摘要
目的 基于网络药理学探讨补中益气汤治疗视网膜色素变性(RP)的机制,并通过体外实验进行验证.方法 通过检索TCMSP筛选补中益气汤组方药物活性成分及其对应靶点,检索GeneCards、OMIM数据库获取RP相关作用靶点,通过Cytoscape 3.7.1软件构建补中益气汤-RP相互作用网络,筛选核心靶点.应用DAVID在线数据库进行GO功能富集和KEGG通路富集分析;检索FerrDb数据库,获得铁死亡调控基因,综合探析补中益气汤活性靶点、RP及铁死亡之间关系并作出预测;通过AutoDock Vina 1.1.2软件进行分子对接,验证结果可靠性.体外实验以小鼠Muller细胞为研究对象,以脂多糖诱导细胞构建体外模型细胞,设置空白组、模型组、阳性对照组及补中益气汤低、中、高剂量组,采用CCK-8法检测细胞活力;使用试剂盒检测细胞中LDH、SOD、MDA水平和铁离子浓度;RT-PCR检测各组细胞中IL-6、HIF-1αmRNA及GPX-4、FTH-1、ACSL4 mRNA水平.结果 获得补中益气汤作用靶点117个,预测得到IL6、IL1β、EGFR、ESR1、PPARG等18个靶点可能是补中益气汤调控铁死亡抗RP的作用靶点,补中益气汤可通过介导MAPK、HIF-1、TNF、AGE-RAGE、Toll样受体、IL-17、PI3K-Akt等信号通路调控铁死亡.分子对接结果提示,补中益气汤中的槲皮素、山柰酚、β-谷甾醇与IL-6、IL-1β、EGFR、ESR1、PPARG等候选核心靶点间存在较好的结合活性.与模型组比较,补中益气汤各剂量组细胞活力、SOD活性增强(P＜0.05),LDH活性、MDA水平、铁离子含量及IL-6、HIF-1 α、ACSL4 mRNA 水平降低(P＜0.05),GPX-4、FTH-1 mRNA 水平升高(P＜0.05).结论 补中益气汤可能通过多成分、多靶点,涉及多条分子通路发挥抗炎、抗铁死亡的作用,从而达到防治RP的作用.
Abstract
Objective To explore the mechanism of Buzhong Yiqi Decoction in the treatment of retinitis pigmentosa(RP)based on network pharmacology;To verify it by in vitro experiments.Methods The effective active components and corresponding targets of Buzhong Yiqi Decoction were screened by retrieving TCMSP database,and the RP-related targets were obtained by searching GeneCards,OMIM and other databases.The interaction network diagram of Buzhong Yiqi Decoction-RP was drawn by Cytascape3.7.1 software,and the core targets were extracted.DAVID online database was used to enrich and analyze gene ontology(GO)and Kyoto Encyclopedia of Genes and Genome(KEGG)pathway;the FerrDb database was searched to obtain the genes that regulate ferroptosis,and finally comprehensively analyze the relationship between the active targets of Buzhong Yiqi Decoction,RP and ferroptosis and make predictions;finally,molecular docking was performed by Auto Dock software to verify the reliability of the results.In vitro experiment,rat Muller cells were used as the research object,and LPS-induced cells were used to construct model cells in vitro.The blank group,model group,Buzhong Yiqi Decoction low-,medium-and high-dosage groups were set up,and the cell proliferation was detected by CCK-8 method;the concentrations of lactate dehydrogenase(LDH),superoxide dismutase(SOD),malondialdehyde(MDA)and ferroptosis in the cells were detected by the kit method;the expressions of HIF-1α MRNA and GPX-4,FTH-1,ACSL4 mRNA levels in cells of each group were detected by RT-PCR.Results Totally 117 effective targets were selected from Buzhong Yiqi Decoction,and IL-6,IL-1β,EGFR,ESR1,PPARG may be the targets of Buzhong Yiqi Decoction in regulating ferroptosis and anti-RP.Buzhong Yiqi Decoction could regulate ferroptosis by mediating MAPK,HIF-1,TNF,AGE-RAGE,Toll-like receptor,IL-17,PI3K-Akt and other signal pathways.Molecular docking virtual results suggested that the quercetin,kaempferol,β-Sitosterol and IL-6,IL-1β,EGFR,ESR1,PPARG and other candidate core targets had good binding activity.Compared with the model group,the cell activity,level of SOD,LDH activity,MDA content and Fe3+content,IL-6 mRNA,HIF1A mRNA,ACSL4 mRNA in each Buzhong Yiqi Decoction group significantly decreased(P＜0.05),the expressions of GPX-4 and FTH-1mRNA significantly increased(P＜0.05).Conclusion Buzhong Yiqi Decoction may play the role of anti-inflammation and anti-ferroptosis through multiple components,multiple targets and multiple molecular pathways,thus achieving the effect of prevention and treatment of RP.
基金项目
国家自然科学基金(82205195)
国家自然科学基金(82205117)
河南省中医临床研究基地科研专项(2022JDZX127)
河南省中医临床研究基地科研专项(2022JDZX135)
NETL
NSTL
万方数据