HMGB1介导NLRP3炎性小体活化诱导肝细胞焦亡在药物性肝损伤中的作用机制研究

Acetaminophen (APAP) is the most frequent drug for the study of drug-induced liver injury (DILI), and it is also the classic animal model of DILI. NLRP3 inflammasome is the key to regulating inflammatory responses and cell fate. HMGB1 is involved in the pathogenesis of NLRP3 inflammasome as an important inflammatory substance. Pyroptosis and the inflammatory factors released are involved in the formation of " pyroptosis-inflammatory response-hepatocyte injury" axis-like pathological process. Previous studies found that HMGB1 and NLRP3 expression are significantly up-regulated during APAP-induced hepatocyte injury, suggesting that they play an important role in APAP-induced liver injury. Therefore, we assume that inhibiting NLRP3 inflammasome activation by blocking HMGB1 to regulate hepatocyte pyroptosis and suppress cellular crosstalk between hepatocytes-kupffer cells, eventually improve inflammatory response and the development of liver injury. Herein, by applying gene knock-down or over-expression cell lines and cell co-culture system, we will elucidate cell type-specific role of HMGB1 mediating NLRP3 inflammasome to regulate hepatocytes pyroptosis; then applying in vivo model of APAP induced DILI and gene knockout mice to investigate the underlying mechanisms of pyroptosis-inflammatory response-hepatocyte injury. Thus, this work will add the new knowledge for prevention and treatment of DILI.

对乙酰氨基酚（APAP）是研究药物性肝损伤最常见的药物，也是经典的肝损伤动物模型。NLRP3炎性小体是调控炎性反应和细胞命运的关键。HMGB1作为一种重要的炎性物质参与NLRP3炎性小体的致病过程。细胞焦亡及其释放的炎性因子共同参与形成“焦亡-炎症反应-肝细胞损伤”轴样病理改变。前期研究发现在APAP诱发的肝细胞损伤过程中，HMGB1、NLRP3表达显著上调，提示二者在APAP肝损伤中起重要的调节作用。由此提出抑制HMGB1介导NLRP3炎性小体活化，进而调控肝细胞焦亡，阻断肝细胞-枯否细胞交叉对话，则能改善肝脏炎症反应及肝损伤进程。本项目拟利用基因过表达和RNA干扰技术及细胞共培养体外模型，明确HMGB1介导NLRP3炎性小体调控肝细胞焦亡多靶向调控网络的关键切入点；采用APAP诱导肝损伤模型和基因敲除小鼠，阐明肝细胞焦亡-炎症反应-肝损伤进程的作用机制，为药物性肝损伤的防治提供新思路。

药物性肝损伤是由于药物自身或其代谢产物对肝脏造成的直接肝毒性损害。临床常用的解热镇痛药对乙酰氨基酚（APAP）是引起药物性肝损伤的常见原因。NLRP3炎性小体是调控炎性反应和细胞命运的关键，其活化导致炎症反应，诱导肝细胞焦亡。HMGB1作为一种重要的炎性物质与caspase-1的下游物质共同参与NLRP3炎性小体通路的致病过程。本项目通过动物模型及细胞模型，对药物性肝损伤的发病机制进行初步研究。利用APAP肝损伤模型以及NLRP3-/-小鼠APAP肝损伤模型，从体内角度深入研究HMGB1介导NLRP3炎性小体通路干预肝细胞-枯否细胞交叉对话调控对乙酰氨基酚肝损伤发生发展的分子机制。体外实验中采用肝细胞、肝脏巨噬细胞培养，给予甘草酸、MCC950预处理，明确HMGB1介导NLRP3炎性小体通路是调控肝细胞焦亡-炎症应答-肝损伤进程的多靶向调控网络的关键位点，进一步阐明HMGB1介导NLRP3炎性小体活化调控肝细胞焦亡和肝损伤进程的作用机制，为提出新的药物性肝损伤的治疗靶点及在药物应用上提供了理论依据。