基于网络药理学及细胞实验验证探讨补肾益心片治疗糖尿病肾病的作用机制

Exploring the Mechanism of Bushen Yixin Tablets in the Treatment of Diabetic Nephropathy Based on Network Pharmacology and Cellular Experimental Validation

【目的】探讨补肾益心片治疗糖尿病肾病的作用机制。【方法】应用网络药理学预测补肾益心片治疗糖尿病肾病的关键靶点。构建高糖处理MPC-5细胞模型,应用酶联免疫吸附法(ELISA)、实时定量聚合酶链反应(RT-qPCR)法和Western Blot法验证补肾益心片治疗糖尿病肾病的潜在作用靶点及通路。【结果】获得补肾益心片24个有效成分。补肾益心片治疗糖尿病肾病涉及靶点131个,通路富集分析结果显示关键靶点可能主要集中在炎症通路、脂质与动脉粥样硬化等。进一步实验验证结果发现:与高糖组比较,补肾益心片含药血清中、高剂量组MPC-5细胞上清中基质金属蛋白酶1(MMP-1)分泌水平升高(P<0.001),组织金属蛋白酶抑制因子1(TIMP-1)和转化生长因子β1(TGF-β1)分泌水平降低(P<0.001);补肾益心片含药血清高剂量组MMP-1和BCL-2蛋白及mRNA表达水平升高(P<0.001),磷脂酰肌醇3-激酶(PI3K)和蛋白激酶B(AKT)蛋白及mRNA表达水平降低(P<0.001)。【结论】补肾益心片可能是通过调节PI3K/AKT/BCL-2通路抑制肾小球系膜细胞增殖,促进细胞外基质的降解,从而发挥对糖尿病肾病的治疗作用。

Objective To investigate the mechanism of Bushen Yixin Tablets in the treatment of diabetic nephropathy(DN).Methods The key targets of Bushen Yixin Tablets for the treatment of DN were predicted by network pharmacology.A high-glucose-processed MPC-5 cell model was constructed,enzyme-linked immunosorbent assay(ELISA),real-time quantitative polymerase chain reaction(RT-qPCR)and Western Blot were applied to verify the potential action targets and pathways of Bushen Yixin Tablets for the treatment of DN.Results Twenty-four active ingredients were obtained from Bushen Yixin Tablets.There were 131 targets involved in the treatment of DN by Bushen Yixin Tablets,and the results of pathway enrichment analysis showed that the key targets might mainly focus on inflammatory pathways,lipids and atherosclerosis.The further experimental validation showed that,compared with the high glucose group,the secretion level of matrix metalloproteinase 1(MMP-1)in the supernatant of MPC-5 cells in the medium-and high-dose groups of Bushen Yixin Tablets medicated serum were increased(P<0.001),and the secretion levels of tissue inhibitor of metalloproteinase 1(TIMP-1)and transforming growth factorβ1(TGF-β1)were decreased(P<0.001).Compared with the high glucose group,the protein and mRNA expression levels of MMP1 and BCL-2 in the high-dose group of Bushen Yixin Tablets medicated serum were increased(P<0.001),and the protein and mRNA expression levels of phosphatidylinositol 3-kinase(PI3K)and protein kinase B(AKT)were decreased(P<0.001).Conclusion Bushen Yixin Tablets may play a protective role against DN by inhibiting the proliferation of glomerular mesangial cells and promoting the degradation of extracellular matrix through PI3K/AKT/BCL-2 pathway.