降脂颗粒通过miRNAs调控LPS-Kupffer信号通路治疗NASH的分子机理

Fatty liver has become China's second largest liver disease, and NSAH attracted widespread attention as the progressed stage of fatty liver. Our group had developed a recipe named Jiangzhi Granula in treating fatty liver disease which was confirmed to be effective and safe in multicenter, randomized, placebo-controlled clinical studies. Animal experiments also proved its action in improving insulin resistance and leptin resistance, and its hepatoprotective role through inhibiting Kupffer cell(KC) activation. Recent studies showed that the KC is one of the key factors in the pathogenesis of NASH, multiple miRNAs can regulate LPS-induced inflammatory cytokines expression caused by KC activation. Here we choose a LPS-KC cell model, using miRNA microarray analysis techniques to identify and verify the differential expression of miRNA; select the DSS or MCDD + LPS induced NASH mice model to analyze the change of pivotal molecules in TLR4 pathway such as IRAK1,TRAF6 and related cytokines by Real time PCR and Western Blot, thus to explore Jiangzhi Granula in regulating miRNA-KC pathway and to elucidate its mechanism in treating NASH. These work will provide valuable experience in further Chinese recipe research.

脂肪肝已经成为中国第二大肝病，NSAH作为脂肪肝恶性化的限速步骤受到广泛关注。课题组研制的降脂颗粒经多中心、随机、安慰剂对照研究证实对脂肪肝安全有效，并可通过改善瘦素和胰岛素抵抗发挥对脂肪肝的治疗作用，通过抑制KC活化发挥保肝降酶作用。新近研究显示，KC是NASH发病的关键环节之一，多个miRNAs能够调控LPS诱导的KC的炎症因子表达。本项目选择LPS-KC细胞模型，利用miRNA芯片分析技术发现并验证差异表达miRNA；选择 LPS-KC反应的经典途径TLR4信号通路和DSS、MCDD+LPS两种NASH经典模型，Real time PCR和Western Blot等技术同步检测信号通路关键蛋白IRAK1、TRAF6的表达以及相关细胞因子的变化，旨在证实降脂颗粒通过miRNA-KC信号通路关键蛋白实现miRNA的差异表达，为阐明降脂颗粒的治疗NASH的机制提供实验依据。

非酒精性脂肪性肝病(Nonalcoholic fatty liver disease，NAFLD)的发病率逐年升高，危害日益严重。非酒精性脂肪肝炎（Nonalcoholic steatohepatitis, NASH）是NAFLD向肝硬化，肝癌进展的重要中间环节，对NASH的早期干预，是防止病变继续恶化的关键节点。MicroRNA(miRNA)是一类新近发现的非编码小RNA，通过与靶基因mRNA的互补发挥调节作用。本项目先后完成了降脂颗粒干预LPS激活Kupffer细胞后miRNA差异表达；降脂颗粒对可能调控的miRNA及其作用位点研究；降脂颗粒对高脂饲料联合硫酸葡聚糖（HFD-DSS）诱导的NASH小鼠的干预研究；降脂颗粒对NASH小鼠的药效机制的探索四部分内容。结果在LPS激活Kupffer细胞发现了降脂颗粒的效应miRNA，分析和验证了miRNA-152-3p在调节Kupffer细胞活化和炎症细胞因子分泌中的作用；通过建立HFD-DSS诱导的NASH小鼠模型，我们评价了降脂颗粒在改善NASH脂肪变和炎症方面的效应；通过靶基因预测，我们分析了miRNA-152-3p相关的TLR/NF-κB的通路在NASH发生中的作用，并探讨了降脂颗粒对其调节的机制以及对肠道环境 (肠道菌群、肠源性内毒素、肠粘膜屏障等) 的改善作用。以上结果通过发现并验证差异表达miRNA，为阐明降脂颗粒抑制Kupffer细胞活化的机制提供实验依据。围绕降脂颗粒调控肠道菌群-肠源性内毒素-炎症通路的级联反应的研究，对深入探索降脂颗粒的药效机制奠定了一定的基础。