Clinical Study Of Huagan-Tongluo And The Mechanism Of Th17 Cell Differentiation Involved In Hepatic Fibrosis

Objective:In clinical research,the present study evaluates the efficacy of HuaGanTongLuoFang(HGTLF)on patients hepatitis B liver fibrosis.In the in vitro study,the rat model of hepatic fibrosis was prepared to investigate the effect of HGTLF on hepatic fibrosis,and the possible mechanism of HGTLF on fibrosis,as well as the potential regulatory mechanism of Thl7 cell differentiation on liver fibrosis was further explored.Methods:1.To evaluate the clinical Effect of HGTLF on patients with Liver FibrosisSixty patients with chronic hepatitis B(CHB)were divided into two groups:control and experiment groups(n=30).experimental group received the treatments of HGTLF combined with entecavir(ETV),control grou)only received the treatment of ETV.After six months,the Syndrome scores,serum liver function index,serum liver fibrosis index were determined.2.Evaluation of the therapeutic effects of HGTLF on rats with liver fibrosis.SD rats(n=72)were randomly divided into 5 groups,including:sham,HGTLF high dose group,HGTLF middle dose group,HGTLF low dose group,and ursodeoxycholic acid(UDC A).The rat model of hepatic fibrosis was established by ligating the common bile duct.After the model was established successfully,serum and liver tissue were collected 8 weeks after drug intervention.The liver function and routine pathology of rats were determined by biochemical methods.The expressions ofTIMP-1 and ICAM-1 were detected by ELISA.The expressions of hepatic stellate cells HSC),TGF-β1,NF-κB,PDGF,p38,ICAM-1 and TIMP-1 were measured by real-time PCR,the Th17 cell ratio was determined using flow cytometry.3.To explore the regulatory mechanism of Th17 cell differentiation in hepatic fibrosis rats3.1 Healthy male C57BL/6 mice,intraperitoneal injection of 10%CCL4 olive oil solution to establish liver fibrosis model,the control rats were received the same amount of olive oil,after 10 weeks,the mice were sacrificed.Serum ALT and AST were measured;the infiltrating CD4+T cells were isolated from liver of mice;the proportion of Th17 cells(CD4+IL-17A+)in CD4+T cells was detected by flow cytometry;the levels of IL-17A and IL-22 were detected using ELISA;The expression of IL-17A and IL-22 was measured by Real-time PCR.3.2 The liver tissues of model mice and control mice were obtained to examine the expression of miR-29a,miR-652 and ARRB1 mRNA using real-time PCR/Western blot.3.3 CD4+T cells that isolated from normal mouse spleen were transfected with pre-NC or miR-29a mimic or miR-652 mimic,and then cultured to induct the production of Th17 cells.The proportion of Th17 cells was analyzed by flow cytometry.The levels of IL-17A and IL-22 was examined by ELISAkit.The expression of IL-17A,IL-22 mRNAand ARRB1 mRNA levels was examined by Western blot.The protein expression of ARRB was examined by Western blot.3.4 HEK293T cells were used to verify the relationship between miR-29a/miR-652 and ARRB1 using dual luciferase reporter.The splenic CD4+T cells of normal mice were isolated and transfected with miR-29a mimic/miR-652 mimic/miR-29a mimic+miR-652 mimic or miR-29a inhibitor/miR-inhibitor/miR-29a inhibitor+miR-652 inhibitor.The expression of ARRB1 was detected by real-time PCR and western blot.3.5 Spleen CD4+T cells were isolated from normal mouse,after transfection with the corresponding vector(miR-29a mimic or miR-652 mimic and pcDNA-ARRB1),and cultured to induce the production of Th17 cells.The content and expression of IL-17A and IL-22 were determined.3.6 The healthy male C57BL/6 mice were intraperitoneally injected with CCL4 for 8 weeks.All mice were divided into 3 grops:control group,Ad-miR-29a group and Ad-miR-652 group.The contents of ALT and AST were measured;the proportion of Th17 cells was measured by flow cytometry;the expression of ARRB1 was detected by real-time PCR and western blot.Results:1.Effects of HGTLF on patients with liver fibrosisCompared with control,the patients in experiment group showed alleviated clinical symptoms,such as pain at right side,dry mouth,bitter taste,abdominal distention,anorexia,yellow urine,constipation,gloomy complexion,purple lips,dark purple tongue,and moss yellow greasy.The liver function indexes,such as ALT,AST,ALP,GGT;the liver fibrosis indexes,such as HA,IVC and PCIII were all alleviated.2.Effects of HGTLF on rats with liver fibrosis2.1 Comparing with model group,the HGTLF treatment significantly decreased the indicators of the liver function,and the inflammatory cells were reduced.2.2 Comparing with model group,the HGTLF treatment significantly decreased the liver fibrosis cytokines,such as TGF-β1,NF-κB,PDGF,p38,ICAM-1 and TIMP-1.2.3 Comparing with model group,the HGTLF treatment significantly decreased the differentiation of TH17.3.The mechanism of Th17 differentiation.3.1 Comparing with control,the levels of ALT and AST,the Th17 cells ratio,were significantly increased,while IL-17Aand IL-22 were decreased in model group.3.2 The expression of miR-29a and miR-652 was decreased,while ARRBI was increased in model group comparing with control.3.3 CD4+T cells transfected with miR-29a mimic or miR-652 mimic significantly decreased the ratio of Th17 cells,decreased the content and level of IL-17A and IL-22 as well as the expression of ARRB1.3.4 Luciferase reporter assay revealed that ARRB1 is the target gene of miR-29a and miR-652.3.5 Overexpressed miR-29a or miR-652 suppressed the level of IL-17A and IL-22,then co-transfected with overexpressed ARRB1 abolished the effects of overexpressed miR-29a or miR-652.3.6 In vivo verified that overexpressed miR-29a or miR-652 suppressed the level of IL-17A,IL-22 and ARRB1.Conclusion1.Effect of HGTLF on Liver Fibrosis Patients.HGTLF combined with ETV and ETV significantly alleviated patients with hepatitis B and liver fibrosis.2.Evaluation of the therapeutic effects of Huagantongluofang on liver fibrosis in rats.HGTLF can dose-dependently reduce liver damage and improve liver fibrosis.The mechanism of anti-hepatic fibrosis may reduce hepatocyte injury through anti-inflammatory and inhibition of hepatic stellate cell activation,in which TGF-β1,NF-κB,PDGF,p38 participate in the process of reversing hepatic fibrosis.At the same time,TH17 cells play an important role in the differentiation of the liver and liver.3.To explore the regulatory mechanism of Th17 cell differentiation in hepatic fibrosis rats CCL4-induced hepatic fibrosis significantly increased the percentage of Th17,the levels of IL-17A,IL-22 and the expression of ALT and AST.Overexpression of miR-29a/miR-652 significantly reduced the levels of ALT,AST,and ARRB1,indicating the protective effect of overexpression of miR-29a/miR-652 on liver fibrosis.Thus,miR-29a and miR-652 ameliorate the progression of hepatic fibrosis by blocking ARRB1-mediated Th17 cell differentiation.