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目的探索Mibefradil在体外对胰岛素抵抗HepG2细胞的作用。方法将HepG2细胞分为对照组、棕榈酸盐(Palmitate,PA)诱导的HepG2细胞胰岛素抵抗模型组、药物溶剂(DMSO)组、低剂量(0.025μmol/L)Mibefradil组、高剂量(0.05μmol/L)Mibefradil组,通过检测各组糖原合成及葡萄糖消耗,观察Mibefradil对胰岛素抵抗HepG2细胞的改善作用。用qRT-PCR、Western blot检测各组Fox O1,糖异生、糖原分解限速酶,即:磷酸烯醇式丙酮酸羧基激酶(PEPCK)、葡萄糖-6-磷酸激酶(G6Pase)的mRNA、蛋白相对表达量,明确转录因子Fox O1及其靶基因的表达水平。结果相比于对照组,HepG2细胞胰岛素抵抗模型组的糖原合成量[(3.28±0.74)μg/μL vs(9.14±0.33)μg/μL,P<0.01]及葡萄糖消耗量[(1.31±0.49)nmol/μg vs(5.87±2.26)nmol/μg,P<0.01]明显下降;经Mibefradil干预后,胰岛素抵抗HepG2细胞的糖原合成及葡萄糖消耗得到明显改善,且高剂量组的糖原合成[(7.09±0.60)μg/μL vs(5.73±0.16)μg/μL,P<0.01)]及葡萄糖消耗[(6.45±0.02)nmol/μg vs(5.61±0.29)nmol/μg,P<0.01]显著高于低剂量组,表明Mibefradil可改善HepG2细胞的胰岛素抵抗。qRT-PCR及Western blot检测结果表明,胰岛素抵抗HepG2细胞的Fox O1、PEPCK、G6Pase的表达量均显著增加(P<0.01);经Mibefradil干预后,其表达量呈剂量依赖性下降(P<0.05)。结论Mibefradil对胰岛素抵抗HepG2细胞具有改善作用,此作用可能与下调Fox O1的表达有关。
Objective To explore the effect of Mibefradil on insulin resistance HepG2 cells in vitro. Methods HepG2 cells were divided into control group, HepG2 cell insulin resistance model group induced by palmitate (PA), drug solution (DMSO) group, Mibefradil low dose (0.025μmol / L) L) Mibefradil group. The effects of Mibefradil on insulin resistance in HepG2 cells were observed by detecting glycogen synthesis and glucose consumption in each group. The mRNA expression of Fox O1, gluconeogenesis, the rate-limiting enzymes of glycogenolysis, ie, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphate kinase (G6Pase) were detected by qRT- Protein relative expression, clear transcription factor FoxO1 and its target gene expression levels. Results Compared with the control group, the glycogen synthesis [(3.28 ± 0.74) μg / μL vs (9.14 ± 0.33) μg / μL, P <0.01] and the glucose consumption [(1.31 ± 0.49 ) (nmol / μg vs (5.87 ± 2.26) nmol / μg, P <0.01], and the glycogen synthesis and glucose consumption of insulin resistant HepG2 cells were significantly improved after Mibefradil intervention. (7.09 ± 0.60) μg / μL vs (5.73 ± 0.16) μg / μL, P <0.01) and glucose consumption (6.45 ± 0.02 nmol / μg vs 5.61 ± 0.29 nmol / μg, P <0.01) Higher than the low dose group, indicating that Mibefradil can improve insulin resistance HepG2 cells. The results of qRT-PCR and Western blot showed that the expressions of FoxO1, PEPCK and G6Pase in HepG2 cells were significantly increased (P <0.01), and the expression of Foxp1, PEPCK and G6Pase was decreased in a dose-dependent manner after Mibefradil treatment (P <0.05) ). Conclusion Mibefradil can improve insulin resistance in HepG2 cells, which may be related to the down-regulation of FoxO1 expression.