基于网络药理学和实验验证研究加味独活寄生合剂治疗腰椎间盘突出症的作用机制  

Mechanism of action of Jiawei Duhuo Jisheng Mixture in treating lumbar disc herniation based on network pharmacology and experimental verification

作　　者：蒋浩波 段嘉豪 刘恩旭 陈茜 李夏 邵乐[2] 杨少锋[2] 张晓[2] JIANG Haobo;DUAN Jiahao;LIU Enxu;CHEN Xi;LI Xia;SHAO Le;YANG Shaofeng;ZHANG Xiao(Hunan University of Chinese Medicine,Changsha,Hunan 410208,China;The First Hospital of Hunan University of Chinese Medicine,Changsha,Hunan 410007,China;School of Public Health,Guangdong Medical University,Dongguan,Guangdong 523808,China)

机构地区：[1]湖南中医药大学,湖南长沙410208 [2]湖南中医药大学第一附属医院,湖南长沙410007 [3]广东医科大学公共卫生学院,广东东莞523808

出　　处：《湖南中医药大学学报》2023年第11期2081-2091,共11页Journal of Hunan University of Chinese Medicine

基　　金：国家自然科学基金项目(82174402);湖南中医药大学校级开放基金(2021XJJJ043);湖南中医药大学研究生创新课题(2022CX152);湖南省自然科学基金项目(2023JJ60345;2023JJ60342);湖南省教育厅科学研究项目(21C0233);湖南省卫健委科研计划项目(202204074858)。

摘　　要：目的通过网络药理学预测加味独活寄生合剂治疗腰椎间盘突出症(lumbar disc herniation,LDH)的相关机制,并通过体外实验验证。方法应用TCMSP、BATMAN-TCM数据库检索加味独活寄生合剂组成药物的活性成分以及潜在靶点。通过GeneCards、OMIM数据库搜集LDH相关的疾病靶点,构建“药物-化合物-疾病靶标”网络模型,获取加味独活寄生合剂干预LDH的交集靶标。运用STRING数据库构建蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络获取核心靶点。使用R语言软件及Perl语言进行GO分析与KEGG通路分析获得可能的关键通路。提取原代终板软骨细胞,进行细胞形态学观察以及Ⅱ型胶原蛋白(CollagenⅡ)免疫荧光染色鉴定。将体外培养的第3代终板软骨细胞随机分成5组:空白对照组、模型对照组、加味独活寄生合剂低剂量组、加味独活寄生合剂中剂量组、加味独活寄生合剂高剂量组。其中后4组使用添加含有10 ng/mL的白细胞介素17(interleukin-17,IL-17)的DMEM培养基对细胞进行诱导退变造模。流式细胞技术检测各组细胞周期;ELISA检测各组细胞IL-6的含量;实时荧光定量PCR检测p-p65、MMP9 mRNA的表达;Western blot法检测p-p65、MMP9蛋白表达情况。结果网络药理学结果显示,根据ADME标准共筛选加味独活寄生汤19种药物共计295种活性成分,与LDH的疾病靶标相匹配得到交集靶标50个。通过PPI提取得到IL-6成为核心靶标可能性最大,GO分析和KEGG通路分析得到IL-17相关信号途径的富集因子较高。体外实验结果:相比于空白对照组,其他各组细胞生长情况明显受到抑制(P<0.05);相比于模型对照组,加味独活寄生合剂各组生长情况明显受到促进(P<0.05)。相比于空白对照组,模型对照组及加味独活寄生合剂各组IL-6、p-p65、MMP9表达显著升高(P<0.05);相比于模型对照组,加味独活寄生合剂各组IL-6、p-p65、MMP9表达显著降低(P<0.05)。�Objective To predict the related mechanism of Jiawei Duhuo Jisheng Mixture(JWDHJSM)in treating lumbar disc herniation(LDH)by network pharmacology and to verify it by in vitro experiments.Methods TCMSP and BATMAN-TCM were used to search for the active ingredients and potential targets of drugs in JWDHJSM.Through the GeneCards and OMIM databases,the LDH-related disease targets were collected,and the“drug-compound-disease target”network model was constructed to obtain the intersection targets of JWDHJSM in the intervention of LDH.The STRING database was utilized to build a protein-protein interaction(PPI)network to obtain core targets.Gene ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were performed using R language software and Perl language to obtain potential key pathways.Primary endplate chondrocytes were extracted for cell morphology observation and identified by type II collagen immunofluorescence staining.The third generation of endplate chondrocytes cultured in vitro were randomized into five groups:blank control,model control,and low-,medium-,and high-dose JWDHJSM groups.The cells in the latter four groups were induced for degeneration modeling by DMEM medium containing 10 ng/mL interleukin-17(IL-17).The cell cycle of each group was determined by flow cytometry,and the content of IL-6 in cells of each group was measured by ELISA.In addition,the mRNA expressions of p-p65 and MMP9 were checked by real-time fluorescence quantitative PCR,and the protein expressions of p-p65 and MMP9 were examined by Western blot.Results Network pharmacology showed that a total of 295 active ingredients of 19 drugs in JWDHJSM were screened according to the ADME criteria,and 50 intersection targets were obtained by matching with the disease targets of LDH.IL-6 was most likely to be the core target by PPI extraction.Besides,GO analysis and KEGG pathway analysis revealed a higher enrichment factor of IL-17-related signaling pathways.In vitro experiments indicated that compared with the b

关 键 词：加味独活寄生合剂 网络药理学 腰椎间盘突出 终板软骨细胞 作用机制 炎症反应 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]