摘要
目的 利用网络药理学与分子对接的方法探讨桔梗皂苷D(PD)治疗肥胖的潜在作用靶点及相关通路,并通过体外实验进行初步验证。方法 通过Gene Card、OMIM和TTD数据库检索肥胖相关靶点,使用CTD、HREB、SEA以及Swisse Target prediction等平台,并结合查阅相关文献获取PD的靶点,利用VENNY作图获取药物与疾病基因交集靶点并获得关键基因;利用STRING、R软件进行蛋白相互作用(PPI)与GO、KEGG通路富集分析,通过微生信平台将结果可视化,并构建药物-靶点-通路网络图;最后采用分子对接技术,将PD分别与AMP依赖的蛋白激酶(AMPK)、固醇调节元件结合蛋白-1c(SREBP-1c)及脂肪酸合成酶(FAS)进行分子对接;以小鼠3T3-L1前脂肪细胞作为研究对象,取对数生长期的3T3-L1前脂肪细胞,分别给予5、10、20、40、60、80μmol/L PD处理24、48及72 h;采用细胞增殖实验(CCK-8)法测定细胞存活率,流式细胞仪检测PD对3T3-L1前脂肪细胞凋亡的影响,采用油红O染色检测PD在3T3-L1前脂肪细胞中脂滴的积累情况,采用实时荧光定量PCR(RT-qPCR)和蛋白免疫印迹(Western blot)实验检测不同浓度PD对固醇调节元件结合蛋白-1c(SREBP-1c)、脂肪酸合成酶(FAS)的m RNA及蛋白表达的影响。结果 获得PD与肥胖交集靶点131个,网络拓扑分析筛选出关键靶点10个;PPI结果显示,PD对应的关键靶点基因为蛋白激酶B-α(AKT1)、肿瘤蛋白p53(TP53)、信号传导与转录激活因子3(STAT3)、白介素6(IL6)、肿瘤坏死因子(TNF)、表皮生长因子受体(EGFR)等;GO富集分析显示,PD与类固醇结合、细胞氧化应激、线粒体生成等相关;KEGG通路分析结果表明,其可调控154条信号通路,包括AMPK信号通路、脂肪细胞因子通路、胰岛素信号通路等;在分子对接中,PD与关键靶点AMPK、SREBP-1c、FAS的对接效果良好,结合能为-10.2~-8.8 kcal/mol;CCK-8结果显示,PD浓度<20μmol/L为安全浓度范围,且不造成细胞凋亡;油红O染色结果显示,PD可抑制细胞分化,并具有浓度依赖效应;PD能够显著抑制SREBP-1c、FAS的表达。结论 PD可以通过调控多靶点、多通路对肥胖产生影响,其抑制3T3-L1前脂肪细胞分化为成熟脂肪细胞的作用机制可能与下调SREBP-1c、FAS蛋白表达有关。
Objective To explore the potential molecular target and mechanism of Platycodin D(PD)in the treatment of obesity,preliminarily verified by in vitro experiments.Methods The disease targets about obesity were collected from GeneCards,OMIM,and TTD database,and PD targets were colected from CTD,HREB,SEA,Swisse Target prediction,and Uniprot database based on literature search.The intersection target genes of PD and obesity were obtained by Venn package.STRING and R programming language were used to construct the protein-protein interaction network diagram and Gene Ontology(GO)function enrichment and Kyoto Encyclopedia of Genes,and Genomes(KEGG),and then the results were visualized as histograms and bubble charts using the Bioinformatics platform.The network of"PD-Core Targets-Signaling Pathways"was mapped.The PD were validated by Autodock software for molecular docking with obesity targets,including AMP-activated protein kinase(AMPK),sterol regulating element binding protein 1c(SREBP-1c),fatty acid synthesis(FAS);validation experiment using mouse 3T3-L1 preadipocytes as a cell model,3T3-L1 cell lines in logarithmic growth phase were treated with no-PD and containing,different concentrations of PD(5,10,20,40,60,and 80μmol/L)for 24 h,48 h,and 72 h,respectively;Cell Counting Kit-8(CCK-8)was used to assess the effect of different concentrations of PD on cell viability;apoptosis rate of 3T3-L1 preadipocytes were detected by flow cytometry after 24 h,48 h,and 72 h of PD treatment;the effects of PD on lipid accumulation and adipogenic differentiation of 3T3-L1 were detected by oil red O staining;meanwhile,the gene expression levels of SREBP-1c,FAS mRNA and protein were determined by RT-qPCR and Western blot,respectively.Result One hundred and thirty-one common targets of disease and drug were obtained,which acted mainly through AKT1,STAT3,TP53,TNF,VEGFA,IL-6,and other key targets.GO enrichment analysis showed that PD was connected with steroid binding,cellular response to oxidative stress,mitochondrial biogenesis,etc.KEGG analysis enriched 154 pathways,including AMPK signaling,adipocytokine,and insulin resistance,etc.The molecular docking revealed that PD could be bound with AMPK,SREBP-1c,FAS,with binding energy between-10.2 kcal/mol and-8.8 kcal/mol;CCK-8 showed that that the PD concentration within 20μmol/L was a safe concentration range and did not cause apoptosis;oil red O stain showed that PD inhibited the differentiation of 3T3-L1 cells and lipid accumulation;PD could inhibit the expression of SREBP-1c and FAS.Conclusion PD can inhibit obesity through multi-target and multi-pathway,and can inhibit the differentiation of 3T3-L1 cells into adipocytes.Its mechanism may be related to the inhibition of SREBP-1c and FAS protein expression.
作者
王贤
钟沁
刘芳
韦睿然
桂黎明
WANG Xian;ZHONG Qin;LIU Fang;WEI Ruiran;GUI Liming(Department of Immunology,School of Basic Medicine,Guizhou Medical University,Guiyang 550000,Guizhou,China;Department of Basic Medical Sciences,Clinical College,Anhui Medical University,Hefei 230031,Anhui,China)
出处
《贵州医科大学学报》
CAS
2022年第10期1125-1136,共12页
Journal of Guizhou Medical University
基金
国家自然科学基金委员会-贵州省人民政府联合基金项目(U812403-4)
贵州省科技厅社会发展科技支撑计划项目(黔科合支撑[2019]2789号)。
作者简介
王贤,贵州医科大学2019级硕士研究生;通信作者:桂黎明,E-mail:416493934@qq.com。
