世界中医药

作者	单位
赵丹丹1，吴瑞2，白颖1，莫芳芳1，马如风1，柳辰玥1，朱如愿1，左加成1，姜广建1，张东伟1，高思华1	1 北京中医药大学中医学院,北京,100029 2 中国中医科学院广安门医院南区内分泌科,北京,1026183

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中文摘要:

目的:观察人参皂苷Rb1对胰岛素抵抗(IR)的C2C12骨骼肌细胞葡萄糖利用的影响,并基于AMPK信号通路探讨其可能的作用机制。方法:将C2C12细胞分为正常组、模型组、人参皂苷Rb1组,模型组以0.25 mmol/L棕榈酸,1%小牛血清白蛋白培养16 h诱导IR,人参皂苷Rb1组在模型组的基础上以1、3、10、30、100 μmol/L浓度的人参皂苷Rb1分别干预24 h和48 h,通过检测培养液中的葡萄糖含量反映葡萄糖消耗量,并应用CCK8试剂盒及光镜观察不同浓度人参皂苷Rb1对C2C12细胞活性及形态的影响,实时荧光定量PCR扩增分析人参皂苷Rb1对C2C12细胞AMPKα、SIRT-1、PGC-1α基因表达的影响。并应用shRNA沉默AMPKα的表达,构建AMPKα低表达的骨骼肌细胞模型,以1、3、10、30、100 μmol/L浓度的人参皂苷Rb1分别干预24 h和48 h,与不含人参皂苷Rb1的培养液比较,再次验证人参皂苷Rb1对C2C12细胞葡萄糖利用的影响及其与AMPK信号通路的关系。结果:1、3、10、30 μmol/L人参皂苷Rb1对C2C12细胞形态及活性无显著影响,而100 μmol/L人参皂苷Rb1造成C2C12细胞活性降低及部分细胞死亡。10,30 μmol/L人参皂苷Rb1能够促进IR的C2C12骨骼肌细胞葡萄糖消耗量,与模型组比较，差异有统计学意义(P<0.05),且人参皂苷Rb1能够上调C2C12细胞AMPKα、SIRT-1、PGC-1α的基因表达(P<0.05)。10、30 μmol/L人参皂苷Rb1亦能增加AMPKα低表达的骨骼肌细胞的葡萄糖消耗量(P<0.05),而该作用程度低于其对棕榈酸诱导的C2C12细胞IR的影响,且RT-PCR结果显示人参皂苷Rb1能上调AMPKα低表达的C2C12细胞SIRT-1、PGC-1α的基因表达。结论:人参皂苷Rb1能够有效促进IR的C2C12骨骼肌细胞葡萄糖消耗,该作用与调节AMPK信号通路有关,但除AMPK信号通路亦有其他作用途径。

English Summary:

To observe the effects of ginsenoside Rb1 on glucose utilization in C2C12 cells with insulin resistance (IR) and to explore its possible mechanism based on AMPK signaling pathway.Methods:C2C12 cells were divided into normal group, model group and ginsenoside Rb1 group.C2C12 cells in model group were incubated in 0.25 mmol/L palmitic acid and 1% bovine serum albumin for 16 hours to induce IR.On the basis of the model group, different concentrations of ginsenoside Rb1 of 1 μmol/L、3 μmol/L、10 μmol/L、30 μmol/L, and 100 μmol/L were added in ginsenoside Rb1 groups for 24 hours and 48 hours respectively.Glucose consumption of the cells in different groups was measured by measuring the glucose content in the nutrient fluid.CCK8 kit and light microscope were used to observe the effect of ginsenoside Rb1 on the vitality and morphology of C2C12 cells with IR.Real-time fluorescence quantitative PCR was used to analyze the effect of ginsenoside Rb1 on the gene expressions of AMPK alpha, SIRT-1 and PGC-1 alpha.In addition, anther IR cell model was established by knocking down expression of AMPK-alpha through shRNA.Ginsenoside Rb1 at concentrations of 1 μmol/L, 3 μmol/L, 10 μmol/L, 30 μmol/L, 100 μmol/L were used to this IR cell model for 24 h and 48 h respectively.Glucose utilization and gene expression were again determined to verify the effects of ginsenoside Rb1 on C2C12 cells and its relationship with AMPK signaling pathway.Results:Ginsenoside Rb1 with concentrations of 1 μmol/L, 3 μmol/L, 10 μmol/L, 30 μmol/L did not influence the morphology and vitality of C2C12 cells significantly.However, high concentrations of ginsenoside Rb1 (100 μmol/L) decreased the vitality of C2C12 cells significantly and caused cell death.Compared with model group, 10 μmol/L and 30 μmol/L ginsenoside Rb1 could promote the glucose consumption of C2C12 cells with IR obviously, and the difference was statistically significant (P<0.05).Ginsenoside Rb1 could up-regulate the gene expressions of AMPK alpha, SIRT-1 and PGC-1 alpha in C2C12 cells (P<0.05).In addition, 10 μmol/L and 30 μmol/L ginsenoside Rb1 could also enhance the glucose utilization of C2C12 cells with AMPK alpha knocked down (P<0.05).However, this function was not as effective as that in the former IR C2C12 cells induced by palmitic acid.RT-PCR results showed that ginsenoside Rb1 could increase the gene expression of SIRT-1 and PGC-1 alpha in C2C12 cells with low expression of AMPK alpha.Conclusion:Ginsenoside Rb1 can effectively promote glucose utilization in C2C12 skeletal muscle cells with insulin resistance, which is related but not limited to the regulation of AMPK signaling pathway.

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