柔肝方下调Th17细胞的IL-17A和FBRS表达抑制HSC活化抗肝纤维化的机制研究

Hepatic fibrosis (HF) in chronic hepatitis B is positively related to the expression of FBRS and Th17. IL-17A and FBRS secreted by Th17 up-regulate the levels of TGFβ receptor and TGF β1 in HSC by activating the MAPK signal and then result in HSC activation. The Rougan Recipe composed of Traditional Chinese Medicine of nourishing yin and promoting blood circulation is efficient in the treatment of Hepatic fibrosis (HF) in chronic hepatitis B, which may inhibit the activation of HSC and decrease Th17 and FBRS expression. The hypothesis is proposed that" Rougan Recipe reduces the level of IL-17A and FBRS expressed by Th17 in the liver and down-regulates the expression of TGFβ receptor and TGF β1 in HSC, and then inhibit the activation of HSC. . The following studies will be carried out: the effects of Rougan Recipe on levels of IL-17A and FBRS expressed by Th17 in the liver, MAPK activation and levels of TGFβ receptor and TGF β1 in HSC in HF model mice induced by ConA will be examined; IL-17A silenced mice FBRS silenced mice will be constructed and then MAPK activation and levels of TGFβ receptor and TGF β1 in HSC were observed interrupting by ConA in vivo and Rougan Recipe's effects ; TGF beta 1, TGF beta receptor expressed by HSC are respectively silenced in vitro, and then MAPK activation and the activation of HSC and Rougan Recipe's effects will be studied. The mechanism of anti-hepatic hepatitis by multi targets will be clarified.

慢乙肝肝纤维化（HF）程度与外周血纤维化蛋白（FBRS）和Th17正相关。Th17分泌IL-17A和FBRS通过MAPK信号分别上调HSC表达TGFβ受体和TGFβ1而致其激活。养阴活血组方的柔肝方是治疗乙肝HF有效验方，其可抑制HSC活化,降低Th17及FBRS表达。提出假说”柔肝方降低肝内Th17的IL-17A和FBRS表达，下调HSC的TGFβ受体和TGFβ1表达，抑制HSC活化”。拟开展：柔肝方对ConA诱导HF小鼠肝内Th17的IL-17A、FBRS表达和HSC的MAPK活化、TGFβ1与TGFβ受体表达的影响；分别构建IL-17A、FBRS沉默小鼠，观察ConA体内干预对HSC的MAPK、TGFβ1、TGFβ受体表达和柔肝方作用；体外沉默小鼠HSC的TGFβ1、TGFβ受体，观察IL-17A、FBRS刺激后MAPK活化、HSC活化和柔肝方作用，明确柔肝方多靶点抗HF免疫机制。

慢乙肝肝纤维化程度与外周血纤维化蛋白(FBRS)和Th17正相关。Th17分泌IL-17A 和FBRS通过MAPK信号分别上调肝星状细胞（HSC）表达TGFβ受体和TGFβ1而致其激活。养阴活血组方的柔肝方是治疗乙肝肝纤维化有效验方,其可抑制HSC活化,降低Th17及FBRS表达。应用 ConA 肝纤维化小鼠模型,观察柔肝方对模型鼠肝内浸润的Th17频数及其表达的IL-17A 、FBRS 、TGFβ1 及 TGFβRII表达水平的影响,发现柔肝方能够明显降低肝纤维化模型小鼠肝内的胶原沉积和肝内纤维化相关蛋白的表达水平，减少肝脏内FBRS和IL-17A的表达水平及肝内浸润的Th17细胞的频数，降低肝星状细胞的FBRS、TGFβ1及 TGFβRII表达水平。进一步分离小鼠肝脏原代肝星状细胞、肝细胞、巨噬细胞和CD4+T细胞，发现ConA肝纤维化模型小鼠肝星状细胞表达的FBRS最高。构建FBRS-siRNA腺病毒相关载体，建立FBRS沉默小鼠，发现沉默FBRS后，ConA诱导的肝纤维化模型小鼠肝组织α-SMA表达明显降低，肝星状细胞的TGFβ1及 TGFβRII表达水平明显降低。应用免疫共沉淀－质谱法筛选出FBRS的潜在结合蛋白FAT1，通过构建FBRS高表达的LX-2细胞系，发现FBRS过表达后FAT1高表达，且α-SMA表达明显增加，提示FAT1可能是FBRS诱导肝纤维化的结合蛋白。体外分别构建TGF-β1和TGF-βRII沉默的JS-1细胞系，发现柔肝方通过降低IL-17A和FBRS表达，抑制TGFβ1/TGFRII通路，减少肝星状细胞的活化发挥抗肝纤维化的作用。本项目发表本项目标注的论文10篇，其中第一作者或通讯作者的SCI论文4篇、中文核心论文4篇。获得科技成果奖励6项，其中第一完成人1项。申请国家发明专利5项，完成成果转让1项。培养毕业硕士研究生3名，协助指导毕业硕、博士研究生6名。