HRC激活NLRP3炎性小体诱导肝细胞焦亡在肝纤维化中的作用及机制研究

Liver fibrosis is a consequence of chronic liver diseases, which will result in cirrhosis. Inflammation in the liver will lead to liver fibrosis. Our previous studies confirmed that HRC (histidine rich calcium-binding protein) is associated with liver fibrosis, but the molecular mechanism is not fully understood. Pyroptosis can induce inflammation by secreting pro-inflammatory cytokines, and plays an important role in liver fibrosis. Recent studies have shown that HRC plays a critical role in regulating intracellular calcium concentration. Moreover, increased [Ca2+]i activated NLRP3 inflammasome, which lead to pyroptosis. Up to now, the relationships among HRC, NLRP3 and pyroptosis have not been investigated. Our preliminary experiment demonstrated that HRC activated NLRP3 and induced hepatocyte pyroptosis, and the effect of HRC-induced hepatocyte pyroptosis was abolished by NLRP3-siRNA treatment. So we suggest that HRC activates NLRP3 by increasing Ca2+, which induces hepatocyte pyroptosis to promote liver fibrosis. This project intends to study the role of HRC and NLRP3 in liver inflammation and fibrosis, explore the molecular mechanism of HRC-activated NLRP3 and HRC-induced hepatocyte pyroptosis, and animal experiment will be used to detect the effect of hepatocyte pyroptosis induced by HRC-mediated NLRP3 inflammosome activation on liver fibrosis. We hope to find potential intervention targets of liver fibrosis.

肝纤维化是慢性肝病进展至肝硬化的中间环节。肝脏只要有炎症，就一定有肝纤维化。我们前期研究证实，钙结合蛋白HRC与肝纤维化相关，但机制未完全阐明。细胞焦亡可分泌炎症因子诱发炎症反应，在肝纤维化中发挥重要作用。研究表明，HRC可调控胞内钙离子浓度；胞内Ca2+增加可激活NLRP3炎性小体，后者可诱导细胞焦亡。肝纤维化中HRC、NLRP3与肝细胞焦亡的关系尚无研究。本项目预实验显示，HRC可激活NLRP3、诱导肝细胞焦亡，干扰NLRP3后HRC诱导肝细胞焦亡的作用减弱。据此推测，HRC通过调控胞内Ca2+激活NLRP3，诱导肝细胞焦亡，促进肝纤维化。本项目拟观察HRC、NLRP3在肝脏炎症、肝纤维化中的作用；研究HRC增加胞内Ca2+激活NLRP3的分子机制；筛选并验证HRC诱导肝细胞焦亡的信号途径；动物实验验证HRC激活NLRP3诱导肝细胞焦亡促进肝纤维化；以期寻找干预肝纤维化进展的潜在靶点。

细胞焦亡是一种新的细胞程序性死亡方式，其主要依赖于炎症小体的激活。肝细胞焦亡在肝星状细胞活化、肝纤维化中发挥重要作用。我们的前期研究表明，钙结合蛋白HRC可激活肝星状细胞促进肝纤维化，但其在肝细胞焦亡中的作用尚不明确。首先，通过实时荧光定量PCR（RT-qPCR）、蛋白电泳（western blot）及免疫组化等方法检测临床肝纤维化组织样本及腹腔注射 CCl4 小鼠肝纤维化模型中HRC、NLRP3及α-SMA的表达，结果证实HRC、NLRP3的阳性表达与肝纤维化的分期呈正相关；其次，通过流式细胞双染技术、乳酸脱氢酶检测、Edu技术、Transwell实验等方法检测肝细胞焦亡、肝星状细胞活化情况，结果证实HRC可诱导肝细胞焦亡，促进肝星状细胞的活化、增殖及迁移，而这一作用又依赖NLRP3/caspase-1信号通路的活化；最后，通过酶联免疫吸附分析（ELISA）等实验方法检测炎性细胞因子水平，结果证实HMGB-1作为NLRP3/caspase-1的下游信号分子激活肝星状细胞。综上所述，本研究发现HRC 通过激活 NLRP3/caspase-1信号诱导肝细胞焦亡，后者释放 HMGB1，进而促进 肝星状细胞的活化、增殖及迁移；阐明了HRC在肝细胞焦亡与肝星状细胞活化“交流对话”中的重要作用，为HRC作为肝纤维化治疗的靶点提供了依据。