基于CD4+T淋巴细胞亚群的变化探讨雄芍汤抗肝纤维化的免疫机制研究

CD4+T lymphocytes include Th1, Th2, Th17, regulatory T cells (Treg) and Th22 cells. The study of Th1/Th2 pattern has been relatively clear. It has also been confirmed that Th17/Treg imbalance exists in mice with liver fibrosis, which potentially promotes liver fibrosis via HSC activation. It has been confirmed that Th17/Th22 expression imbalance exists in liver failure patients. But the relationship between Th17/Th22 imbalance, HSC activation and liver fibrosis is unclear and requires further study. . The applicant has achieved the satisfactory efficacy by treating liver fibrosis with Xiongshao decoction for many years, and the preliminary experiments also confirmed that Xiongshao decoction can inhibit HSC activation and proliferation, but the related immune research has not been done. The hypothesis that Xiongshao decoction inhibits HSC activation by adjusting CD4+T lymphocyte subsets needs the further research. We propose the hypothesis that advanced liver fibrosis is accompanied by Th17/Treg imbalance and Th17/Th22 imbalance, and Xiongshao decoction inhibits HSC activation by restoring Th17/Treg and Th17/Th22 balance. It will be verified through experiments in vivo and in vitro, and the immune mechanism of Xiongshao decoction on hepatic fibrosis will be revealed.

CD4+T淋巴细胞包括Th1、Th2、Th17、Treg及Th22细胞等。其中，Th1/Th2模式的研究已较为明确，Th17/Treg也已被证实，Th17/Treg失衡激活HSC促进了肝纤维化（HF）。有关Th17/Th22的研究，已证实肝衰竭患者存在Th17/Th22表达失衡。而Th17/Th22失衡与HSC活化及HF的关系，目前相关机制尚不清楚，需要进一步研究。. 课题组多年来采用临床验方雄芍汤治疗HF，疗效满意，前期实验证实雄芍汤能抑制HSC的活化与增殖，而雄芍汤是否通过调节CD4+T淋巴细胞亚群来抑制HSC活化，还需要进一步研究。为此，本课题提出HF的进展期伴随着Th17/Treg失衡和Th17/Th22失衡，其通过激活HSC促进HF，雄芍汤通过恢复二者平衡而抑制HSC活化，从而发挥其抗HF作用的假设，并针对该假设通过体内外实验进行验证，以揭示雄芍汤抗HF的免疫机制。

CD4+T淋巴细胞包括Th1、Th2、Th17、Treg及Th22细胞等。其中，Th1/Th2模式的研究已较为明确，而Th17/Treg失衡与肝纤维化（HF）的关系仍存在争议，Th17/Th22失衡与HSC活化及HF的关系，目前相关机制尚未阐明。已证实雄芍汤能抑制HSC活化与增殖，它是否通过调节CD4+T淋巴细胞来抑制HSC活化仍有待研究。因此，本课题提出HF进程伴随着Th17/Treg失衡和Th17/Th22失衡，其通过激活HSC促进HF，雄芍汤通过恢复二者平衡而抑制HSC活化，从而发挥其抗HF作用的假设，通过体内外实验进行验证。120只SPF级SD雄性大鼠随机分为正常组40只、模型组40只、雄芍汤组40只。采用50%CCl4橄榄油溶液腹腔注射法制备HF大鼠模型，雄芍组第4、8、12周大鼠每日分别给予雄芍汤药液（7.1875 g·kg -1）灌胃，第16周大鼠在成模后每日灌胃，共4周。采用ELISA法检测各组大鼠血清IL-17、TGF-β及IL-22水平，Western blot检测各组大鼠肝组织IL-17、TGF-β及IL-22蛋白表达，流式细胞仪检测脾Th17、Treg及Th22细胞比例。结果显示，在CCl4诱导的大鼠HF模型中，HF进展期和恢复期并没有出现Th17/Treg失衡。而在HF进展后期和恢复期Th17细胞比例及IL-17均明显升高，Th22细胞比例及IL-22均显著降低，α-SMA与Th17细胞比例呈显著正相关，而与Th22细胞比例呈显著负相关，提示HF进展后期和恢复期Th17/Th22失衡通过激活HSC促进HF的发生发展。雄芍汤可能通过在肝纤维化进展后期和恢复期下调Th17细胞比例，抑制促纤维化细胞因子IL-17生成，并上调Th22细胞比例，促进IL-22的生成，恢复Th17/Th22平衡从而抑制HSC活化来发挥防治肝纤维化的作用。体外实验从小鼠肝组织中分离HSC，从小鼠脾脏中分离Th17、Treg和Th22细胞，采用Western blot和免疫荧光染色法测定各组HSC中α-SMA的表达。结果显示，Th17细胞可以促进HSC的活化，而Th22细胞可以抑制HSC的活化，雄芍汤抑制Th17细胞对HSC活化的促进作用，增强Th22细胞对HSC活化的抑制作用。结论：雄芍汤通过恢复Th17/Th22平衡而抑制HSC活化，从而发挥其抗HF作用。