小檗碱对体外HepG2细胞IR模型抗IR的效应及机制
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摘要
目的:探讨小檗碱对HepG2细胞胰岛素抵抗(IR)模型的改善作用及机制。方法:HepG2细胞采用高糖(25 mmol·L~(-1))和高胰岛素(1×10~(-6)mol·L~(-1))联合诱导24 h,建立体外IR模型。模型培养液中分别加入1×10~(-5),1×10~(-6)mol·L~(-1)小檗碱,设1×10-3mol·L~(-1)二甲双胍作为阳性药,孵育24 h。葡萄糖氧化酶法检测培养液葡萄糖含量,计算细胞葡萄糖消耗量,细胞增殖活性检测(CCK-8)测定细胞增殖活性;生化法检测细胞丙酮酸激酶(PK)活性,酶联免疫吸附试验(ELISA)检测细胞葡萄糖激酶(GCK)和葡萄糖转运蛋白2(GLUT2)的活性;实时荧光定量聚合酶链式反应(Real-time PCR)检测细胞胰岛素受体(InsR),磷脂酰肌醇-3激酶(PI3K),丝氨酸/苏氨酸蛋白激酶B(Akt)和GLUT2 mRNA表达水平,蛋白免疫印迹法(Western blot)检测各组细胞胰岛素受体底物-1(IRS-1),磷酸化的磷脂酰肌醇-3激酶(p-PI3K)和磷酸化的丝氨酸/苏氨酸蛋白激酶B(p-Akt)的表达水平。结果:与正常组比较,模型组细胞葡萄糖消耗量显著降低(P<0.01),PK,GCK,GLUT2蛋白活性显著降低(P<0.01),InsR,PI3K,Akt,GLUT2 mRNA表达水平显著降低(P<0.01),IRS-1,p-PI3K,p-Akt蛋白表达水平显著降低(P<0.01)。与模型组比较,小檗碱组和二甲双胍组的细胞葡萄糖消耗量明显增加(P<0.01),PK,GCK,GLUT2蛋白活性显著升高(P<0.01),InsR,PI3K,Akt,GLUT2 mRNA和IRS-1,p-PI3K,p-Akt1蛋白表达水平均明显升高(P<0.05,P<0.01)。结论:小檗碱体外对HepG2细胞IR模型有显著的改善作用,其机制可能通过调节IRS-1/PI3K/Akt信号通路促进肝脏葡萄糖转运和改善葡萄糖代谢。
Objective: To investigate the effect of berberine on HepG2 insulin resistance( IR) model and its mechanism. Method: The IR model was established by combining high-glucose( 25 mmol·L~(-1)) with highinsulin( 1 × 10~(-6) mol ·L~(-1)) for 24 h. And then, high-dose( 1 × 10-5 mol·L~(-1)) berberine, low-dose( 1 × 10~(-6) mol·L~(-1)) berberine,and melbine( 1 × 10~(-3) mol·L~(-1),positive control) were respectively added in medium and cultured for another 24 h. At last,medium and HepG2 cells were collected. Glucose level was detected by glucose oxidase detection method,and cell reproductive capacity was detected by cell counting kit-8( CCK-8); pyruvate kinase( PK) was detected by biochemical analyzer; glucokinase( GCK) and glucose transporter 2( GLUT2) were detected by enzyme linked immunosorbent assay( ELISA) kits. The mRNA expressions of insulin receptor( InsR),phosphatidylinositol-3-kinase( PI3 K),protein kinase B( Akt),GLUT2 were detected by Real-time PCR. The proteins expressions of insulin receptor substrate-1( IRS-1),PI3 K,and pAkt were detected by Western blot. Result: Compared with normal group,the glucose consumption of the model group was significantly decreased( P < 0. 01),and the activities of PK,GCK and GLUT2 were significantly decreased( P < 0. 01). Compared with model group,Berberine and metformin groups significantly increased cell glucose consumption,and PK,GCK and GLUT2 protein activities notably increased( P < 0. 01). Compared with normal group,the mRNA expressions of InsR,PI3 K,Akt and GLUT2 and the protein expressions of IRS-1,PI3 K and Akt were significantly decreased( P < 0. 01). Compared with model group,berberine and metformin groups significantly increased protein expression of InsR,PI3 K,Akt and GLUT2( P < 0. 05,P < 0. 01). Conclusion:Berberine has a noteworthy therapeutic effect on the insulin resistance of HepG2 cells. Its mechanism may be correlated with the promotion of hepatic glucose metabolism by regulating IRS-1/PI3 K/Akt signaling pathway.
Objective: To investigate the effect of berberine on HepG2 insulin resistance( IR) model and its mechanism. Method: The IR model was established by combining high-glucose( 25 mmol·L~(-1)) with highinsulin( 1 × 10~(-6) mol ·L~(-1)) for 24 h. And then, high-dose( 1 × 10-5 mol·L~(-1)) berberine, low-dose( 1 × 10~(-6) mol·L~(-1)) berberine,and melbine( 1 × 10~(-3) mol·L~(-1),positive control) were respectively added in medium and cultured for another 24 h. At last,medium and HepG2 cells were collected. Glucose level was detected by glucose oxidase detection method,and cell reproductive capacity was detected by cell counting kit-8( CCK-8); pyruvate kinase( PK) was detected by biochemical analyzer; glucokinase( GCK) and glucose transporter 2( GLUT2) were detected by enzyme linked immunosorbent assay( ELISA) kits. The mRNA expressions of insulin receptor( InsR),phosphatidylinositol-3-kinase( PI3 K),protein kinase B( Akt),GLUT2 were detected by Real-time PCR. The proteins expressions of insulin receptor substrate-1( IRS-1),PI3 K,and pAkt were detected by Western blot. Result: Compared with normal group,the glucose consumption of the model group was significantly decreased( P < 0. 01),and the activities of PK,GCK and GLUT2 were significantly decreased( P < 0. 01). Compared with model group,Berberine and metformin groups significantly increased cell glucose consumption,and PK,GCK and GLUT2 protein activities notably increased( P < 0. 01). Compared with normal group,the mRNA expressions of InsR,PI3 K,Akt and GLUT2 and the protein expressions of IRS-1,PI3 K and Akt were significantly decreased( P < 0. 01). Compared with model group,berberine and metformin groups significantly increased protein expression of InsR,PI3 K,Akt and GLUT2( P < 0. 05,P < 0. 01). Conclusion:Berberine has a noteworthy therapeutic effect on the insulin resistance of HepG2 cells. Its mechanism may be correlated with the promotion of hepatic glucose metabolism by regulating IRS-1/PI3 K/Akt signaling pathway.
引文
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[11]LUO Z,ZHANG Y,LI F,et al.Resistin induces insulin resistance by both AMPK-dependent and AMPKindependent mechanisms in Hep G2 cells[J].Endocrine,2009,36(1):60-69.
[12]黎宇,罗新新,严奉东,等.葛根上调肝胰岛素抵抗Hep G2细胞OB-R,IRS2,GLUT1和GLUT2蛋白调节糖代谢的研究[J].中国中药杂志,2017,42(10):1939-1944.
[13]Clementi E.Role of nitricoxide and it s in tracellular signalling pathways in the control of Ca2+homeostasis[J].Biochem Pharmacol,1998,55(6):713-718.
[14]Malabu U H,Dryden S,Mccarthy H D,et al.Effects of chronic vanadate administration in the STZ-induced diabetic rat[J].Diabetes,1994,43(1):9-15.
[15]Seoane J,BarberàA,Télémaque-Potts S,et al.Glucokinase over expression restores glucose utilization and storage in cultured hepatocytes from male Zucker diabetic fatty rats[J].J Biol Chem,1999,274(45):31833-31838.
[16]Cantley L C.The phosphoinositide 3-kinase pathway[J].Science,2002,296(5573):1655-1657.
[17]王慧莲.大黄素和小檗碱对胰岛素抵抗Hep G2细胞的实验研究[D].太原:山西医科大学,2009.
[18]李芬.小檗碱改善胰岛素抵抗作用分子机制的研究[D].武汉:华中科技大学,2016.
[19]王小兰,幸尚平,肖建勇,等.左金方及其主要成分小檗碱对人胃癌细胞SGC7901上皮间质转化的影响[J].中国实验方剂学杂志,2018,24(12):62-67.
[20]涂珺,罗新新,李冰涛,等.小檗碱对脂肪胰岛素抵抗细胞PPARγ及脂肪分泌因子表达的影响[J].中国中药杂志,2016,41(11):1983-1989.
[2]LIU W H,HEI Z Q,NIE H,et al.Berberine ameliorates renal injury in streptozotocin induced diabetic rats by suppression of both oxidative stress and aldose reductase[J].Chin Med J:Engl,2008,121(8):706-712.
[3]张彦,陈靖,王超凡.小檗碱降血糖作用的实验研究[J].中国民族民间医药,2008,17(12):24-26.
[4]LENG S H,LU F E,XU L J.Therapeutic effects of berberine in impaired glucose tolerance rats and its influence on insulin secretion[J].Acta Pharmacol Sin,2004,25(4):496-502.
[5]殷峻,胡仁明,唐金凤,等.小檗碱的体外降糖作用[J].上海第二医科大学学报,2001,21(5):425-427.
[6]张敬升,王晓婉,张广霞,等.小檗碱对3T3-L1前脂肪细胞及其胰岛素抵抗模型的影响[J].中国实验方剂学杂志,2015,21(22):145-149.
[7]张汝学,贾正平,李茂星,等.体外胰岛素抵抗细胞模型的建立及在药物筛选中的应用[J].中国药理学通报,2008,24(7):971-976.
[8]方飞,吴新荣,罗明俐,等.Hep G2细胞胰岛素抵抗模型的建立及在筛选桑叶有效部位中的应用[J].医药导报,2012,31(6):691-694.
[9]龚菂,李芬,邹欣,等.Hep G2细胞胰岛素抵抗模型建立及盐酸小檗碱改善胰岛素抵抗的实验研究[J].中国药理学通报,2016,32(12):1750-1754.
[10]龚又明,郑显辉,谢鸣坤,等.小檗碱对Hep G2胰岛素抵抗细胞PI3K/Akt1/GLUT1信号通路的调控作用[J].新中医,2017,49(8):1-4.
[11]LUO Z,ZHANG Y,LI F,et al.Resistin induces insulin resistance by both AMPK-dependent and AMPKindependent mechanisms in Hep G2 cells[J].Endocrine,2009,36(1):60-69.
[12]黎宇,罗新新,严奉东,等.葛根上调肝胰岛素抵抗Hep G2细胞OB-R,IRS2,GLUT1和GLUT2蛋白调节糖代谢的研究[J].中国中药杂志,2017,42(10):1939-1944.
[13]Clementi E.Role of nitricoxide and it s in tracellular signalling pathways in the control of Ca2+homeostasis[J].Biochem Pharmacol,1998,55(6):713-718.
[14]Malabu U H,Dryden S,Mccarthy H D,et al.Effects of chronic vanadate administration in the STZ-induced diabetic rat[J].Diabetes,1994,43(1):9-15.
[15]Seoane J,BarberàA,Télémaque-Potts S,et al.Glucokinase over expression restores glucose utilization and storage in cultured hepatocytes from male Zucker diabetic fatty rats[J].J Biol Chem,1999,274(45):31833-31838.
[16]Cantley L C.The phosphoinositide 3-kinase pathway[J].Science,2002,296(5573):1655-1657.
[17]王慧莲.大黄素和小檗碱对胰岛素抵抗Hep G2细胞的实验研究[D].太原:山西医科大学,2009.
[18]李芬.小檗碱改善胰岛素抵抗作用分子机制的研究[D].武汉:华中科技大学,2016.
[19]王小兰,幸尚平,肖建勇,等.左金方及其主要成分小檗碱对人胃癌细胞SGC7901上皮间质转化的影响[J].中国实验方剂学杂志,2018,24(12):62-67.
[20]涂珺,罗新新,李冰涛,等.小檗碱对脂肪胰岛素抵抗细胞PPARγ及脂肪分泌因子表达的影响[J].中国中药杂志,2016,41(11):1983-1989.