人参皂苷Rb1对C2C12骨骼肌细胞葡萄糖利用及AMPK信号通路相关基因表达的影响
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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、100μmol/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、100μmol/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、30μmol/L人参皂苷Rb1对C2C12细胞形态及活性无显著影响,而100μmol/L人参皂苷Rb1造成C2C12细胞活性降低及部分细胞死亡。10,30μmol/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、30μmol/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。
Objective: 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 AMPKalpha 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 30μmol/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.
Objective: 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 AMPKalpha 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 30μmol/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.
引文
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[13]Xie J T,Mchendale S,Yuan C S.Ginseng and diabetes[J].Am JChin Med,2005,33(3):397-404.
[14]Yuan H D,Kim J T,Kim S H,et al.Ginseng and Diabetes[J].Journal of Ginseng Research,2012,36(3):27-39.
[15]左加成.降糖消渴颗粒及益气温阳组分对肥胖小鼠C57BL/6J白色脂肪棕色化的影响研究[D].北京:北京中医药大学,2017.
[16]Shang W,Yang Y,Jiang B,et al.Ginsenoside Rb1 promotes adipogenesis in 3T3-L1 cells by enhancing PPARgamma2 and C/EBPalpha gene expression[J].Life Sci,2007,80(7):618-625.
[17]赵文惠,杨文英.PGC-1α与糖脂代谢[J].国外医学:内分泌学分册,2005,25(5):355-357.
[18]Wu Z,Puigserver P,Andersson U,et al.Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1[J].Cell,1999,98(1):115-124.
[19]CantóC,Auwerx J.PGC-1α,SIRT1 and AMPK,an energy sensing network that controls energy expenditure[J].Current Opinion in Lipidology,2009,20(2):98-105.
[20]Ke R,Xu Q,Li C,et al.Mechanisms of AMPK in the maintenance of ATP balance during energy metabolism[J].Cell Biol Int,2018,42(4):384-392.
[21]Kukidome D,Nishikawa T,Sonoda K,et al.Activation of AMP-activated protein kinase reduces hyperglycemia-induced mitochondrial reactive oxygen species production and promotes mitochondrial biogenesis in human umbilical vein endothelial cells[J].Diabetes,2006,55(1):120-127.
[2]Dimopoulos N,Watson M,Sakamoto K,et al.Differential effects of palmitate and palmitoleate on insulin action and glucose utilization in rat L6 skeletal muscle cells[J].Biochem J,2006,399(3):473-481.
[3]赵丹丹,穆倩倩,方心,等.降糖消渴颗粒含药血清对C2C12细胞胰岛素抵抗的影响[J].中华中医药杂志,2014,29(5):1577-1579.
[4]Shen L,Haas M,Wang DQ,et al.Ginsenoside Rb1 increases insulin sensitivity by activating AMP-activated protein kinase in male rats[J].Physiol Rep,2015,3(9):e12543.
[5]Mu Q,Fang X,Li X,et al.Ginsenoside Rb1 promotes browning through regulation of PPARγin 3T3-L1 adipocytes[J].Biochem Biophys Res Commun,2015,466(3):530-535.
[6]Tabandeh MR,Hosseini SA,Hosseini M.Ginsenoside Rb1 exerts antidiabetic action on C2C12 muscle cells by leptin receptor signaling pathway[J].J Recept Signal Transduct Res,2017,37(4):370-378.
[7]Tabandeh MR,Jafari H,Hosseini SA,et al.Ginsenoside Rb1 stimulates adiponectin signaling in C2C12 muscle cells through up-regulation of AdipoR1 and AdipoR2 proteins[J].Pharm Biol,2015,53(1):125-132.
[8]Hosseini S A,Tabandeh M R,Mesbah Namin S A.Promoting Effect of Ginsenoside Rb1 for GLUT-4 Gene Expression and Cellular Synthesis in C2Cl2 Muscle Cells[J].International Journal of Pharmaceutical Research&Allied Sciences,2016,5(2):151-158.
[9]Tangeman L,Wyatt CN,Brown TL.Knockdown of AMP-activated protein kinase alpha 1 and alpha 2 catalytic subunits[J].J RNAi Gene Silencing,2012,8:470-478.
[10]Lam CK,Chari M,Rutter GA,et al.Hypothalamic nutrient sensing activates a forebrain-hindbrain neuronal circuit to regulate glucose production in vivo[J].Diabetes,2011,60(1):107-113.
[11]Kim S,Shin BC,Lee MS,et al.Red ginseng for type 2 diabetes mellitus:a systematic review of randomized controlled trials[J].Chin JIntegr Med,2011,17(12):937-944.
[12]盖鑫,弓晓杰,鲁明明,等.人参治疗糖尿病有效成分研究[J].长春中医药大学学报,2013,29(3):539-540.
[13]Xie J T,Mchendale S,Yuan C S.Ginseng and diabetes[J].Am JChin Med,2005,33(3):397-404.
[14]Yuan H D,Kim J T,Kim S H,et al.Ginseng and Diabetes[J].Journal of Ginseng Research,2012,36(3):27-39.
[15]左加成.降糖消渴颗粒及益气温阳组分对肥胖小鼠C57BL/6J白色脂肪棕色化的影响研究[D].北京:北京中医药大学,2017.
[16]Shang W,Yang Y,Jiang B,et al.Ginsenoside Rb1 promotes adipogenesis in 3T3-L1 cells by enhancing PPARgamma2 and C/EBPalpha gene expression[J].Life Sci,2007,80(7):618-625.
[17]赵文惠,杨文英.PGC-1α与糖脂代谢[J].国外医学:内分泌学分册,2005,25(5):355-357.
[18]Wu Z,Puigserver P,Andersson U,et al.Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1[J].Cell,1999,98(1):115-124.
[19]CantóC,Auwerx J.PGC-1α,SIRT1 and AMPK,an energy sensing network that controls energy expenditure[J].Current Opinion in Lipidology,2009,20(2):98-105.
[20]Ke R,Xu Q,Li C,et al.Mechanisms of AMPK in the maintenance of ATP balance during energy metabolism[J].Cell Biol Int,2018,42(4):384-392.
[21]Kukidome D,Nishikawa T,Sonoda K,et al.Activation of AMP-activated protein kinase reduces hyperglycemia-induced mitochondrial reactive oxygen species production and promotes mitochondrial biogenesis in human umbilical vein endothelial cells[J].Diabetes,2006,55(1):120-127.