基于波形蛋白介导的NLRP3/Caspase-1炎症信号通路研究健脾益气方防治肝癌的机理

Chronic inflammation has been increasingly recognized to occur during the progression and metastasis of various carcinomas such as primary liver cancer (hepatocellular carcinoma), which is one of the most common cancers. JPYQD can reduce liver cell necrosis and the invasion of hepatocellular carcinoma cells, and induce the apoptosis of hepatocellular carcinoma cells. In our previous studies, the overexpression of vimentin in DEN-induced rat hepatoma was observed, while significant reduction in the production of vimentin was examined by JPYQD. Furthermore, proteolytic digestion of vimentin by Caspase-3, and subsequent decreased caspase-1 level were observed in TGF-β1-induced SMMC-7721 cells, after treatement with JPYQD. Importantly, vimentin can regulate activation of the NLRP3/caspase-1 inflammasome pathway by protein-protein interaction between NLRP3 and vimentin. We speculate that down-regulating activation of the NLRP3/caspase-1 inflammasome pathway will be observed in liver cancer cells, when the expression level of vimentin decreases by JPYQD. Therefore, vimentin-activating NLRP3/caspase-1 inflammasome pathway will be examined in diethylnitrosamine (DEN)-induced hepatocarcinogenic rat models after treatment by JPYQD, and the same experiments will do in vitro. The role of decreased stringency of vimentin-activating NLRP3/caspase-1 inflammasome pathway will be elucidated after treatment by JPYQD. The results will provide new evidences for the clinic usage of the JPYQD.

肝癌是临床常见难治的恶性肿瘤，慢性炎症与肝癌的发生发展及转移密切相关。前期研究发现健脾益气方（JPYQD）可降低肝细胞炎性坏死程度及肝癌细胞的侵袭能力、诱导肝癌细胞凋亡；其防治肝癌的核心靶点之一是波形蛋白，后者水平下调可明显降低Caspase-1表达。波形蛋白是联系炎癌转化的重要纽带，且能通过与NLRP3直接作用驱动NLRP3/Caspase-1炎症信号通路。推测JPYQD可能通过下调波形蛋白水平，下调NLRP3/Caspase-1信号通路，干预肝癌炎症微环境。项目拟采用DEN诱导的大鼠肝炎-肝硬化-肝癌模型和LPS+ATP联合刺激的人肝癌细胞HepG2，结合通路抑制剂，综合应用PCR、Western blot、ELISA等技术，观察JPYQD下调波形蛋白表达对NLRP3/Caspase-1炎症信号通路的影响，深入探讨JPYQD防治肝癌的机理，为健脾类方药进一步的开发应用提供基础研究支持。

肝癌是临床常见难治的恶性肿瘤，慢性炎症与肝癌的发生发展及转移密切相关。前期研究发现健脾益气方可降低肝细胞炎性坏死程度及肝癌细胞的侵袭能力、诱导肝癌细胞凋亡；其防治肝癌的核心靶点之一是Vimentin（波形蛋白），后者水平下调可明显降低Caspase-1表达。Vimentin是联系“炎癌转化”的重要纽带，且能通过与NLRP3炎性小体作用驱动NLRP3/Caspase-1炎症信号通路。因此，我们推测健脾益气方可能通过下调Vimentin水平，下调NLRP3/Caspase-1信号通路，干预肝癌炎症微环境。本项目分别采用DEN诱导的大鼠肝癌模型和LPS+ATP联合诱导的人肝癌细胞HepG2，结合通路抑制剂，综合应用生物化学、分子生物学、病理学等现代研究技术，观察健脾益气方下调Vimentin表达对NLRP3/Caspase-1炎症信号通路的影响，深入探讨健脾益气方防治肝癌的机理。研究结果表明，腹腔注射DEN可成功制备大鼠肝癌模型，其中Caspase-1及其相关炎症因子IL-1β、IL-18、NFκB可作为DEN诱癌过程中肝细胞损伤的标志因子。健脾益气方（尤其是高剂量的健脾益气方）可减弱DEN肝癌大鼠肝功能的恶化，降低这些炎性因子的表达，并抑制人肝癌细胞HepG2的增殖和侵袭、诱导肝癌细胞凋亡；明显降低DEN肝癌大鼠肝组织及HepG2细胞中Vimentin、NLRP3、ASC、Caspase-1、IL-1β等的表达水平。证实健脾益气方的作用机制可能是通过下调Vimentin蛋白的表达，抑制NLRP3/Caspase-1炎症信号通路的激活，进而通过调节肝癌炎症微环境抑制肝癌的发生发展。研究结果为健脾治法的灵活运用及健脾益气方进一步开发应用提供基础研究支持。