基于PI3K/AKT/mTOR信号通路研究黄莪胶囊抗良性前列腺增生的作用及机制

Study on the effect and mechanism of anti-benign prostatic hyperplasia of Huange Capsule based on PI3K/AKT/mTOR signaling pathway

关键词:  良性前列腺增生  黄莪胶囊  网络药理学  PI3K/AKT/mTOR信号通路

Key words:  benign prostatic hyperplasia  huange capsule  network pharmacology  PI3K/AKT/mTOR signalling pathway

摘要:

目的 基于网络药理学方法探讨黄莪胶囊（Huange Capsule,HE）通过PI3K/AKT/mTOR信号通路抗良性前列腺增生（benign prostatic hyperplasia,BPH）的作用及其机制。方法 构建丙酸睾酮诱导BPH大鼠模型，观察大鼠体重、前列腺湿重、前列腺指数，评价黄莪胶囊对BPH的抑制作用；苏木素-伊红染色实验观察大鼠前列腺腺腔，检测各组大鼠上皮细胞厚度、腺腔面积及乳头突起的长度；ELISA检测大鼠血清中前列腺特异性抗原（PSA）的表达；借助TCMSP、TCMID数据库筛选黄莪胶囊主要化学成分和潜在靶点；整合OMIM、GeneCards、DisGeNET数据库获得BPH相关靶点；利用Cytoscape3.7.2构建蛋白相互作用网络；采用Metascape数据库进行GO和KEGG通路富集分析；通过AutoDock软件进行分子对接，并用Pymol进行结果可视化；免疫印记法验证黄莪胶囊抗前列腺增生KEGG筛选的作用机制。结果 黄莪胶囊降低BPH大鼠前列腺湿重、指数，显著改善前列腺组织的形态，使前列腺的上皮细胞厚度减少、乳头突起减少、腺腔面积减小；黄莪胶囊降低了BPH模型大鼠PSA的表达。筛选得到黄莪胶囊85个有效药效成分；与BPH相关的137个潜在有效靶点，相关作用靶点为TP53、AKT1、ESR1、CASP3、JUN、MYC、TNF、IL6，治疗BPH有效成分槲皮素、山奈酚、木犀草素、β-谷甾醇；富集分析分别得到GO功能2193条，KEGG富集分析195条，其抗BPH关键信号通路主要涉及癌症通路、磷脂酰肌醇3-激酶（Phosphatidylinositol 3-kinase,PI3K）/蛋白激酶B（Protein kinase B, AKT）信号通路、脂质与动脉粥样化、乙型肝炎、人巨细胞感染、疱疹病毒感染等。通过对前列腺组织进行蛋白表达验证发现黄莪胶囊能降低其PI3K/AKT/mTOR信号通路相关蛋白的表达。结论 黄莪胶囊通过抑制PI3K/AKT/mTOR信号通路改善BPH。

Abstract:

Objective A network-based pharmacological approach was used to investigate the anti-benign prostatic hyperplasia (BPH) effect of Huange Capsule (HE) and its mechanism through the PI3K/AKT/mTOR signalling pathway.Methods Testosterone propionate-induced BPH was established as a rat model, and body weight, prostate wet weight, and prostate index were observed. The body weight, prostate wet weight, and prostate index of the rats were measured to evaluate the inhibitory effect of the capsule on BPH. H&E staining was performed to observe the glandular cavity of the rat prostate, and the thickness of the epithelium, glandular cavity area, and length of the papillae were detected in each group. ELISA was performed to examine the expression of prostate-specific antigens in the sera of rats, and TCMSP and TCMID databases were used to screen for the main chemical components and potential targets of the capsule. Integration of OMIM, GeneCards, and DisGeNET databases to obtain BPH-related targets, construction of protein interaction networks using Cytoscape 3.7.2, GO and KEGG pathway enrichment analysis using the Metascape database, validation of the mechanism of action of huange capsule in anti-prostate hyperplasia KEGG screening by immunoblotting.Results The capsule reduced the wet weight and index of the prostate gland in BPH rats, and significantly improved the morphology of prostate tissue, which resulted in a reduction in the thickness of the epithelium of the prostate, a reduction in the number of papillae protrusions, an increase in the area of the gland lumen, and reduced the expression of prostate-specific antigens in BPH model rats. The 85 active ingredients of the capsule were screened; 137 potentially effective targets were related to BPH, and the relevant targets were TP53, AKT1, ESR1, CASP3, JUN, MYC, TNF, and IL6.The active ingredients for the treatment of BPH are quercetin, kaempferol, luteolin and β-sitosterol, which yielded 2,193 GO functions and 195 KEGG functions. The key signalling pathways of anti-BPH were mainly involved in the phosphatidylinositol 3-kinase, PI3K, protein kinase B, and AKT signalling pathways. The key anti-BPH signalling pathways are mainly related to the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signalling pathway. The protein expression in prostate tissues was verified,it was found that huange capsules could significantly reduce the expression of PI3K/AKT/mTOR signalling pathway-related proteins in prostate tissues.Conclusion Huange Capsule can improve benign prostatic hyperplasia by the mechanism of anti-prostatic hyperplasia through PI3K/AKT/mTOR signalling pathway.