黄柏碱-11-O-β-D-葡萄糖醛酸苷的制备及对胰岛素抵抗HepG2细胞糖脂代谢的影响

Preparation of Phellodendrine-11-O-β-D-Glucuronide and Its Effects on Glycolipid Metabolism in Insulin-Resistant HepG2 Cells

    摘要
  • 摘要:
    目的 建立黄柏碱(PHE)代谢产物黄柏碱-11-O-β-D-葡萄糖醛酸苷(M1)的制备方法，探讨M1调节胰岛素抵抗(IR)-HepG2细胞糖脂代谢紊乱的作用及机制。
    方法 利用肠微粒体体外孵育法结合高效液相、质谱、核磁共振(NMR)等技术完成对M1的制备、提取、分离纯化及鉴定。以二甲双胍为阳性对照，考察M1低(25 μmol·L-1)、中(50 μmol·L-1)、高(100 μmol·L-1)剂量给药IR-HepG2细胞24 h对其葡萄糖消耗量、糖吸收、甘油三酯(TG)、胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)的影响。通过分子对接技术探讨M1与IRS1/PI3K/AKT通路各关键因子胰岛素表面受体(INSR)、胰岛素受体底物1(IRS1)、PI3K、AKT2的结合能力。采用qPCR法及Western blot法分别检测M1对IRS1/PI3K/AKT通路各关键因子mRNA及蛋白表达的影响。
    结果 经NMR确认分离纯化所得产物为黄柏碱-11-O-β-D-葡萄糖醛酸苷(纯度为95%)。与模型组相比，M1在各给药浓度均可显著提高IR-HepG2细胞葡萄糖消耗量(P＜0.01，P＜0.001)及糖吸收水平(P＜0.001)，且作用比PHE更强(P＜0.001);在中、高给药浓度可显著降低细胞TG及TC含量(P＜0.001)，其中在TG水平上其改善作用优于PHE(P＜0.01);在各浓度均明显降低LDL-C含量(P＜0.001)，提高HDL-C含量(P＜0.01，P＜0.001)。分子对接结果显示M1与通路关键因子INSR、IRS1、PI3K、AKT2具有良好结合活性。与模型组相比，M1给药组不同程度提高INSR、IRS1、PI3K、AKT2、GLUT2 mRNA的表达，并且下调p-IRS1/IRS1蛋白表达(P＜0.001)，上调PI3K、p-AKT/AKT、p-GSK3β/GSK3β蛋白表达(P＜0.05，P＜0.001)。
    结论 建立的方法可用于制备高纯度M1, M1通过调控IRS1/PI3K/AKT信号通路改善糖脂代谢紊乱，从而缓解IR。

     

    Abstract:
    OBJECTIVE To establish a preparation method for Phellodendrine-11-O-β-D-glucuronide (M1), a metabolite of Phellodendrine(PHE), and to evaluate its regulatory effects on glycolipid metabolism disorder in insulin-resistant HepG2 (IR-HepG2) cells.
    METHODS The preparation, extraction, separation, purification and identification of M1 were completed by in vitro incubation of intestinal microsomes combined with liquid phase, mass spectrometry and NMR. With Metformin as positive control, IR-HepG2 cells were treated with low (25 μmol ·L-1), medium (50 μmol ·L-1) and high (100 μmol ·L-1) doses of M1 for 24 h. Glucose consumption, glucose uptake, triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were quantified. The binding ability of M1 to the key targets INSR, IRS1, PI3K and AKT2 in the IRS1/PI3K/AKT pathway was explored through molecular docking technology. The effects of M1 on the mRNA and protein expression of these key targets in the IRS1/PI3K/AKT pathway were detected by qPCR and Western blot, respectively.
    RESULTS The purified product was confirmed by NMR as Phellodendrine-11-O-β-D-glucuronide (95% purity). Compared to the model group, all M1 doses significantly enhanced glucose consumption (P < 0.01, P < 0.001) and uptake (P < 0.001) in IR-HepG2 cells, outperforming PHE (P < 0.001). At medium and high doses, it could significantly reduce the content of TG and TC in cells (P < 0.001), and its improvement effect at the TG level was better than that of PHE (P < 0.01);while all concentrations decreased LDL-C (P < 0.001) and increased HDL-C (P < 0.01, P < 0.001). Molecular docking revealed strong binding interactions between M1 and INSR, IRS1, PI3K, and AKT2. Compared with the model group, M1 administration group increased the expression of INSR, IRS1, PI3K, AKT2, and GLUT2 mRNA to varying degrees, downregulated the protein expression of p-IRS1/IRS1 (P < 0.001), and upregulated the protein expression of PI3K, p-AKT/AKT, and p-GSK3β/GSK3β.
    CONCLUSION The established protocol enables high purity M1 preparation. M1 alleviates IR by regulating IRS1/PI3K/AKT signaling pathway to improve the disorder of glycolipid metabolism.

     

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