基于PD-L1/PD-1信号通路研究丹参调节自然杀伤细胞功能抗肝纤维化的作用机制

The functions of natural killer cells are inhibited by the up-regulation of PD-L1/PD-1 singal pathway, which is the key pathomechanism during the development of liver fibrosis. In our previous studies, Salvia miltiorrhiza (SM) was found to reverse liver fibrosis, down-regulate the expression of PD-1 on the surface of NK cells and up-regulate the numbers of NK cells. Hence we put forward the hypothesis that the pharmacological mechanism of SM against liver fibrosis is involved with promoting the functions of NK cells to accelerate the apoptosis of activated HSC and regulating the PD-L1/PD-1 signal pathway, especially inhibiting the expression of PD-1, to strengthen the functions of NK cells. This project will be developed as follows: Firstly, with the positive control of ASGM-1 (NK inhibitor) and anti-PD-L1 antibody (PD-L1/PD-1 blocker) applied, anti-fibrotic effects of SM on inhibiting the PD-L1/PD-1 signal pathway to promote the functions of NK cells are investigated in vivo. Secondly, primary NK cells are isolated and then are exhausted by ASGM-1. With the positive control of anti-PD-L1 antibody and construction of over-expressed PD-1 gene, NK cell lines are pre-incubated with SM before co-culturing with hepatic stellate cell (HSC), which intend to prove the anti-fibrotic mechanism of SM on promoting the functions of NK cells to accelerate the apoptosis of activated HSC by blocking PD-L1/PD-1 signal pathway. This project will reveal the immunologic mechanism of SM against the liver fibrosis.

研究证实PD-L1/PD-1信号通路上调抑制NK细胞功能是肝纤维化发展的重要病理机制，丹参有良好的抗纤维化作用及抑制PD-1表达并上调NK细胞数量的前期工作基础。为此提出“增强NK细胞功能是丹参促进活化的HSC凋亡而发挥抗肝纤维化作用的重要药理机制，调节PD-L1/PD-1信号通路尤其是抑制PD-1分子的表达是丹参促进NK细胞功能的分子药理机制”之科学假说，拟开展：①体内实验设NK抑制剂(ASGM-1)、抗-PD-L1抗体对照，观察丹参抑制PD-L1/PD-1信号通路、促进NK细胞功能与抗肝纤维化的作用；②体外实验分离NK细胞，ASGM-1模拟NK细胞“衰竭”，设抗-PD-L1抗体对照；构建人NK细胞株过表达PD-1，予丹参孵育后再与HSC共培养，验证丹参下调PD-L1/PD-1信号通路促进NK细胞功能而诱导HSC凋亡的抗肝纤维化机制。该研究将进一步揭示丹参防治肝纤维化的免疫作用机制。

NK细胞是肝内重要的天然免疫细胞，可杀伤活化的HSC而抗肝纤维化。但随着肝纤维化进展，NK细胞的功能受到抑制，对活化的HSC“杀伤”能力减弱，因此恢复并促进NK细胞功能可能是治疗肝纤维化的重要途径之一。本课题基于PD-L1/PD-1信号通路探讨丹参调节肝脏NK细胞功能抗肝纤维化的作用机制；并拓展观察丹参抑制促炎型巨噬细胞和NLRP3炎症小体抗肝损伤的作用机制，以此工作基础成功申请到国家自然科学基金面上项目（No.81874346）。首先体内复制CCl4染毒小鼠肝纤维化模型，丹参干预治疗；体外分离原代肝脏NK细胞，将原代NK细胞和JS-1肝星状细胞（HSC）分别与丹参共孵育；采用NK细胞抑制剂ASGM-1诱导NK细胞衰竭模型，并采用抗-PD-L1抗体阻断；将预孵育的NK细胞及其细胞上清分别与HSC共培养，观察NK细胞和HSC活性与功能及PD-L1/PD-1表达，结果发现丹参在体内和体外均可促进NK细胞功能而发挥抗肝纤维化作用，其机制与下调NK细胞表达PD-1和抑制活化HSC表达PD-L1相关。其次，体内观察CCl4染毒小鼠肝内炎症因子，体外采用LPS诱导促炎型巨噬细胞活化，观察丹参在体内和体外对巨噬细胞相关炎症因子的影响。结果发现丹参可抑制多种促炎因子表达。最后体内复制DDC染毒胆汁淤积性模型，丹参干预治疗，观察丹参抗胆汁淤积性肝损伤的作用，结果发现丹参通过抑制肝脏NLRP3炎症小体表达而抗肝损伤。该工作的完成，为肝纤维化的发病机制增添基础，并为其治疗提供新的有效的靶点；同时本课题研究证实调节PD-L1/PD-1是丹参增强NK细胞功能抗肝纤维化的重要作用环节，有助于发展中医药防治肝纤维化的新策略与新理论。本课题已发表论文4篇，其中SCI期刊收录3篇；参加国际学术会议交流2次；协助培养博士研究生2名。