基于网络药理学和实验验证探讨补肾痹通方治疗膝骨关节炎的作用机制

Exploration of the mechanism of Bushen Bitong Prescription in the treatment of knee osteoarthritis based on network pharmacology and experimental verification

目的运用网络药理学和实验验证探讨补肾痹通方治疗膝骨关节炎(KOA)的作用机制。方法运用中药系统药理学数据库与分析平台获取补肾痹通方的有效成分与作用靶点,运用GeneCards、OMIM、PharmGkb、TTD、DrugBank数据库检索KOA疾病相关靶点,将药物和疾病靶点取交集,将获得的有效成分及相关靶点导入Cytoscape软件,绘制“补肾痹通方-有效成分-KOA作用靶点”的网络图,筛选关键有效成分,将潜在靶点导入STRING数据库绘制蛋白质-蛋白质相互作用网络图。筛选补肾痹通方治疗KOA的核心靶点,进行GO富集与KEGG注释分析。选取人软骨细胞设置对照组、损伤组、给药组,对照组正常培养,损伤组和给药组在含白细胞介素(IL)-1β(10 ng/ml)DMEM/F12培养基中培养,给药组给予0.4447μg/ml补肾痹通方药液。培养24 h后,观察三组细胞形态,采用酶联免疫吸附试验检测炎症因子水平,qRT-PCR与免疫印迹法检测三组软骨修复因子的表达。结果共筛选出补肾痹通方有效成分186个,主要为槲皮素、木犀草素、山柰酚等,主要通过TNF、MMP1、CXCL2、IL1B等关键靶点和PI3K/Akt、T细胞受体、Toll样受体、低氧诱导因子-1、TNF、IL-17等信号通路发挥作用,涉及氧化应激反应、细胞凋亡、炎症反应等生物过程。显微镜下可见,损伤组细胞数量减少,给药组细胞数量增加。损伤组肿瘤坏死因子-α(TNF-α)、IL-1β、IL-6水平高于对照组,转化生长因子-β(TGF-β)、胰岛素样生长因子-1(IGF-1)、碱性成纤维细胞生长因子(BFGF)水平及SOX9、COL2A1基因和蛋白的表达水平均低于对照组(P<0.05)。给药组TNF-α、IL-1β、IL-6水平低于损伤组,TGF-β、IGF-1、BFGF及SOX9、COL2A1基因和蛋白的表达水平均高于损伤组(P<0.05)。结论补肾痹通方可能通过缓解炎症细胞浸润并参与SOX9、COL2A1调控来促进损伤软骨细胞修复。

Objective To explore the mechanism of Bushen Bitong Prescription in the treatment of knee osteoarthritis(KOA)by network pharmacology and experimental verification.Methods Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform were used to obtain the active ingredients and targets of Bushen Bitong Prescription.GeneCards,OMIM,PharmGkb,TTD,and DrugBank databases were used to search KOA disease related targets,and the intersection of drug and disease targets was selected.The obtained active ingredients and related targets were introduced into Cytoscape software,and the network map of“Bushen Bitong Prescription-active ingredients-KOA targets”was drawn to screen the key active ingredients.Potential targets were imported into the STRING database to plot the protein-protein interaction network.The core targets of Bushen Bitong Prescription in the treatment of KOA were screened,and GO enrichment and KEGG annotation were analyzed.Human chondrocytes were selected and set up in control group,injury group,and administration group.The control group was cultured normally,the injury group and administration group were cultured in the medium containing interleukin(IL)-1β(10 ng/ml)DMEM/F12,and the administration group was treated with 0.4447μg/ml Bushen Bitong Prescription liquid.After 24 h of culture,the cell morphology of the three groups was observed.The levels of inflammatory factors were detected by enzyme-linked immunosorbent assay,and the expression of cartilage repair factors in the three groups was detected by qRT-PCR and Western blot.Results A total of 186 active ingredients of Bushen Bitong Prescription were screened,mainly including quercetin,luteolin,kaempferol,etc.,mainly through the key targets of TNF,MMP1,CXCL2,IL1B,and signaling pathways of PI3K/Akt,T cell receptors,toll-like receptors,hypoxy-inducer factor-1,TNF,IL-17,and others.It involved,oxidative stress,apoptosis,inflammation,and other biological processes.Microscopically,the number of cells decreased in the injury group and increased in the administration group.The levels of tumor necrosis factor-α(TNF-α),IL-1β,and IL-6 in injury group were higher than those in control group,and the levels of transforming growth factor-β(TGF-β),insulin-like growth factor-1(IGF-1),basic fibroblast growth factor(BFGF),and the gene and protein expression levels of SOX9 and COL2A1 were lower than those in control group(P<0.05).The levels of TNF-α,IL-1β,and IL-6 in administration group were lower than those in injury group,and the levels of TGF-β,IGF-1,BFGF,and the gene and protein expression levels of SOX9 and COL2A1 were higher than those in injury group(P<0.05).Conclusion Bushen Bitong Prescription may promote the repair of injured chondrocytes by relieving inflammatory cell infiltration and participating in the regulation of SOX9 and COL2A1.