基于网络药理学及实验验证探究甘松治疗帕金森病伴发焦虑的作用机制

Effect of Nardostachys jatamansi in treatment of Parkinson’s disease with anxiety based on network pharmacological analysis and experimental verification

目的基于网络药理学方法预测甘松Nardostachys jatamansi治疗帕金森病伴发焦虑(Parkinson’s disease with anxiety,PDA)的作用靶点和信号通路,并通过PDA模型大鼠进行药效学和关键靶点的验证。方法通过中药系统药理学数据库分析平台(TCMSP)和文献挖掘获得甘松主要化学成分,利用SwissTargetPrediction、GeneCards数据库获得甘松化学成分治疗PDA的潜在靶点,对其进行基因本体(gene ontology,GO)功能富集分析和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,利用Cytoscape 3.7.2构建"成分-靶点-通路"网络。大鼠采用颈背部sc鱼藤酮制备PD模型,并通过旷场实验筛选出PDA模型大鼠;设置对照组、模型组、甘松(620 mg/kg)组、左旋多巴(49 mg/kg)组,利用转棒实验、旷场实验和免疫组化法对甘松治疗PDA的药效学进行评价;并对关键蛋白和大鼠脑内神经递质含量进行测定。结果 "成分-靶点-通路"网络中包含甘松抗PDA的12个有效成分如甘松新酮、诺卡酮、金合欢素、β-谷甾醇等,丝裂原活化蛋白激酶3(mitogen-activated protein kinases 3,MAPK3)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和信号传导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)等99个靶点,主要涉及神经活性配体-受体相互作用、雌激素、FoxO、TNF等信号通路。实验结果显示,甘松延长了PDA大鼠在转棒上的停留时间,增加了其在旷场的总活动距离及在中央区的活动时间和距离,提高了黑质区酪氨酸羟化酶阳性表达(P<0.05);甘松显著降低大鼠纹状体MAPK3、TNF-α含量以及STAT3蛋白表达水平(P<0.05、0.01),提高了多巴胺、3,4-二羟基苯乙酸、高香草酸、5-羟色胺和5-羟吲哚乙酸水平(P<0.01、0.001)。结论甘松中的有效成分可能是通过MAPK3、TNF-α、STAT3等靶点调控多条信号通路来抑制神经炎症反应,提高神经递质水平以治疗PDA疾病。

Objective To explore the targets and signal pathways of Nardostachys jatamansi in the treatment of Parkinson’s disease with anxiety(PDA) by network pharmacology, and verified the pharmacodynamics and key targets through the PDA model rats.Methods Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) database and literature mining were used to obtain the main chemical ingredients of N. jatamansi. SwissTargetPrediction and GeneCards database were used to obtain potential targets for the treatment of PDA with the chemical components of N. jatamansi. Gene ontology(GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways enrichment analysis were performed for potential targets. “Ingredients-targets-pathways” network was constructed by using Cytoscape 3.7.2. The model rats of PD were established by sc rotenone on the back of neck, and the model rats of PDA were screened out by open field experiment. Conytol group, model group, N. jatamansi(620 mg/kg) group and levodopa(49 mg/kg) group were set up respectively. The pharmacodynamics of N. jatamansi in the treatment of PDA was evaluated by rod rotation test, open field test and immunohistochemistry. The contents of key proteins and neurotransmitters in the brain of rats were detected. Results There were 12 active ingredients including narcinone, nootkatone, acanthine and β-sitosterol of N. jatamansi anti-PDA in “ingredients-targets-pathways” network, 99 targets such as mitogen-activated protein kinases 3(MAPK3), tumor necrosis factor-α(TNF-α), signal transducer and activator of transcription 3(STAT3), mainly involved in neuroactive ligand-receptor interaction,estrogen, FoXO and TNF signaling pathways. The results showed that N. jatamansi extended the PDA rats in turn on the rod retention time, increased the total activity distance of PDA rats in the open field and the activity time and distance in the central area,improved the substantia nigra area tyrosine hydroxylase positive expression(P < 0.05). N. jatamansi also reduced MAPK3 and TNF-α levels and STAT3 protein expression(P < 0.05, 0.01), increased the content of dopamine, 3,4-dihydroxyphenylacetic acid,homovanillic acid, 5-hydroxytryptamine and 5-hydroxyindolacetic acid(P < 0.01, 0.001). Conclusion The active ingredients in N.jatamansi may regulate multiple signal pathways through MAPK3, TNF-α, STAT3 and other targets to reduce neuroinflammatory response and increase neurotransmitters level to treat PDA disease.