Herbal formula Jiawei Xiaochengqi decoction prevents postoperative abdominal adhesion in a rat model through inhibition of CXCL2-CXCR2 pathway.

BACKGROUND: Postoperative abdominal adhesion (PAA) is the most common complication after abdominal surgeries, which can lead to intestinal obstruction, chronic abdominal pain or female infertility. Jiawei Xiaochengqi decoction (JWXCQ) is a hospital preparation widely used for PAA treatment in Nanfang Hospital of Southern Medical University for more than twenty years. PURPOSE: This study aimed to investigate the therapeutic effects and potential mechanism of JWXCQ against PAA and provide beneficial information for its clinical application. METHODS: The main active components of JWXCQ were identified using ultra high performance liquid chromatography (UHPLC) combined with standard substance comparison. The efficacy and underlying mechanism of JWXCQ were evaluated through in vivo experiments with a postsurgical-induced peritoneal adhesion rat model, and in vitro studies with LPS-stimulated Raw 264.7 macrophages and primary fibroblasts. H&E and Masson staining were performed to assess histopathological changes. The levels of cytokines/proteins-associated with inflammation and degradation of extracellular matrix as well as CXCL2-CXCR2 pathway-related proteins were determined by ELISA, qRT-PCR, western blot assays or immunohistochemistry, respectively. Furthermore, siCXCR2 transfection was used to validate the mechanism of action of JWXCQ. RESULTS: JWXCQ treatment significantly reduced the formation of PAA, inhibited the inflammation and collagen deposition, and facilitated the secretion of MMP9, decreased the levels of IL-1ß, IL-6, TIMP1, COL-1, and suppressed the CXCL2-CXCR2 pathway in PAA rats. Furthermore, JWXCQ inhibited its downstream pathways, the JAK2-STAT3 and PI3K-AKT signaling, as indicated by the suppression of the phosphorylation levels of STAT3 and AKT. In vitro cell experiments revealed that JWXCQ reduced IL-1ß and IL-6 secretion in Raw 264.7 macrophages and COL-1 in primary fibroblasts. The CXCL2-CXCR2, JAK2-STAT3 and PI3K-AKT pathways were also inhibited after JWXCQ treatment, which were consistent with the in vivo results. More importantly, silence of CXCR2 eliminated the regulatory effects of JWXCQ. CONCLUSION: JWXCQ could effectively prevent the PAA formation by alleviating inflammation and collagen deposition, which was associated with the inhibition of CXCL2-CXCR2 pathway. This study investigated the relevant pharmacological mechanisms of JWXCQ, providing further evidence for the application of JWXCQ in clinical PAA treatment.

Resveratrol Inhibits High Glucose-Induced H9c2 Cardiomyocyte Hypertrophy and Damage via RAGE-Dependent Inhibition of the NF-κB and TGF-ß1/Smad3 Pathways.

Hyperglycaemia is associated with the development of cardiac vascular disease. Resveratrol (RES) is a naturally occurring polyphenolic compound that possesses many biological properties, including anti-inflammatory properties and antioxidation functions. Our study aimed to explore the RES's protective roles on high glucose (HG)-induced H9c2 cells and the underlying mechanisms. Small-molecule inhibitors, western blotting (WB), as well as reverse-transcription PCR (RT-PCR) were employed to investigate the mechanisms underlying HG-induced damage in H9c2 cells. RES (40 µg/mL) treatment significantly alleviated HG-induced cardiac hypertrophy and cardiac dysfunction. RES abated the HG-induced increase in the levels of extracellular matrix (ECM) components and inflammatory cytokines, reducing ECM accumulation and inflammatory responses. Additionally, RES administration prevented HG-induced mitochondrion-mediated cardiac apoptosis of myocardial cells. In terms of mechanisms, we demonstrated that RES ameliorated the HG-induced overexpression of receptor for advanced glycation endproducts (RAGE) and downregulation of NF-κB signalling. Moreover, RES inhibited HG-induced cardiac fibrosis by inhibiting transforming growth factor beta 1 (TGF-ß1)/Smad3-mediated ECM synthesis in cultured H9c2 cardiomyocytes. Further studies revealed that the effects of RES against HG-induced upregulation of NF-κB and TGF-ß1/Smad3 pathways were similar to those of FPS-ZM1, a RAGE inhibitor. Collectively, the results implied that RES might help alleviate HG-induced cardiotoxicity via RAGE-dependent downregulation of the NF-κB and TGF-ß/Smad3 pathways. This study provided evidence that RES can be developed as a promising cardioprotective drug.

Transcriptome sequencing analysis of primary fibroblasts: a new insight into postoperative abdominal adhesion.

PURPOSE: The specific genes or pathways in fibroblasts responsible for the pathogenesis of postoperative abdominal adhesion (PAA) remain to be elucidated. We aim to provide a new insight into disease mechanisms at the transcriptome level. METHODS: Male Sprague-Dawley rats were used to establish a PAA model. Primary fibroblasts were separated from normal peritoneal tissue (NF) and postoperative adhesion tissue (PF). RNA sequencing was used to analyze the transcriptome in NF and PF. RESULTS: One thousand two hundred thirty-five upregulated and 625 downregulated DEGs were identified through RNA-Seq. A pathway enrichment analysis identified distinct enriched biological processes, among which the most prominent was related to immune and inflammatory response and fibrosis. HE staining and Masson's trichrome staining histologically validated the RNA-Seq results. Six hub genes, ITGAM, IL-1ß, TNF, IGF1, CSF1R and EGFR were further verified by RT-PCR. CONCLUSIONS: Our study revealed the roles of the immune and inflammatory responses and fibrosis in the process of PAA. We also found six hub genes that may be potential therapeutic targets for PPA.

Aldose Reductase Inhibitor Engeletin Suppresses Pelvic Inflammatory Disease by Blocking the Phospholipase C/Protein Kinase C-Dependent/NF-κB and MAPK Cascades.

Pelvic inflammatory disease (PID) is a common inflammation in the upper reproductive tract in women and may cause serious and costly consequences without effective treatment. Engeletin is a flavanonol glycoside and a naturally derived aldose reductase (AR) inhibitor that is widely distributed in vegetables, fruits, and plant-based foods. The present study investigated the anti-PID activity of engeletin in a mucilage-induced rat model of PID and LPS-stimulated RAW 264.7 macrophages. Engeletin significantly reduced inflammation and ameliorated the typical uterine pathological changes in PID rats. Engeletin also inhibited AR-dependent PLC/PKC/NF-κB and MAPK inflammatory pathways, as indicated by the suppression of the phosphorylation levels of PLC, PKC, p38, ERK, and JNK and the nuclear translocation of NF-κB p65. In vitro studies demonstrated that engeletin significantly inhibited inflammatory mediator expression and enhanced the phagocytic ability of LPS-induced RAW 264.7 macrophages. RNA interference of AR prevented the engeletin-induced inhibition of inflammatory mediators. Engeletin also inhibited AR-dependent PLC/PKC/NF-κB and MAPK inflammatory pathways, which was consistent with the in vivo results. These findings support engeletin as a potential agent for prevention or treatment of PID.

CXC Chemokine Receptors in the Tumor Microenvironment and an Update of Antagonist Development.

Chemokine receptors, a diverse group within the seven-transmembrane G protein-coupled receptor superfamily, are frequently overexpressed in malignant tumors. Ligand binding activates multiple downstream signal transduction cascades that drive tumor growth and metastasis, resulting in poor clinical outcome. These receptors are thus considered promising targets for anti-tumor therapy. This article reviews recent studies on the expression and function of CXC chemokine receptors in various tumor microenvironments and recent developments in cancer therapy using CXC chemokine receptor antagonists.

Application of Traditional Chinese Medicines in Postoperative Abdominal Adhesion.

Adhesion is a frequent complication after abdominal surgery. Although various methods have been applied to prevent and treat postoperative abdominal adhesion (PAA), few modern drugs designed for clinical applications have reached the expected preventive or therapeutic effect so far. There is an imperative to develop some new strategies for the treatment of PAA. Traditional Chinese medicine (TCM) has been widely practiced for thousands of years and played an indispensable role in the prevention and treatment of diseases. Modern medicine researchers have accepted the therapeutic effects of many active components derived from Chinese medicinal herbs. The review stresses the most commonly used TCM treatment, including Chinese medicinal herbals and monomers, TCM formulas, and acupuncture treatment.

Sodium aescinate significantly suppress postoperative peritoneal adhesion by inhibiting the RhoA/ROCK signaling pathway.

BACKGROUND: Although mechanical barriers and modern surgical techniques have been developed to prevent postoperative adhesion formation, high incidence of adhesions still represents an important challenge in abdominal surgery. So far, there has been no available therapeutic drug in clinical practice. PURPOSE: In this study, we explored the efficacy of sodium aescinate (AESS) treatment against postoperative peritoneal adhesions, the potential molecular mechanism was also investigated. STUDY DESIGN AND METHODS: Sixty male Sprague-Dawley rats were randomly divided into 6 groups for the study: the blank, vehicle, positive control and three AESS administration groups (0.5, 1 and 2 mg/kg/d, intravenous administration for 7 days). Adhesions were induced by discretely ligating peritoneal sidewall. An IL-1ß-induced HMrSV5 cell model was also performed to explore possible functional mechanism. RESULTS: The results indicated that the incidence and severity of peritoneal adhesions were significantly lower in the AESS-treated groups than that in the vehicle and positive control group. AESS-treated groups showed that the secretion, activity, and expression of tPA in rat peritoneum were notably increased. The FIB levels in rat plasma were decreased. The immunohistochemical staining analysis demonstrated that collagen I and α-SMA deposition were significantly attenuated in AESS-treated peritoneal tissues. Besides, we found that AESS treatment reduced the protein levels of p-MYPT1. To further explore the mechanisms of AESS, both activator and inhibitors of RhoA/ROCK pathway were employed in this study. It was found that AESS-induced up-regulation of tPA was reversed by activator of ROCK, but the effects of ROCK inhibitors were consistent with AESS. CONCLUSION: Taken together, the findings of in vivo and in vitro experiments proved that AESS could significantly suppress postoperative peritoneal adhesion formation through inhibiting the RhoA/ROCK signaling pathway. Our researches provide important pharmacological basis for AESS development as a potential therapeutic agent on peritoneal adhesions.

The flavonoid-enriched extract from the root of Smilax china L. inhibits inflammatory responses via the TLR-4-mediated signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Smilax china L. has been used clinically to treat various inflammatory disorders with a long history. AIM OF THE STUDY: To investigate the mechanisms underlying anti-inflammatory action of the extract from the herb. MATERIALS AND METHODS: The extract was identified and quantified using the Ultra Performance Liquid Chromatography-Photo Diode Array-Mass Spectrometer method. The anti-inflammatory activities were examined in xylene-induced mouse ear edema and cotton ball-induced rat granuloma. The inflammatory mediators, pro-inflammatory cytokines and TLR-4-mediated signals in LPS-stimulated RAW264.7 macrophages were determined using ELISA, real-time PCR, Western blot and/or immunofluorescence, respectively. RESULTS: The extract was found to enrich flavonoids (44.3%, mainly astilbin, engeletin, isoastilbin, cinchonain Ia, quercetin-3-O-a-L-rhamnopyranoside and chlorogenic acid). The flavonoid-enriched extract (FEE) inhibited xylene-induced mouse ear edema and cotton ball-induced rat granuloma, and suppressed LPS-induced over-release and/or overexpression of tumor necrosis factor-α, cyclooxygenase-2, inducible nitric oxide synthase, interleukin-1ß and interleukin-6 in RAW264.7 macrophages. Mechanistically, FEE suppressed protein overexpression of TLR-4 and its downstream signals, MyD88 protein, phosphorylated inhibitory κB-α, NF-κB-P65 and MAPK p38, as well as phosphorylation of phosphoinositide 3-kinase (PI3K) p85α at Tyr607 and Akt at Ser473 in LPS-stimulated macrophages. The mode of the anti-inflammatory action of FEE was similar to that of TAK-242 (a selective TLR-4 inhibitor). CONCLUSIONS: The present results demonstrate that FEE inhibit inflammatory responses via the TLR-4-mediated signaling pathway. Our findings go a new insight into the mechanisms underlying anti-inflammatory action of the herb, and provide a better understanding of its use for inflammatory diseases.

Yinning Tablet, a hospitalized preparation of Chinese herbal formula for hyperthyroidism, ameliorates thyroid hormone-induced liver injury in rats: Regulation of mitochondria-mediated apoptotic signals.

ETHNOPHARMACOLOGICAL RELEVANCE: Hyperthyroidism is closely associated with liver injury. The preliminary clinical observation suggests that Yinning Tablet, a hospitalized preparation of traditional Chinese formula for hyperthyroidism, improves not only hyperthyroidism, but also hyperthyroidism-associated liver injury. AIM: To evaluate the effect and underlying mechanisms of Yinning Tablet on thyroid hormone-induced liver injury. MATERIALS AND METHODS: Female rats were orally administered L-thyroxine (1 mg/kg) once daily for 60 days, and co-treated with the carefully identified Yinning Tablet extract (0.6-2.4 g/kg) during the last 30 days. Blood and liver variables were determined enzymatically, histologically, by ELISA, radioimmunoassay, Real-Time PCR or Western blot, respectively. RESULTS: Co-treatment with the extract attenuated L-thyroxine-induced increases in serum alanine transaminase and aspartate transaminase activities, the ratio of liver weight to body weight, cytoplasmic vacuolization in hepatocytes, infiltrated inflammatory cells and confused structures in liver tissue, accompanied by attenuation of increased serum triiodo-l-thyronine concentration and hepatic deiodinase type I overexpression in rats. Importantly, Yinning Tablet suppressed L-thyroxine-triggered hepatic Bax, cleaved caspases-3, -8 and -9 protein overexpression, and Bcl-2 protein downregulation. Furthermore, the increases in cytochrome c protein expression, Ca2+-ATPase activity and malondialdehyde content, and decreases in activities of Na+/K+-ATPase, catalase, superoxide dismutase and glutathione peroxidase, and total antioxidant capacity in liver tissue were attenuated. CONCLUSION: The present results suggest that Yinning Tablet ameliorates thyroid hormone-induced liver injury in rats by regulating mitochondria-mediated apoptotic signals. Our findings go insight into the pharmacological basis of the hospitalized preparation for treatment of hyperthyroidism-associated liver injury.

[Up-regulation of atrial natriuretic peptide and GATA3 levels in peripheral blood of children with respiratory syncytial virus bronchiolitis].

Objective To investigate the relationship between changes in atrial natriuretic peptide (ANP) signal and GATA3 expression in peripheral blood of children with bronchiolitis, and to explore the possible mechanism of ANP signal in the pathogenesis of bronchiolitis.Methods 20 normal children, 16 children with mild bronchiolitis, and 14 medium-severe children were enrolled. ELISA was used to detect the level of ANP and interleukin-4(IL-4) in plasma. Real-time fluorescent quantitative PCR was performed to determine the mRNA expression of natriuretic peptide receptor A(NPRA)and GATA3 in peripheral blood mononuclear cells(PBMCs) .Western blot analysis was used to determine the protein expression of NPRA and GATA3. Results As the degree of inflammation of bronchiolitis increases, the level of ANP and IL-4 in plasma increased significantly, and the mRNA and protein expression of NPRA and GATA3 in PBMCs also increased. Conclusion The levels of ANP, NPRA, IL-4 and GATA3 increased in peripheral blood of children with bronchiolitis.

The Changes of Th17/Treg and Related Cytokines: IL-17, IL-23, IL-10, and TGF-ß in Respiratory Syncytial Virus Bronchiolitis Rat Model.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and hospitalization that lead to high morbidity and mortality among young infants. T helper 17 (Th17) cells and regulatory T cells (Tregs) play essential roles in the pathogenesis of autoimmune, cancer, and inflammatory diseases. However, whether changes in T-cell subsets are related to the systemic immune responses in RSV-caused bronchiolitis merit further investigation. Three-week-old Sprague Dawley (SD) rats were randomly divided into the normal control (NC) and RSV bronchiolitis (RSV-B) groups. An RSV-B model was successfully established using nasal drip containing RSV. Furthermore, pathological changes in the lung tissues were observed using hematoxylin and eosin staining. Flow cytometry determined the levels of Th17 and Treg subsets. The related cytokines were measured using enzyme-linked immunosorbent assay (ELISA). The expression levels of related transcription factors, such as RORγt and FOXP3, were examined using real-time quantitative PCR and western blot analysis. The RSV-B group exhibited pulmonary interstitial hyperemia and edema, inflammatory cell infiltration, wide alveolar septa, and bronchial collapse and deformation. The percentage of Th17 cells in RSV-B group was about 2.3 fold higher than that of NC group, and the concentration of IL-17, IL-23 and RORγt was higher than in NC group. In contrast, the percentage of Treg cells in the RSV-B group was approximately 0.7 fold lower than that in the NC group, and the levels of IL-10, TGF-ß, and FOXP3 in the RSV-B group were lower than those in the NC group. The above results were statistically significant. The changes of Th17/Treg, and their associated cytokines, specific transcription factors, are present in RSV bronchiolitis model rats, which may play an important role in the pathogenesis of RSV bronchiolitis.

Placenta­derived mesenchymal stem cells improve airway hyperresponsiveness and inflammation in asthmatic rats by modulating the Th17/Treg balance.

Mesenchymal stem cells (MSCs) possess reparative and immunoregulatory properties, representing a hope for stem cell­based treatments. However, the mechanisms by which transplanted MSCs affect T helper (Th)17/regulatory T cell (Treg) balance in asthma patients remain unclear. The aim of the present study was to assess the therapeutic effects of human placenta MSCs (hPMSCs) in asthma, and explore the underlying mechanisms; in addition, the impact of hPMSCs transplantation on Th17/Treg balance in lymph and serum samples from asthmatic animals was evaluated. Sprague­Dawley rats were sensitized and challenged with ovalbumin (OVA). Administration of hPMSCs from human placenta resulted in increased Th17 and Treg in lymph samples compared with peripheral blood specimens. Enhanced pause values in OVA­treated animals were significantly higher than those in the control and hPMSCs treatment groups. The numbers of total cells, macrophages, neutrophils, and eosinophils were markedly increased in the OVA group compared with those of control + hPMSCs and control groups. In addition, interleukin 10, forkhead box P3 (Foxp3) and Treg levels in lymph, peripheral blood and lung tissue samples from asthma rats were increased significantly following hPMSC transplantation. Furthermore, Foxp3 protein levels increased, while those of RAR­related orphan receptor γ (RORγt) decreased after hPMSCs transplantation compared with the asthma group. Reduced IL­17, RORγt and Th17 levels were accompanied by reduced inflammatory cell infiltration, sub­epithelial smooth layer attenuation and mucus production in lung tissues. These results suggest that hPMSCs may improve airway hyperresponsiveness and inflammation by regulating the Th17/Treg balance in rats with asthma.

[γ-secretase inhibitor MW167 reduces the level of GATA3 in peripheral blood mononuclear cells in children with respiratory syncytial virus bronchiolitis].

Objective To study the changes of GATA3 in peripheral blood mononuclear cells (PBMCs) of children with respiratory syncytial virus (RSV) bronchiolitis after γ-secretase inhibitor (GSI) MW167 blocks Notch signaling. Methods The study enrolled 30 patients with RSV bronchiolitis and 25 normal controls. PBMCs were separated and divided into normal control group, RSV group, and MW167 treated group. The level of interleukin-4 (IL-4) in supernatant was detected by ELISA; level of GATA3 mRNA was determined by real-time fluorescent quantitative PCR; and the expressions of Notch1 intracellular domain (NICD) and GATA3 proteins were examined by Western blotting. Results Compared with RSV group, the levels of IL-4 decreased, and the levels of GATA3 mRNA, NICD and GATA3 proteins also significantly decreased in MW167 treated group. Conclusion GSI can block the Notch signaling pathway and decrease the level of GATA3.

[The level of RORγt increases in rat lung tissues of bronchiolitis caused by respiratory syncytial virus].

OBJECTIVE: To study the level of retinoic acid receptor-related orphan receptor γt (RORγt) in rat lung tissues of bronchiolitis caused by respiratory syncytial virus (RSV) and its implication. METHODS: The rats were randomly divided into normal group and bronchiolitis group. After the model of bronchiolitis was established successfully by nasal dripping, the pathological changes of lung tissues were detected by HE staining; the plasma levels of interleukin 23 (IL-23), IL-17 were detected by ELISA; the level of RORγt mRNA in lung tissues and peripheral blood mononuclear cells (PBMCs) were detected by real-time quantitative PCR; the level of RORγt protein in lung tissues was examined by Western blotting. RESULTS: Compared with the normal group, the rats with bronchiolitis presented with pulmonary interstitial hyperemia and edema, more inflammatory cell infiltration, wider alveolar septa and bronchial collapse and deformation. Compared with the normal group, the level of RORγt mRNA in the lung tissues and PBMCs increased in rats with bronchiolitis. The level of RORγt protein in lung tissues and the plasma levels of IL-23 and IL -17 were higher in rats with bronchiolitis than in normal rats. CONCLUSION: The level of RORγt was elevated in the lung tissues of rats with RSV-induced bronchiolitis.

[Changes and the clinical significance of CD4⁺ CD25⁺ regulatory T cells and Th17 cells in peripheral blood of infants with respiratory syncytial virus bronchiolitis].

AIM: To observe the percentages of CD4(+);CD25(+); regulatory T cells (Tregs) and Th17 cells and the levels of IL-10, TGF-ß and IL-17 in peripheral blood of infants with respiratory syncytial virus (RSV) bronchiolitis. The relationship between above cells, cytokines and RSV bronchiolitis was determined. METHODS: Thirty-three infants with RSV bronchiolitis, twenty-eight infants with non-RSV pneumonia and twenty-six healthy infants were enrolled. The percentages of Tregs and Th17 cells in peripheral blood were detected by flow cytometer (FCM), and the levels of IL-10, TGF-ß and IL-17 in plasma were determined by ELISA. RESULTS: The percentage of Tregs and the levels of IL-10 and TGF-ß in infants with RSV bronchiolitis were significantly lower than those in infants with non-RSV pneumonia and healthy infants (P<0.05), while the percentage of Th17 cells and the level of IL-17 in infants with RSV bronchiolitis were significantly higher than those in infants with non-RSV pneumonia and healthy infants (P<0.05). CONCLUSION: The imbalance between Tregs and Th17 cells in peripheral blood of infants with RSV bronchiolitis may be one of the pathogenesis of RSV bronchiolitis.

[Relationship between variation of coxsackievirus B3 VP1 sequence from cerebrospinal fluid of children and severity of damage to central nervous system].

OBJECTIVE: To investigate the possible relationship between variation of coxsackievirus B3 (CoxB3) VP1 sequence from cerebrospinal fluid of children with severe and mild central nervous system (CNS) infection and damage to CNS in children from Shandong province. METHODS: The enteroviruses were detected using VP1 typing and sequencing primer for enteroviruses from 73 enterovirus-infected cases confirmed by detection of cerebrospinal fluid by enteroviruses common primer. VP1 sequences (450 nucleotides) were determined and analyzed for 21 CoxB3 enteroviruses strains isolated in Qingdao and Binzhou, and were compared with that of BLAST search procedures from GeneBank in NCBI. The variation of VP1 gene and amino acids sequence of CoxB3 enteroviruses was analyzed for severe and mild CNS infection. RESULTS: The nucleotide homogeneity of these CoxB3 appeared to be 97% - 99%, however, the homogeneity among different genotypes were 83% - 76%. Replacement of glutamine by histidine at amino acid locus 856 of VP1 CoxB3 was found in 4 cases with severe encephalitis. There were different variation in VP1 nucleotide sequence of CoxB3 in 3 cases with mild encephalitis and 14 cases with meningitis, but amino acids sequences had no regular variation. The modified Glasgow's coma score was below 7 in all the 4 cases with severe encephalitis. Of these 4 cases, 3 had consciousness disturbance for less than 3 days. Lethargy, restlessness and psychiatric symptoms were major manifestations, of whom 3 also had dysphagia, 1 had encephalatrophy obviously, Glasgow's coma score was 3, deep coma lasted for 9 days, and had concomitant fatal epileptic attacks. Of these 4 cases, 2 completely recovered, 1 had high muscle tone, 1 remained under anti-epileptic drug treatment at follow-up 6 months later. CONCLUSION: There were a small epidemic of CoxB3 CNS infection in children in 2005 in this area. The amino acid variation of CoxB3 VP1 possibly caused increased viral virulence and caused damage to CNS.