基于网络药理学研究玉屏风颗粒调节IgA肾病模型小鼠Th1/Th2平衡的作用机制

The Mechanism of Yupingfeng Granules(玉屏风颗粒)in Regulating Th1/Th2 Balance in Mice with IgA Nephropathy Model Based on Network Pharmacology

目的:基于网络药理学预测玉屏风颗粒治疗IgA肾病(IgAN)的作用机制并进行机制验证。方法:通过中药系统药理学数据库与分析平台(TCMSP)数据库检索玉屏风颗粒有效成分;从GeneCards、OMIM、TTD数据库获取IgAN靶点,运用Venny2.1.0、Cytoscape 3.7.2软件分别获取玉屏风颗粒与IgAN两者交集靶点,构建“活性成分-交集靶点-疾病”关系网络;使用String数据库构建蛋白互作网络图,运用DAVID数据库进行GO、KEGG富集分析;通过AutoDock Vina软件对玉屏风颗粒中的主要活性成分与关键靶点进行分子对接验证。选取60只雄性昆明小鼠随机分为正常组10只,造模组50只,造模成功后随机分为玉屏风颗粒高、中、低剂量组及西药对照组,给药8周。免疫荧光法检测IgA在肾组织中的沉积情况;HE染色、糖原染色观察肾组织病理学改变;生化法检测24 h-UTP、BUN、Scr;ELISA法检测血清中IgA,Th1相关因子TNF-α、INF-γ、IL-2表达,以及Th2相关因子IL-4、IL-6表达。结果:网络药理学部分,筛选出玉屏风颗粒活性成分34个,药物靶点222个;疾病靶点1436个;交集靶点102个;主要活性成分为槲皮素、山柰酚、汉黄芩素等;关键靶点有TNF、IL-6、IL-4、IL-2等。分子对接结果表明,主要活性成分与关键靶点均能较好的结合,所有结合能均≤-6.0 kJ/mol;GO富集分析得到生物过程689条、细胞组分54条、分子功能115条;KEGG富集分析得到IL-17、NF-κB等信号通路。动物实验部分,肾组织IgA免疫荧光显示,与正常组比较,模型组肾小球系膜区可见强绿色团块状IgA荧光信号;与模型组比较,各治疗组IgA沉积情况均有所改善,其中玉屏风颗粒高剂量组改善较明显。HE染色显示,与正常组比较,模型组可见肾小球肿胀,系膜细胞、内皮细胞增生,细胞外基质增多,系膜区扩张;与模型组比较,各治疗组肾组织病理损伤均有所减轻;糖原染色显示,与正常组比较,模型组可见系膜细胞增生,细胞外基质增多,给予药物治疗后,各治疗组肾组织病变程度由中重度变为中轻度。与正常组比较,模型组24 h-UTP、BUN、Scr明显升高(P<0.01);与模型组比较,各治疗组肾功能均有所改善(P<0.01或P<0.05)。与正常组比较,模型组血清中IgA表达明显升高,与模型组比较,各治疗组血清中IgA表达均有所降低(P<0.05)。与正常组比较,模型组血清中Th1相关因子TNF-α、INF-γ、IL-2表达水平明显降低(P<0.05),各治疗组血清中Th1相关因子TNF-α、INF-γ、IL-2均有所上调。与正常组比较,模型组血清中Th2相关因子IL-4、IL-6表达水平明显升高(P<0.05),使用药物治疗后,各治疗组血清中Th2相关因子IL-4、IL-6表达水平均有所下调。与正常组比较,模型组血清中Th1/Th2比值明显下降(P<0.05),各治疗组血清中Th1/Th2比值均有所上调(P<0.05)。结论:玉屏风颗粒对IgAN模型小鼠具有较好的治疗作用,其机制可能与纠正Th1/Th2失衡有关。

Objective:To predict the mechanism of Yupingfeng granules for the treatment of IgA nephropathy(IgAN)based on network pharmacology and to perform mechanism validation.Methods:The active ingredients of Yupingfeng granules were retrieved from TCMSP database.The IgAN targets were obtained from GeneCards,OMIM and TTD databases,and the intersecting targets of Yupingfeng granules and IgAN were obtained by using the software of Venny 2.1.0 and Cytoscape 3.7.2 to construct the"active ingredient-intersecting target".DAVID database was used to carry out GO and KEGG enrichment analysis.String database was used to construct protein-protein interaction network diagram,and AutoDock Vina software was applied to validate the molecular docking between the main active ingredients and the key targets of Yupingfeng granules.The molecular docking verification was carried out by AutoDock Vina software.Totally 60 male Kunming mice were randomly divided into normal group(n=10)and modelling group(n=50).After successful modelling,the mice were randomly divided into Yupingfeng granules high,medium and low groups,and western medicine group.The mice were administered for eight weeks.Immunofluorescence method was used to detect the deposition of IgA in renal tissues;HE staining and glycogen staining were used to observe the pathological changes of renal tissues;Biochemical method was used to detect 24 h-UTP,BUN and Scr;ELISA method was used to detect the expression of IgA,Th1-related factors,including TNF-α,INF-γand IL-2,and the expression of Th2-related factors,including IL-4 and IL-6.Results:In the cyberpharmacology part,34 active ingredients of Yupingfeng granules were screened,and there were 222 drug targets,1436 disease targets,and 102 intersecting targets.The main active ingredients were quercetin,kaempferol,wogonin and so on.The key targets were TNF,IL-6,IL-4,IL-2 and so on.The results of molecular docking showed that the main active ingredients and key targets could be well combined,and the binding energies were≤-6.0 kJ/mol;GO enrichment analysis obtained 689 biological processes,54 cellular components and 115 molecular functions;KEGG enrichment analysis obtained IL-17,NF-κB and other signaling pathways.In animal experiments,IgA immunofluorescence of renal tissues showed that compared with normal group,the model group showed strong green IgA fluorescence signals in the mesangial area,and that compared with model group,the IgA deposition improved in each treatment group,among which,the improvement was more obvious in Yupingfeng granules high dose group.HE staining showed that compared with normal group,the model group showed glomerular swelling,proliferation of mesangial cells and endothelial cells,increase in extracellular matrix,and expansion of the mesangial area,and that compared with model group,the pathological damage of renal tissue was reduced in each treatment group.Glycogen staining showed that compared with normal group,model group showed proliferation of mesangial cells and increase in extracellular matrix,and that the degree of renal pathological changes of treatment groups changed from moderately severe to moderately mild after the administration of the drug.Compared with the normal group,24 h-UTP,BUN and Scr increased in model group(P<0.01).Compared with model group,renal function was improved in treatment groups(P<0.01 or P<0.05).Compared with normal group,IgA expression in serum increased in model group.Compared with model group,IgA expression in serum was reduced in treatment groups(P<0.05).Compared with normal group,the expression levels of Th1-related factors,including TNF-α,INF-γ,and IL-2,were significantly reduced in model group(P<0.05),and the Th1-related factors,including TNF-α,INF-γand IL-2 were up-regulated in treatment groups.Compared with normal group,the expression levels of Th2-related factors,including IL-4 and IL-6,increased in model group(P<0.05),and the expression levels of Th2-related factors,including IL-4 and IL-6 were down-regulated after the use of drug treatment in treatment groups.Compared with normal group,the serum Th1/Th2 ratio was significantly decreased in model group(P<0.05),and the serum Th1/Th2 ratio was up-regulated in treatment groups(P<0.05).Conclusion:Yupingfeng granules have better therapeutic effect on IgAN model mice,and the mechanism may be related to the correction of Th1/Th2 imbalance.