基于网络药理学及实验验证探究阳和汤治疗膝骨关节炎的作用机制

Mechanism of Yanghe Decoction in Treatment of Knee Osteoarthritis Based on Network Pharmacology and Cell Experiments

目的通过网络药理学分析阳和汤治疗(Knee osteoarthritis,KOA)的作用机制研究。方法选择TCMSP数据库和Batman数据库获取阳和汤的活性成分和靶点。网络药理学方法涉及“药物-成分-靶点”网络图的构建和靶点预测。使用京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)数据库确定阳和汤和膝骨关节炎集群网络的重要信号通路。体外细胞实验验证阳和汤对LPS诱导的大鼠原代软骨细胞的凋亡与相关蛋白表达的影响。结果筛选出槲皮素(Quercetin)、油酸(Oleic acid)、山奈酚(Kaempferol)、β-谷甾醇(Beta-sitosterol)、柚皮素(Naringenin)等139个有效成分,对应于阳和汤的276个靶点。此外,2092个基因与阳和汤密切相关,其中有127个与阳和汤的靶点重叠,被认为与治疗相关。功能富集分析表明,阳和汤主要通过调节AMPK信号通路发挥其在膝骨关节炎中的药理作用,并且与AMPK/SIRT3通路相关。体外细胞实验验证提示,与对照组比较,阳和汤含药血清组软骨细胞活性增强,差异有统计学意义(P<0.05)、凋亡水平明显降低,差异有统计学意义(P<0.01)以及软骨细胞Caspase 3明显降低,差异有统计学意义(P<0.01)、p-AMPK、SIRT3蛋白表达明显升高,差异有统计学意义(P<0.01)。结论阳和汤可能通调节AMPK/SIRT3信号通路与线粒体凋亡途径治疗KOA。

Objective To decipher the mechanism of Yanghe Decoction in the treatment of knee osteoarthritis based on network pharmacology.MethodsThe active ingredients and targets of Yanghe Decoction were obtained from the Traditional Chinese Medicine Systems Pharmacology Databased and Analysis Platform(TCMSP)and the Batman.The“drug-component-target”network was established and the key targets were predicted.The Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis was carried out to predict the key signaling pathways in the treatment of knee osteoarthritis with Yanghe Decoction.The cell experiments were carried out to examine the effects of Yanghe Decoction on the apoptosis and related protein expression in the lipopolysaccharide-induced primary chondrocytes of rats.Results A total of 139 active ingredients including quercetin,oleic acid,kaempferol,β-sitosterol,and naringenin of Yanghe Decoction were screened out,corresponding to 276 targets.In addition,2092 genes were closely associated with Yanghe Decoction,of which 127 genes overlapping with the targets of Yanghe Decoction were considered to be therapeutically relevant.The KEGG enrichment analysis showed that Yanghe Decoction exerted the pharmacological effect on knee osteoarthritis mainly by modulating the AMPK signaling pathway and was associated with the AMPK-SIRT3 feedback loop.The cell experiments showed that compared with the control group,the Yanghe Decoction-containing serum enhanced the activity(P<0.05),reduced the apoptosis(P<0.01),down-regulated the expression of caspase 3(P<0.01),and up-regulated the expression of p-AMPK and SIRT3(P<0.01)in the chondrocytes.Conclusion Yanghe Decoction may regulate the AMPK-SIRT3 feedback loop and mitochondrial apoptosis pathway to treat knee osteoarthritis.