Ruan Shuiliang, Han Chenyang, Sheng Yongjia, Wang Jin, Zhou Xiaohong, Guan Qiaobing, Li Wenyan, Zhang Caiqun, Yang Yi
Department of Center Laboratory, The Second Affiliated Hospital of Jiaxing University, China.
Department of Pharmacy, The Second Affiliated Hospital of Jiaxing University, China.
Int Immunopharmacol. 2021 Nov;100:108126. doi: 10.1016/j.intimp.2021.108126. Epub 2021 Sep 4.
Pyroptosis, a pattern of inflammatory death, is regulated by NLRP3-Caspase-1 inflammasome and GSDMD-FL protein. Antcin A is a small triterpenoid molecule. In this study, Kupffer cells (KC) were used for in vitro model, which were treated with LPS and Nigericin (L/N) to induce pyroptosis. ELISA was used to determine the influence of Antcin A on the expression of inflammatory factors, IF was utilized to investigate NLRP3 and Caspase-1, PI staining was used to detect the opening level of membrane pores in KCs, C57BL/6J wild-type mice were fed with high-fat diet to construct a NAFLD model, and were simultaneously treated with Antcin A. H&E staining was used to detect hepatic pathological changes in mice, oil red staining was utilized to detect hepatic fat deposits in mice, IHC was used to detect the expression of NLRP3 and Caspase-1, Western blot was used to detect the expression levels of NLRP3 inflammasome (including NLRP3, ASC, Caspase-1, GSDMD-FL and GSDMD-NT). Pull-down assay and immunoprecipitation assay were used to detect the binding between Antcin A and NLRP3. As a result, Antcin A could significantly inhibit the occurrence of pyrolysis, decrease the expression of inflammatory factors, inhibit the activation and assembly of NLRP3 inflammasome, and significantly down-regulate the expression of NLRP3, Caspase-1 and GSDMD-NT in KCs. In NAFLD mice, Antcin A could suppress the inflammatory response in liver tissues of mice, reduce lipid deposition, down-regulate the levels of ALT and AST, and improve liver function in mice. Antcin A could also inhibit the activation of NLRP3 inflammasome in liver tissue and decrease the level of inflammatory factors. In the study of mechanism, we revealed that Antcin A could inhibit the assembly and activation of NLRP3 inflammasome by binding with NLRP3. In summary, in this study, we found that Antcin A could inhibit pyroptosis in KC and alleviate the inflammatory response of liver tissue in NAFLD by targeting NLRP3 inflammasome, which was one of the mechanisms of Anctin A in protecting liver.
细胞焦亡是一种炎症性死亡模式，受NLRP3 - 半胱天冬酶 - 1炎性小体和GSDMD - FL蛋白调控。蚁酸A是一种小三萜类分子。在本研究中，采用库普弗细胞（KC）建立体外模型，用脂多糖（LPS）和尼日利亚菌素（L/N）处理以诱导细胞焦亡。采用酶联免疫吸附测定（ELISA）来确定蚁酸A对炎性因子表达的影响，采用免疫荧光（IF）来研究NLRP3和半胱天冬酶 - 1，采用碘化丙啶（PI）染色来检测KC中膜孔的开放水平，给C57BL/6J野生型小鼠喂食高脂饮食以构建非酒精性脂肪性肝病（NAFLD）模型，并同时用蚁酸A进行处理。采用苏木精 - 伊红（H&E）染色来检测小鼠肝脏的病理变化，采用油红染色来检测小鼠肝脏中的脂肪沉积，采用免疫组织化学（IHC）来检测NLRP3和半胱天冬酶 - 1的表达，采用蛋白质免疫印迹法（Western blot）来检测NLRP3炎性小体（包括NLRP3、凋亡相关斑点样蛋白（ASC）、半胱天冬酶 - 1、GSDMD - FL和GSDMD - NT）的表达水平。采用下拉试验和免疫沉淀试验来检测蚁酸A与NLRP3之间的结合。结果显示，蚁酸A可显著抑制细胞焦亡的发生，降低炎性因子的表达，抑制NLRP3炎性小体的激活和组装，并显著下调KC中NLRP3、半胱天冬酶 - 1和GSDMD - NT的表达。在NAFLD小鼠中，蚁酸A可抑制小鼠肝脏组织中的炎症反应，减少脂质沉积，下调谷丙转氨酶（ALT）和谷草转氨酶（AST）水平，并改善小鼠肝功能。蚁酸A还可抑制肝脏组织中NLRP3炎性小体的激活并降低炎性因子水平。在机制研究中，我们发现蚁酸A可通过与NLRP3结合来抑制NLRP3炎性小体的组装和激活。综上所述，在本研究中，我们发现蚁酸A可通过靶向NLRP3炎性小体抑制KC中的细胞焦亡并减轻NAFLD中肝脏组织的炎症反应，这是蚁酸A保护肝脏的机制之一。
