网络药理学-分子对接分析及实验验证蒙药高尤的肾功能保护作用机制

Network pharmacology-molecular docking analysis and experimental validation to explore the protective mechanism of Mongolian medi-cine Gaoyou on renal function

目的:通过网络药理学-分子对接及动物实验探讨蒙药高尤保护肾功能的作用机制。方法:运用中药相关数据库获取高尤作用于急性肾损伤(AKI)的有效活性成分和靶点、AKI疾病的相关靶点,把靶点和成分相互构建,作用于网络,富集分析药物及疾病的信号通路,并对网络中度值较高的药效成分与核心作用靶点进行分子对接验证研究。在此基础上通过建立急性肾损伤大鼠模型给予高尤干预后检测大鼠血清中肌酐(Cr)、尿素氮(BUN)、肿瘤坏死因子-α(TNF-α)、肾损伤分子-1(Kim-1)、胱抑素C(Cys-C)、中性粒细胞明胶酶相关脂质运载蛋白(NGAL)含量并HE染色观察肾组织切片验证蒙药高尤对急性肾损伤大鼠肾功能保护作用。通过蛋白免疫印迹法(Westernblot)验证预测信号通路。结果:网络药理学及分子对接分析得到关键靶点40个,并对关键靶点进行KEGG和GO富集分析,预测出ALB、AKT1、VEGFA、IL-6、CASP3等为关键靶点,癌症信号通路、TNF信号通路、PI3K/AKT信号通路等为关键通路。分子对接结果显示高尤主要的活性成分与相关作用靶点有较好的结合活性。动物实验结果表明高尤具有很好的肾功能保护作用,Cr、BUN明显低于模型组(P<0.05);TNF-α含量明显降低(P<0.05);急性肾损伤标志物Kim-1、Cys-C、NGAL均显著降低(P<0.05)。HE染色结果表明高尤能明显改善急性肾损伤的病理学状态。急性肾损伤模型大鼠肾组织中AKT1、p-AKT1、CASP3表达升高(P<0.05);与模型组比较,高尤组大鼠肾组织中AKT1、p-AKT1、CASP3表达显著降低(P<0.05)。结论:高尤可通过多组分、多靶点的特点,从抗炎、抗氧化、抗血栓,抗凋亡等多通路途径发挥保护肾损伤大鼠肾功能作用,干预PKA3/AKT信号通路与细胞凋亡因子可能是其主要作用机制之一。

AIM:To explore the protective mecha-nism of Mongolian medicine Gaoyou on renal function through network pharmacology molecular docking and animal experiments.METHODS:The effective active components and targets of Gao you acting on acute kidney injury(AKI)and the related targets of AKI disease were obtained by using the relevant database of traditional Chinese medicine.The targets and components were mutually constructed to act on the network,and the signal path-ways of drugs and diseases were enriched and analyzed.And the molecular docking validation study was carried out on the pharmacodynamic components with high moderate value in the network and the core action target.On this basis,after establishing the rat model of acute renal injury and giving Gaoyou intervention,the contents of creatinine(CR),urea nitrogen(BUN)and tumor necrosis factor-A(TNFα),Kidney damage molecule-1(Kim-1),cystatin C(Cys-C),neutrophil gelatinase related lipid transporter(NGAL)in serum of rats were detected,and renal tissue sections were observed with HE staining to verify the protective effect of Mon-golian medicine Gaoyou on renal function of rats with acute renal injury.The predicted signal path-way was verified by Western blot.RESULTS:40 key targets were obtained by network pharmacology and molecular docking analysis.KEGG and go enrichment analysis were carried out on the key targets.ALB,AKT1,VEGFA,IL-6,CASP3 and other key targets were predicted,and cancer signal pathway,TNF signal pathway,PI3K/AKT signal pathway and other key pathways were predicted.The results of molecular docking showed that the main active components of Gaoyou had good binding activity with related targets.The results of animal experi-ments showed that Gao you had a good protective effect on renal function,and Cr and BUN were sig-nificantly lower than those in the model group(P<0.05).The content of TNF-αdecreased significantly(P<0.05).Kim-1,Cys-C and NGAL decreased signifi-cantly(P<0.05).HE staining showed that Gao can obviously improve the pathological state of acute renal damage.The expression of AKT1,p-AKT1 and CASP3 in renal tissue of rats with acute renal injury increased(P<0.05).Compared with the model group,the expression of AKT1,p-AKT1 and CASP3 in renal tissue of rats in Gaoyou group decreased significantly(P<0.05).CONCLUSION:Gaoyou can protect the renal function of rats with renal injury through multiple pathways such as anti-inflamma-tory,antioxidant,antithrombotic,anti apoptotic and so on through the characteristics of multi-component,multi target.Intervention of PKA3/AKT signal pathway and apoptosis factor may be one of its main mechanisms.