糖利平胶囊干预2型糖尿病的作用机制研究

Mechanistic study of Tangliping Capsule in the intervention of type 2 diabetes mellitus

关键词:  糖利平胶囊  2型糖尿病  网络药理学  磷脂酰肌醇3激酶/蛋白激酶B信号通路

Key words:  Tangliping Capsule  type 2 diabetes mellitus  network pharmacology  PI3K/Akt signaling pathway

摘要:

[目的] 运用网络药理学和分子对接技术探究糖利平胶囊治疗2型糖尿病（T2DM）的潜在作用机制，并通过体外实验进行初步验证。[方法] 利用TCMSP数据库筛选糖利平胶囊的活性成分，并结合Swiss Target Prediction数据库预测和筛选出活性成分的作用靶点。通过Genecard、OMIM、TTD数据库检索T2DM相关作用靶点，将糖利平胶囊活性成分的作用靶点和T2DM的相关作用靶点导入到VENN在线工具，获得共同靶点，利用STRING在线数据库对共同靶点进行蛋白相互作用（PPI）网络构建并进行分析，筛选出糖利平胶囊治疗T2DM的核心靶点。利用David数据库进行基因本体论（GO）和京都基因与基因组百科全书（KEGG）通路富集分析。采用AutoDockTools对核心靶点与活性成分进行分子对接验证。并进一步采用细胞实验进行验证：选取HepG2细胞，采用胰岛素诱导胰岛素抵抗（IR）模型，蛋白免疫印迹（Western blot）法检测磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）信号通路相关蛋白的表达情况。[结果] 共筛选出34个糖利平胶囊的活性成分和649个成分作用靶点，T2DM相关作用靶点有1 562个；得到193个药物与疾病的共同靶点；通过构建PPI网络获得核心靶点30个；GO功能富集分析共获取334条结果，KEGG通路富集分析得到138条结果，主要包括PI3K/Akt信号通路、糖尿病并发症中的晚期糖基化终末化产物（AGE）/晚期糖基化终末产物（RAGE）受体信号通路、表皮生长因子受体（EGFR）酪氨酸激酶抑制剂耐药性、低氧诱导因子-1（HIF-1）信号通路等。分子对接结果显示，核心靶点Akt1和EGFR与29个活性成分均具有良好的亲和力及结合活性。进一步通过细胞实验验证，糖利平胶囊可以提高IR模型HepG2细胞葡萄糖消耗量（P＜0.01），升高PI3K和Akt蛋白磷酸化水平（P＜0.01），验证了网络药理学的部分预测结果。[结论] 糖利平胶囊可能通过PI3K/Akt信号通路增加IR细胞对葡萄糖的摄取能力，发挥其抗T2DM作用，为后续临床应用与研究提供理论参考和科学依据。

Abstract:

[Objective] Predicting the potential mechanism of action of Tangliping Capsule in the treatment of type 2 diabetes mellitus(T2DM) using network pharmacology methods and molecular docking techniques,and to conduct the preliminary verification through in vitro experiments. [Methods] The active ingredients of Tangliping Capsule were screened using TCMSP database,and the targets of active ingredients were predicted and screened through the Swiss Target Prediction database. The targets related to T2DM were searched through Genecard,OMIM and TTD databases,and the common targets of Tangliping Capsule and T2DM were obtained by VENN online tool. Then,the common targets were imported into the STRING online database for protein interaction(PPI) network construction and analysis,in order to screen out the core targets of Tangliping Capsule for the treatment of T2DM. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis were performed using the David database. The core target and the active ingredients were validated using molecular docking with AutoDockTools. Cell experiments were further used to verify:HepG2 cells were selected,insulin-induced insulin resistance(IR) model was used with high concentration of insulin. The expression of PI3K/Akt signaling pathway-related proteins was detected by Western blotting methods. [Results] A total of 34 active components and 649 target components of Tangliping Capsule were obtained. The 1 562 disease targets of T2DM were obtained,and 193 common targets of drugs and diseases were obtained. A total of 30 core targets are obtained by constructing protein interaction network diagram. A total of 334 results were obtained from GO enrichment and 138 results were obtained from KEGG pathway enrichment analysis. The main pathways included PI3K-Akt signaling pathway,AGE-RAGE signaling pathway in diabetic complications,EGFR tyrosine kinase inhibitor resistance,HIF-1 signaling pathway. Molecular docking results showed that the core targets(Akt1 and EGFR) and 29 active components had good affinity and binding activity. Results of the experimental testing prove that Tangliping Capsule could significantly elevate the glucose consumption in insulin resistance cell models(P<0.01),enhance the p-PI3K and p-Akt protein expression(P<0.01). [Conclusion] Tangliping Capsule may increase the glucose uptake of insulin resistance cells through PI3K/Akt signaling pathway and exert its anti-type 2 diabetes effect,providing theoretical reference and scientific basis for subsequent clinical application and research.