Rapamycin Ameliorates CCl4-induced Liver Fibrosis In Mice Through Reciprocal Regulation Of Th17/Treg Balance

Part one Rapamycin inhibits the development of CCl4-induced liver fibrosisObjective: To observe the therapeutic effect of rapamycin on CCl4- induced liver fibrosis.Methods: Thirty male C57BL/6 mice were randomly divided into control group, model group, and treatment group. To induce liver fibrosis, mice were injected intraperitoneally, twice a week, for 8 weeks, with 5 ml/g of 20% CCl4 dissolved in olive oil. Negative control mice were given the same volume of olive oil alone for the indicated time intervals. Starting 0 day after CCl4 injection, in the treatment group mice were treated IP with rapamycin for the length of the study. In addition, mice were not treated with rapamycin but received equivalent volumes of PBS during the experiment. After 8 weeks, liver tissues were obtained from each group for H&E staining and Masson staining to observe the degree of hepatic fibrosis. The levels of TGF-β1 and α-SMA proteins were measured by immunohistochemistry.Results: Liver function tests showed that rapamycin significantly reduced the concentrations of total bilirubin and aminotransferases in CCl4-induced liver fibrosis model. After CCl4 administration, the livers of mice treated with PBS exhibited a distorted architecture with extensive fibrosis combined with development of micronodules throughout the liver parenchyma as shown by H&E staining. Pathological progression in liver fibrosis was attenuated by adding rapamycin, and showed fewer and smaller fibrotic nodules. TGF-β and α-SMA expression was significantly elevated in mice with CCl4-induced liver fibrosis, and was reduced following rapamycin administration.Conclusion: Rapamycin treatment alleviates liver injury caused by CCl4 injection, and restrains the development of hepatic fibrosis in mice.Part two Rapamycin inhibits the responds of Th17 cells in liver fibrosisObjective: To observe the changes of Th17 cells after the administration of rapamycin.Methods: Liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride(CCl4). Treatment group mice were received rapamycin treatment as described in previous section. The numbers of Th17 cells in liver and spleen were detected by FACSCalibur flow cytometer. The expression levels of ROR-γt in liver tissue were evaluated by Western Blot.Results: Following rapamycin administration, the percentages of Th17 cells were significantly lower than that of the PBS-treated mice, although higher than that of the negative control. Meanwhile, as the crucial transcription factor of Th17 cells differentiation, ROR-γt expression in rapamycin-treated mice was decreased when compared to PBS-treated mice.Conclusion: Rapamycin can suppress Th17 cells involved in mediating the responds of inflammation, consequently inhibits the development of liver fibrosis.Part three Rapamycin expands regulatory T cells with suppressive ability in hepatic fibrosisObjective: To explore the immune effect of rapamycin on the TregsMethods: Liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride(CCl4). Treatment group mice were received rapamycin treatment as described in previous section. The numbers of Tregs cells in liver and spleen were detected by FACSCalibur flow cytometer. The expression levels of Foxp3 in liver tissue were evaluated by Western Blot. Tregs form rapamycin-or PBS-treated mice were co-cultured with CD4+CD25- T cells for 3 days. Following a 18-h pulse with [3H] thymidine at 1 μCi/well, proliferation was analyzed using a scintillation counter. In addition, Tregs form rapamycin-or PBS-treated mice were co-cultured with HSCs from healthy mice. Immunofluorescence staining and Western Blot were used to detect the expression levels of α-SMA.Results: The frequency of Treg cells in spleen and liver from rapamycin-treated mice was significantly higher than that of the PBS-treated mice. Consistent with the results of flow cytometric analysis, the expression of Fox P3 in liver from rapamycin-treated mice was markedly enhanced relative to that from PBS-treated mice. Furthermore, the suppressive activity of Treg cells in spleen from rapamycin-treated mice was obviously stronger in comparison with that of the PBS-treated mice. immunofluorescence staining and Western Blot showed that α-SMA expression was significantly reduced after exposure to Treg cells from rapamycin-treated mice when compared with PBS-treated mice.Conclusion: Rapamycin not only promote the proliferation of Tregs, but also make their suppressive power stronger.