葛根素改善3T3-L1脂肪细胞葡萄糖摄取和胰岛素抵抗

Puerarin Improves the Glucose Uptake and Insulin Resistance of 3T3-L1 Adipocytes

目的:探讨葛根素对3T3-L1脂肪细胞胰岛素抵抗(Insulin resistance,IR)模型葡萄糖利用和IR的影响及其作用机制。方法:诱导分化3T3-L1前脂肪细胞为成熟脂肪细胞;用地塞米松诱导建立3T3-L1脂肪细胞胰岛素抵抗模型。成功建立模型后,将实验分为5组进行,分别为空白组、模型组、葛根素10 mg·L^-1组、葛根素100 mg·L^-1组、葛根素200 mg·L^-1组;其中,空白组不建模,模型组建模但不用药物干预,各葛根素组为在建立模型后,给予不同浓度的葛根素干预。用葛根素干预24小时后,以葡萄糖氧化酶-过氧化物酶法测定细胞培养基中葡萄糖浓度,四甲基偶氮唑蓝[3-(4,5-dimehyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide, MTT]比色法检测细胞增值活力;然后分别用Real time PCR、Western Blot方法检测细胞葡萄糖转运蛋白4(glucose transporter type 4, GLUT4)、磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase, PI3K)、蛋白激酶B(protein kinase B, PKB/AKT)、腺苷酸活化蛋白激酶(adenosine monophosphate activated protein kinase, AMPK)、过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma, PPARγ)mRNA基因和蛋白表达。结果:葛根素干预后,细胞培养基中葡萄糖浓度均较模型组降低(P <0.01);GLUT4、PI3K、AKT、AMPK、PPARγmRNA水平及蛋白表达较模型组均增加(P <0.05)。结论:葛根素能增加3T3-L1脂肪细胞胰岛素抵抗模型葡萄糖利用、改善IR,其作用机制可能与上调P13K、AKT、AMPK、PPARγ基因和蛋白表达,进而增加葡萄糖运转有关。

Objective: To observe the effects of puerarin on glucose utilization and insulin resistance(IR) in 3T3-l1 adipocyte IR model, and to explore its mechanism. Methods: The induced differentiation of 3T3-ll preadipocytes into mature adipocytes was followed by the induction of dexamethasone to establish the insulin resistance model of 3T3-l1 adipocytes. After the successful establishment of the model, the experiment was divided into 5 groups: a blank group, a model group, a puerarin 10 mg?L-1 group, a puerarin 100 mg?L-1 group, and a puerarin 200 mg?L-1 group. Among them,the blank group did not establish model. The model was constructed without drug intervention in the model group, and puerarin groups were given different concentrations of puerarin intervention after the model was established. After 24 hours of intervention with puerarin, glucose concentration in cell culture medium was determined by glucose oxidaseperoxidase method, and cell proliferation activity was detected by tetramethylazazole blue [MTT] colorimetric method.Then, the expressions of glucose transporter type 4(GLUT4), phosphatidylinositol 3-kinase(PI3K), protein kinase B(PKB/AKT), denosine monophosphate activated protein kinase(AMPK), and peroxisome proliferator-activated receptor gamma(PPARγ) mRNA were detected by Real time PCR and Western Blot. Results: After puerarin intervention, the glucose concentration in cell culture medium was decreased than that in the model group(P<0.01). The mRNA levels and protein expressions of GLUT4, PI3K, AKT, AMPK and PPARγ were all increased compared with the model group(P<0.05). Conclusion: Puerarin can increase the utilization of glucose and improve IR in the insulin resistance model of 3T3-l1 adipocytes, and its mechanism may be related to the up-regulation of P13K, AKT, AMPK, PPAR γ gene and protein expression, thus increasing glucose metabolism.