Caspase-11/GSDMD非经典细胞焦亡途径介导Kupffer细胞焦亡在非酒精性脂肪性肝炎发病机制及化痰祛湿活血方干预作用

The involvement of inflammatory factors as "second hit" is the key link in the development of Non-alcoholic steatohepatitis (NASH). Studies have shown that during the formation of NASH, Caspase-11/GSDMD inflammasome signaling mediates the release of inflammatory factors from Kupper cells (KCs) in the form of pyroptosis. “TanShiYu” is the common foundation of KCs pyroptosis and NASH. Previous studies have confirmed that Huatanqushihuoxue Prescription has effective anti-NASH effect, and the mechanism is related to the inhibition of inflammatory factor production. We hypothesized that Huatanqushihuoxue Prescription can regulate the Caspase-11/ GSDMD inflammasome signaling to regulate the pyroptosis of KCs, reduce the production of inflammatory factors, and interfere with the development of NASH. To verify the above hypothesis, this project intends to replicate NASH mice and LPS-induced activation of RAW264.7 cell model, intervene with Huatanqushihuoxue Prescription, observe the Phenotype of KCs by Flow Cytometry and ultrastructure of KCs by electron microscopy, RT-PCR and Western Blot were used to detect the protein and genes expression of LBP、Caspase-11、Caspase-1、NLRP3、GSDMD、GSDMD-N, and detect the expression of inflammatory factors by ELISA. To elucidate the intervention mechanism of KCs cell pyroptosis mediated by Caspase-11/GSDMD non-canonical inflammasome signaling in NASH pathogenesis and the intervention of Huatanqushihuoxue Prescription, provide a theoretical basis for clinical prevention and treatment of NASH.

炎症因子参与“二次打击”是非酒精性脂肪性肝炎（NASH）发病的关键环节，研究表明Caspase-11/GSDMD通路调控库普弗细胞（KCs）焦亡释放炎症因子，痰湿瘀是KCs焦亡和NASH的共同基础。前期研究证实化痰祛湿活血方具有抑制炎症因子抗NASH作用，我们假说化痰祛湿活血方可以调节Caspase-11/GSDMD通路抑制KCs焦亡，减少炎症因子生成，干预NASH发病。本项目拟复制NASH小鼠模型及LPS诱导RAW264.7细胞模型，以化痰祛湿活血方干预，流式细胞术及电镜观察KCs表型及超微结构，RT-PCR、Western Blot检测LBP、Caspase-11、Caspase-1、NLRP3、GSDMD、GSDMD-N，ELISA检测下游炎症因子以验证假说。阐明Caspase-11/GSDMD通路介导KCs焦亡在NASH发病机制及化痰祛湿活血方干预作用，为临床防治NASH提供依据。

非酒精性脂肪性肝炎(NASH)发病率高，是向肝硬化、肝癌发展的重要环节,尚缺乏有效西药，炎症因子参与“二次打击”是NASH发展核心环节。NASH过程中肝巨噬细胞(KCs)通过Caspase-11/GSDMD非经典焦亡途径介导的细胞焦亡释放炎症因子参与NASH发展。本项目研究内容：①复制NASH小鼠模型，观察化痰祛湿活血方抗NASH作用及对Caspase-11/GSDMD信号通路影响；②制备LPS诱导RAW264.7细胞焦亡模型，沉默Caspase-11基因，观察化痰祛湿活血方对Caspase-11/GSDMD信号通路影响、下游炎症因子表达、细胞形态等的影响，阐明Caspase-11/GSDMD途径介导的细胞焦亡是化痰祛湿活血法抗NASH的中心环节。重要结果及关键数据：①化痰祛湿活血法可降低NASH小鼠血清ALT、AST、TC、TG水平，改善肝组织脂肪变、炎症及纤维化程度，减少库普弗细胞浸润，下调肝组织中NLRP3、Caspase-1、Caspase-11、GSDMD mRNA及蛋白的表达，降低肝组织促炎因子IL-18、IL-1β水平。②建立了以化痰祛湿活血方中柴胡皂苷a、大黄酚等20种成分的炮制前后及不同煎煮方法的指纹图谱。③化痰祛湿活血方可降低LPS诱导RAW264.7细胞焦亡模型细胞上清液NLRP3、Caspase-1、Caspase-11、GSDMD基因及蛋白表达，减少炎症因子IL-1β、IL-18表达，降低GSDMD-N表达，改变细胞焦亡形态，发挥与类似基因沉默Caspase-11表达作用。科学意义：证明 Caspase-11/ GSDMD细胞焦亡途径是化痰祛湿活血方抗 NASH 的关键作用靶点，揭示化痰祛湿活血方干预Caspase-11/GSDMD焦亡途径介导KCs焦亡在NASH发生发展中的作用机制靶点，为中医药防治NASH提供理论基础，为后续中药新药研发提供重要理论依据。