油酸诱导胰岛素抵抗HepG2细胞模型优化及小檗碱、黄芩苷、葛根素和甘草苷的体外降糖作用研究

Optimization of oleic acid-induced insulin resistance HepG2 cell model and anti-hyperglycemic effect of berberine,baicalin,puerarin and liquiritin

目的以油酸诱导脂代谢紊乱建立肝胰岛素抵抗(IR)细胞模型,研究中药单体成分小檗碱、黄芩苷、葛根素和甘草苷的体外降糖作用,为中药降糖组方或成分配伍优化提供研究基础。方法采用油酸不同浓度(0.1、0.2、0.5、1.0 mmol/L)及不同作用时间点(24、36、48 h)诱导HepG2细胞,建立IR-HepG2细胞模型,葡萄糖氧化酶法测定细胞葡萄糖消耗量,CCK-8法检测细胞活力,确立模型的油酸最佳诱导浓度及最佳作用时间;蒽酮法和三酰甘油氧化酶法检测IR模型细胞内肝糖原和三酰甘油水平;油红O染色检测细胞形态的变化;观察葡萄糖消耗量检测IR模型的稳定性。采用油酸最佳诱导浓度及最佳作用时间建立IR-HepG2细胞模型,研究不同剂量小檗碱、葛根素、黄芩苷和甘草苷对细胞葡萄糖消耗量、糖原含量、三酰甘油及细胞活力的影响。结果油酸诱导IR-HepG2细胞模型最佳浓度1 mmol/L,最佳作用时间24 h,此时与对照组比较,HepG2细胞肝糖原含量明显下降(P<0.001),三酰甘油显著上升(P<0.01),模型建立后可至少持续稳定36 h以上。与IR模型组比较,给药干预24 h后,不同浓度小檗碱(5、10、20、50μmol/L)、黄芩苷(1、5、10、20、50μmol/L)、葛根素(20、40、80、160μmol/L)和1μmol/L甘草苷显著增加葡萄糖消耗量(P<0.05、0.01、0.001)。与IR组比较,不同浓度小檗碱(10、20、50μmol/L)、黄芩苷(20、50μmol/L)、葛根素(10、20、80、160μmol/L)显著提高肝糖原含量(P<0.001);甘草苷升高糖原含量但是差异不显著。与对照组比较,除160μmol/L葛根素和1μmol/L黄芩苷显著抑制细胞活力(P<0.05)外,其他组对细胞活力的影响均不显著,结论 1 mmol/L油酸诱导24 h后能够建立稳定IR-HepG2细胞模型,适合作为高脂饮食诱导2型糖尿病的体外肝IR细胞模型。小檗碱、黄芩苷和葛根素显著增加油酸诱导的IR-HepG2细胞葡萄糖消耗量和糖原合成,进而改善脂代谢诱发的肝IR。

Objective To investigate the anti-hyperglycemic effect of the Chinese medicine ingredients such as berberine, baicalin, puerarin and liquiritin on the optimal insulin resistance （IR）-HepG2 cell model by oleic acid. It would provide the theoretical basis for the optimization of Chinese medicine prescription or anti-hyperglycemic components combination. Methods Different concentrations （0.1, 0.2, 0.5, and 1.0 mmol/L） of oleic acid were used to induce HepG2 cells for different time （24, 36 and 48 h）, the glucose consumption was measured by glucose oxidase assay, and cell viability was detected by CCK-8 assay to define the optimal inducing concentration and time for IR-HepG2 cell model. Then the cell morphological changes were detected by oil red O staining. Finally, the stability of IR-HepG2 cell model was tested. After the IR-HepG2 model was optimally established, the glucose consumption, glycogen content and cell viability were detected after 24 h administration with different concentrations of berberine, baicalin, puerarin and liquiritin by anthrone method, glycerol phosphate oxidase assay and CCK-8 assay respectively. Results The optimal oleic acid-induced concentration was 1 mmol/L and the optimal induced time was 24 h for the IR-HepG2 cell model that could keep stable more than 36 h. Comparing with IR model group, berberine, puerarin and baicalin significantly increased the glucose consumption, whereas liquiritin did not show significant change in the glucose consumption except for 1 ~trnol/L. Only 160 Ixmol/L puerarin and 1 μmol/L baicalin significantly inhibited IR-HepG2 cell viability. Moreover, berberine, puerarin, and baicalin significantly elevated the glycogen content; Liquiritin did not change glycogen content significantly. Conclusion The IR-HepG2 cell model could be stably established with 24 h treatment of 1 mmol/L oleic acid. Berberine, puerarin, and baicalin significantly increased the glucose consumption and glycogen content in the IR-HepG2 cells. The results suggest that berberine, baicalin and puerarin maybe perform different pathways of anti-hyperglycemic effects due to different incentives ofIR.