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.