基于肠道菌群和系统药理学探讨藏药十五味乳鹏丸抗高尿酸血症肾病的作用机制

Mechanism of Tibetan medicine Shiwuwei Rupeng Pills on hyperuricemia nephropathy based on intestinal flora and systematic pharmacology

目的基于肠道菌群、网络药理学和分子对接技术多维度挖掘藏药十五味乳鹏丸(Shiwuwei Rupeng Pills,SRP)抗高尿酸血症肾病(hyperuricemic nephropathy,HN)的作用机制。方法腺嘌呤联合乙胺丁醇构建HN大鼠模型,设置对照组、模型组、别嘌醇(50 mg/kg)组和SRP高、低剂量(1.2、0.4 g/kg)组。连续给药14 d后,检测大鼠血清中尿酸(uric acid,UA)、肌酐(creatinine,CREA)及尿素氮(urea nitrogen,BUN)水平;采用苏木素-伊红(HE)染色法观察肾组织病理变化。获取SRP和HN核心靶点,构建“药物-活性成分-作用靶点”和蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络,并对潜在靶点进行基因本体(gene ontology,GO)功能及京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析;采用分子对接技术模拟关键成分与核心靶点的结合活性。采用qRT-PCR法验证各组大鼠肾组织中关键靶点的mRNA表达;收集大鼠粪便,采用16S rDNA高通量测序法检测肠道菌群变化。结果SRP显著降低HN大鼠血清中UA、CREA及BUN水平(P<0.05),并改善肾组织病理损伤。SRP可能通过作用于晚期糖基化终末化产物(advanced glycation end products,AGE)-晚期糖基化终末产物受体(receptor for advanced glycation end products,RAGE)、白细胞介素-17(interleukin-17,IL-17)和肿瘤坏死因子(tumor necrosis factor,TNF)等信号通路发挥抗HN作用。白蛋白(albumin,ALB)、TNF、过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma,PPARG)、信号传导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)和骨髓细胞瘤病毒癌基因(myelocytomatosis viral oncogene,MYC)与木犀草素、槲皮素、山柰酚、表没食子酸儿茶素没食子酸酯和儿茶素有较好的对接活性。SRP显著下调HN大鼠肾组织中TNF、STAT3和ALB mRNA表达水平(P<0.05),显著上调PPARG mRNA表达水平(P<0.05)。与模型组比较,SRP组大鼠肠道菌群丰富度和多样性显著升高,且在门水平上降低拟杆菌门和厚壁菌门的比例;主要降低Acteroides、Akkermansia、Ralstonia及Prevotellaceae Ga6A1 group菌属,增加Lactobacillus及Ruminococcaceae UCG-014的相对丰度。结论SRP可以通过调节肠道菌群结构,调控AGE-RAGE、IL-17、TNF等信号通路及相关靶点的表达发挥抗HN作用,具有多成分、多靶标及多通路的治疗特点。

Objective To explore the mechanism of Tibetan medicine Shiwuwei Rupeng Pills(十五味乳鹏丸,SRP)on hyperuricemic nephropathy(HN)from multiple dimensions based on intestinal flora,network pharmacology and molecular docking technology.Methods HN rats model was established by adenine combined with ethambutol,and rats were divided into control group,model group,allopurinol(50 mg/kg)group,SRP high-and low-dose(1.2,0.4 g/kg)groups.After continuous administration for 14 d,levels of uric acid(UA),creatinine(CREA)and urea nitrogen(BUN)in serum of rats were detected;Pathological changes of kidney tissue was observed by hematoxylin-eosin(HE)staining.SRP and HN core targets were obtained,“drug-active ingredient-targets”and protein-protein interaction(PPI)networks were constructed,gene ontology(GO)function and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis on potential targets were performed;Molecular docking technology was used to simulate the binding activity of key components and core targets.qRT-PCR was used to verify the mRNA expression of key targets in kidney tissue of rats in each group;Rat feces were collected,and 16S rDNA high-throughput sequencing was used to detect changes in intestinal flora.Results SRP significantly decreased the levels of UA,CREA and BUN in serum of HN rats(P<0.05),ameliorated the pathological damage of renal tissue.SRP played an anti-HN role by acting on advanced glycation end products(AGE)-receptor for advanced glycation end products(RAGE),interleukin-17(IL-17),tumor necrosis factor(TNF)and other signaling pathways.Albumin(ALB),TNF,peroxisome proliferator-activated receptor gamma(PPARG),signal transducer and activator of transcription 3(STAT3)and bone marrow Myelocytomatosis viral oncogene(MYC)had good docking activity with luteolin,quercetin,kaempferol,epigallocatechin gallate and catechin.SRP significantly down-regulated the mRNA expression levels of TNF,STAT3 and ALB in kidney tissue of HN rats(P<0.05),and significantly up-regulated the expression level of PPARG mRNA(P<0.05).Compared with model group,SRP significantly increased the richness and diversity of intestinal flora,and decreased the proportions of bacteroidetes and firmicutes at phylum level;mainly decreased acteroides,akkermansia,ralstonia and prevotellaceae Ga6A1 group,increased the relative abundance of Lactobacillus and Ruminococcaceae UCG-014.Conclusion SRP can exert anti-HN effect by regulating the structure of intestinal flora,regulating expression of AGE-RAGE,IL-17,TNF and other signaling pathways related targets,and has the characteristics of multi-component,multi-target and multi-pathway treatment.