Mechanism Of QingChangHuaShi Method Mediating MSCs Regulating MiR-21 In The Treatment Of Ulcerative Colitis

Objectives To clarify the specific mechanism of treating ulcerative colitis by regulating miR-21 expression through IL-6/STAT3 pathway mediated MSCs by QingChangHuaShi,and to analyze the mechanism of action of gallic acid,the main active ingredient of its prescription drugs,so as to further explore the scientific connotation of QingChangHuaShi in treating ulcerative colitis.Methods:In vitro experiment:MSCs from rat bone marrow were isolated and cultured.The cell morphology was observed by inverted biological microscope.Cell surface markers were identified by flow cytometry,and adipogenic differentiation was induced.The cell types were identified.The proliferation of self-extracted MSCs cells was detected by CCK8 method to determine the intervention concentration of compound prescription for QingChangHuaShi in subsequent experiments.The following experiments were divided into MSCs blank group,inflammation model group induced by LPS,traditional Chinese medicine group obtained by using compound prescription of QingChangHuaShi to intervene MSCs inflammation model,inhibitor group obtained by treating MSCs cells with STAT3 inhibitor NSC74859,model+inhibitor group obtained by treating MSCs inflammation model with STAT3 inhibitor NSC74859.As well as the traditional Chinese medicine+inhibitor group obtained by treating MSCs inflammation model together with the compound prescription for QingChangHuaShi and the STAT3 inhibitor NSC74859,the expression levels of IL-6,IL-1?,IL-17,TNF-?,IL-10,TGF-?inflammatory factors in cell supernatants of each group were detected by ELISA method,the expression levels of miR-21,STAT3 mRNA in cells of each group were detected by RT-qPCR method,and the expression of p-STAT3 protein in cells of each group was detected by Western Blot method.HPLC method was used to simultaneously determine berberine hydrochloride,gallic acid and baicalin in Qingchang Huashi compound,and CCK8 method was used to detect IC50 value of gallic acid to deterrmine the concentration of high and low traditional Chinese medicine group in subsequent experiments.A blank control group and an LPS-induced MSCs inflammation model group were established.Flow cytometry was used to detect the cell cycle and apoptosis of each group.Cell scratch and Transwell test were used to detect the cell migration and invasion ability of each group.ELISA was used to detect the expression levels of IL-6,IL-1?,IL-17,TNF-?,IL-10 and TGF-P in cell supernatants of each group.RT-qPCR method was used to detect the expression level of miR-21 and STAT3 mRNA in each group of cells,and Western Blot method was used to detect the expression of p-STAT3 protein in each group of cells.In vivo experiment:TNBS method established rat ulcerative colitis model.Sixty rats were randomly divided into blank group and TNBS induced model group.On the basis of the model group,1 ml of 1 ×10h/ml MSCs cell suspension of the third generation was infiused via tail vein into MSCs group.On the basis of MSCs group,13.6g/kg of Qingchang Huashi compound water decoction was given to the compound group,and 5mg/kg STAT3 inhibitor NSC74859 compound+inhibitor group was injected intraperitoneally on the basis of the compound group.The gross morphology and histopathological changes of colon mucosa were observed.The expression of IL-6 in serum of rats in each group was detected by ELISA method,the expression levels of miR-21 and STAT3 mRNA in colon tissue of rats in each group were detected by RT-qPCR method,and the expression of p-STAT3 protein in colon tissue of rats in each group was detected by Western Blot method.Results:In vitro experiment:After identification,the self-extracted cells conform to MSCs cell characteristics.Compared with the blank group,the expression of pro-inflammatory cytokines IL-6,IL-I(3,IL-17 and TNF-a in the model group increased after LPS induction,while the expression of irnflammatory inhibitory factors IL-10 and TGF-P decreased,the expression levels of miR-21 and STAT3 mRNA increased,and the expression level of p-STAT3 protein increased.Compared with the model group,the traditional Chinese medicine group can reduce the expression level of IL-6,IL-1?,IL-17,TNF-?,increase the expression level of IL-10 and TGF-?,reduce the expression level of miR-21,STAT3 mRNA,and reduce the expression level of P-STAT3 protein after the compound intervention therapy of QingChangHuaShi.The HPLC system conditions for simultaneous determination of berberine hydrochloride,gallic acid and baicalin in Qingchang Huashi compound were successfully established.Compared with the blank group,the model group can cause S phase arrest of MSCs cell cycle,increase apoptosis,and decrease migration and invasion ability.Compared with the model group,the traditional Chinese medicine group treated with GA intervention can relieve cell cycle arrest of MSCs,reduce apoptosis and enhance its migration and invasion ability.At the same time,it can reduce the expression levels of IL-6,IL-1?,IL-17 and TNF-?,increase the expression levels of IL-10 and TGF-?,reduce the expression levels of miR-21 and STAT3 mRNA,and reduce the expression level of p-STAT3 protein in a dose-dependent effect.In vivo experiment:Compared with the model group and MSCs group,the compound group has better relieving effect on the general condition and histopathological changes of colon mucosa,and can better reduce the expression of serum IL-6,miR-21,STAT3 mRNA and p-STAT3 protein in colon tissue.Conclusion:The isolation and culture system of rat bone marrow MSCs has been successfully constructed and optimized.It is proved that QingChangHuaShi method can alleviate the inflammatory reaction of MSCs inflammatory model,reduce the expression of inflammatory promoting factors,improve the release of inflammatory inhibiting factors,and maintain its immune regulation function.At the same time,QingChangHuaShi can mediate MSCs.The specific mechanism may be to regulate miR-21 expression through IL6/STAT3 pathway,thus improving the therapeutic effect on ulcerative colitis.GA,the main active ingredient of QingChangHuaShi,can improve cell cycle arrest of MSCs inflammatory cell model,reduce cell apoptosis,enhance migration and invasion ability,and may mediate MSCs to regulate miR-21 expression through IL-6/STAT3 pathway,thus providing scientific basis for clinical treatment of ulcerative colitis with traditional Chinese medicine.