Study On The Immunoregulatory Mechanism Of Fuzheng Huayu Recipe Against Liver Fibrosis Based On The Changes Of CD8~+T Lymphocyte Subsets

Objective:To observe the effect of liver CD8~+T lymphocytes and their subpopulations on co-cultured hepatic stellate cells after Fuzheng Huayu Recipe,and to explore the mechanism of Fuzheng Huayu Recipe against liver fibrosis.METHODS:Flow cytometry was used to sort CD8~+T lymphocytes in liver of mice with acute liver injury and immunomagnetic beads method.Liver transplantation for liver cirrhosis was performed.CD8~+T and its subpopulations were discarded.Fuzheng Huayu Recipe was given in vitro.Serum intervention or pre-mouse administration,and co-culture with hepatic stellate cell line(JS 1,LX-2)in a 96-well plate at a ratio of 2:1,using the q PCR method to detect Col.I m RNA The expression of cytokines in the co-culture supernatants was determined by ELISA.The differences between the groups were statistically compared.RESULTS:1.In the first part of the experiment,CD8~+T lymphocytes were isolated from JS 1 in mice with acute liver injury,showing that the FZ-group(CD8~+T lymphocytes were first incubated with drug serum,and the normal rat serum was changed during co-culture)JS 1 expression?-SMA m RNA was significantly reduced compared with the control group of single JS 1 culture(P<0.05);compared with X group(both CD8~+T lymphocytes pre-incubation and co-culture with normal rat serum),FZ-group JS 1 expression Col.I m RNA was significantly inhibited,and the difference between the groups was statistically significant(P<0.001).After incubation with the drug serum,the factor of promoting liver fibrosis in the co-culture supernatant was decreased,and the FZ-group and FZ groups(FZHY drug serum were used for pre-incubation and co-culture of CD8~+T lymphocytes)IL-13 content The levels of IL-1?in the FZ group were significantly lower than those in the X group(P<0.01),while the cytokine levels in the liver fibrosis were increased.For example,IL-10 levels in the FZ-group and FZ groups increased,but only the IL-10 levels in the FZ group were significantly different from those in the X group(P<0.05).The content of granzyme B in FZ group increased significantly,and the difference between group and group X was statistically significant(P<0.05).2.The second part of the experiment showed that the ALT and AST activities of the acute liver injury model(M)group were significantly increased,the difference was statistically significant(all P<0.0001).The increase of ALT activity in FZ group(previously treated with Fuzheng Huayu Recipe for 5 days)was significantly lower than that of M group,the difference was statistically significant(P<0.001).The CD8~+T lymphocytes isolated from the liver of each group were co-cultured with JS 1 for 48 h.The expression of?-SMA m RNA was significantly increased in M group compared with Control group(JS 1 single culture)(P<0.01);Col.I m RNA The expression was also significantly higher than that of Control group and N group(normal mouse liver CD8~+T lymphocytes co-cultured with JS 1),the difference was statistically significant(all P<0.001).The expression of?-SMA m RNA and Col.I m RNA in FZ group was significantly lower than that in group M(P<0.01).A variety of cytokine levels were detected in the 48h co-culture supernatant,but these results did not support Fuzheng Huayu Recipe to regulate HSC by affecting CD8~+T lymphocyte secretion cytokines.3.The third part of the experiment was to isolate CD8~+T,CD8~+CD28~+T and CD8~+CD28~-T lymphocytes from liver cirrhosis patients and transplant them with LX-2.The results showed that compared with the X group that was incubated with normal rat serum,the CD8~+T,CD8~+CD28~+T and CD8~+CD28~-T lymphocytes of the FZ-group(CD8~+T lymphocytes and subpopulations were first incubated with drug serum and replaced with normal rat serum)both reduced the?-SMA m RNA expression of LX-2,and CD8~+CD28~+T lymphocytes significantly decreased the?-SMA m RNA expression of LX-2(P<0.0001).CD8~+T,CD8~+CD28~+T and CD8~+CD28~-T lymphocytes in the FZ group(both CD8~+T lymphocytes and their subpopulations were pre-incubated and co-cultured)significantly reduced LX-2 The expression of?-SMA m RNA was significantly different from that of group X and FZ-group(P<0.05,P<0.001 or P<0.0001).CD8~+CD28~+T lymphocytes in FZ-and FZ groups inhibited the expression of Col.I m RNA in LX-2,and the difference was significant between the two groups(all P<0.05);CD8~+T lymphocytes in FZ group The expression of Col.I m RNA in LX-2 was decreased,and the difference was statistically significant compared with FZ-group(P<0.05).Conclusion:1.Fuzheng Huayu Recipe can inhibit the activated HSC indirectly by changing the functional phenotype of CD8~+T lymphocytes in mice,which has little effect on the role of secreted cytokines.2.In addition to directly inhibiting human HSC,Fuzheng Huayu Recipe may indirectly inhibit HSC activation mainly through CD8~+CD28~+T lymphocytes of CD8~+T lymphocytes in the liver of patients with liver cirrhosis.