基于ADPN/PI3K/AKT信号通路抑制炎症反应的化痰祛湿活血法治疗NASH的机制

Non-alcoholic steatohepatitis (NASH) with a highh mortality rate is critical in the development of hepatic fibrosis and cirrhosis. The mechanisms for NASH is complicated, and the imblance of anti-inflammatory and proinflammatory adipokines play a key role in the development of NASH. ADPN is one of the important anti-inflammatory adipokines. The phosphorylation of PI3K/AKT induced by ADPN receptor activation plays a central role in the ADPN-mediated signaling pathway, it may be a potential therapeutic target for the treatment of NASH. Currently, the effective therapeutic intervention for NASH is not available in clinical practice. Our previous studies show that Huatanqushihuoxue intervention protects against NASH and prvents inflammatory responses possibly via the activation of ADPN ,but as for the effect on the ADPN downstresm signaling pathway is not clear. To clarify the role of ADPN-mediated signaling pathway in Huatanqushihuoxue-induced protection against NASH, we investigate the effects of Huatanqushihuoxue intervention on inflammatory responses, the levels of ADPN, ADPN receptor expression, and the levels of PI3K/AKT phosphorylation in the liver of NASH rats. Moreover, we will further determine the effects of the serum derived from the Huatanqushihuoxue-treated rats on ADPN-meidated signaling pathway and NF-κB, and the effects of PI3K/AKT blockade on Huatanqushihuoxue-mediated protection in hepatocyte culture. Our studies will clarify the pharmacological mechanisms for Huatanqushihuoxue-mediated protection against NASH, and provide the theoretical basis for clinical application on NASH.

NASH发病率高，是向肝纤维化、肝硬化发展的重要环节。其发病机制复杂，抗炎促炎脂肪因子失衡是NASH发生发展的病理基础。ADPN是重要的抗炎脂肪因子，其受体活化与PI3K/AKT磷酸化是信号传导的中心环节，亦是抗NASH的关键作用靶点。目前临床尚无有效抑制NASH药物，前期研究发现化痰祛湿活血法具有良好抗NASH作用，其抗炎机制与活化ADPN效应有关，但对ADPN介导下游信号通路的影响尚不明确。本项目拟通过化痰祛湿活血法干预NASH大鼠模型，观察肝组织炎症程度、ADPN蛋白与受体表达、PI3K/AKT磷酸化水平等影响；复制脂肪变肝细胞模型，分别以阻滞剂阻断ADPN信号传导及AKT磷酸化，经化痰祛湿活血法药物血清干预，观察其对PI3K/AKT磷酸化，核转录因子NF-κB活性影响，炎症因子表达。阐明化痰祛湿活血法通过调控ADPN信号转导抗NASH关键分子作用机制及靶点，为临床治疗提供理论依据。

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,抗炎促炎因子失衡是NASH发生发展的病理基础，脂联素（ADPN）对NASH具有很强的保护作用。ADPN与其受体AdipoR2结合，活化信号通路PI3K/AKT，抑制下游靶蛋白NF-κB核转录，减少TNF-α、IL-1、IL-6等促炎因子的表达，从而发挥抗炎作用。研究内容：①复制大鼠NASH模型，观察化痰祛湿活血法抗NASH作用及对ADPN信号通路影响。②复制脂肪变肝细胞模型，沉默AdipoR2基因，观察化痰祛湿活血法对PI3K/AKT磷酸化，核转录因子NF-κB活性，炎症因子的表达等进而阐明化痰祛湿活血法作用机制。③AKt抑制剂阻断ADPN/PI3K/AKT信号通路，最佳药物浓度干预脂肪变肝细胞模型，观察对PI3K/AKT磷酸化、核转录因子NF-κB活性、炎症因子表达等的影响，阐明PI3K/AKT通路活化是化痰祛湿活血法基于ADPN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大鼠血清ALT、AST、ALP、TC、TG、GLU水平，减少肝组织及血清促炎因子TNF-α、IL-1α、IL-6及氧化应激因子MCP-1、PTGS2、NOS2、MDA含量，提高抗炎因子IL-10含量，减少库普弗细胞浸润，改善肝组织炎症及纤维化程度，上调肝组织中ADPN、AdipoR2、PI3K 、AKT2 mRNA及蛋白的表达，下调NF-κB mRNA及蛋白、pNF-κB蛋白的表达。②化痰祛湿活血方可降低脂肪变肝细胞模型细胞上清液ALT、AST、TG水平，减少细胞裂解液iNOS水平，减少炎症因子TNF-α、IL-1、IL-6 mRNA的表达，升高抗炎因子IL-10及AdipoR2 mRNA的表达，上调AdipoR2、pAkt蛋白的表达，下调pNF-κB蛋白的表达。③ADPN/PI3K/AKT信号通路阻断后，与中药组相比，中药+ AKt抑制剂组细胞上清液ALT、AST、TG水平升高，细胞裂解液iNOS含量增加，炎症因子TNF-α、IL-1、IL-6升高，抗炎因子IL-10降低，pAkt、pNF-κB蛋白表达升高。科学意义：找到ADPN/PI3K/AKT信号通路关键靶点，明确化痰祛湿活血法防治NASH的作用，为临床治疗NASH提供实验基础，作为进一步开发新药的主要理论依据。