通心络胶囊对脑缺血再灌注损伤小鼠的保护作用及作用机制

Protective Effect of Tongxinluo Capsule on Cerebral Ischemia-reperfusion Injury and Its Mechanism of Action

目的:研究通心络胶囊对脑缺血再灌注损伤的保护作用,并通过网络药理学探讨通心络胶囊治疗脑缺血的作用机制。方法:C57BL/6小鼠采用改良线栓法制作小鼠中动脉闭塞(MCAO)模型,分为假手术组、模型组、通心络低、中、高剂量组(1,2,4 g·kg^-1,灌胃给药)、阿司匹林组(2.055 g·L^-1,腹腔注射),于小鼠脑缺血再灌注24,48,72 h参照Longa评分法对小鼠进行神经功能评分,苏木素-伊红(HE)染色法观察脑组织病理学变化情况,2,3,5-氯化三苯基四氮唑(TTC)染色法测定小鼠脑梗死面积。首先从BATMAN-TCM数据库中筛选通心络胶囊化学成分并分析作用靶点,然后分析靶点富集的信号通路,之后构建中药-活性成分-靶点网络,最后预测通心络胶囊治疗脑缺血的多维药理作用机制。结果:Longa评分法,HE染色和TTC染色法都提示通心络胶囊可改善脑缺血再灌注小鼠的脑损伤,并且通心络胶囊对脑损伤的改善程度随着通心络胶囊剂量升高而逐渐升高。在通心络胶囊所含12味中药中筛选得到132个有效活性成分及环磷酸腺苷(cAMP)依赖性蛋白激酶催化亚基(PRKACA),腺苷酸环化酶1(ADCY1),丝氨酸/苏氨酸激酶1(Akt1),多巴胺受体D2(DRD2),Discs大同源物4(DLG4)等240个交集靶点。基因本体(GO)富集于阳离子通道活性、离子门控通道活性、门控通道活性、神经递质受体活性、离子通道活性等。京都基因与基因组百科全书(KEGG)富集于cAMP信号通路,钙信号通路,神经活性配体-受体相互作用,环磷酸鸟苷(cGMP)/蛋白激酶G(PKG)信号通路、多巴胺能突触信号通路。结论:通心络胶囊可减轻脑缺血再灌注小鼠的脑损伤,其通过多靶点、多环节实现脑保护作用;网络药理学揭示了通心络胶囊治疗脑缺血的有效成分、靶点及通路,为通心络胶囊治疗脑缺血的作用机制提供理论支持。

Objective:To study the protective effect of Tongxinluo capsule on cerebral ischemiareperfusion injury,and explore the mechanism of Tongxinluo capsule in treating cerebral ischemia through network pharmacology.Method:The C57BL/6 mouse middle cerebral artery occlusion(MCAO)model was established by improved suture method and divided into sham operation group,model group,low,medium and high-dose Tongxinluo groups(crude drug 1,2,4 g·kg^-1,intragastric administration),Aspirin group(2.055 g·L^-1,intraperitoneal injection).Then,neurological function score and 2,3,5-triphenyltetrazole chloride(TTC)staining method were used to determine the infarct size of mice at 24,48,72 h by cerebral ischemia-reperfusion.First,chemical constituents of Tongxinluo capsule were screened from the BATMANTCM database,and the targets were analyzed.Then,gene ontology(GO)enrichment and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis were performed,and traditional Chinese medicine(TCM)-active ingredient-target network was constructed.Finally,the multi-dimensional pharmacological mechanism of Tongxinluo capsule in the treatment of cerebral ischemia was predicted.Result:Longa score,HE staining and TTC staining all suggested that Tongxinluo capsule could alleviate brain injury in mice after cerebral ischemia and reperfusion,and the improvement degree of Tongxinluo capsule on brain injury was gradually enhanced with the increase of Tongxinluo capsule dose.A total of 132 active components and 240 intersection targets,including cyclic adenosine monophosphate(cAMP)-dependent protein kinase catalytic subunit alpha(PRKACA),adenylate cyclase 1(ADCY1),serine/threonine kinase 1(Akt1),dopamine receptor D2(DRD2)and discs large homolog 4(DLG4)were screened from 12 TCM in Tongxinluo capsule.GO was enriched in cationic channel activity,ion gated channel activity,gate channel activity,neurotransmitter receptor activity,ion channel activity,etc.KEGG was enriched in cAMP signaling pathway,calcium signaling pathway,neuroactive ligand-receptor interaction,cyclic guanosine monophosphate(cGMP)/protein kinase G(PKG)signaling pathway and dopaminergic synaptic signaling pathway.Conclusion:Tongxinluo capsule can alleviate brain damage in mice with cerebral ischemia-reperfusion,and achieve brain protection through multiple targets and multiple links.Network pharmacology reveals effective components,targets and pathway of Tongxinluo capsule in the treatment of cerebral ischemia,which provides theoretical support for the mechanism of Tongxinluo capsule in the treatment of cerebral ischemia.