基于JNK/MAPK信号通路分析脊痛消胶囊治疗腰椎间盘突出症的作用机制

Mechanism of Jitongxiao Capsule in Treating Lumbar DiscHerniation Based on JNK/MAPK Signaling Pathway

目的:利用网络药理学方法和实验验证分析脊痛消胶囊治疗腰椎间盘突出症(lumbar disc herniation,LDH)的作用机制。方法:从中药系统药理学(Traditional Chinese Medicine Systems Pharmacology,TCMSP)、中药分子机理的生物信息学分析工具(Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine,BATMAN-TCM)、中医药资料@Taiwan和Swiss数据库中检索并筛选脊痛消胶囊的有效成分及其作用靶点,并使用GeneCards、人类孟德尔遗传(Online Mendelian Inheritance in Man,OMIM)和DisGeNET数据库预测LDH的疾病靶点,再将活性成分作用靶点与疾病靶点进行映射,得到脊痛消胶囊治疗LDH潜在靶点,继续在STRING数据库中进行蛋白互作分析(protein-protein interaction,PPI),将结果导入Cytoscape软件获取PPI网络图和“药物-活性成分-潜在靶点”网络图。利用clusterProfiler包对潜在靶点进行基因本体(Gene Ontology,GO)功能、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析,用Autodock Vina和Discovery Studio软件对活性成分和关键靶点进行分子对接验证。最后以脊痛消胶囊为干预因素,在LDH模型大鼠进行实验验证。结果:网络药理学结果显示,共收集到脊痛消胶囊活性成分139种、靶点479个,获得LDH靶点590个,映射得到84个交集靶点,通过PPI网络筛选得到脊痛消胶囊治疗LDH的关键治疗靶点10个:信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3)、转录因子JUN(transcription factor Jun-1,JUN)、白细胞介素(interleukin,IL)-6、IL-10、丝裂原活化蛋白激酶1(mitogen-activated protein kinase 1,MAPK1)、FOS蛋白(protein c-fos,FOS)、连环素(catenin beta-1,CTNNB1)、丝裂原活化蛋白激酶14(MAPK14)、蛋白激酶B(protein kinase B,PKB/AKT1)和肿瘤坏死因子(tumor necrosis factor,TNF),GO功能富集分析显示交集靶点涉及2163种生物学过程、36种细胞组分和102种分子功能,KEGG通路富集分析显示,共涉及155条信号通路,分子对接结果显示核心靶点与成分对接结合良好。实验验证结果显示,脊痛消胶囊可显著降低LDH模型大鼠血清IL-6和TNF-α水平,提高血清IL-10水平,降低髓核组织p-JUN/JUN、p-FOS/FOS、p-JNK/JNK和p-p38MAPK/p38MAPK水平。结论:脊痛消胶囊可以通过抑制炎症反应和JNK/MAPK信号通路的激活从而起到保护LDH模型大鼠的作用。

Objective:Analyze the mechanism of Jitongxiao Capsule in treatment of lumbar disc herniation(LDH)using network pharmacology and experimental verification.Methods:Retrieve and screen the effective ingredients and targets of Jitongxiao Capsule from Traditional Chinese Medicine Systems Pharmacology(TCMSP),Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine(BATMAN-TCM),Traditional Chinese Medicine Information@Taiwan and Swiss databases.Use Genecards,online Mendelian inheritance in man(OMIM)and DisGeNET databases to predict the disease targets of LDH,and then map the active ingredient action targets with the disease targets to obtain potential targets for the treatment of LDH with Jitongxiao capsules.Protein interaction analysis(PPI)is performed in the STRING database,and the results are imported into Cytoscape software to obtain PPI network diagrams and"drug-active ingredient-potential targets"Network diagram.Use the clusterProfiler software to perform gene ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis on potential targets.Conduct molecular docking validation of active ingredients and key targets using Autodock Vina and Discovery Studio software.Finally,experimental validation was conducted on LDH model rats with Jitongxiao capsule being intervention factor.Results:Network pharmacology results showed that a total of 139 active ingredients and 479 targets of Jitongxiao Capsules were collected,and 590 LDH targets as well as 84 intersection targets with mapping were obtained.Through PPI network screening,10 key therapeutic targets for treating LDH with Jitongxiao Capsule were identified as follows:signal transducer and activator of transcription 3(STAT3),transcription factor jun-1(JUN),Interleukin-6(IL-6),Interleukin-10(IL-10),mitogen activated protein kinase 1(MAPK1),protein c-fos(FOS),catenin beta-1(CTNNB1),mitogen activated protein kinase 14(MAPK14),protein kinase B(PKB/AKT1)and tumor necrosis factor(TNF).GO analysis shows that the intersecting targets involves 2163 biological processes,36 cell components and 102 molecular functions.KEGG analysis shows that there are 155 signaling pathways involved in and molecular docking results showed good binding between the core target and components.The experimental verification shows that Jitongxiao Capsule can significantly reduce serum IL-6 and TNF levels in LDH rats,while increase serum IL-10 level,and reduce levels of p-JUN/JUN,p-FOS/FOS,p-JNK/JNK and p-p38MAPK/p38MAPK in the nucleus pulposus tissue.Conclusion:Jitongxiao Capsule can protect LDH rat by inhibiting inflammatory response and activating the JNK/MAPK signaling pathway.