小檗碱对体外HepG2细胞IR模型抗IR的效应及机制

Effect of Berberine on HepG2 IR Model and Its Mechanism

目的：探讨小檗碱对HepG2细胞胰岛素抵抗（IR）模型的改善作用及机制。方法：HepG2细胞采用高糖（25 mmol·L（-1））和高胰岛素（1×10（-6）mol·L（-1））联合诱导24 h,建立体外IR模型。模型培养液中分别加入1×10（-5）,1×10（-6）mol·L（-1）小檗碱,设1×10-3mol·L（-1）二甲双胍作为阳性药,孵育24 h。葡萄糖氧化酶法检测培养液葡萄糖含量,计算细胞葡萄糖消耗量,细胞增殖活性检测（CCK-8）测定细胞增殖活性;生化法检测细胞丙酮酸激酶（PK）活性,酶联免疫吸附试验（ELISA）检测细胞葡萄糖激酶（GCK）和葡萄糖转运蛋白2（GLUT2）的活性;实时荧光定量聚合酶链式反应（Real-time PCR）检测细胞胰岛素受体（InsR）,磷脂酰肌醇-3激酶（PI3K）,丝氨酸/苏氨酸蛋白激酶B（Akt）和GLUT2 mRNA表达水平,蛋白免疫印迹法（Western blot）检测各组细胞胰岛素受体底物-1（IRS-1）,磷酸化的磷脂酰肌醇-3激酶（p-PI3K）和磷酸化的丝氨酸/苏氨酸蛋白激酶B（p-Akt）的表达水平。结果：与正常组比较,模型组细胞葡萄糖消耗量显著降低（P〈0.01）,PK,GCK,GLUT2蛋白活性显著降低（P〈0.01）,InsR,PI3K,Akt,GLUT2 mRNA表达水平显著降低（P〈0.01）,IRS-1,p-PI3K,p-Akt蛋白表达水平显著降低（P〈0.01）。与模型组比较,小檗碱组和二甲双胍组的细胞葡萄糖消耗量明显增加（P〈0.01）,PK,GCK,GLUT2蛋白活性显著升高（P〈0.01）,InsR,PI3K,Akt,GLUT2 mRNA和IRS-1,p-PI3K,p-Akt1蛋白表达水平均明显升高（P〈0.05,P〈0.01）。结论：小檗碱体外对HepG2细胞IR模型有显著的改善作用,其机制可能通过调节IRS-1/PI3K/Akt信号通路促进肝脏葡萄糖转运和改善葡萄糖代谢。

Objective： To investigate the effect of berberine on HepG2 insulin resistance（ IR） model and its mechanism. Method： The IR model was established by combining high-glucose（ 25 mmol·L（-1）） with highinsulin（ 1 × 10（-6） mol ·L（-1）） for 24 h. And then, high-dose（ 1 × 10-5 mol·L（-1）） berberine, low-dose（ 1 × 10（-6） mol·L（-1）） berberine,and melbine（ 1 × 10（-3） mol·L（-1）,positive control） were respectively added in medium and cultured for another 24 h. At last,medium and HepG2 cells were collected. Glucose level was detected by glucose oxidase detection method,and cell reproductive capacity was detected by cell counting kit-8（ CCK-8）; pyruvate kinase（ PK） was detected by biochemical analyzer; glucokinase（ GCK） and glucose transporter 2（ GLUT2） were detected by enzyme linked immunosorbent assay（ ELISA） kits. The mRNA expressions of insulin receptor（ InsR）,phosphatidylinositol-3-kinase（ PI3 K）,protein kinase B（ Akt）,GLUT2 were detected by Real-time PCR. The proteins expressions of insulin receptor substrate-1（ IRS-1）,PI3 K,and pAkt were detected by Western blot. Result： Compared with normal group,the glucose consumption of the model group was significantly decreased（ P〈 0. 01）,and the activities of PK,GCK and GLUT2 were significantly decreased（ P〈 0. 01）. Compared with model group,Berberine and metformin groups significantly increased cell glucose consumption,and PK,GCK and GLUT2 protein activities notably increased（ P〈 0. 01）. Compared with normal group,the mRNA expressions of InsR,PI3 K,Akt and GLUT2 and the protein expressions of IRS-1,PI3 K and Akt were significantly decreased（ P〈 0. 01）. Compared with model group,berberine and metformin groups significantly increased protein expression of InsR,PI3 K,Akt and GLUT2（ P〈 0. 05,P〈 0. 01）. Conclusion：Berberine has a noteworthy therapeutic effect on the insulin resistance of HepG2 cells. Its mechanism may be correlated with the promotion of hepatic glucose metabolism by regulating IRS-1/PI3 K/Akt signaling pathway.