目的 通過網(wǎng)絡(luò)藥理學(xué)研究預(yù)測分心木總黃酮（TFDJF）的主要活性成分和作用靶點，探討其多成分-多靶點-多通路的抗2型糖尿病（T2DM）的作用機(jī)制；并選取AKT/FoxO1信號通路進(jìn)行細(xì)胞實驗驗證。方法 基于文獻(xiàn)檢索構(gòu)建分心木黃酮類成分的化學(xué)成分?jǐn)?shù)據(jù)庫，并以"類藥五原則"篩選潛在的活性成分，采用PharmMapper服務(wù)器反向藥效團(tuán)匹配方法預(yù)測活性成分可能的作用靶標(biāo)，并根據(jù)TTD、OMIM數(shù)據(jù)庫中與T2DM相關(guān)的靶點，篩選分心木黃酮類成分抗T2DM的作用靶標(biāo)，通過對靶標(biāo)的基因本體（GO）功能富集分析和KEGG通路分析，構(gòu)建活性成分-抗T2DM作用靶點-代謝通路網(wǎng)絡(luò)圖，探討TFDJF抗T2DM的作用機(jī)制。采用系統(tǒng)對接網(wǎng)站進(jìn)行GSK3B和AKT1對山柰酚的分子對接。并進(jìn)一步采用細(xì)胞實驗進(jìn)行驗證：選取HepG2細(xì)胞，運用高濃度胰島素誘導(dǎo)胰島素抵抗（IR）模型，150 μg/mL TFDJF處理細(xì)胞36 h，氧化酶法檢測各組細(xì)胞的葡萄糖消耗量，Western blotting法檢測AKT-FoxO信號通路相關(guān)蛋白的表達(dá)。結(jié)果 共獲得分心木潛在的黃酮類活性成分10個，預(yù)測成分作用靶標(biāo)共104個，篩選出與抗T2DM相關(guān)的關(guān)鍵靶點15個，KEGG通路富集分析發(fā)現(xiàn)TFDJF抗T2DM的作用機(jī)制可能與胰島素、FoxO、AMPK和PI3K-AKT等信號通路有關(guān)。山柰酚與GSK3B有強(qiáng)烈的結(jié)合活性，與AKT1有較好的結(jié)合活性。進(jìn)一步通過細(xì)胞實驗驗證，TFDJF可顯著提高HepG2細(xì)胞IR模型葡萄糖消耗量（P<0.01），顯著升高AKT蛋白磷酸化水平（P<0.01），顯著降低FoxO1蛋白表達(dá)水平（P<0.01），驗證了網(wǎng)絡(luò)藥理學(xué)的部分預(yù)測結(jié)果。結(jié)論 TFDJF抗T2DM的作用機(jī)制可能與胰島素、FoxO、AMPK和PI3K-AKT等信號通路有關(guān)。

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.

國家自然科學(xué)基金資助項目（81373546、30472125、81273660）