Protective Role And The Mechanism Of Rapamycin On Concanavalin A(ConA)-induced Autoimmune Hepatitis Mice

Part 1 Protective role and the mechanism of Rapamycin on Concanavalin A(Con A) induced acute liver injuryObjectiveTo investigate the therapeutic effects of rapamycin(RAPA) on concanavalin A-induced acute liver injury and the underlying mechanism.MethodsEighteen eight-week-old male C57BL/6 were randomly allocated into three groups: control group, Con A model group, Con A~+RAPA treatment group. Liver damage of mice with hepatitis was evaluated with respect to serum levels of alanine transaminase(ALT) and Aspartate aminotransferase(AST) and HE staining. Splenocytes was harvested 24 h after Con A injection. Subsequently, the frequencies of splenic DCs, CD4~+ T cells, CD8~+T cells, CD4~+CD25~+Treg,CD4~+CXCR5~+Tfh, and the expression of co-stimulatory molecules(CD40, CD80,CD86) on DCs were analyzed by flow cytometry.ResultsThe levels of ALT and AST of RAPA treatment group were significantly lower than those of Con A model group; H&E staining showed liver damage of RAPA treatment group was less severe than that of Con A model group. The proportion of splenic CD4~+ and CD8~+ T cells in RAPA treatment group were statistically lower than those of Con A model group. The proportion of spleen CD4~+CD25~+Treg among CD4~+ T cells were significantly higher than that of Con A model group.Expression of CD80 and CD86 on splenic DCs of RAPA treatment group were lower than those of Con A model group.The differences between CD80 and CD86 were statistically significant.However, no statistically significant difference in the proportion of splenic DCs and the expression of CD40 was noted between RAPA treatment group and Con A model group.ConclusionRAPA exerts protective effects on ConA-induced hepatitis, and its mechanism might involve modulation of immune cells in the spleen, including dendritic cells and T cells.This study might provide new ideas for the prevention and treatment of autoimmune hepatitis.Part 2 Preventive effects of Rapamycin against Concanavalin A(Con A) induced Autoimmune Hepatitis and liver fibrosisObjectiveTo investigate the attenuating effects of rapamycin on the development of liver fibrosis in Con-A induced chronic hepatitis and the underlying mechanism.MethodEight-week-old female C57BL/6 mice were randomly allocated into three groups: control group, Con A model group, Con A~+RAPA treatment group. Liver damage of mice with hepatitis was evaluated with respect to serum levels of alanine transaminase(ALT),aspartate aminotransferase(AST),HE and Masson staining which by Points-scoring system of Knodell HAI and Ishak. Liver mononuclear cell were isolated by density gradient centrifugation 24 h after the last Con A injection. The frequencies of liver CD4~+ T cells, CD8~+ T cells and the expression of IFN-?, IL-10, IL-4, and TGF-? were analyzed by flow cytometric analysis.ResultsCompared with model group, RAPA treatment group showed significantly lower serum levels of ALT( p<0.05); less severe liver inflammation and fibrosis by H&E and Masson staining; the expression of TGF-? in RAPA treatment group were significantly lower than model group(16.65±2.05)%,p<0.05;the frequencies of liver CD4~+ T,CD8~+T and T helper cell 1(Th1) were significantly lower than model group(p<0.05);but higher frequencies of Th3,Tr1 cells than model group(p<0.05)ConclusionsRAPA exerted significant protective effects against the progress of liver inflammation and fibrosis in Con A induced chronic hepatitis, and its action mechanism might be associated with promotion of Th3/Tr1 cells and inhibition of the expression of TGF-? in liver mononuclearcell.Part 3 Rapamycin-conditioned dendritic cells alleviate Autoimmune Hepatisis through induced immune toleranceObjectiveTo investigate the effect of tolerogenic dendritic cells(Tol-DC) generated by Rapamycin(Rapa) on the phenotype,the velocity and the Cytokine and explore the possible mechanism of tolerance induction.MethodThe purity dc were obtained by macs, the expression of Co-stimulatory molecules and Chemokine receptor,such as CD86, CCR7,CD40 and CD80 was analyzed by flow cytometry,and compare the ability of motility by Confocal microscopy. We observe the CFSE-based T-cell proliferation by co-culture with DC in vitro.ResultsThe immunephenotypic analysis showed that in comparison with the RAPA group the expression of the co-stimulatory molecules CD40 and CD80 were significantly lower than the control group. The ability to stimulate proliferation of T cells of the same genotype in the Rapa-group was significantly inhibited(P<0.01). RAPA conditioned Tol-DC can indued CD4~+CD25~+Treg from CD4~+CD25-T cells.ConclusionsTol-DC generated by Rapamycin can significantly induce immune tolerance through up-regulate Tregs and down-regulate proliferation of T cells.