摘要
目的基于网络药理学分析及实验验证探讨五苓散治疗尿酸性肾病(UAN)的作用机制。方法(1)利用TCMSP、Swiss Target Prediction数据库筛选五苓散的活性成分及其作用靶点;利用OMIM、GeneCard、TTD数据库检索UAN疾病相关靶点;对活性成分作用靶点与UAN疾病相关靶点取交集,所得交集靶点即为五苓散治疗UAN的潜在作用靶点。通过STRING数据库构建潜在作用靶点蛋白互作(PPI)网络,筛选出关键靶点;利用Cytoscape 3.6.1软件构建“中药-活性成分-关键靶点”网络。通过DAVID分析平台对关键靶点进行GO功能及KEGG通路富集分析;筛选出核心靶点及其对应的关键活性成分,并进行分子对接验证。(2)采用大鼠灌胃给予氧嗪酸钾(1.5 g·kg^(-1))+腺嘌呤(0.1 g·kg^(-1))溶液,每日1次,连续28 d,复制UAN大鼠模型。将SD大鼠随机分为正常组、模型组、别嘌醇组(0.03 g·kg^(-1))和五苓散低、中、高剂量组(2.88、5.76、11.52 g·kg^(-1)),每组10只,灌胃给药(10 mL·kg^(-1)),每天1次,连续28 d。采用ELISA法检测大鼠血尿酸(SUA)、血肌酐(SCR)、血尿素氮(BUN)及尿尿酸(UUA)、尿肌酐(UCR)、尿尿素氮(UUN)的水平;HE染色法观察大鼠肾脏组织病理变化;免疫组化法检测大鼠肾脏组织中MMP2、P-IGF1R、P-SRC、P-PIK3CA、P-ERα蛋白表达水平。结果(1)共得到五苓散治疗UAN的潜在作用靶点124个,关键靶点46个。关键靶点的生物过程涉及对MAPK级联正调控、肽基酪氨酸磷酸化、磷脂酰肌醇3-激酶信号传导正调控等;KEGG通路主要涉及癌症发病途径、内分泌抵抗、AGE-RAGE信号通路在糖尿病并发症中的作用等。内分泌抵抗途径是五苓散治疗UAN的关键通路,涉及5个核心靶点包括MMP2、IGF1R、SRC、PIK3CA、ESR1;7个关键活性成分包括泽泻醇B、泽泻醇B乙酸酯、16α-羟基松苓新酸、去氢土莫酸、猪苓酸C、啤酒甾醇、(22E,24R)-麦角甾-6-烯-3β,5α,6β-三醇。7个关键活性成分与5个核心靶点均有较强的结合力。(2)与正常组比较,模型组大鼠的SUA、SCR、BUN水平均明显升高(P<0.05),UUA、UCR、UUN水平均明显降低(P<0.05),肾脏系数明显升高(P<0.05);肾小球局部硬化、萎缩,肾小管出现不规则扩张或萎缩,部分肾小管有明显炎性细胞浸润;肾脏组织中MMP2、P-IGF1R、P-SRC、P-PIK3CA、P-ERα蛋白表达水平均明显升高(P<0.05)。与模型组比较,各给药组大鼠的SUA、SCR、BUN水平均明显降低(P<0.05),UUA、UUN水平均明显升高(P<0.05);别嘌醇组和五苓散中、高剂量组大鼠的UCR水平明显升高(P<0.05);别嘌醇组及五苓散低、高剂量组大鼠的肾脏系数明显降低(P<0.05);各给药组大鼠的肾小球、肾小管病变均有不同程度改善;肾脏组织中MMP2、P-IGF1R、P-SRC、P-PIK3CA、P-ERα蛋白表达水平均明显降低(P<0.05)。结论五苓散能够降低UAN大鼠的SUA、SCR、BUN水平,改善其肾脏组织病理损伤,内分泌抵抗途径可能是五苓散治疗UAN的关键通路,涉及MMP2、IGF1R、SRC、PIK3CA、ESR1等核心靶点。
Objective To investigate the mechanism of Wuling San in treating uric acid nephropathy(UAN)through network pharmacology analysis and experimental validation.Methods(1)Active components and their targets of Wuling San were screened using the TCMSP and Swiss Target Prediction databases.Disease-related targets of UAN were retrieved from OMIM,GeneCards,and TTD databases.The intersection of component targets and disease targets was identified as potential therapeutic targets for Wuling San against UAN.A protein-protein interaction(PPI)network of potential targets was constructed using STRING,and core targets were filtered.A“herbal medicine-active componentscore targets”network was built with Cytoscape 3.6.1.GO functional and KEGG pathway enrichment analyses of core targets were performed via the DAVID platform.Core targets and their corresponding key active components were selected for molecular docking validation.(2)A UAN rat model was established by intragastric administration of potassium oxonate(1.5 g·kg^(-1))+adenine(0.1 g·kg^(-1))once daily for 28 days.SD rats were randomly divided into normal,model,allopurinol(0.03 g·kg^(-1)),and low-,medium-,and high-dose Wuling San groups(2.88,5.76,11.520.03 g·kg^(-1)),with 10 rats per group.Intragastric administration(10 mL·kg^(-1))was performed once daily for 28 days.Serum uric acid(SUA),serum creatinine(SCR),blood urea nitrogen(BUN),urinary uric acid(UUA),urinary creatinine(UCR),and urinary urea nitrogen(UUN)levels were measured by ELISA.Renal histopathological changes were observed via HE staining.Immunohistochemistry was used to detect renal protein expression of MMP2,P-IGF1R,P-SRC,P-PIK3CA,and P-ERα.Results(1)A total of 124 potential therapeutic targets and 46 core targets were identified.Key biological processes included positive regulation of MAPK cascade,peptidyl-tyrosine phosphorylation,and phosphatidylinositol 3-kinase signaling.KEGG pathways primarily involved cancer pathways,endocrine resistance,and AGE-RAGE signaling in diabetic complications.The endocrine resistance pathway was identified as the critical pathway for Wuling San against UAN,involving 5 core targets(MMP2,IGF1R,SRC,PIK3CA,ESR1)and 7 key active components[alisol B,alisol B acetate,16α-hydroxy-trametenolic acid,dehydrotumulosic acid,polyporenic acid C,ergosta-7,and(22E,24R)-ergosta-6-ene-3β,5α,6β-triol].Molecular docking confirmed strong binding affinity between the 7 key active components and 5 core targets.(2)Compared with the normal group,the model group exhibited significantly elevated SUA,SCR,BUN levels(P<0.05),reduced UUA,UCR,UUN levels(P<0.05),increased kidney coefficient(P<0.05),local glomerular sclerosis and atrophy,tubular atrophy/dilation,inflammatory cell infiltration,and upregulated protein expression of renal MMP2,P-IGF1R,P-SRC,P-PIK3CA,and P-ERα(P<0.05).Compared with the model group,all treatment groups showed reduced SUA,SCR,BUN levels(P<0.05),increased UUA and UUN levels(P<0.05),elevated UCR levels in the allopurinol and medium-and high-dose Wuling San groups(P<0.05),decreased kidney coefficient in the allopurinol and low-and high-dose Wuling San groups(P<0.05),improved renal histopathology,and downregulated protein expression of renal MMP2,P-IGF1R,P-SRC,P-PIK3CA,and P-ERα(P<0.05).Conclusion Wuling San reduces SUA,SCR,and BUN levels,alleviates renal histopathological damage in UAN rats,and likely exerts therapeutic effects via the endocrine resistance pathway targeting MMP2,IGF1R,SRC,PIK3CA,and ESR1.
作者
唐雨
谢秉恒
吴刘俊
吴星霏
张家乐
申艳朵
张明昊
马丽亚
TANG Yu;XIE Bingheng;WU Liujun;WU Xingfei;ZHANG Jiale;SHEN Yanduo;ZHANG Minghao;MA Liya(The Second Clinical Medical College of Henan University of Chinese Medicine,Zhengzhou 450046 Henan,China;The Fifth Clinical Medical College of Henan University of Chinese Medicine,Zhengzhou 450002 Henan,China;Medical College of Henan University of Traditional Chinese Medicine,Zhengzhou 450046 Henan,China;Administrative Office of Henan University of Traditional Chinese Medicine,Zhengzhou 450046 Henan,China)
出处
《中药新药与临床药理》
北大核心
2025年第7期1071-1083,共13页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
河南省重点研发与推广(科技攻关)项目(222102310139)
河南省自然科学基金面上项目(242300420168)
河南省中医药科学研究专项课题(2023ZYZD09)
河南省中医药科学研究专项课题重点课题(20-21ZY1053)。
关键词
五苓散
尿酸性肾病
网络药理学
作用机制
内分泌抵抗途径
大鼠
实验验证
Wuling San
uric acid nephropathy
network pharmacology
mechanism
endocrine resistance pathway
rats
experimental vaidation
