基于生物信息学及体外实验研究降香治疗缺血再灌注损伤作用机制

Mechanism of Rosewood Heart Wood Treating Ischemia-reperfusion Injury Based on Bioinformatics and in Vitro Experiments

目的 运用生物信息学及细胞实验方法研究降香治疗心肌缺血再灌注损伤(myocardial ischemia-reperfusion injury, MIRI)的作用机制。方法 在中医药系统药理学数据库分析平台(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, TCMSP)收集降香主要活性成分;再通过Swiss Target Prediction选取降香作用靶点。在DisGeNET、人类孟德尔遗传数据库(Online Mendelian Inheritance In Man, OMIM)、治疗靶点数据库(Therapeutic Target Database, TTD)筛选MIRI有关靶点;在Venny得到相同作用靶点。在String输入构筑蛋白质-蛋白质相互作用(protein-protein interaction, PPI)网络模型。在Matescape及微生信对降香抑制MIRI损伤的作用靶点进行基因本体(Gene Ontology, GO)分析及京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)信号通路富集分析。细胞实验,按实验方案分为空白组、模型组、降香高组、降香低组。测定各组细胞活力;测定各组乳酸脱氢酶(lactate dehydrogenase, LDH)、肿瘤坏死因子α(tumor necrosis factor alpha, TNF-α)、白细胞介素1β(interleukin 1 beta, IL-1β)及IL-6的含量;测定各组Src激酶、磷脂酰肌醇3-激酶(phosphatidylinositol-3-kinase, PI3K)、细胞外调节蛋白激酶(extracellular regulated protein kinases, ERK1/2)、磷酸化细胞外调节蛋白激酶(phosphorylation-extracellular regulated protein kinases, p-ERK1/2)蛋白表达。结果 得到相同作用靶点229个,其中Src、丝裂原活化蛋白激酶1(mitogen-activated protein kinase 1,MAPK1)、热休克蛋白α(heatshockprotein alpha, HSPα)、血管内皮生长因子(vascular endothelial growth factor, VEGF)、PI3K为关键靶点。通过GO和KEGG分析,降香最可能通过凋亡信号通路抑制MIRI。降香组比模型组细胞增殖率显著升高(P<0.05)。降香组比模型组LDH、TNF-α、IL-1β、IL-6含量显著降低(P均<0.05)。降香组比模型组Src、PI3K表达显著升高,p-ERK1/2蛋白表达显著降低(P均<0.05)。结论 降香通过调节Src、PI3K、ERK1/2、p-ERK1/2表达,作用于凋亡信号通路减轻MIRI损伤。

Objective To study the mechanism of the therapeutic effects of Rosewood Heart Wood on myocardial ischemia-reperfusion injury(MIRI) using bioinformatics and cellular experimental methods. Methods The main active ingredients of Rosewood Heart Wood in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) was collected;the target points for Rosewood Heart Wood effect were selected through the Swiss Target Prediction. MIRI related targets were screened on DisGeNET, Online Mendelian Inheritance In Man(OMIM) and Therapeutic Target Database(TTD);the same target at Venny was obtained. Protein-protein interaction(PPI) network model with String input was constructed. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) signaling pathway enrichment analysis were conducted on the targets of Matescape and Microbioinformatics in inhibiting MIRI damage of Lignum Dalbergiae. Cell experiments were divided into blank group, model group, high Rosewood Heart Wood group and low Rosewood Heart Wood group according to the experimental plan. The cell viability of each group was measured. The content of lactate dehydrogenase(LDH), tumor necrosis factor alpha(TNF-α), interleukin 1 beta(IL-1β) and IL-6 in each group were measured. The protein expression of non-receptor Src kinase, phosphatidylinositol-3-kinase(PI3K), extracellular regulated protein kinases(ERK1/2) and phosphorylation-extracellular regulated protein kinases(p-ERK1/2) were detected in each group. Results A total of 229 identical targets were obtained, including Src, mitogen-activated protein kinase 1(MAPK1), heatshockprotein alpha(HSPα), vascular endothelial growth factor(VEGF) and PI3K were key targets;Through GO and KEGG analysis, it was most likely that Rosewood Heart Wood inhibited MIRI through the apoptotic signaling pathway. The cell proliferation rate of the Rosewood Heart Wood group was significantly higher than that of the model group(P<0.05). The content of LDH, TNF-α, IL-1β, IL-6 in Rosewood Heart Wood group was significantly higher than that in the model group(all P<0.05). Compared with the model group, the expression of Src and PI3K in Rosewood Heart Wood group significantly increased, while the expression of p-ERK1/2 protein significantly reduced(all P<0.05). Conclusion Rosewood Heart Wood alleviates MIRI by regulating the expression of Src, PI3K, ERK1/2 and p-ERK1/2 through the apoptotic signaling pathway.