黑树莓花色苷对HepG2细胞胰岛素抵抗作用研究

Anti-insulin resistance activity of Rubus occidentalis anthocyanins in HepG2 cells

目的探讨黑树莓花色苷(ROAs)对HepG2细胞胰岛素抵抗(IR)的影响。方法MTT法检测3.75、7.50、15.00、30.00、60.00μg·mL^(-1)的ROAs作用24、48 h对HepG2细胞存活率的影响,确定ROAs的给药条件;含高糖(4.5 g·L^(-1))、高胰岛素(1.0×10^(-7)、1.0×10^(-6)、1.0×10^(-5)、1.0×10^(-4) mol·L^(-1))的DMEM培养基培养HepG2细胞24或48 h,采用葡萄糖氧化试剂盒检测每孔上清液中葡萄糖水平,计算每孔细胞葡萄糖消耗量,确定IR细胞模型的建立条件;HepG2细胞分为对照组、模型组、二甲双胍(阳性药,0.01 mol·L^(-1))组和ROAs(7.5、15.0、25.0、30.0μg·mL^(-1))组,除对照组外,各组细胞培养24 h后建立IR模型,模型建立后药物处理24 h,试剂盒法检测葡萄糖消耗量,HE染色评价细胞形态学改变。结果选择7.5、15.0、25.0、30.0μg·mL^(-1)作为ROAs后续给药质量浓度,给药时间为24 h;HepG2细胞IR模型建立条件为:含高糖(4.5 g·L^(-1))、高胰岛素(1.0×10^(-4) mol·L^(-1))的DMEM培养基培养24 h。与对照组比较,模型组细胞葡萄糖的消耗量明显降低,具有统计学差异(P<0.001);与模型组比较,质量浓度为15.0、25.0、30.0μg·mL^(-1)的ROAs组细胞葡萄糖消耗量均显著增加,具有统计学意义(P<0.05、0.001)。与对照组比较,模型组细胞的形态不固定,细胞核圆形,胞浆不成型,脂滴明显增多;ROAs对HepG2细胞的胞浆形态有改善作用并且使脂滴明显减少。结论ROAs能够改善高糖、高胰岛素诱导的HepG2细胞IR。

Objective To investigate the effect of Rubus occidentalis anthocyanins(ROAs)on insulin resistance(IR)in HepG2 cells.Methods MTT assay was used to detect the effects of ROAs of 3.75,7.50,15.00,30.00,and 60.00μg·mL^(-1) on the survival rate of HepG2 cells for 24 and 48 h,and to determine the administration conditions of ROAs.HepG2 cells were cultured in DMEM medium containing high glucose(4.5 g·L^(-1))and high insulin(1.0×10^(-7),1.0×10^(-6),1.0×10^(-5),1.0×10^(-4) mol·L^(-1))for 24 or 48 h.The glucose level in the supernatant of each well was detected by glucose oxidation kit,and the glucose consumption of cells in each well was calculated,to establish the conditions of IR cell model.HepG2 cells were divided into control group,model group,metformin(positive drug,0.01 mol·L^(-1))group and ROAs(7.5,15.0,25.0,30.0μg·mL^(-1))group.Except for the control group,the IR model was established after 24 h of cell culture.After the establishment of the model,cells were treated with drugs for 24 h,Glucose consumption was detected by kit method,and morphological changes were evaluated by HE staining.Results 7.5,15.0,25.0,30.0μg·mL^(-1) were selected as the following concentrations of ROAs,and the administration time was 24 h.The IR model of HepG2 cells was established under the following conditions:DMEM medium containing high glucose(4.5 g·L^(-1))and high insulin(1.0×10^(-4) mol·L^(-1))for 24 h.Compared with the control group,the consumption of glucose in IR model group was significantly decreased(P<0.001).Compared with the model group,the glucose consumption of cells in the ROAs groups with concentrations of 15.0,25.0,and 30.0μg·mL^(-1) was significantly increased(P<0.05,0.001).Compared with control group,the morphology of IR model cells was not fixed,the nucleus was round,the cytoplasm was not formed,and the lipid droplets increased significantly.ROAs could improve the cytoplasmic morphology of HepG2 cells and significantly reduce lipid droplets.Conclusion ROAs can improve the insulin resistance of HepG2 cells induced by high glucose and high insulin.