基于整合网络药理学和实验验证的人参皂苷Rb1抗代谢相关脂肪性肝病的作用机制探讨

Integrating network pharmacology and experimental verification to explore mechanism of ginsenoside Rb1 in treating metabolic associated fatty liver disease

目的探究人参皂苷Rb_(1)(ginsenoside Rb_(1),Rb_(1))治疗大鼠代谢相关脂肪性肝病(MAFLD)的作用靶点及机制。方法采用网络药理学方法系统地预测Rb_(1)治疗MAFLD的核心靶点,通过STRING平台和Cytoscape3.9.1软件分别绘制蛋白质相互作用(PPI)网络图谱和“Rb_(1)-MAFLD靶点”网络模型,利用DAVID数据库对核心靶点进行基因本体(GO)功能富集与京都基因与基因组百科全书(KEGG)通路富集分析。动物体内实验进行验证,高脂饲料诱导建立实验性MAFLD大鼠模型。测定各组大鼠体质量、肝脏系数、肝功能指标[天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)]及血脂指标[三酰甘油(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)];HE染色观察肝组织病理学变化;酶联免疫吸附(ELISA)和实时荧光定量聚合酶链式反应(qRT-PCR)法分别检测大鼠血清和肝脏中炎症因子[白细胞介素-6(IL-6)、IL-10、肿瘤坏死因子-α(TNF-α)和转化生长因子-β1(TGF-β1)]表达水平以验证网络药理学预测结果;16 S rDNA测序分析评估Rb_(1)对MAFLD大鼠肠道菌群组成的影响。结果获得134个Rb_(1)治疗MAFLD的潜在靶点,富集分析显示Rb_(1)干预MAFLD的生物学过程与转录调控、细胞增殖、炎症反应等相关,作用的通路主要为癌症、炎症、脂质和动脉粥样硬化、糖尿病等途径。动物实验结果显示,与对照组相比较,MAFLD模型组大鼠肝脏系数、肝功能指标、炎症因子水平显著升高,血脂代谢紊乱,且肠道菌群结构紊乱。Rb_(1)可改善肝组织中脂肪变性及炎症细胞浸润,显著降低血清中AST、ALT、TC、TG、LDL-C水平,增加HDL-C水平,且有效抑制MAFLD大鼠中炎症因子的过表达;此外,Rb_(1)改变了MAFLD大鼠的肠道菌群的组成,以拟杆菌门、乳杆菌属、布劳特氏菌属富集和脱硫弧菌减少为特征。结论Rb_(1)能通过改善血脂紊乱与肝功能、抑制炎症反应和调节肠道菌群的稳态,最终发挥抗MAFLD的作用。

Objective This study was carried out to investigate the targets and mechanisms of action of ginsenoside Rb_(1)(Rb_(1))in the therapy of metabolic associated fatty liver disease(MAFLD)in rats.Methods The network pharmacology methods were used to systematically predict the core targets of ginsenoside Rb_(1) for the treatment of MAFLD.PPI network and"Rb_(1)-MAFLD targets"networks were plotted using STRING platform and Cytoscape3.9.1 software,respectively.GO functional enrichment and KEGG pathway enrichment analysis were performed on core targets using DAVID database.Further in vivo animal experiments were conducted for validation,an experimental MAFLD model was developed in rats by feeding them high-fat diet.Body mass,liver coefficient,liver function index(AST,ALT)and blood lipid index(TG,TC,LDL-C,HDL-C)were determined.HE staining was used to observe the pathological changes of liver tissue.The expression levels of inflammatory factors(IL-6,IL-10,TNF-α,TGFβ1)in serum and liver of rats were detected by ELISA and real-time PCR,respectively,to validate the predicted results of network pharmacology.16S rDNA sequencing analysis was used to evaluate the effect of Rb_(1) on gut microbiota composition in MAFLD rats.Results A total of 134 potential targets for Rb_(1) treatment of MAFLD were obtained.Enrichment analysis indicated that Rb_(1) interferes with the biological process of MAFLD,which is related to transcriptional regulation,cell proliferation and inflammatory response,and the action pathways are mainly cancer,inflammation,lipid and atherosclerosis and diabetes.The results of animal experiments indicated that compared with the normal group,rats in the MAFLD model group had conspicuously elevated liver coefficients,liver function indexes,inflammatory factors,disturbed lipid metabolism,and structural disorders of the intestinal microbiota.Rb_(1) improved steatosis and inflammatory cell infiltration in liver tissues,conspicuously reduced serum levels of AST,ALT,TC,TG,and LDL-C,increased HDL-C levels,and effectively suppressed the overexpression of inflammatory factors in MAFLD rats.Furthermore,Rb_(1) altered the composition of the gut microbiota in MAFLD rats,characterized by the enrichment of Bacteroidota,Lactobacillus,Blautia and the reduction of Firmicutes/Bacteroidota and Desulfovibrio.Conclusion Rb_(1) can ultimately exert anti-MAFLD effects by improving dyslipidemia and liver function,suppressing inflammatory responses and modulating the homeostasis of the gut microbiota.