基于网络药理学探讨苍附导痰汤加减方对多囊卵巢综合征大鼠胰岛素抵抗的作用机制

Investigation on Mechanism of action of Modified cangfu daotan decoction on insulin resistance in Polycystic Ovarian Syndrome rats based on network pharmacology

关键词:  多囊卵巢综合征  苍附导痰汤加减方  网络药理学  胰岛素抵抗  生殖激素

Key words:  polycystic ovarian syndrome  modified cangfu daotan decoction  network pharmacology  insulin resistance  reproductive hormone

摘要:

目的 基于网络药理学探讨苍附导痰汤加减方(modified Cangfu Daotan decoction，CFDTD)对多囊卵巢综合征(polycystic ovarian syndrome，PCOS)大鼠胰岛素抵抗(insulin resistance，IR)的影响。方法 利用TCMSP、TCM Database@Taiwan、Drugbank、GeneCards数据库检索CFDTD中的活性化合物并筛选作用靶点；从TTD、PubMed及PharmGKB数据库检索与PCOS相关的疾病靶点，成分靶点与疾病靶点映射后使用Cytoscape 3.7.1软件构建药物有效成分-靶点蛋白相互作用网络。然后，采用STRING和Cytoscape软件构建蛋白相互作用网络并筛选核心靶点。随后，利用Cytoscape软件中的ClueGO插件进行Kyoto Encyclopedia of Genes and Genomes(KEGG)通路分析。最后，采用来曲唑联合高糖高脂饮食构建PCOS伴IR大鼠模型加以验证；给药组分别给予15，30 g·kg^-1·d^-1CFDTD及50 mg·kg^-1·d^-1盐酸二甲双胍(metformin hydrochloride，MH)灌胃；对照组和PCOS组给予等体积生理盐水灌胃，干预4周。全自动化学分析仪检测大鼠空腹血糖(fasting blood glucose，FPG)、空腹胰岛素(fasting insulin，FINS)、促卵泡生长激素(follicle stimulating hormone，FSH)、促黄体生成素(luteinizing hormone，LH)、睾酮(testosterone，T)、雌二醇(estradiol，E₂)含量差异并计算胰岛素抵抗指数(homeostatic model assessment insulin resistance index，HOMA-IR)。HE染色观察大鼠卵巢组织病理变化。Western blotting检测大鼠卵巢组织胰岛素受体(insulin receptor，INSR)、胰岛素样生长因子-I(insulin-like growth factor-I，IGF-I)、胰岛素样生长因子I受体(insulin-like growth factor-I receptor，IGF-IR)、胰岛素(insulin，INS)、肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)、白介素-6(interleukin-6，IL-6)蛋白表达水平。结果 CFDTD筛选得到86个活性成分及126个CFDTD治疗PCOS潜在相关靶点。KEGG分析结果显示，潜在靶点主要涉及insulin resistance，steroid hormone biosynthesis，ovarian steroidogenesis，estrogen signaling pathway，PI3K-Akt signaling pathway，type II diabetes mellitus等信号通路。蛋白相互作用分析提示INS、TNF-α及IL-6为蛋白相互作用网络中的关键靶点。与PCOS组相比，给药组大鼠血清FPG、FINS、HOMA-IR、LH、T、E₂的含量明显减少(P均<0.01)及卵巢组织形态明显恢复。Western blotting检测结果显示CFDTD显著降低PCOS大鼠卵巢组织INSR、IGF-IR、INS、TNF-α及IL-6蛋白表达。结论 CFDTD可能通过下调INSR、IGF-IR、INS、TNF-α及IL-6蛋白表达，改善PCOS大鼠IR。

Abstract:

OBJECTIVE To explore the effects of modified Cangfu Daotan decoction(CFDTD) on insulin resistance(IR) in polycystic ovarian syndrome(PCOS) rats based on network pharmacology analysis. METHODS TCMSP, TCM database@Taiwan, Drugbank and GeneCards databases were used to screen out the active chemical components and action targets of CFDTD. TTD, PubMed, and PharmGKB databases were used to screen out the disease targets associated with PCOS. After mapping the component target with the disease targets, the Cytoscape 3.7.1 software was used to construct the component-target interaction network of CFDTD. Next, STRING and Cytoscape software was used to construct protein-protein interaction network and identify the hub targets. Subsequently, Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis was performed by using the ClueGO plug-in tool in Cytoscape. Furthermore, experimental validation was conducted in the PCOS with IR model rats induced by letrozole combined with high-fat and high-sugar diet. The treated groups were given CFDTD at a dose of 15 and 30 g·kg^-1·d^-1, and metformin hydrochloride(MH, 50 mg·kg^-1·d^-1) respectively, the control and PCOS groups were given equivoluminal normal saline for 4 weeks. The serum levels of fasting blood glucose(FPG), fasting insulin(FINS), follicle stimulating hormone(FSH), luteinizing hormone(LH), testosterone(T) and estradiol(E₂) of rats were detected by automatic chemical analyzer, and the homeostatic model assessment insulin resistance index(HOMA-IR) was then calculated. HE staining was used to observe the morphological changes of ovarian tissues. The protein expression levels of insulin receptor(INSR), insulin-like growth factor-I(IGF-I), insulin-like growth factor-I receptor(IGF-IR), insulin(INS), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) were detected by Western blotting. RESULTS The 86 active components of CFDTD and 126 potential targets related to PCOS were screened by network pharmacology analysis. KEGG analysis showed that the potential targets were closely related to signaling pathway such as insulin resistance, steroid hormone biosynthesis, ovarian steroidogenesis, estrogen signaling pathway, PI3K-Akt signaling pathway, type II diabetes mellitus, etc. Protein interaction analysis indicated that INS, TNF-α, and IL-6 were the hub targets of protein interaction network. Compared with the PCOS group, the levels of FPG, FINS, HOMA-IR, LH, T and E₂ in the treated groups decreased significantly(P all<0.01), and the damaged degree of ovarian tissue was significantly reduced. Western blotting showed that CFDTD could significantly decrease the protein expression levels of INSR, IGF-IR, INS, TNF-α, and IL-6. CONCLUSION CFDTD can improve the IR in PCOS rats by downregulate the protein expression levels of INSR, IGF-IR, INS, TNF-α, and IL-6.