基于网络药理学探讨身痛逐瘀汤治疗腰椎间盘突出症的作用机制

Mechanism of Shentong Zhuyu Decoction against Lumbar Disc Herniation: Based on Network Pharmacology

目的:运用网络药理学方法探讨身痛逐瘀汤治疗腰椎间盘突出症(LDH)的作用机制。方法:利用中药系统药理学数据库与分析平台(TCMSP)、化学专业数据库获取身痛逐瘀汤中各味中药的相关化学成分,于SwissTargetPrediction平台实现活性成分靶点预测;分别于人类基因数据库(GeneCards)、在线人类孟德尔遗传数据库(OMIM)、DisGeNET数据库检索LDH疾病靶点;将中药成分靶点与LDH靶点取交集,即身痛逐瘀汤治疗LDH的潜在作用靶点;应用Cytoscape软件构建身痛逐瘀汤治疗LDH的中药-化学成分-交集靶点网络图;利用String数据库完成蛋白质-蛋白质相互作用分析及关键靶点的筛选,运用Cytoscape构建网络图;通过DAVID平台对关键靶点进行基因本体(GO)富集分析和京都基因和基因组百科全书(KEGG)富集分析,初步揭示其作用机制。以髓核细胞作为实验研究对象,分为单纯压力组与中药组(加入身痛逐瘀汤中药血清干预),置于静水压加载装置,1 MPa加压干预下作用2、4、6 h,应用凋亡试剂盒检测髓核细胞的凋亡情况,通过蛋白质印迹法检测髓核细胞核因子κB p65、基质金属蛋白酶-13(MMP-13)、胱天蛋白酶3蛋白的表达情况。结果:筛选得到身痛逐瘀汤121个活性成分和871个作用靶点,LDH疾病相关靶点438个,身痛逐瘀汤治疗LDH的潜在作用靶点75个,关键作用靶点69个;GO及KEGG富集分析显示关键靶点主要通过参与癌症通路、促分裂原活化的蛋白激酶信号通路、磷脂酰肌醇-3-激酶-蛋白激酶B信号通路、肿瘤坏死因子信号通路、核因子κB信号通路等信号通路对LDH进行调控。身痛逐瘀汤可降低髓核细胞凋亡,降低核因子κB p65、MMP-13、胱天蛋白酶3的蛋白表达水平(P<0.05)。结论:身痛逐瘀汤通过多成分、多靶点、多通路治疗LDH,其作用机制可能与核因子κB信号通路的抑制以及细胞外基质的分解代谢相关。

Objective:To explore the mechanism of Shentong Zhuyu Decoction against lumbar disc herniation(LDH) by means of network pharmacology.Methods:The active components of the medicinals in the decoction were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and chemical databases, and the targets of the active component were predicted by SwissTargetPrediction.LDH-related targets were searched from GeneCards, Online Mendelian Inheritance in Man(OMIM),and DisGeNET,respectively.Thereby, the common targets of the disease and the decoction were screened out, which were the potential targets of the decoction against LDH.Cytoscape was employed to construct the Chinese medicinal-chemical component-common target network, and String to analyze the protein-protein interaction(PPI) and screen key targets.Cytoscape was adopted to construct the network, followed by the Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of key targets by DAVID.Thereby, the mechanism was preliminarily revealed.The nucleus pulposus cells were classified into the simple pressure group and the Chinese medicine group(with the intervention of Shentong Zhuyu Decoction-containing serum) and placed in a hydrostatic pressure loading device for intervention under 1 MPa pressure for 2,4,and 6 h, separately.Apoptosis of nucleus pulposus cells was detected with a kit, and expression of nuclear factor kappa B(NF-κB) p65,matrix metalloproteinase-13(MMP-13),and cysteine-aspartic acid protease(Caspase3) in the cells by Western Blotting.Results:A total of 121 active ingredients and 871 targets of Shentong Zhuyu Decoction, 438 LDH-related targets, 75 potential targets of Shentong Zhuyu Decoction against LDH,and 69 key targets were screened out.The key targets were involved in the GO terms and KEGG pathways of cancer pathway, mitogen-activated protein kinase(MAPK) signaling pathway, phosphatidylinositol-3-kinase-protein kinase B(PI3 K-AKT) signaling pathway, tumor necrosis factor(TNF) signaling pathway, and NF-κB signaling pathway.The experimental results showed that Shentong Zhuyu Decoction could reduce the apoptosis of nucleus pulposus cells and the protein expression of NF-κB p65,MMP-13,and Caspase3(P<0.05).Conclusion:Shentong Zhuyu Decoction exerts therapeutic effect on LDH through multiple components, multiple targets and multiple pathways.The mechanism is related to the inhibition of NF-κB signaling pathway and the catabolism of extracellular matrix.