基于网络药理学和实验验证探究尿石素A治疗肾纤维化的作用机制

Investigation of the mechanism of urolithin A in the treatment of renal fibrosis based on network pharmacology and experimental verification

目的:基于网络药理学、分子对接及动物实验探究尿石素A(urolithin A,UA)治疗单侧输尿管结扎(unilateral ureteral obstruction,UUO)诱导的肾纤维化的疗效及潜在作用机制。方法:采用网络药理学技术分析UA改善肾纤维化的作用机制;使用Auto Dock Tool软件对UA与改善肾纤维化关键靶点进行分子对接。采用UUO建立大鼠肾纤维化模型,纤维化标志蛋白的免疫组化染色检测UA对纤连蛋白和胶原蛋白Ⅰ的调控作用,免疫荧光染色和Western blot检测UA对p-Akt1和Akt1的调控作用,Western blot检测UA对Akt1上游PI3K、p-PI3K和下游GSK3β、p-GSK3β蛋白表达的影响。结果:通过网络药理学技术共筛选获得21个UA抗纤维化核心靶点。通过PPI网络拓扑分析、GO生物功能注释和KEGG通路富集分析发现UA可以通过PI3K-Akt、p53等信号通路及Akt1、CCND1、CDK4、EGFR、CDK2、ERBB2、CDK6、GSK3B、BCL2L1、PTK2、ESR1、PTGS2等关键核心靶点发挥治疗肾纤维化的作用,其中Akt1是此次网络研究中节点程度最高的关键靶点。分子对接进一步验证了UA和Akt1的对接亲和度较高。动物实验结果表明,UA可显著降低UUO大鼠的纤连蛋白和胶原蛋白Ⅰ的表达(P<0.01),并抑制p-Akt1的表达(P<0.01),同时抑制Akt1上游PI3K和下游GSK3β蛋白的磷酸化(P<0.01)。结论:UA可通过抑制PI3K/Akt1/GSK3β信号通路,降低大鼠肾脏纤连蛋白和胶原蛋白Ⅰ的表达,有效缓解肾纤维化,可为后续尿石素A治疗肾纤维化的深入研究提供科学依据。

OBJECTIVE To investigate the efficacy and potential mechanism of urolithin A(UA)in the treatment of renal fibrosis induced by unilateral ureteral obstruction(UUO)based on network pharmacology,molecular docking and animal experiment.METHODS Network pharmacology techniques were used to analyze the mechanism of improving renal fibrosis action of UA.Auto Dock Tool software was used to perform molecular docking of UA with key targets to improve renal fibrosis.UUO was used to establish a rat renal fibrosis model.The regulatory effect of UA on renal fibrosis marker proteins was verified by immunohistochemical staining of fibronectin and collagen Ⅰ.The regulatory effects of UA on p-Akt1 and Akt1 were examined by immunofluorescence staining and Western blot.The effects of UA on the expression of PI3K and p-PI3K upstream,and GSK3β and p-GSK3β downstream of Akt1 were examined by Western blot.RESULTS A total of 21 core targets were obtained by screening through network pharmacology techniques.Through PPI network topology analysis,GO biological function annotation and KEGG pathway enrichment analysis,it was found that UA could play a role in the treatment of renal fibrosis through signal pathways such as PI3K-Akt,p53 and key core targets such as Akt1,CCND1,CDK4,EGFR,CDK2,ERBB2,CDK6,GSK3B,BCL2L1,PTK2,ESR1 and PTGS2.Akt1 was the key target with the highest node degree in the PPI network study.Molecular docking further verified the high docking affinity of UA and Akt1.Animal experiments showed that UA could significantly reduce the expression of fibronectin and collagenⅠin UUO rats(P<0.01),and inhibit the expression of p-Akt1(P<0.01),as well as inhibited the phosphorylation of PI3K upstream and GSK3β downstream of Akt1(P<0.01).CONCLUSION UA can effectively alleviate renal fibrosis by inhibiting PI3K/Akt1/GSK3βsignal pathway,and effectively reduce the expression of fibronectin and collagenⅠin rat kidney,which provide scientific basis for further study of UA in the treatment of renal fibrosis.