芪明颗粒对糖尿病引起的视网膜病变的治疗作用研究

Study on the Therapeutic Effect of Qiming Granule on Retinopathy Caused by Diabetes

关键词:  芪明颗粒  糖尿病视网膜病变  有效成分  靶点  通路

Key words:  Qiming granule  diabetic retinopathy  active ingredients  targets  pathways

摘要:

目的 基于网络药理学技术探讨芪明颗粒治疗糖尿病视网膜病变（diabetic retinopathy，DR）的作用机制并进行初步实验验证。方法 通过TCMID、TCMSP数据库及文献挖掘筛选芪明颗粒有效成分，利用Drugbank、SwissTargetPrediction及ChEMBL数据库预测筛选成分相关靶点，CTD、GeneCards、Pubmed数据库预测筛选DR相关靶点，重合靶点获取芪明颗粒有效成分治疗DR的相关靶点；采用Cytoscape软件构建靶点蛋白互作网络；分子对接验证核心靶点及对应成分并进行基因本体论及通路注释分析。此外，利用链脲佐菌素（streptozotocin，STZ）诱导建立糖尿病大鼠模型，随机分为模型组（等量生理盐水）和芪明颗粒低、中、高剂量组（3.75，7.5，15 g·kg^-1·d^–1芪明颗粒）4组，同时选取6只正常SD大鼠设为空白组（等量生理盐水），均连续灌胃干预4周。每周监测大鼠体质量、血糖浓度变化，并采用qRT-PCR检测视网膜组织中ALB、TNF-α、IL6、VEGFA、NOS3、MAPK3的mRNA水平。结果 芪明颗粒中共筛选出33个活性成分，包括黄芪苷、异鼠李素、葛根素、梓醇、水蛭素等，与治疗DR的59个靶点相关，其中包含TNF、IL6、VEGFA、TGF-β、NOS3、MAPK3、PTGS2等核心靶点。分子对接结果显示核心靶点与对应活性成分具有良好的结合力。针对靶点的基因本体论及KEGG通路富集分析表明这些靶点涉及了信号传导、糖尿病代谢、免疫系统、肿瘤等生物学过程，以及AGE-RAGE signaling pathway in diabetic complications、Type I diabetes mellitus、Type II diabetes mellitus、VEGF signaling、TGF-β signaling pathways等与DR发生发展直接密切相关的代谢或信号通路。动物实验验证结果显示芪明颗粒可有效提高糖尿病大鼠体质量，降低血糖浓度，同时可显著上调视网膜组织中ALB、VEGFA、NOS3、MAPK3 mRNA表达，下调TNF-α、IL6 mRNA表达，且呈剂量依赖性。结论 芪明颗粒可通过其有效成分上调ALB、VEGFA、NOS3、MAPK3 mRNA表达，下调TNF-α、IL6 mRNA表达发挥治疗DR的作用。

Abstract:

OBJECTIVE To explore the mechanism and conduct preliminary experimental validation of Qiming granules in the treatment of diabetic retinopathy(DR) using the network pharmacology. METHODS The effective components of Qiming granules were screened by TCMID and TCMSP databases and literature mining. The target targets of the components were predicted by Drugbank, SwissTargetPrediction and ChEMBL database. CTD, GeneCards and Pubmed databases were used to predict and screen the DR related targets, and the corresponding targets of Qiming granules for the treatment of DR were obtained by overlapping targets. Cytoscape software was used to construct the target protein interaction network. Core targets and corresponding components were verified by molecular docking, and gene ontology and pathway annotation were analyzed. In addition, streptozotocin(STZ) induction was used to establish a diabetic rat model, diabetic rats were randomly divided into four groups:model group(equal volume of saline), low-, medium- and high-dose group of Qiming granula(3.75, 7.5 and 15 g·kg^-1·d^-1 Qiming granula), meanwhile, six normal SD rats were selected as the blank group(equal volume of normal saline), all of which were continuously gavaged with intervention for four weeks. The rats were monitored weekly for body weight and blood glucose concentration changes, and the mRNA levels of ALB, TNF-α, IL6, VEGFA, NOS3 and MAPK3 in the retinal tissues were determined by qRT-PCR. RESULTS A total of 33 active ingredients including astragaloside Ⅳ, isorhamnetin, puerarin, catalpol, hirudin, and 59 related targets for DR treatment including TNF, IL6, VEGFA, TGF-β, NOS3, MAPK3, PTGS2 of Qiming granule were obtained with the network pharmacology analysis. Molecular docking results demonstrated good binding score between hub targets and relative compounds. GO and KEGG pathway analysis revealed that these targets involved in the multiple signaling pathways, diabetes metabolism, immune system, cancers, etc. What's more, AGE-RAGE signaling pathway in diabetic complications, Type I diabetes mellitus, Type II diabetes mellitus, VEGF signaling, TGF-β signaling pathways were directly related with the occurrence and development of DR. Animal validation results showed that Qiming granules effectively increased the body weight and decreased the blood glucose concentration of diabetic rats, while significantly upregulating ALB, VEGFA, NOS3 and MAPK3 mRNA expression and downregulating TNF-α and IL6 mRNA expression in retinal tissues, and in a dose-dependent manner. CONCLUSION Qiming granula could play a role in the treatment of DR by up regulating ALB, VEGFA, NOS3 and MAPK3 mRNA expression and down regulating TNF-α and IL6 mRNA expression through its active ingredients.