白子菜对HepG2细胞胰岛素抵抗作用的分子机制
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摘要
目的:观察白子菜对胰岛素抵抗人肝癌Hep G2细胞的胰岛素受体(insulin receptor,InsR),葡萄糖转运蛋白4(glucose transporter4,GLUT4)mRNA表达和蛋白激酶B(protein kinase B,PKB),糖原合成激酶-3β(glycogen synthase kinase-3β,GSK-3β)蛋白表达的影响,探讨其干预胰岛素抵抗的分子机制。方法:用胰岛素诱导Hep G2细胞使其产生胰岛素抵抗,实验分为空白组,模型组,白子菜水提物(Gynura divaricata hot water extracts,GDE)高、低质量浓度(1.0,0.5 g·L-1)组,白子菜总黄酮(G.divaricata flavonoids,GDF)高、低质量浓度(0.2,0.1 g·L-1)组,白子菜总生物碱(G.divaricata alkaloid,GDA)高、低质量浓度(0.1,0.05 g·L-1)组,二甲双胍(1×10-3mol·L-1)组,葡萄糖氧化酶法检测Hep G2细胞培养液上清液中葡萄糖的含量,实时荧光定量聚合酶反应(Real-time PCR)检测Hep G2细胞InsR,GLUT4 mRNA表达,蛋白免疫印迹法(Western blot)检测Hep G2细胞PKB,GSK-3β的蛋白表达。结果:与模型组比较,1.0,0.5 g·L-1GDE组上清液葡萄糖含量极显著降低(P<0.01),0.2 g·L-1GDF组上清液葡萄糖含量显著降低(P<0.05);GDE,GDF组InsR,GLUT4的mRNA表达显著增加(P<0.05);GDE组PKB的蛋白表达显著增加(P<0.05),GSK-3β的蛋白表达显著降低(P<0.05),GDF组PKB的蛋白表达极显著增加(P<0.01),GSK-3β的蛋白表达极显著降低(P<0.01)。结论:白子菜可改善胰岛素抵抗Hep G2细胞对葡萄糖的摄取,其机制可能与上调胰岛素抵抗Hep G2细胞InsR,GLUT4 mRNA表达和PKB的蛋白表达及降低GSK-3β的蛋白表达相关。
Objective: To observe the effect of Gynura divaricata on mRNA expressions of insulin receptor( InsR) and glucose transporter 4( GLUT4),and protein expressions of protein kinase B( PKB) and glycogen synthase kinase-3β( GSK-3β) on insulin resistance in Hep G2 cells,in order to discuss the molecular mechanism of insulin resistance after intervention. Method: Insulin-induced Hep G2 cells were used to produce insulin resistance. The experiment included blank control group, model group, high-dose and low-dose G.divaricata hot water extracts( GDE,1. 0,0. 5 g·L-1) groups,high-dose and low-dose G. divaricata flavonoids( GDF,0. 2,0. 1 g·L-1) groups,high-dose and low-dose G. divaricata alkaloid( GDA,0. 1,0. 05 g·L-1)groups. Glucose oxidase method was used to detect the content of glucose in supernatant of Hep G2 cell culture medium; mRNA expressions of insulin receptor( InsR) and glucose transporter 4( GLUT4) in Hep G2 cells were detected by Real-time PCR; and Western blot was used to detect protein expressions of kinase B( PKB) and GSK-3β in Hep G2 cells. Result: Compared with model control group, the glucose content of supernatant was significantly decreased in 1. 0,0. 5 g·L-1 GDE groups( P < 0. 01),and the glucose content was significantly decreased in supernatant of 0. 2 g·L-1 GDF group( P < 0. 05); the mRNA expressions of INSR and GLUT4 in GDE and GDF groups increased significantly( P < 0. 05); the protein expression of PKB was significantly increased( P < 0. 05),and the protein expression of GSK-3β was significantly reduced in GDE group( P < 0. 05),the protein expression of PKB was significantly increased( P < 0. 01) and the protein expression of GSK-3β decreased significantly in GDF group( P < 0. 01). Conclusion: Gynura divaricata may improve insulin resistance Hep G2 cells' uptake of glucose. Its mechanism may be related to the up-regulation of InsR,GLUT4 mRNA expression and PKB protein expression,and the reduction of GSK-3β protein expression in insulin resistant Hep G2 cells.
Objective: To observe the effect of Gynura divaricata on mRNA expressions of insulin receptor( InsR) and glucose transporter 4( GLUT4),and protein expressions of protein kinase B( PKB) and glycogen synthase kinase-3β( GSK-3β) on insulin resistance in Hep G2 cells,in order to discuss the molecular mechanism of insulin resistance after intervention. Method: Insulin-induced Hep G2 cells were used to produce insulin resistance. The experiment included blank control group, model group, high-dose and low-dose G.divaricata hot water extracts( GDE,1. 0,0. 5 g·L-1) groups,high-dose and low-dose G. divaricata flavonoids( GDF,0. 2,0. 1 g·L-1) groups,high-dose and low-dose G. divaricata alkaloid( GDA,0. 1,0. 05 g·L-1)groups. Glucose oxidase method was used to detect the content of glucose in supernatant of Hep G2 cell culture medium; mRNA expressions of insulin receptor( InsR) and glucose transporter 4( GLUT4) in Hep G2 cells were detected by Real-time PCR; and Western blot was used to detect protein expressions of kinase B( PKB) and GSK-3β in Hep G2 cells. Result: Compared with model control group, the glucose content of supernatant was significantly decreased in 1. 0,0. 5 g·L-1 GDE groups( P < 0. 01),and the glucose content was significantly decreased in supernatant of 0. 2 g·L-1 GDF group( P < 0. 05); the mRNA expressions of INSR and GLUT4 in GDE and GDF groups increased significantly( P < 0. 05); the protein expression of PKB was significantly increased( P < 0. 05),and the protein expression of GSK-3β was significantly reduced in GDE group( P < 0. 05),the protein expression of PKB was significantly increased( P < 0. 01) and the protein expression of GSK-3β decreased significantly in GDF group( P < 0. 01). Conclusion: Gynura divaricata may improve insulin resistance Hep G2 cells' uptake of glucose. Its mechanism may be related to the up-regulation of InsR,GLUT4 mRNA expression and PKB protein expression,and the reduction of GSK-3β protein expression in insulin resistant Hep G2 cells.
引文
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[16]马正东,陈磊,宋洪涛,等.白背三七水提取物对2型糖尿病大鼠的降血糖作用及其机制[J].中草药,2010,41(4):623-626.
[17]贺克,刘姣,刘丽华,等.白背三七总黄酮对2型糖尿病大鼠胰岛素抵抗的影响[J].中成药,2014,37(11):2501-2504.
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[19]昊霞,张钱钱,王忠震,等.白子菜叶片中酸性多糖的降血糖作用及其对相关指标的影响[J].植物资源与环境学报,2015,24(2):115-117.
[20]俞浩,毛斌斌,周国梁,等.白背三七总黄酮对糖尿病大鼠的降血糖作用[J].食品科学,2013,34(15):295-298.
[21]韦乃球,冼寒梅,杨柯,等.白背三七对Hep G2细胞胰岛素抵抗改善作用的实验研究[J].时珍国医国药,2011,22(6):1395-1396.
[22]韦乃球,杨柯,冼寒梅,等.白子菜水提液对胰岛素抵抗Hep G2细胞关键酶活性的影响[J].医药导报,2013,32(3):281-284.
[23]郭丽民.地黄寡糖对Hep G2细胞(人肝癌细胞株)胰岛素抵抗的改善作用及机制研究[D].兰州:兰州大学,2007.
[24]Knowles B B,Howe C C,Aden D P.Human hepatocellular careinoma cell lines secrete the major plasma protein and hepatitis B surface antigen[J].Science,1980,209(4455):497-499.
[25]Czauderna F,Fechtner M,Aygun H,et al.Functional studies of the PI(3)-kinase signalling pathway employing synthetic and expressed siRNA[J].Nucleic Acids Res,2003,31(2):670-682.
[26]Hers I,Vincent E E,Tavare J M.Akt signalling in health and disease[J].Cell Signal,2011,23(10):1515-1527.
[27]WANG H,ZHANG Q,WEN Q,et al.Proline-rich Akt substrate of 40k Da(PRAS40):a novel downstream target of PI3k/Akt signaling pathway[J].Cell Signal,2012,24(1):17-24.
[28]Brozinick J T,Roberts B R,Dohm G L.Defective signaling through Akt-2 and-3 but not Akt-1 in insulinresistant human skeletal muscle:potential role in insulin resistance[J].Diabetes,2003,52(4):935-941.
[29]HU X,WANG S,XU J,et al.Triterpenoid saponins from Stauntonia chinensis ameliorate insulin resistance via the AMP-activated protein kinase and IR/IRS-1/PI3K/Akt pathways in insulin-resistant Hep G2 cells[J].Int J Mol Sci,2014,15(6):10446-10458.
[30]吕娟,陈莉娜,马欣.PI3K/Akt信号通路与胰岛素抵抗相关疾病的关系[J].国外医学:医学地理分册,2012,33(2):127-131.
[31]韩海峰,张雪英,申红立,等.白背三七对胰岛素抵抗大鼠肝脏胰岛素受体表达的影响[J].中药药理与临床,2014,30(5):102-105.
[32]黄桂红,刘天旭,朱钊铭,等.鬼针草黄酮对Hep G2胰岛素抵抗细胞PI3K/AKT1/GLUT4信号通路的调控作用[J].实用医学杂志,2016,32(24):3994-3998.
[33]刘颐轩,臧莎莎,宋光耀,等.津力达对胰岛素抵抗大鼠肝脏PI3K/AKT信号通路的影响[J].中国实验方剂学杂志,2015,21(12):72-76.
[34]金鑫,赵海燕,马永平.甘草黄酮对2型糖尿病大鼠肝脏中GSK-3β蛋白表达的影响[J].天然产物研究与开发,2014,26(3):419-422.
[2]Reaven G M.Insulin resistance:the link between obesity and cardiovascular disease[J].Med Clin N Am,2011,95(5):875-892.
[3]Cook J R,Langlet F,Kido Y,et al.Pathogenesis of selective insulin resistancein isolated hepatocytes[J].J Biol Chem,2015,290(22):13972-13980.
[4]HAN J H,Kim I S,Jung S H,et al.The effects of propionate and valerate on insulin responsiveness for glucose uptake in 3T3-L1 adipocytes and C2Cl2myotubes via G proteincoupled receptor 41[J].PLo S One,2014,9(4):e95268.
[5]Bence K K.Hepatic PTP1B deficiency:the proise of a treatment formetabolic syndrome[J].Am J Med,1998,105:331-345.
[6]Albury-Warren T M,Pandey V,Spinel L P,et al.Prediabetes linked to excess glucagon in transgenic mice with pancreatic active AKT1[J].J Endocrinol,2016,228(1):49-59.
[7]唐海双,王清松,焦炳华,等.GLUT4在胰岛素调控葡萄糖转运中作用[J].生命的化学,2014,34(2):285-290.
[8]XIAO X,HE Y,LI C,et al.Nicastrin mutations in familial acne inversa impact keratinocyte proliferation and differentiation through the Notch and PI3K/Akt signaling pathways[J].Br J Dermatol,2016,174(3):522-532.
[9]CHI M,YE Y,ZHANG X D,et al.Insulin induces drug resistance in melanoma through activation of the PI3K/Akt pathway[J].Drug Des Devel Ther,2014,8(default):255-262.
[10]Eldar-Finkelman H,Schreyer S A,Shinohara M M,et al.Increased glycogen synthase kinase-3 activity in diabetes and obesity-prone C57BL/6J mice[J].Diabetes,1999,48(3):1662-1666.
[11]Nikoulina S E,Ciaraldi T P,Mudaliar S,et al.Potential role of glycogen synthase kinase-3 in skeletal muscle insulin resistance of type 2 diabetes[J].Diabetes,2000,49(1):263-271.
[12]陈忠云,杨叔禹.中医药改善2型糖尿病胰岛素抵抗的病理生理学机制研究进展[J].福建医药杂志,2017,39(3):140-142.
[13]广西壮族自治区革委会卫生局.广西本草选编[M].南宁:广西人民出版社,1974:1826.
[14]江苏新医学院.中药大词典[M].上海:上海人民出版社,1977:749.
[15]韩海峰,张雪英,申红立,等.白背三七对胰岛素抵抗大鼠肝脏胰岛素受体表达的影响[J].中药药理与临床,2014,30(5):102-105.
[16]马正东,陈磊,宋洪涛,等.白背三七水提取物对2型糖尿病大鼠的降血糖作用及其机制[J].中草药,2010,41(4):623-626.
[17]贺克,刘姣,刘丽华,等.白背三七总黄酮对2型糖尿病大鼠胰岛素抵抗的影响[J].中成药,2014,37(11):2501-2504.
[18]袁六六,杨娴,张国彬,等.白子菜提取物对糖尿病小鼠血脂代谢和肝脏保护作用[J].中国医院药学杂志,2014,34(18):1572-1576.
[19]昊霞,张钱钱,王忠震,等.白子菜叶片中酸性多糖的降血糖作用及其对相关指标的影响[J].植物资源与环境学报,2015,24(2):115-117.
[20]俞浩,毛斌斌,周国梁,等.白背三七总黄酮对糖尿病大鼠的降血糖作用[J].食品科学,2013,34(15):295-298.
[21]韦乃球,冼寒梅,杨柯,等.白背三七对Hep G2细胞胰岛素抵抗改善作用的实验研究[J].时珍国医国药,2011,22(6):1395-1396.
[22]韦乃球,杨柯,冼寒梅,等.白子菜水提液对胰岛素抵抗Hep G2细胞关键酶活性的影响[J].医药导报,2013,32(3):281-284.
[23]郭丽民.地黄寡糖对Hep G2细胞(人肝癌细胞株)胰岛素抵抗的改善作用及机制研究[D].兰州:兰州大学,2007.
[24]Knowles B B,Howe C C,Aden D P.Human hepatocellular careinoma cell lines secrete the major plasma protein and hepatitis B surface antigen[J].Science,1980,209(4455):497-499.
[25]Czauderna F,Fechtner M,Aygun H,et al.Functional studies of the PI(3)-kinase signalling pathway employing synthetic and expressed siRNA[J].Nucleic Acids Res,2003,31(2):670-682.
[26]Hers I,Vincent E E,Tavare J M.Akt signalling in health and disease[J].Cell Signal,2011,23(10):1515-1527.
[27]WANG H,ZHANG Q,WEN Q,et al.Proline-rich Akt substrate of 40k Da(PRAS40):a novel downstream target of PI3k/Akt signaling pathway[J].Cell Signal,2012,24(1):17-24.
[28]Brozinick J T,Roberts B R,Dohm G L.Defective signaling through Akt-2 and-3 but not Akt-1 in insulinresistant human skeletal muscle:potential role in insulin resistance[J].Diabetes,2003,52(4):935-941.
[29]HU X,WANG S,XU J,et al.Triterpenoid saponins from Stauntonia chinensis ameliorate insulin resistance via the AMP-activated protein kinase and IR/IRS-1/PI3K/Akt pathways in insulin-resistant Hep G2 cells[J].Int J Mol Sci,2014,15(6):10446-10458.
[30]吕娟,陈莉娜,马欣.PI3K/Akt信号通路与胰岛素抵抗相关疾病的关系[J].国外医学:医学地理分册,2012,33(2):127-131.
[31]韩海峰,张雪英,申红立,等.白背三七对胰岛素抵抗大鼠肝脏胰岛素受体表达的影响[J].中药药理与临床,2014,30(5):102-105.
[32]黄桂红,刘天旭,朱钊铭,等.鬼针草黄酮对Hep G2胰岛素抵抗细胞PI3K/AKT1/GLUT4信号通路的调控作用[J].实用医学杂志,2016,32(24):3994-3998.
[33]刘颐轩,臧莎莎,宋光耀,等.津力达对胰岛素抵抗大鼠肝脏PI3K/AKT信号通路的影响[J].中国实验方剂学杂志,2015,21(12):72-76.
[34]金鑫,赵海燕,马永平.甘草黄酮对2型糖尿病大鼠肝脏中GSK-3β蛋白表达的影响[J].天然产物研究与开发,2014,26(3):419-422.