基于Hippo/AMPK/AKT信号通路探究十二味疏肝利胆颗粒防治胆固醇结石的机制

Mechanism of Twelve-Flavor Shugan Lidan Granule in Prevention and Treatment of Cholesterol Stones: A Study Based on the Hippo/AMP-activated Protein Kinase/Protein Kinase B Signaling Pathway

目的探究十二味疏肝利胆颗粒(Twelve-Flavor Shugan Lidan Granule,TSLG)降低胆固醇累积预防结石生成的分子机制。方法基于药理学数据分析获取TSLG的活性成分与靶点;从Genebass数据库获取与胆囊结石疾病相关的基因,通过Cytoscape软件构建TSLG调控网络并进行核心基因鉴定;采用GO与KEGG通路富集分析对TSLG调控网络中的核心基因及肝组织转录组差异基因进行生物功能与通路注释;利用分子对接方法模拟关键生物标志物与TSLG核心成分的对接模式。采用油酸构建胆固醇累积的细胞模型,应用Western blot法、免疫荧光法检测磷酸化腺苷酸活化蛋白激酶(phosphorylated AMP-activated protein kinase,p-AMPK)、肝脏X受体α(liver X receptorsα,LXRα)、ATP结合盒转运蛋白(ATP-binding cassette sub-family G member,ABCG)5、ABCG8、胆固醇调节元件结合蛋白2(sterol regulatory element-binding protein 2,SREBP2)、磷酸化蛋白激酶B(phosphorylated protein kinase B,p-AKT)、磷酸化叉头盒蛋白O1a(phosphorylated forkhead box O1 a,p-FOXO1A)的表达水平;Seahorse细胞能量代谢仪检测细胞线粒体耗氧率和细胞外酸化率;RT-qPCR检测三羧酸循环(tricarboxylic acid cycle,TAC)关键酶的mRNA表达水平。结果网络药理学分析获得了41个TSLG调控疾病的相关靶点。分子生物学实验结果表明,TSLG抑制p-AMPK、LXRα、ABCG5、ABCG8、SREBP2的表达,增强p-AKT、p-FOXO1A的表达,TSLG抑制糖酵解并增强氧化磷酸化,TSLG抑制TAC循环酶活性,改善肝脏糖脂代谢,从而预防胆固醇结石的生成。结论TSLG中主要成分可能通过靶向AMPK、AKT及Hippo信号通路调节肝脏代谢,以达到降低胆固醇累积、预防结石生成的作用。

Objective To investigate the molecular mechanism of Twelve-Flavor Shugan Lidan Granule(TSLG)in reducing cholesterol accumulation and preventing the formation of stones.Methods Pharmacology data were analyzed to obtain the active components of TSLG and their corresponding targets;Genebass database was used to obtain the genes associated with gallstones,and Cytoscape software was used to construct a TSLG regulatory network and identify core genes;GO and KEGG pathway enrichment analyses were used to perform the annotation of biological functions and pathways for the core genes in the TSLG regulatory network and the differentially expressed genes in liver transcriptome;molecular docking was used to simulate the docking pattern between key biomarkers and TSLG core components.Oleic acid was used to establish a cellular model of cholesterol accumulation,and Western blot and immunofluorescence assay were used to measure the expression levels of phosphorylated AMP-activated protein kinase(p-AMPK),liver X receptor alpha(LXRα),ATP-binding cassette sub-family G member 5(ABCG5),ATP-binding cassette sub-family G member 8(ABCG8),sterol regulatory element-binding protein 2(SREBP2),phosphorylated protein kinase B(p-AKT),and phosphorylated forkhead box O1 a(p-FOXO1A);Seahorsecellular energy metabolism assay was used to measure mitochondrial oxygen consumption rate and extracellular acidification rate;RT-qPCR was used to measure the mRNA expression levels of the key enzymes involved in tricarboxylic acid cycle.Results The network pharmacology analysis identified 41 targets associated with disease regulation by TSLG.Molecular biology experiments showed that TSLG inhibited the expression of p-AMPK,LXRα,ABCG5,ABCG8,and SREBP2 and enhanced the expression of p-AKT and p-FOXO1A,and it also inhibited glycolysis,enhanced oxidative phosphorylation,and improved glucose and lipid metabolism in the liver,thereby preventing the formation of cholesterol stones.Conclusion The main components of TSLG can reduce cholesterol accumulation and prevent the formation of stones by targeting the AMPK,AKT,and Hippo signaling pathways to regulate liver metabolism.