| Background:Autoimmune hepatitis（AIH）is a chronic autoimmune liver disease whose pathogenesis has not been fully elucidated.Impaired immunoregulatory function of regulatory T cell（Treg）and increased differentiation of pathogenic T helper cell 17（Th17）play a central role in the occurrence and development of AIH and are closely related to disease severity.Therefore,increasing the number of Treg cells and/or maintaining their functional stability and rebuilding the Treg/Th17 cell balance may provide a practical therapeutic approach for AIH patients.All-trans retinoic acid（at RA）has a wide range of biological functions and has been proved to play an important role in regulating the body’s self-immune tolerance system.However,whether rebuilding Treg/Th17 cell balance in AIH immune microenvironment can improve immune liver injury in AIH mice is not clear.Objective:To establish an AIH mouse disease model induced by liver autoantigen S100 protein,observe the therapeutic effect of at RA on AIH,explore whether at RA can alleviate the severity of the disease by regulating the phenotype and function of lymphocytes in AIH mice,and explore the molecular biological mechanism of at RA regulating Treg/Th17 cell balance.Method:（1）Animal experiment:30 male 4-6 week-old C57BL/6 mice were randomly divided into control group（Control）,model group（Model）and at RA treatment group（at RA）,10 mice in each group.The AIH mouse model was induced by intraperitoneal injection of S100 protein immunization agent in the model group and the treatment group.The treatment group was intraperitoneally injected with at RA（25 mg/kg）every other day for 30 consecutive days.The control group and the model group were intraperitoneally injected with the same concentration of drug solvent during the same period.The general conditions of the mice in each group,such as body weight,food intake,water intake,hair color,activity and mental state,were compared and observed since the model establishment.After the intervention,the mice were executed.An automatic biochemical analyzer was used to detect liver function to evaluate the degree of liver injury,HE staining was used to evaluate the histopathological manifestations of the liver,and the Knodell histological activity index（HAI）score was performed.The ratio of Treg/Th17 cells was detected by flow cytometry in spleen and then calculated,and the frequency of CD4+T cells in liver tissue was detected by immunofluorescence staining;in addition,the content of serum IL-6 was detected by ELISA,the relative m RNA expression of IL-6,RORγt,Foxp3,STAT3 in liver tissue were detected by q RT-PCR and the ratio of Foxp3/RORγt were calculated.The protein expression changes of RORγt and STAT3 in liver tissue of mice in each group were further observed by western blotting,and the effect of at RA on IL-6/STAT3 signaling pathway in AIH mice was studied.（2）Cell experiment:First,the mouse spleen CD4+CD62L+na（?）ve T cells were sorted by immunomagnetic beads method,and the purity of the obtained cells was identified by flow cytometry,and then Th17 and Treg cells were induced and differentiated under the intervention of at RA.The effects of atRA on the differentiation of Th17 and Treg cells in vitro were observed.Results:（1）Compared with the control group,the general condition of the mice in the AIH model group was worse,the spleen was severely swollen,the liver texture was rough and hard,the liver and spleen index increased（P<0.01）,the body weight decreased（P<0.01）,and the serum AST,ALT and GLB were significantly increased（P<0.05）.Compared with the mice in the model group,the general condition of the mice in the at RA treatment group was improved,the swelling of the spleen was significantly relieved,the liver capsule was smooth,clear in outline and soft in texture,and the liver and spleen index was significantly decreased（P<0.05）,body weight was increased（P<0.05）,and the above serum liver function indexes were significantly decreased（P<0.05）.The results of HE staining of liver tissue further showed that a large number of lymphocytes infiltrated in the portal area of hepatic lobules of mice in the model group,some hepatocytes in the lobules showed ballooning degeneration and fatty degeneration,and some hepatocytes were lytic and necrotic,with disordered arrangement.The infiltration of inflammatory cells in the liver tissue portal area of the mice in the at RA treatment group was significantly reduced,and various pathological indicators were significantly improved compared with the model group,the HAI score was significantly decreased（P<0.01）,which proved that at RA could improve the liver immune damage and alleviate the severity of the disease in AIH mice.（2）Compared with the control group,the number of lymphocytes in the spleen of the AIH model group was significantly increased（P<0.001）,the proportion of Th17cells was increased（P<0.001）,the proportion of Treg cells was decreased,and the ratio of Treg/Th17 cells was decreased.（P<0.01）,after atRA treatment,the number of lymphocytes recruited in the spleen decreased（P<0.01）,the proportion of Th17 cells decreased（P<0.01）,while the proportion of Treg cells increased and the ratio of Treg/Th17 cells increased（P<0.05）;The results of IF staining of liver tissue showed that the CD4+T cells infiltrated in the liver tissue of the model group were significantly increased compared with the control group,while the treatment group was significantly reduced（P<0.001）.The results of cell experiments further showed that compared with at RA 0μM group,the proportion of Th17 cells decreased significantly（P<0.05）and the proportion of Treg cells increased significantly（P<0.01）in at RA 500μM group,indicating that ATRA has a certain regulatory effect on the balance of Treg/Th17 cells in AIH mice.（3）Compared with the control group,the serum IL-6 content and the relative m RNA expressions of IL-6,RORγt,Foxp3,and STAT3 in the AIH model group were significantly increased（P<0.05）.The content of serum IL-6 and the m RNA expression level of liver IL-6,RORγt,STAT3 in ATRA treatment group decreased significantly（P<0.01）,while the expression level of Foxp3 m RNA increased（P<0.01）,foxp3/RORγt ratio increased（P<0.001）;Western Blot results showed that the protein expressions of RORγt and p-STAT3 in the liver tissue of the AIH model group were significantly higher than those of the control group（P<0.05）,while at RA could down-regulate the protein expressions of RORγt and p-STAT3 in the liver tissue（P<0.05）.,indicating that the immunoregulatory effect of at RA on AIH mice may be related to the activation of IL-6/STAT3 signaling pathway.Conclusions:atRA can improve liver inflammatory injury and reduce the severity of the disease by rebuilding the balance of Treg/Th17 cells in the immune microenvironment of AIH mice induced by S100 protein;the immunoregulatory effect of at RA on AIH mice may partly be achieved by regulating the IL-6/STAT3 signaling pathway. |
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