南苜蓿总皂苷对HepG2细胞及糖尿病大鼠外周组织胰岛素抵抗的作用及机制研究

Hypoglyceminc Effects of Total Saponins from Medicago polymorpha on HepG2 Cells in Vitro and Ameliorating Insulin Resistance of Peripheral Tissuesin Type 2 Diabetic Rats

目的考察南苜蓿总皂苷提取物对体内外降糖作用的特点。方法采用胰岛素持续作用于HepG2细胞复制细胞胰岛素抵抗模型。测定单位细胞葡萄糖的消耗量,检测细胞内丙二醛(MDA)含量、超氧化物歧化酶(SOD)含量,谷胱甘肽过氧化物酶(GSH-Px)活性。将10只SD大鼠设为对照组、其余60只大鼠采用高糖高脂饮食和链脲佐菌素复制糖尿病大鼠模型,成模后进行分组,随机分为模型组、二甲双胍组、南苜蓿总皂苷高、低剂量组(以提取物计分别为1.4、0.7 g/kg),每组10只。各给药组灌胃相应药物,连续给药30 d。末次给药后,取肝脏和骨骼肌组织检测MDA、SOD、GSH-Px、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)的水平;采用Western blot法检测骨骼肌组织磷酸化磷脂酰肌醇3-激酶(p-PI3K)、磷脂酰肌醇3-激酶(PI3K)、磷酸化蛋白激酶B(p-AKT)、蛋白激酶B(AKT)、葡萄糖转运体4(GLUT4)蛋白的表达。结果与模型组比较,200、100μg/mL浓度组能增加胰岛素抵抗HepG2细胞葡萄糖消耗量和SOD、GSH-Px含量,降低MDA含量(P<0.05或P<0.01);与模型组比较,高剂量组能使模型大鼠的肝脏和骨骼肌中IL-1β、TNF-α、MDA水平降低;升高SOD、GSH-Px水平(P<0.05);骨骼肌组织中p-PI3K、PI3K、p-AKT、AKT、GLUT4的蛋白表达量升高(P<0.05)。结论南苜蓿总皂苷体外促进胰岛素抵抗HepG2细胞的葡萄糖消耗;体内通过减少对肝脏和骨骼肌细胞的氧化损伤和炎症反应,增强骨骼肌中胰岛素介导的信号传导,达到减轻胰岛素抵抗的作用,可能是其体内降糖机制。

Objective To evaluate thehypoglycemic effect of total saponins from Medicago polymorpha(TSMP)in vitro and in vivo.Methods HepG2 cells were treated with insulin continuously to induce insulin resistance.They were treated with relevant medicine and the glucose consumption of unit cell was determined.The activities of malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in each groupwere measured.T2 DM rats were induced by high fat diet feedingcombined with exposure to streptozotocin(30 mg/kg).Rats were randomly divided into control group,diabetic group,two TSMP treated groups with different doses(0.7 or 1.4 g/kg)and metformin(0.2 g/kg)group.After the last administration,the levels of MDA,IL-1β,TNF-α,SOD and GSH-Px in rat liver and muscle tissues were determined.The expressions of p-PI3 K,PI3 K,p-AKT,AKT and GLUT4 in muscle tissue were measured by Western Blot.Results The 200,100μg/mL of TSMP could increase the glucose consumption,SOD and GSH-Px activities and decrease the content of MDA of unit cell in insulin resistant HepG2 cells(P<0.05 or P<0.01).The levels of MDA,IL-1βand TNF-αwere significantly reduced in liver and muscle homogenate in highdose group than those in diabetic group.And the levels of SOD and GSH-Px in liver and muscle homogenate were significantly increased in the highdose group than those in the diabetic group.Also the highdose of TSMP increased the expressions of p-PI3 K,PI3 K,p-AKT,AKT and GLUT4 proteins in rats muscle.Conclusion TSMP could simulate the HepG2 cells to consume glucose.And TSMP has a hypoglycemic activity in rats.Its anti-hyperglycemic activity is probably associated with ameliorating insulin resistance in liver and muscle,potentially via regulating oxidative stress,inflammation and insulin signaling pathway.