PDF(Pubmed)
Abstract：
Acetaminophen (APAP) overdose is the primary cause of drug‑induced acute liver failure in numerous Western countries. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation serves a pivotal role in the pathogenesis of various forms of acute liver injury. However, the cellular source for NLRP3 induction and its involvement during APAP‑induced hepatotoxicity have not been thoroughly investigated. In the present study, hematoxylin and eosin staining was performed to assess histopathological changes of liver tissue. Immunohistochemistry staining(NLRP3, Caspase‑1, IL‑1β, GSDMD and Caspase‑3), western blotting (NLRP3, Caspase‑1, IL‑1β, GSDMD and Caspase‑3) and RT‑qPCR (NLRP3, Caspase‑1 and IL‑1β) were performed to assess the expression of NLRP3/GSDMD signaling pathway. TUNEL staining was performed to assess apoptosis of liver tissue. The serum expression levels of inflammatory factors (IL‑6, IL‑18, IL‑1β and TNF‑α) were assessed using ELISA and inflammation of liver tissue was assessed using immunohistochemistry (Ly6G and CD68) and RT‑qPCR (TNF‑α, Il‑6, Mcp‑1, Cxcl‑1, Cxcl‑2). A Cell Counting Kit‑8 was performed to assess cell viability and apoptosis. Protein and gene expression were analyzed by western blotting (PCNA, CCND1) and RT‑qPCR (CyclinA2, CyclinD1 and CyclinE1). Through investigation of an APAP‑induced acute liver injury model (AILI), the present study demonstrated that APAP overdose induced activation of NLRP3 and cleavage of gasdermin D (GSDMD) in hepatocytes, both in vivo and in vitro. Additionally, mice with hepatocyte‑specific knockout of Nlrp3 exhibited reduced liver injury and lower mortality following APAP intervention, accompanied by decreased infiltration of inflammatory cells and attenuated inflammatory response. Furthermore, pharmacological blockade of NLRP3/GSDMD signaling using MCC950 or disulfiram significantly ameliorated liver injury and reduced hepatocyte death. Notably, hepatocyte Nlrp3 deficiency promoted liver recovery by enhancing hepatocyte proliferation. Collectively, the present study demonstrated that inhibition of the NLRP3 inflammasome protects against APAP‑induced acute liver injury by reducing hepatocyte pyroptosis and suggests that targeting NLRP3 may hold therapeutic potential for treating AILI.
摘要：
在许多西方国家，对乙酰氨基酚（APAP）过量是药物引起的急性肝衰竭的主要原因。NLR家族含pyrin结构域3（NLRP3）炎性体激活在各种形式的急性肝损伤的发病机理中起关键作用。然而,NLRP3诱导的细胞来源及其在APAP诱导的肝毒性过程中的参与尚未得到彻底研究。在本研究中,进行苏木精和伊红染色以评估肝组织的组织病理学变化。免疫组织化学染色（NLRP3，Caspase‑1，IL‑1β，GSDMD和Caspase‑3)，蛋白质印迹（NLRP3，Caspase-1，IL-1β，进行GSDMD和Caspase‑3）和RT‑qPCR（NLRP3，Caspase‑1和IL‑1β）以评估NLRP3/GSDMD信号通路的表达。进行TUNEL染色以评估肝组织的凋亡。使用ELISA评估血清炎症因子（IL‑6，IL‑18，IL‑1β和TNF‑α）的表达水平，并使用免疫组织化学（Ly6G和CD68）和RT‑qPCR评估肝组织的炎症（TNF‑α，Il‑6、Mcp‑1、Cxcl‑1、Cxcl‑2)。进行细胞计数试剂盒-8以评估细胞活力和凋亡。蛋白质和基因表达通过蛋白质印迹分析(PCNA，CCND1）和RT‑qPCR（CyclinA2，CyclinD1和CyclinE1）。通过对APAP诱导的急性肝损伤模型（AILI）的研究，本研究表明，APAP过量诱导肝细胞中NLRP3的激活和gasderminD（GSDMD）的裂解，体内和体外。此外,肝细胞特异性敲除Nlrp3的小鼠在APAP干预后表现出减少的肝损伤和较低的死亡率，伴有炎症细胞浸润减少和炎症反应减弱。此外，使用MCC950或双硫仑对NLRP3/GSDMD信号传导的药物阻断可显着改善肝损伤并减少肝细胞死亡。值得注意的是,肝细胞Nlrp3缺乏通过增强肝细胞增殖促进肝脏恢复。总的来说，本研究表明,抑制NLRP3炎性体可通过减少肝细胞焦凋亡来预防APAP诱导的急性肝损伤,并提示靶向NLRP3可能具有治疗AILI的治疗潜力.
