基于网络药理学的雷公藤抗肾纤维化作用及实验验证  

Mechanism of Tripterygium wilfordii in treatment of renal fibrosis based on network pharmacology and experimental verification

作　　者：刘馨月 田杰[2] 李赟[2] 周雪娇 李维鹏 李贤霖 郑晓俊[2] LIU Xinyue;TIAN Jie;LI Yun;ZHOU Xuejiao;LI Weipeng;LI Xianlin;ZHENG Xiaojun(School of Pharmacy,Shanxi Medical University,Taiyuan 030001,China;Department of Pharmacy,First Hospital of Shanxi Medical University)

机构地区：[1]山西医科大学药学院,太原030001 [2]山西医科大学第一医院药剂科

出　　处：《山西医科大学学报》2023年第4期535-543,共9页Journal of Shanxi Medical University

基　　金：山西省卫生健康委员会青年项目(2023121)。

摘　　要：目的运用网络药理学探究雷公藤中药缓解肾纤维化(renal fibrosis,RF)的核心成分及相关信号通路,并进一步针对肾纤维化关键信号通路之一Wnt信号通路进行细胞实验验证。方法运用网络药理学筛选雷公藤核心成分的潜在靶点,构建“雷公藤核心成分-潜在靶点”及蛋白质互作网络分析图,进行京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析。将不同浓度的山柰酚(0,5,10,15,20μmol/L)、β-谷甾醇(0,5,10,15,20μmol/L)、雷公藤甲素(0,5,10,15,20 nmol/L)作用于人肾小管上皮细胞(human embryonic kidney epithelial cells,HKC),通过CCK-8实验检测存活率,存活率最高的药物浓度作为后续实验的给药浓度。蛋白免疫印迹法及q-PCR检测空白组、山柰酚组、β-谷甾醇组、雷公藤甲素组磷脂酶D1(phospholipase D1,PLD-1)表达水平,筛选影响Wnt通路的核心成分。进一步将细胞分组:对照组、雷公藤甲素组、TGF-β组、TGF-β+雷公藤甲素组,观察各组细胞形态改变,蛋白免疫印迹法及q-PCR法检测各组细胞α-平滑肌肌动蛋白(α-SMA)表达水平。结果网络药理学表明雷公藤通过WNT/β-catenin信号通路发挥抗肾纤维化作用,主要核心成分为山柰酚、β-谷甾醇、雷公藤甲素。CCK-8实验结果显示,与0μmol/L相比,山柰酚浓度≥15μmol/L时,HKC细胞存活率降低(P<0.01);β-谷甾醇浓度≥15μmol/L时,HKC细胞存活率降低(P<0.01);雷公藤甲素浓度≥10 nmol/L时,HKC细胞存活率降低(P<0.01)。故选择山柰酚10μmol/L、β-谷甾醇10μmol/L和雷公藤甲素5 nmol/进行后续实验研究。与空白组相比,雷公藤甲素组PLD-1蛋白及mRNA表达量明显降低(P<0.01),山柰酚组及β-谷甾醇组PLD-1蛋白及mRNA表达量差异均无统计学意义(P>0.05)。细胞形态学结果表明,与对照组相比,TGF-β组梭形转化增多(P<0.01);与TGF-β组相比,TGF-β+雷公藤甲素组梭形转化减少(P<0.01)。与对照组相Objective To investigate the core components and related signalling pathways of Tripterygium wilfordii in improvement of renal fibrosis(RF)using network pharmacology,and further validate the Wnt signalling pathway,one of the key signalling pathways in renal fibrosis in cellular experiments.Methods The database was used to screen the potential targets of core components of Tripterygium wilfordii.“Core components of Tripterygium wilfordii-potential target”network analysis and PPI network were constructed.Kyoto encyclopedia of genes and genomes(KEGG)pathway was used to complete enrichment analysis.Human embryonic kidney epithelial cells(HKC)were respectively treated with different concentrations of kaempferol(0,5,10,15,20μmol/L),β-sitosterol(0,5,10,15,20μmol/L)and triptolide(0,5,10,15,20 nmol/L)to screen the drug concentration with the highest survival rate for subsequent experiments.The expression levels of phospholipase D1(PLD-1)were detected in blank group,kaempferol group,β-sitosterol geoup and triptolide group by Western blot test and q-PCR to screen the core components affecting the Wnt pathway.Cells were further divided into control group,triptolide group,TGF-βgroup,and TGF-β+triptolide group.Morphological changes of cells in each group were observed.The expression level ofα-smooth muscle actin(α-SMA)was detected by Western blotting and q-PCR.Results Network pharmacology showed that Tripterygium wilfordii exerted anti-renal fibrosis effect through Wnt/β-catenin signaling pathway,and the main core components of Tripterygium wilfordi were kaempferol,β-sitosterol and triptolide.CCK-8 assay showed that compared with 0μmol/L,HKC cell survival was reduced at kaempferol concentration≥15μmol/L(P<0.01),β-sitosterol concentration≥15μmol/L(P<0.01),and triptolide concentration≥10 nmol/L(P<0.01).Therefore,10μmol/L kaempferol,10μmol/Lβ-sitosterol and 5 nmol/L triptolide were selected for the subsequent experimental study.Compared with blank group,PLD-1 protein and mRNA expression levels in tripto

关 键 词：雷公藤 肾纤维化 WNT/Β-CATENIN信号通路 网络药理学 雷公藤甲素 

分 类 号：R285[医药卫生—中药学]