread moxibustion can effectively improve the activity of antioxidant enzymes, reduce the production of lipid peroxide, and regulate the expression of antioxidant related proteins and genes, which may be one of the mechanisms in treating DGP.

Effects and molecular mechanism of exogenous L-carnitine on excessive endoplasmic reticulum stress-mediated hepatic pyroptosis in severely scald rats

Effects and molecular mechanism of exogenous L-carnitine on excessive endoplasmic reticulum stress-mediated hepatic pyroptosis in severely scald rats

The role of miR-135b-5p in inhibiting mice acute lung injury (ALI) induced by sepsis and its mechanism

Objective: To investigate the expression status of miR-135b-5p in the sepsis induced acute lung injury (ALI) mice and its effects on inflammatory responses and cell pyroptosis in mice pulmonary tissues. Methods: The cecal ligation puncture (CLP) method was employed to construct sepsis-induced ALI mice models. The C57BL/6 mice were randomly divided into 6 groups with 8 mice in each group. The sepsis mouse models were constructed by performing CLP surgery: mice were anesthetized by intraperitoneal injection of 0.1 mg/kg barbital, the abdomen was cut longitudinally to expose the cecum, the cecum was ligated and perforated with syringe needle, the wound was sutured after extruding part of the intestinal contents. The sham operation group (Sham group) did not undergo any treatment and suture wounds after laparotomy, and no CLP operation was performed. The treatment groups were divided into CLP+NC mimic group, CLP+ miR-135b-5p mimic group, CLP+NC mimic+ Empty vector group, CLP+GSDMD group, and CLP+ miR-135b-5p mimic+GSDMD group. One week before CLP surgery, mice in the treatment group were injected subcutaneously with 200 μ L NC mimic (200 nmol/L), miR-135b-5p mimic (200 nmol/L), Empty vector (100 nmol/L), GSDMD Vector (100 nmol/L), and miR-135b-5p mimic (200 nmol/L), once a day for a week. The euthanasia was performed 24 h after operation by carbon dioxide asphyxiation. The qRT-PCR was utilized to determine miR-135b-5p and GSDMD expressions;HE staing assay was performed to observe the pathological changes of pulmonary tissues. The mice right lung tissues were flushed with 5 ml saline for 3 times, and each flush lasted for 3~5 min to collect the BALF, and the levels of GSDMD, IL-1β and IL-18 in BALF were determined by ELISA. The protein levels of NLRP3, caspase 1 and cleaved GSDMD in mice lung tissues were examined by immunoblotting analysis; Dual-luciferase reporter gene system assay was employed to validate the targeting relationship of miR-135b-5p and GSDMD. Results: Compared with the control group mice, there were a large number of inflammatory cell infiltration, alveolar damage, interstitial edema and alveolar collapse in the lung tissues of the CLP group mice (P<0.01), and the expression levels of the pyroptosis-associated proteins (NLRP3, caspase-1 and GSDMD) were all increased, while miR-135b-5p was down-regulated in the CLP group (P<0.01). Further experiments confirmed that overexpression of miR-135b-5p significantly reduced CLP-induced pyroptotic cell death in mice lung tissues (P<0.01), and dual-luciferase reporter gene system assay confirmed that miR-135b-5p targeted GSDMD for its degradation. Moreover, the rescuing experiments validated that up-regulation of GSDMD abrogated the inhibition effects of miR-135b-5p overexpression on cell pyroptosis in CLP mice lung tissues (P<0.01). Consistently, we verified that miR-135b-5p also suppressed the expression levels of IL-1β and IL-18 in mice BALF via degrading GSDMD (P<0.05). Conclusion: Overexpression of miR-135b-5p attenuated sepsis-induced ALI by inhibiting GSDMD-mediated pyroptotic cell death, and this study provided potential therapeutic target and theoretical foundation for sepsis-induced ALI treatment.

Saikosaponin d Alleviates Liver Fibrosis by Negatively Regulating the ROS/NLRP3 Inflammasome Through Activating the ERβ Pathway.

Background and aims: Saikosaponin d (SSd) has a steroidal structure and significant anti-inflammatory effects. The purpose of this study was to explore the mechanism underlying SSd’s inhibitory effects on liver fibrosis. Methods: Wild-type and estrogen receptor knockout (ERKO) mice were treated with CCl4 to establish liver fibrosis mouse models. The effects of SSd on hepatic fibrogenesis were studied in these mouse models. Hepatic stellate cells (HSCs) were activated by H2O2 to investigate the potential molecular mechanisms. The establishment of the models and the degrees of inflammation and liver tissue fibrosis were evaluated by detecting changes in serum liver enzymes and liver histopathology. The expression of α-SMA and TGF-β1 was determined by immunohistochemistry. The expression and significance of NLRP3 inflammasome proteins were explored by RT-PCR and Western blotting analyses. The mitochondrial ROS-related indexes were evaluated by MitoSOX Red. Results: In wild-type and ERKO mice treated with CCl4, the fluorescence expression of mitochondrial ROS was up-regulated, while the mitochondrial membrane potential and ATP content were decreased, suggesting that the mitochondria were damaged. In addition, the expression of NLRP3 inflammatory bodies and fibrosis markers (α-SMA, TGF-β, TIMP-1, MMP-2, and Vimentin) in liver tissue increased. Furthermore, the above indexes showed the same expression trend in activated HSCs. In addition, the peripheral serum ALT and AST levels increased in CCl4-induced liver injury model mice. And HE staining showed a large number of inflammatory cell infiltration in the liver of model mice. Picric acid-Sirius staining and Masson staining showed that there was significant collagen fibrous tissue deposition in mice liver sections. IHC and WB detection confirmed that the expression of α-SMA and TGF-β1 increased. Liver fibrosis scores were also elevated. Then, after SSd intervention, the expression of ROS in wild-type mice and αERKO mice decreased, mitochondrial membrane potential recovered, ATP level increased, NLRP3 inflammasome and fibrosis indexes decreased, liver enzyme levels decreased, and liver pathology showed liver inflammation. The damage and collagen deposition were significantly relieved, the expression of α-SMA and TGF-β1 was decreased, and the fibrosis score was also decreased. More importantly, the effect of SSd in alleviating liver injury and liver fibrosis had no effect on βERKO mice. Conclusion: SSd alleviated liver fibrosis by negatively regulating the ROS/NLRP3 inflammasome through activating the ERβ pathway. By establishing liver fibrosis models using wild-type and ERKO mice, we demonstrated that SSd could alleviate liver fibrosis by inhibiting the ROS/NLRP3 inflammasome axis through activating the ERβ pathway.

Dexmedetomidine alleviates hyperalgesia in arthritis rats through inhibition of the p38MAPK signaling pathway

Background Dexmedetomidine (DEX) has showed significant analgesic effects in neuropathic pain, but the underlying mechanism has remained elusive. Our present study aimed to explore the effect of DEX on hyperalgesia with the involvement of p38MAPK signaling pathway in a rat model of monoarthritis (MA). Methods MA rat model was induced by injection of Complete Freund’s Adjuvant (CFA). Pathological changes of MA rats were observed by HE staining and Safranin-O/Fast Green staining. Ankle circumference, paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were measured to judge the degree of hyperalgesia in MA rats. Immunohistochemistry and ELISA were applied to observe the degree of inflammation in rats. Western blot analysis was conducted to detect expression of p38MAPK signaling pathway-related factors. The mechanism of p38MAPK signaling pathway in MA rats was observed via treatment of Anisomycin or SB203580 combined with DEX. Results After 8 h of CFA induction, joint swelling and hyperalgesia occurred in rats. There were obvious pathological changes in the joint cavity, the joint cavity space became narrow and synovial bursa became rough. A large number of inflammatory cell infiltration was observed under microscope. After injection of DEX and SB203580, PWT and PWL were prolonged, the expression of serum inflammatory factors was decreased, and the expression of p38MAPK signaling pathway-related factors was decreased; while all the detected indexes were recovered in MA rats after treated with DEX and Anisomycin. Conclusions Our study provided evidence that DEX could alleviate hyperalgesia in arthritis rats through inhibition of the p38MAPK signaling pathway.

Effect of Aerobic Treadmill Exercise Regulating Glucose Transporter 9 on Renal Function in Hyperuricemia Rats

<bold>Objective</bold> To explore the possible mechanism of aerobic treadmill exercise in improving renal function in hyperuricemia (HUA) rats. <bold>Methods</bold> A total of 24 male SD rats were randomly divided into the Control group, the Model group and the Exercise group according to the random number table method, with 8 rats in each group. The Control group was given 2 g/(kg·d) distilled water intragastric administration, the Model group and the Exercise group were given 2 g/(kg·d) oxonic aid potassium salt (OAPS) intragastric administration, continuous intragastric administration for 10 days. From the 11th day, the Control group was given distilled water intragastric administration every other day, and the Model group was given OAPS intragastric administration every other day to maintain high uric acid status, with a dose of 2 g/(kg·d). In both groups, diet and water were not restricted, exercise freely, without other intervention, lasted for three weeks. The Exercise group was given intragastric administration as same as the Model group, and was given aerobic treadmill training simultaneously, the speed was 12 m/min, 20 minutes a time, once a day, continuous training for six days and rest for one day, continuous training for three weeks. Hematoxylin-eosin (HE) was used to stain the renal histopathological section, and the microscope (×400) was used to observe the structural changes of renal tubules and glomerulus; the peroxidase method was used to detect the renal function indexes such as uric acid (UA), creatinine (Cre) and urea nitrogen (BUN); enzyme-linked immunosorbent assay (ELISA) method was used to determine the contents of interleukin-1β (IL-1β) in serum and kidney tissues; Western blot was used to detect the expressions of Caspase-1, IL-1β and glucose transporter 9 (GLUT-9) in the kidney tissues. <bold>Results</bold> ① Pathological examination results: there was not obvious pathological changes were observed in the kidney tissues of the Control group; in the Model group, the renal tissue capsule was incomplete, the glomerulus was deformed and atrophic, and the renal tubules were disordered; in the Exercise group, the number of inflammatory cell infiltration was significantly decreased, the renal tubular structure was relatively intact, and the pathological changes such as cytoplasmic vacuolation of renal tubular epithelial cells were significantly decreased. ② UA, Cre and BUN content of serum: compared with the Control group, UA, BUN and Cre content in serum of the Model group and Exercise group was significantly increased (<italic>P</italic>&lt;0.05); compared with the Model group, UA, BUN and Cre content in serum of the Exercise group were significantly decreased (<italic>P</italic>&lt;0.05). ③ Serum and kidney IL-1β content: compared with the Control group, the expression of IL-1β in serum and kidney of the Model group and the Exercise group were significantly increased (<italic>P</italic>&lt;0.05); compared with the Model group, the expression of IL-1β in serum and kidney of the Exercise group was significantly decreased (<italic>P</italic>&lt;0.05). ④ Expressions of Caspase-1, IL-1β and GLUT-9 protein: compared with the Control group, the expressions of Caspase-1, IL-1β and GLUT-9 protein in kidney tissues of the Model group and the Exercise group were significantly increased (<italic>P</italic>&lt;0.05); compared with the Model group, the expressions of Caspase-1, IL-1β, and GLUT-9 protein in kidney tissues of the Exercise group were significantly decreased (<italic>P</italic>&lt;0.05). <bold>Conclusion</bold> Aerobic treadmill exercise can effectively reduce the level of uric acid and inflammatory response of hyperuricemia rats, and improve renal function, which may be related to the regulation of GLUT-9 protein expression.

Moxibustion regulates the polarization of macrophages through the IL-4/STAT6 pathway in rheumatoid arthritis.

To observe the effects of moxibustion on "Shenshu" and "Zusanli" on macrophage polarization and IL-4/STAT6 signaling pathway in rats with rheumatoid arthritis (RA). To further explore the possible anti-inflammatory mechanism of moxibustion in the treatment of RA. The rats' right hind paws were injected with freund's complete adjuvant (FCA) to establish the model of RA. Seven days after the injection of FCA, moxibustion therapy was performed on the acupoints of Shenshu (BL23) and Zusanli (ST36) once a day for three weeks. The researchers measured the thickness of the foot pad. ELISA and Histological Analysis were performed to observe the anti-inflammatory effect of moxibustion. Then researchers detected the expression of macrophage phenotype and the expression of IL-4/STAT6 signaling pathway related molecules. It was observed that after the injection of FCA, the rats' feet showed obvious symptoms of redness and swelling. But the symptoms were significantly improved when moxibustion was employed. The study found lower IL-23 and higher IL-4 level in the serum of FCA-injected rats after moxibustion treatment. HE staining showed that the synovium of the RA group was hyperemia and edema, with a large number of inflammatory cells infiltration and vascular dilatation. In the moxibustion group, the degree of synovial hyperemia and edema was improved, and the infiltration of inflammatory cells and vascular dilation were reduced. The study also found that there wer differences among the expressions of macrophage phenotypes in RA, and this was shown by the high expression of CD86 and low expression of CD206. However, the polarization of macrophages in the moxibustion group changed, and that was manifested by enhanced M2-polarized Mφs and inhibited M1-polarized Mφs. Meanwhile, moxibustion suppressed the activation of JAK1, JAK3 and STAT6 in the IL-4/STAT6 signaling pathway, which contributed to the polarization of M2 . The results demonstrate that moxibustion not only suppresses the polarization of M1, but also promotes the polarization of M1. The anti-inflammatory effect of moxibustion may be related to the regulation of macrophage polarization through IL-4/STAT6 signaling pathway.

Effect of Houpo-Mahuang Decoction on aggravated asthma induced by cigarette smoke and the expression of TRPA1 and tight junctions in mice

Ethnopharmacological relevanceCigarette smoke (CS) is a common environmental irritant and a risk factor for asthma, as it induces as well as aggravates asthmatic attacks. The injured airway epithelial tight junctions (TJs) aggravate asthma. CS can aggravate asthma by activating the transient receptor potential ankyrin A1 (TRPA1) channel and enhancing TJs destruction. Houpo Mahuang decoction (HPMHD) is a classic traditional Chinese prescription for the treatment of asthma. However, its underlying action mechanism is unclear. Aim of the studyThe present study aimed to evaluate the effect of HPMHD on the asthma phenotype and the regulation of TRPA1 and TJs in a CS-induced mouse model of aggravated asthma. Materials and methodsUnder optimized chromatographic and mass spectrometry conditions, the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) technique was used to detect and analyze the major chemical components of HPMHD. C57BL/6 female mice were randomly divided into seven groups, viz, normal saline (NS) group, ovalbumin (OVA) + CS group, dexamethasone group, HPMHD high-dose group and low-dose groups, n-butanol extract group, and ethyl acetate extract group, with 10 mice in each group. OVA sensitization and challenge, and CS exposure were used to establish the aggravated asthma model. As the main indices to evaluate the protective effect of HPMHD, the eosinophils count in peripheral blood, percentages of inflammatory cells classified and the levels of interleukin (IL)-4, IL-5, IL-13 in the bronchoalveolar lavage fluid (BALF), airway responsiveness enhanced pause (Penh), and changes in lung histopathology were determined and compared among the groups. The mRNA and protein expression of TRPA1 and TJs in lung tissue was also examined. ResultsUsing UPLC-QTOF-MS, the chemical components of HPMHD, including ephedrine, pseudoephedrine, laetrile, and amygdalin amide, were identified by 51 signal peaks. Compared with those in the NS group, the eosinophil number in the peripheral blood and the eosinophils and neutrophils percentages in BALF of the OVA + CS group were remarkably increased. Following the inhalation of 50 μl of acetylcholine chloride (ACH) at doses of 25 and 50 mg/mL, the Penh increased significantly (p < 0.01). Moreover, in the OVA + CS group, hematoxylin and eosin (H&E) staining of lung tissue showed a significant number of infiltrated inflammatory cells, increased mucus secretion in the lumen, damaged bronchial mucosa, increased thickness of tracheal wall, and increased score of lung damage (p < 0.01). The IL-4/5/13 levels were also remarkably increased (p < 0.01). The protein as well as gene expression of both ZO-1 and occludin decreased markedly in the lung tissue, while the expression of TRPA1 and claudin-2 was increased (p < 0.05, p < 0.01). Next, the OVA + CS group and the treatment groups were compared. The inflammatory cells, Penh value, and levels of IL-4/5/13 were significantly reduced, and less lung injury was observed in the treatment groups. The gene and protein levels of TRPA1 and TJs were corrected (p < 0.05, p < 0.01); the effects on the HPMHD high-dose and ethyl acetate extract groups were particularly remarkable. ConclusionsHPMHD reduced airway hyperresponsiveness, inflammatory cell recruitment and Th2 cytokine secretion in CS-induced aggravated asthma mice, in a manner potentially dependent on regulation of the expression of TRPA1 and TJ proteins. Both the n-butanol and ethyl acetate extracts contained the active ingredients, especially the ethyl acetate extract.

Effect of Zhuanggu Jianxi Decoction on Synovitis in Knee Osteoarthritis Rabbits via LXRs/NF-&amp;kappa;B Pathway

<sec><title>Objective</title> To study the intervention effect and mechanism of Zhuanggu Jianxi decoction on synovitis in knee osteoarthritis (KOA) rabbits via the inflammatory regulation of synovial tissue and the expression of key node proteins in LXRs/NF-κB pathway. </sec><sec><title>Methods</title> Blank serum and drug-containing serum of rabbits were prepared for the intervention of synovial tissue. Twenty-two rabbits were randomly divided into two groups, with 16 in the model group and 6 in the blank group. The KOA model was injected with papain into the knee joint of the right hind limb. After the model was identified successfully, the synovial tissue of the right hind limb knee was cut into small pieces with a size about 1-2 mm³ under aseptic operation, 60 pieces were collected and randomly divided into model group, Zhuanggu Jianxi decoction group, LXRα inhibitor group and N-CoR inhibitor group with 15 pieces each, the synovial tissue pieces in each group were cultured in 6 well plates with every 3 pieces in each well, the four groups were intervened with 10% blank serum, 10% drug-containing serum, 10% drug-containing serum+5CPPSS-50 and 10% drug-containing serum+ML-792 respectively; the synovium of the blank group was prepared by the same method and cultured with 10% blank serum for 5 wells. After 7 days, synovial tissue was collected. Two wells of synovial tissue were taken from each group for HE staining, and the remaining synovial tissue was detected by Western blot and ELISA. </sec><sec><title>Results</title> 1) Morphology: compared with the blank group, HE staining in the model group showed the proliferation and disorder of synovial lining cells, a large number of inflammatory cell infiltration and fibrous tissue hyperplasia, and the morphological score was significantly increased (<italic>P</italic><0.05). Compared with the model group, mild hyperplasia and disorder of synovial lining cells, sparse scattered inflammatory cell infiltration and a small amount of fibrous tissue hyperplasia were observed in the Zhuanggu Jianxi decoction group, and the morphological score was significantly decreased (<italic>P</italic><0.05). Compared with the Zhuanggu Jianxi decoction group, the morphological score of pathological changes in the LXRα and N-CoR inhibitor groups was significantly increased (<italic>P</italic><0.05). 2) Inflammation-related indicators: compared with the blank group after intervention, the contents of IL-1β, TNF-α, MMP-3 and MMP-13 in the model group were significantly increased (<italic>P</italic><0.05); compared with the model group, the indexes above in the LXRα inhibitor group, N-CoR inhibitor group and Zhuanggu Jianxi decoction group were significantly decreased (<italic>P</italic><0.05); compared with the Zhuanggu Jianxi decoction group, the indexes above in the LXRα and N-CoR inhibitor groups were significantly increased (<italic>P</italic><0.05). Before and after intervention, the contents of IL-1β and TNF-α in the Zhuanggu Jianxi decoction group were significantly decreased (<italic>P</italic><0.05). 3) Protein expression: Compared with the blank group, the expressions of LXRα and N-CoR proteins in the model group were significantly decreased (<italic>P</italic><0.05), and the expression of P50 and P65 proteins were significantly increased (<italic>P</italic><0.05). Compared with the model group, LXRα and N-CoR protein in the LXRα inhibitor group and N-CoR protein in the N-CoR inhibitor group were not significantly decreased (<italic>P</italic>>0.05), P50 and P65 protein expression were significantly decreased (<italic>P</italic><0.05). The protein expression of LXRα and N-CoR in the Zhuanggu Jianxi decoction group were significantly increased (<italic>P</italic><0.05), and the protein expression of P50 and P65 were significantly decreased (<italic>P</italic><0.05). Compared with the Zhuanggu Jianxi decoction group, the protein expression of LXRα and N-CoR in both the LXRα and N-CoR inhibitor groups were significantly decreased (<italic>P</italic><0.05), and the protein expression of P50 and P65 were significantly increased (<italic>P</italic><0.05). </sec><sec><title>Conclusion</title> Zhuanggu Jianxi decoction could improve the pathological manifestations of synovial lining cell hyperplasia and the disorder arrangement, inflammatory cell infiltration and fibrous tissue hyperplasia, reduce the content of inflammatory related indexes in tissues, so as to reduce the inflammatory response of KOA synovial tissue in rabbits, which may be related to the up-regulation of LXRα and N-CoR protein expression in synovial cells via LXRs/NF-κB pathway in synovial tissue, thereby down-regulating P50 and P65 protein to reduce IL-1β, TNF-α, MMP-3 and MMP-13. </sec>

Phytosomal curcumin alleviates collagen-induced arthritis by downregulating Th17 and upregulating Treg cell responses in rats

Objective: To explore the effects of a nano-formulation of curcumin (phytosomal curcumin) on the clinical and pathological symptoms of collagen-induced arthritis (CIA) in rats.Methods: Forty male Wistar rats were immunized with an emulsion containing bovine type II collagen and incomplete Freund's adjuvant and then administered phytosomal curcumin post-immunization. Clinical symptoms and histological analysis of the synovial tissues were performed. The effect of phytosomal curcumin on Th17 and Treg parameters was also evaluated.Results: Phytosomal curcumin reduced the clinical severity and paw swelling in CIA-induced rats, which was accompanied by a reduction in the number of inflammatory cell infiltration in the synovial tissue. Additionally, treatment with phytosomal curcumin significantly inhibited CIA-associated mediators as well as increased the anti-inflammatory mediators in comparison to the control groups.Conclusions: Phytosomal curcumin could improve CIA autoimmune responses and can be considered a potential candidate for the treatment of rheumatoid arthritis.

Effects of reactive oxygen species-responsive antibacterial microneedles on the full-thickness skin defect wounds with bacterial colonization in diabetic mice

Effects of reactive oxygen species-responsive antibacterial microneedles on the full-thickness skin defect wounds with bacterial colonization in diabetic mice