| Part I The research of splenectomy on prevention and Therapeutic function in the murine model of AIH liver fibrosisBackground and objects:(1) To set up autoimmune hepatitis (AIH) liver fibrosis murine model by injecting Concanvalin A (ConA) repeatedly.(2) To observe the effect of splenectomy on the progress of AIH liver fibrosis in mice.(3) To observe the therapeutic effect of splenectomy on AIH liver fibrosis in mice and improve the treatment outcome of splenectomy and azathioprine combination therapy.Methods:(1) Mice of Balb/c received injection of10mg/kg,12.5mg/kg and15mg/kg dose of ConA through tail vein once a week, whereas the controls with PBS. After injection for3,5,7,9weeks, the serum levels of ALT and AST were tested by automatic biochemistry analyzer, liver pathogenic changes were observed by HE (hematoxylin and eosin) staining and Masson staining.(2)The mice were randomly divided into4groups, mice in splenectomy+ConA group were giving splenectomy before injecting ConA, mice in sham-operation+ConA group were giving sham operation before injecting ConA, mice in ConA group were untreated before injecting ConA, and control group. After injection for1,4,7weeks, the serum levels of ALT and AST were tested by automatic biochemistry analyzer, liver pathogenic changes were observed by HE (hematoxylin and eosin) staining and Masson staining.(3)The mice were randomly divided into5groups, mice in splenectomy therapy group were giving splenectomy after murine model were established, mice in sham operation therapy group were giving sham operation after murine model were established, mice in splenectomy+azathioprine (azathioprine, AZA) therapy group were giving splenectomy and intragastric administrated with AZA after murine model were established, mice in sham-operation+AZA therapy group were giving sham operation and intragastric administrated with AZA after murine model were established, mice in control group were untreated after murine model were established. After treatment for1,4,8weeks, blood and livers were extracted from the mice so as to test blood routine examination and observe the histology of the livers.Results:(1) In contrast with the control group, the levels of ALT and AST in experimental group were significantly increased, and the higher the dose, the greater the number(P <0.05). Pathological results showed that in10mg/kg group catched liver fibrosis in9weeks, but not obviously;12.5mg/kg group catched typical liver fibrosis in7weeks;15mg/kg group catched obvious liver fibrosis in5weeks, but the group with a high mortality.(2) In contrast with normal value, the levels of ALT and AST in splenectomy+ConA group were increased, but were lower than the sham-operation+ConA group and control group significantly(P<0.05). Pathological results showed that the degrees of liver inflammation and lymphocyte infiltration in splenectomy+ConA group of1,4weeks were lighter than the sham operation+ConA group and control group; Mice in splenectomy+ConA group did not catch liver fibrosis, but mice in the sham-operation+ConA group and control group had typical liver fibrosis in7weeks.(3) After1week treatment, the levels of WBC, PLT in splenectomy+AZA therapy group and splenectomy therapy group were still low. After4weeks treatment, the levels rised to the top, and then declined. The levels of WBC, PLT in control group, sham-operation therapy group and the sham-operation+AZA therapy group were declined with treatment. The levels of liver fibrosis in splenectomy therapy group and splenectomy+AZA therapy group became lower and lower as time going, and the splenectomy+AZA therapy group reduced more obviously. The levels of liver fibrosis in sham operation therapy group and control group did not see significant change. In contrast with splenectomy+AZA therapy group, the sham-operation+AZA therapy group with a higher mortality.Conclusions:(1) Mice of Balb/c received injection of12.5mg/kg dose of ConA through tail vein weekly, injection of seven weeks could establish animal model of liver fibrosis.(2) Splenectomy can prevent the progress of AIH liver fibrosis in mice.(3) Splenectomy could not only cure AIH liver fibrosis in mice, but improve the treatment outcome of AZA.Part II The implications of splenectomy on Th2cytokines in the murine model of AIH liver fibrosisBackground and objects:(1) To study the role of Th2cytokines in ConA-mediated autoimmune hepatitis.(2) To study the role of Th2cytokines in the murine model of AIH liver fibrosis, and the implications of Splenectomy on Th2cytokines in the murine model of AIH liver fibrosis.Methods:(1) About36female BaLb/C mice received injection of ConA (15mg/Kg) through tail vein, whereas the controls with PBS. After injection for1h,2h,4h,6h,12h,24h, livers were extracted from6mice randomly so as to test the mRNA expression of IL-4, IL-5and IL-13by RT-PCR.(2) The mice were randomly divided into4groups, mice in splenectomy+ConA group were giving splenectomy before injecting ConA, mice in sham-operation+ConA group were giving sham operation before injecting ConA, mice in ConA group were untreated before injecting ConA, mice in control group were untreated all the time. After injection ConA for1,4,7weeks, livers were extracted from mice randomly so as to test the mRNA expression of IL-4, IL-5and IL-13by RT-PCR.Results(1) By RT-PCR analysis, we demonstrated that the mRNA expression of IL-4, IL-5and IL-13were increased in ConA-induced acute hepatitis, higher than the control group (P<0.05). The peak mRNA expression time point for IL-4were6h after ConA asministration, and the peak mRNA expression time point for IL-5and IL-13were2h.(2) In the murine model of AIH liver fibrosis, the mRNA expression of IL-4, IL-5and IL-13were increased, highest in7weeks after ConA injection. Mice in splenectomy+ConA group, the mRNA expression of IL-4, IL-5and IL-13were increased. In contrast with the ConA group, the levels of IL-4and IL-13in splenectomy+ConA group were significantly lower, while the levels of IL-5were no difference between the two groups.Conclusions:(1) Th2cytokines played an important role in ConA-induced acute hepatitis and liver fibrosis model.(2) The spleen could play an immunoregulated role to ConA-induced acute hepatitis and liver fibrosis model by inhibiting the secretion of Th2cytokines. |
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