Research On The Treatment Of 1,25(OH)₂D₃ On Liver Fibrosis In Mice Induced By CCl₄ Through Regulating Th17 Cell Differentiation

OBJECTIVES:Establish the liver fibrosis model,study and confirm the 1,25(OH)₂D₃effect on the treatment of liver fibrosis and regulating the differentiation of Th17 cells in vivo.Study on the 1,25(OH)₂D₃inhibit the differentiation of Th17 cells through STAT proteins in the process of induce CD4⁺T cells which obtained by sorting of the spleen lymphocytes differentiate to Th17.METHODS:1)Mice were administrated by 1,25(OH)₂D₃ with intraperitoneal injection as liver fibrosis model was induced and set up by CCl₄.Liver fibrosis condition was evaluated through pathological inspection of tissue slice and blood biochemical examination of liver function index.Immunohistochemical was used to detect expression of?-SMA,TGF-?and collagen I with a purpose of observe hepatic stellate cells activation level.Lymphocytes were separated from the liver and spleen,and analysis of the change of CD4⁺T cells subgroup with flow cytometry.The level of cytokines and transcription factors of CD4⁺T cells subgroup were detected by ELISA and RT-PCR,respectively,and,comprehensive measurement of 1,25(OH)₂D₃ effects on CD4⁺T cells subgroup.2)Lymphocytes which separated from spleen were go through with CD4⁺T cells sorting.The different concentration of 1,25(OH)₂D₃ was added to the CD4⁺T cells.Th17 cell differentiation was induced under the effect of inducing factor.The level of IL-17A and IL-22 which belong to the cytokines of Th17 cells were detected by ELISA.Flow cytometry was used to analysis the proportion of Th17 cells while RT-PCR was used to detect the expression of transcription factors of th17 cells to identify the inhibiting effect of 1,25(OH)₂D₃ on the Th17 cell differentiation.3)CD4⁺T cells were administrated with 1,25(OH)₂D₃ and/or STAT3 inhibitors.The change of Th17 cytokines(IL-17A,IL-22)were detected by ELISA,and,STAT3 phosphorylation level was examined by cell immunofluorescence,while WB was used to detect STAT3 protein expression level,in order to make sure 1,25(OH)₂D₃ inhibit Th17 cell differentiation through down-regulated STAT3.4)Using ELISA,WB and immunofluorescence to study and confirm the 1,25(OH)₂D₃ inhibit Th17 cell differentiation by up-regulated STAT5 similarly.RESULT:1)Histopathological detection showed that hepatocyte in control group are normal,and have distinct cytoplasm and nucleus,uniform size distribution and no visible fibrosis tissue.While the nucleus boundary is not clear in model group,and a large number of vacuoles and fibrosis tissue were produced.The recovering of cells in 1,25(OH)₂D₃ treatment group are well,and existing a small amount of vacuoles and fibrosis tissue.2)Serum ALT,AST and TBIL level of liver fibrosis mice was higher,while ALB was lower than that of normal mice(P<0.01).ALT and AST level of 1,25(OH)₂D₃ treatment group was lower,and ALB was higher than that of liver fibrosis(P<0.01).3)Immunofluorescence detection revealed that the expression of collagen I,TGF-?and?-SMA were increased significantly,however,was decreased in 1,25(OH)₂D₃treatment group.4)Liver fibrosis mice increased while 1,25(OH)₂D₃ treatment mice reduced IFN?,IL-17A and IL-22 concentration.On the contrary,IL-4 was reduced in liver fibrosis mice while increased in 1,25(OH)₂D₃ treatment mice.All differences between them are significantly.5)The m RNA levels of ROR?t and T-bet were significantly higher whereas GATA3 m RNA level was markedly lower in the liver fibrosis mice than those of control mice.The m RNA levels of ROR?t and T-bet were significantly reduced whereas GATA3 m RNA level was markedly increased in1,25(OH)₂D₃ treatment mice compared with liver fibrosis mice(P<0.01).6)Flow cytometry analysis of CD4⁺T cells in the liver and spleen lymphocytes subgroupphenotypic results show that the rates of Th1 cells and Th17 cells increased while Th2cells decreased in liver fibrosis group,however,1,25(OH)₂D₃ alter aforementioned rates of Th cells.7)In the study of 1,25(OH)₂D₃ inhibit Th17 cell differentiation in vitro,the result of cytokines detection in culture supernatant by ELISA shown that IL-17A and IL-22 concentration reducing following with 1,25(OH)₂D₃concentration increasing.The proportion of Th17 cells was the most in control group after differentiation inducing,was reduced in 1,25(OH)₂D₃treatment group and present a dose dependent.The m RNA levels of IL-17A,IL-22,ROR?t and ROR?were significantly decrease in 1,25(OH)₂D₃ than those of control mice(P<0.01).8)1,25(OH)₂D₃or/and STAT3 inhibitor were added in the process of CD4⁺T cells differentiate into Th17 cells.The detection of ELISA had show that the concentration of IL-17A and IL-22 were significantly lower than that of control group.The concentration of IL-17A and IL-22 in the group of 1,25(OH)₂D₃ combined with STAT3 inhibitor were lower than those of 1,25(OH)₂D₃and STAT3inhibitor alone group with statistical significance(P<0.01).The result of immunofluorescence shown the expression of p-STAT3 in control group was the most.The expression of p-STAT3 was lower in 1,25(OH)₂D₃ group and STAT3 inhibitor group compared with control group,while the lowest expression was in the combine group.The expression of STAT3 protein was detected through WB.The result showed that STAT3protein expression was significantly lower than control group(P<0.01),and expression in1,25(OH)₂D₃combined with STAT3 inhibitor was lower than those of 1,25(OH)₂D₃and STAT3 inhibitor alone group with statistical significance(P<0.01).9)1,25(OH)₂D₃ or/and STAT5 inhibitor were added in the process of CD4⁺T cells differentiate into Th17cells.The detection of ELISA showed that the concentration of IL-17A and IL-22 were significantly lower than that of control group,while concentration were significantly increased in STAT5 inhibitor group.The concentration in the group of 1,25(OH)₂D₃ combined with STAT3 inhibitor was significantly higher than that of 1,25(OH)₂D₃group whereas lower than that of STAT5 inhibitor group(P<0.01).The result of immunofluorescence shown the expression of p-STAT5 in 1,25(OH)2D3 group was higher than that of control group.Compared with the control group,the expression of p-STAT5 was lower in the group of 1,25(OH)2D3 combined with STAT3 inhibitor,while the expression was the lowest in STAT5 inhibitor group.The result of WB showed that STAT5 protein expression in 1,25(OH)₂D₃ group was significantly higher whereas in 1,25(OH)₂D₃combined with STAT3 inhibitor group was significantly lower than control group(P<0.01),and the lowest expression was in STAT5 inhibitor group.CONCLUSION:1)The treatment of 1,25(OH)₂D₃ can alleviate the damage caused by liver fibrosis,reduce the activating level of hepatic stellate cell.Th17 cells involved in the development of liver fibrosis,and 1,25(OH)₂D₃can regulates the differentiation of Th17cells in liver fibrosis mice via change the proportion of Th17/Th1/Th2 cells,the secretion of cytokines and the expression of specific transcription factor.2)Experiment in vitro showed that 1,25(OH)₂D₃play a role in inhibit the differentiation of Th17 cells,and present a decreasing in the proportion of Th17 cells,the secretion of cytokines and the expression of specific transcription factor.3)STAT3 and STAT5 could promote and inhibit the cell differentiation of Th17,respectively.4)The inhibition of 1,25(OH)₂D₃on Th17 cell differentiation was done through the role of STAT3 and STAT5,namely,STATs signal pathway may participate in the regulation of 1,25(OH)₂D₃on Th17 cells.