Gu Lei, Deng Wen-Sheng, Sun Xiao-Fei, Zhou Hong, Xu Qing
Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, P.R. China.
Department of Gastrointestinal Surgery, Yantai Yuhuangding Hospital, Yantai, Shandong 264000, P.R. China.
Mol Med Rep. 2016 Aug;14(2):1153-61. doi: 10.3892/mmr.2016.5392. Epub 2016 Jun 10.
Previous investigations have suggested that the activation of Th17 cells and/or deficiency of regulatory T cells (Tregs) are involved in the pathogenesis of liver fibrosis. The aim of the present study was to investigate the effect of rapamycin on immune responses in a carbon tetrachloride (CCl4)-induced murine liver fibrosis model. Liver fibrosis was induced by intraperitoneal administration with CCl4. Following injection of CCl4, the mice were treated intraperitoneally with rapamycin (1.25 mg/kg/day) for 8 weeks. Hematoxylin and eosin staining and Masson's trichrome staining were used for histological examination. The protein levels of forkhead/winged helix transcription factor P3, retinoic-acid-related orphan receptor (ROR)‑γt in liver tissue were determined by western blotting, the frequency of Th17 and Treg cells in the liver was evaluated by flow cytometry, and a suppression assay was measured by incorporating [3H]‑thymidine. In addition, to explore the effect of Tregs expanded with rapamycin on hepatic stellate cells (HSC), HSCs were co‑cultured with Tregs from rapamycin or phosphate‑buffered saline‑treated mice. It was found that rapamycin treatment led to a significant reduction in the number of Th17 cells and in the expression levels of ROR‑γt in the liver tissues. Simultaneously, the results of the present study showed a significant increase in the frequency of Tregs and a marked enhancement in the expression of forkhead/winged helix transcription factor P3 in the rapamycin‑treated mice. Furthermore, the Tregs in rapamycin‑treated mice had significantly higher suppressive effects, compared with the cells from mice treated with phospphate‑buffered saline. Consequently, rapamycin treatment prevented the development of CCl4-induced hepatic fibrosis, which was shown by its histological appearances. These results suggested that the immunosuppressive effect of rapamycin on liver fibrosis was associated with the suppression of hepatic fibrogenesis and regulation of the Th17/Treg cell balance.
先前的研究表明，Th17细胞的激活和/或调节性T细胞（Tregs）的缺陷参与了肝纤维化的发病机制。本研究的目的是探讨雷帕霉素对四氯化碳（CCl4）诱导的小鼠肝纤维化模型免疫反应的影响。通过腹腔注射CCl4诱导肝纤维化。注射CCl4后，小鼠腹腔注射雷帕霉素（1.25mg/kg/天），持续8周。采用苏木精-伊红染色和Masson三色染色进行组织学检查。通过蛋白质印迹法测定肝组织中叉头/翼状螺旋转录因子P3、维甲酸相关孤儿受体（ROR）-γt的蛋白水平，通过流式细胞术评估肝脏中Th17和Treg细胞的频率，并通过掺入[3H]-胸腺嘧啶进行抑制试验测定。此外，为了探讨用雷帕霉素扩增的Tregs对肝星状细胞（HSC）的影响，将HSCs与来自雷帕霉素或磷酸盐缓冲盐水处理小鼠的Tregs共培养。发现雷帕霉素治疗导致肝脏组织中Th17细胞数量和ROR-γt表达水平显著降低。同时，本研究结果显示，雷帕霉素治疗的小鼠中Tregs频率显著增加，叉头/翼状螺旋转录因子P3的表达显著增强。此外，与磷酸盐缓冲盐水处理的小鼠细胞相比，雷帕霉素治疗的小鼠中的Tregs具有显著更高的抑制作用。因此，雷帕霉素治疗可预防CCl4诱导的肝纤维化的发展，这从组织学表现中得到证实。这些结果表明，雷帕霉素对肝纤维化的免疫抑制作用与抑制肝纤维化形成和调节Th17/Treg细胞平衡有关。
