基于网络药理学和验证实验探讨蒲黄治疗脊髓损伤的分子机制

Molecular mechanism of Typhae Pollen in treatment of spinal cord injury based on network pharmacology and validation experiments

目的利用网络药理学和实验验证探讨蒲黄治疗脊髓损伤的分子机制。方法使用TCMSP数据库及文献资料筛选蒲黄的有效成分及其对应靶点,使用DrugBank、OMIM、TTD、GeneCard数据库获得脊髓损伤相关靶点。Cytoscape构建蒲黄抗脊髓损伤靶点蛋白相互作用(PPI)网络图及“药物–化合物–靶点”网络图,根据网络图中各靶点的拓扑学参数筛选出蒲黄抗脊髓损伤关键靶点及关键成分。使用DAVID数据库对蒲黄抗脊髓损伤靶点进行基因本体论(GO)功能和京都基因与基因组百科全书(KEGG)富集分析。采用Autodock Vina软件对关键化合物与关键靶点蛋白进行分子对接验证。动物实验观察蒲黄对脊髓损伤大鼠后肢运动神经功能的影响及对关键靶点蛋白mRNA表达的影响。结果共筛选出花生四烯酸、异鼠李素、β谷甾醇、山柰酚、棕榈酸睾酮、山柰酚-3-O-α-L-鼠李糖基(1→2)-β-D-葡萄糖苷、槲皮素、异鼠李素-3-O-新橙皮苷、香蒲新苷等活性成分。PPI网络分析显示,肿瘤坏死因子(TNF)、白细胞介素(IL)-6、蛋白激酶B(AKT1)、IL-1B、胱天蛋白酶3(CASP3)等蛋白可能在脊髓损伤的治疗中起关键作用。蒲黄抗脊髓损伤靶点的GO分析显示生物过程主要涉及对脂多糖的反应、对细菌源性分子的反应、细胞对化学应激的反应、活性氧代谢过程的调控过程等。KEGG富集分析显示富集显著性较高的通路主要有TNF信号通路、TLR信号通路、MAPK信号通路等。分子对接显示关键活性成分与关键靶蛋白分子之间亲和力良好。动物实验结果表明,与模型组相比,蒲黄能有效促进脊髓损伤大鼠模型动物的后肢运动恢复,降低TNF、IL-6、IL-1B、CASP3 mRNA表达,升高AKT1 mRNA表达(P<0.05、0.01)。结论蒲黄中槲皮素、山柰酚、异鼠李素、β-谷甾醇等活性成分可能通过作用于TNF、IL-6、AKT1、IL-1B、CASP3等靶点,进而影响炎症及凋亡相关的通路发挥治疗脊髓损伤的作用。

Objective To explore the molecular mechanism of Typhae Pollen in treatment of spinal cord injury by using network pharmacology and validation experiments.Methods TCMSP database and literature data were used to screen the active components and corresponding targets of Typhae Pollen.DrugBank,OMIM,TTD,and GeneCard databases were used to obtain the targets of spinal cord injury.Cytoscape constructed PPI network diagram and drug–compound–target network diagram,and screened the key targets and components of Typhae Pollen against cord injury according to topological parameters of each target in the network diagram.DAVID database was used to analyze the GO function and KEGG enrichment of the anti-spinal cord injury targets of Typhae Pollen.Autodock Vina software was used for molecular docking verification of key compounds and key target proteins.Animal experiments were conducted to observe the effect of Typhae Pollen on the motor nerve function of hind limbs and the expression of key target protein mRNA in rats with spinal cord injury.Results A total of arachidonic acid,isorhamnetin,βsitosterol,kaempferol,testosterone palmitate,kaempferol-3-O-α-L-rhamnosyl(1→2)-β-D-glucoside_qt,quercetin,isorhamnetin-3-O-neohesperidoside,ctyphloside and other active ingredients were screened.PPI network analysis showed that TNF,IL-6,AKT1,IL-1B,CASP3 and other proteins may play a key role in the treatment of spinal cord injury.Gene ontology(GO)analysis of the anti-spinal cord injury targets of Typhae Pollen showed that the biological processes mainly involve the reaction to lipopolysaccharide,the reaction to bacterial molecules,the cellular response to chemical stress,and the regulation of reactive oxygen metabolism.The enrichment analysis of Kyoto Encyclopedia of Genes and Genomes(KEGG)showed that the pathways with high enrichment significance mainly included TNF signaling pathway,TLR signaling pathway,and MAPK signaling pathway.Molecular docking showed that the affinity between key active components and key target protein molecules was good.Animal experimental results showed that compared with the model group,Typhae Pollen could effectively promote the recovery of hind limb movement,decrease the mRNA expression of TNF,IL-6,IL-1B and CASP3,and increase the mRNA expression of AKT1 in rats with spinal cord injury(P<0.05,0.01).Conclusion Quercetin,kaempferol,isorhamnetin,β-sitosterol and other active ingredients in Typhae Pollen may affect inflammatory and apoptosis-related pathways by acting on TNF,IL-6,AKT1,IL-1B,CASP3 and other targets.