[关键词]
[摘要]
目的 通过网络药理学研究预测分心木总黄酮(TFDJF)的主要活性成分和作用靶点,探讨其多成分-多靶点-多通路的抗2型糖尿病(T2DM)的作用机制;并选取AKT/FoxO1信号通路进行细胞实验验证。方法 基于文献检索构建分心木黄酮类成分的化学成分数据库,并以"类药五原则"筛选潜在的活性成分,采用PharmMapper服务器反向药效团匹配方法预测活性成分可能的作用靶标,并根据TTD、OMIM数据库中与T2DM相关的靶点,筛选分心木黄酮类成分抗T2DM的作用靶标,通过对靶标的基因本体(GO)功能富集分析和KEGG通路分析,构建活性成分-抗T2DM作用靶点-代谢通路网络图,探讨TFDJF抗T2DM的作用机制。采用系统对接网站进行GSK3B和AKT1对山柰酚的分子对接。并进一步采用细胞实验进行验证:选取HepG2细胞,运用高浓度胰岛素诱导胰岛素抵抗(IR)模型,150 μg/mL TFDJF处理细胞36 h,氧化酶法检测各组细胞的葡萄糖消耗量,Western blotting法检测AKT-FoxO信号通路相关蛋白的表达。结果 共获得分心木潜在的黄酮类活性成分10个,预测成分作用靶标共104个,筛选出与抗T2DM相关的关键靶点15个,KEGG通路富集分析发现TFDJF抗T2DM的作用机制可能与胰岛素、FoxO、AMPK和PI3K-AKT等信号通路有关。山柰酚与GSK3B有强烈的结合活性,与AKT1有较好的结合活性。进一步通过细胞实验验证,TFDJF可显著提高HepG2细胞IR模型葡萄糖消耗量(P<0.01),显著升高AKT蛋白磷酸化水平(P<0.01),显著降低FoxO1蛋白表达水平(P<0.01),验证了网络药理学的部分预测结果。结论 TFDJF抗T2DM的作用机制可能与胰岛素、FoxO、AMPK和PI3K-AKT等信号通路有关。
[Key word]
[Abstract]
Objictive This paper aimed to investigate the anti-T2DM activity of total flavonoids of Diaphragma Juglandis Fructus (TFDJF), screen anti-T2DM compounds and predict targets of anti-T2DM to explore its multi-component, multi-target and multipathway anti-T2DM mechanism. AKT/FoxO1 signaling pathway was selected for cell experiment. Methods A database of chemical components of flavonoids of Diaphragma Juglandis Fructus was established through literature retrieval, and then the data were combined with five principles of drug absorption to screen active constituents. The targets of active constituents were predicted by using the reverse pharmacophore matching method and screened according to the anti-T2DM related targets in the databases such as TTD, OMIM, et al, and further analyzed the gene GO function and the KEGG pathway enrichment. And the active constituentstargets-pathways network model was also established to study the mechanisms of anti-T2DM of TFDJF. The molecular docking of kaempferol between GSK3B and AKT1 was carried out on the system docking website. 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 cells were treated with 150 μg/mL TFDJF for 36 hours. The glucose consumption of each group was measured by oxidase method, and the expression of AKT-FoxO signal pathway-related proteins was detected by Western blotting methods. Results This study screened 10 active constituents of flavonoids in Diaphragma Juglandis Fructus Totally 104 targets were predicted, and 15 targets related to the anti-T2DM effects were screened. The potential regulatory pathways included Insulin signaling pathway, FoxO signaling pathway, AMPK signaling pathway, PI3K-AKT signaling pathway and so on. Kaempferol has strong binding activity with GSK3B and good binding activity with AKT1. Results of the experimental testing prove that TFDJF could significantly elevate the glucose consumption in insulin resistance cell models (P<0.01), enhance the p-AKT protein expression and decrease the FoxO1 protein expression (P<0.01). Conclusion The anti-T2DM mechanism of TFDJF may be related to the signaling pathways of insulin, FoxO, AMPK and PI3K-AKT.
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[基金项目]
国家自然科学基金资助项目(81373546、30472125、81273660)