Wan Mei-Xuan, Huang Xian-Jun, Li Xue, Suan Juan, Xu Li
College of Basic Medicine, Dali University, Dali, China.
College of Pharmaceutical Science, Dali University, Dali, China.
Front Pharmacol. 2022 Oct 10;13:1006660. doi: 10.3389/fphar.2022.1006660. eCollection 2022.
This study was designed to evaluate the pharmacological mechanisms of puerarin against oliguria in acute alcoholism network pharmacology analysis combined with experimental verification. First, this study established an acute alcoholism rat model, compared the changes in urine volume in each group, and observed the therapeutic effect of puerarin by H&E staining, biochemical, RT-qPCR, and immunohistochemical analyses. Second, puerarin-related targets were searched in TCMS, PubChem, CNKI, Wanfang, PubMed, and GeenMedical Academic databases. Also, potential disease targets were obtained from the GeneCards, MalaCards, and NCBI-gene databases and genes with puerarin target gene intersections were screened out. The interaction network for co-predicted targets was obtained using the STRING database, and the core targets were imported into Cytoscape for visualization using DAVID Bioinformatics Resources 6.8. The essential genes were subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway enrichment analyses to predict related biological processes and significant signaling pathways. Finally, molecular docking was used to examine the interaction of puerarin with key targets, and the core targets were validated further by RT-qPCR and Western blotting. Compared to the model group, the urine volume of the rats was significantly increased after puerarin treatment, and the levels of anti-diuretic hormone (ADH) and aquaporin 2 (AQP) expression were decreased. Searching the intersection of puerarin and acute alcoholism targets yielded 214 potential targets, 837 biological processes, and 185 signaling pathways involved. The molecular docking results indicated a good affinity between puerarin and key targets (cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), cAMP-response element-binding protein (CREB), and c-Fos). RT-qPCR and Western blotting further verified that puerarin could down-regulate the expression of cAMP/PKA/CREB/c-Fos. This study identified the potential targets of puerarin against oliguria in rats with acute alcoholism using network pharmacology and animal experiments. The mechanism may be closely related to the cAMP signaling pathway.
本研究旨在通过网络药理学分析结合实验验证，评估葛根素治疗急性酒精中毒性少尿的药理机制。首先，本研究建立急性酒精中毒大鼠模型，比较各组尿量变化，并通过苏木精-伊红染色、生化检测、逆转录-定量聚合酶链反应（RT-qPCR）和免疫组织化学分析观察葛根素的治疗效果。其次，在中药系统药理学数据库（TCMS）、化学物质数据库（PubChem）、中国知网（CNKI）、万方数据库、美国国立医学图书馆生物医学数据库（PubMed）和医脉通学术数据库（GeenMedical）中检索葛根素相关靶点。此外，从基因卡片数据库（GeneCards）、疾病卡片数据库（MalaCards）和美国国立生物技术信息中心基因数据库（NCBI-gene）中获取潜在疾病靶点，并筛选出与葛根素靶基因有交集的基因。使用STRING数据库获得共预测靶点的相互作用网络，并将核心靶点导入Cytoscape中，利用DAVID生物信息学资源6.8进行可视化。对关键基因进行京都基因与基因组百科全书（KEGG）和基因本体论（GO）通路富集分析，以预测相关生物学过程和重要信号通路。最后，采用分子对接研究葛根素与关键靶点的相互作用，并通过RT-qPCR和蛋白质免疫印迹法进一步验证核心靶点。与模型组相比，葛根素治疗后大鼠尿量显著增加，抗利尿激素（ADH）和水通道蛋白2（AQP）表达水平降低。检索葛根素与急性酒精中毒靶点的交集，得到214个潜在靶点、837个生物学过程和185条信号通路。分子对接结果表明葛根素与关键靶点（环磷酸腺苷（cAMP）、蛋白激酶A（PKA）、cAMP反应元件结合蛋白（CREB）和原癌基因c-Fos）之间具有良好的亲和力。RT-qPCR和蛋白质免疫印迹法进一步验证了葛根素可下调cAMP/PKA/CREB/c-Fos的表达。本研究通过网络药理学和动物实验确定了葛根素治疗急性酒精中毒大鼠少尿的潜在靶点。其机制可能与cAMP信号通路密切相关。
