过表达SIRT1基因通过PI3K/AKT信号通路抑制高糖刺激心肌H9c2细胞凋亡和活性氧水平
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摘要
目的:探讨过表达沉默信息调节因子1(SIRT1)对高糖刺激心肌H9c2细胞凋亡和活性氧簇(ROS)水平的影响。方法:分别用空载质粒(pcDNA3.1-NC)和SIRT1过表达质粒(pcDNA3.1-SIRT1)转染心肌细胞,并进行高糖诱导。实验分为对照组、高糖组、高糖+pcDNA3.1-NC组和高糖+pcDNA3.1-SIRT1组,用qPCR和Western blot法分别检测各组心肌H9c2细胞的SIRT1表达,噻唑蓝(MTT)法检测细胞的活力,流式细胞术检测细胞凋亡,DCFH-DA检测细胞内ROS水平,Western blot检测细胞中磷脂酰肌醇3-激酶(PI3K)、磷酸化PI3K、AKT和磷酸化AKT的蛋白水平。结果:与对照组相比,高糖诱导的心肌H9c2细胞中SIRT1表达显著降低,细胞活力显著下降,ROS水平和细胞凋亡率增加,PI3K和AKT磷酸化蛋白水平下调(P<0.05);过表达SIRT1可诱导高糖刺激的心肌H9c2细胞活力增加,ROS水平和凋亡率降低,PI3K和AKT磷酸化蛋白水平上调(P<0.05)。结论:过表达SIRT1可通过调控PI3K/AKT信号通路逆转高糖刺激心肌H9c2细胞活力的降低及凋亡率和氧化应激的增加。
AIM: To investigate the effects of silent information regulator 1(SIRT1) over-expression on the apoptosis and the level of reactive oxygen species(ROS) in high glucose-induced H9 c2 cardiomyocytes. METHODS: H9 c2 cardiomyocytes were transfected with empty plasmid(pcDNA3.1-NC) and SIRT1 over-expression plasmid(pcDNA3.1-SIRT1), and then stimulated by high glucose. The H9 c2 cells were divided into control group, high glucose group, high glucose + pcDNA3.1-NC group and high glucose + pcDNA3.1-SIRT1 group. The expression of SIRT1 at mRNA and protein levels in each group was determined by qPCR and Western blot. The viability of the cells was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The protein levels of phosphatidylinositol 3-kinase(PI3 K), phosphorylated PI3 K, AKT and phosphorylated AKT were examined by Western blot. RESULTS: SIRT1 was significantly decreased in high glucose-induced H9 c2 cardiomyocytes, the cell viability was significantly decreased compared with control group, while the ROS levels and apoptotic rate were increased, and the phosphorylated PI3 K and AKT protein levels were down-regulated(P<0.05). Over-expression of SIRT1 significantly promoted the viability of H9 c2 cardiomyocytes induced by high glucose, decreased the ROS levels and apoptotic rate, and up-regulated phosphorylated PI3 K and AKT protein levels(P<0.05). CONCLUSION: SIRT1 over-expression reverses the decrease in the viability of high glucose-stimulated H9 c2 cardiomyocytes, and the increases in apoptotic rate and oxidative stress by regulating PI3 K/AKT signaling pathway.
AIM: To investigate the effects of silent information regulator 1(SIRT1) over-expression on the apoptosis and the level of reactive oxygen species(ROS) in high glucose-induced H9 c2 cardiomyocytes. METHODS: H9 c2 cardiomyocytes were transfected with empty plasmid(pcDNA3.1-NC) and SIRT1 over-expression plasmid(pcDNA3.1-SIRT1), and then stimulated by high glucose. The H9 c2 cells were divided into control group, high glucose group, high glucose + pcDNA3.1-NC group and high glucose + pcDNA3.1-SIRT1 group. The expression of SIRT1 at mRNA and protein levels in each group was determined by qPCR and Western blot. The viability of the cells was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The protein levels of phosphatidylinositol 3-kinase(PI3 K), phosphorylated PI3 K, AKT and phosphorylated AKT were examined by Western blot. RESULTS: SIRT1 was significantly decreased in high glucose-induced H9 c2 cardiomyocytes, the cell viability was significantly decreased compared with control group, while the ROS levels and apoptotic rate were increased, and the phosphorylated PI3 K and AKT protein levels were down-regulated(P<0.05). Over-expression of SIRT1 significantly promoted the viability of H9 c2 cardiomyocytes induced by high glucose, decreased the ROS levels and apoptotic rate, and up-regulated phosphorylated PI3 K and AKT protein levels(P<0.05). CONCLUSION: SIRT1 over-expression reverses the decrease in the viability of high glucose-stimulated H9 c2 cardiomyocytes, and the increases in apoptotic rate and oxidative stress by regulating PI3 K/AKT signaling pathway.
引文
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[14] Delbridge LMD,Benson VL,Ritchie RH,et al.Diabetic cardiomyopathy:the case for a role of fructose in disease etiology[J].Diabetes,2016,65(12):3521-3528.
[15] Oblong JE.The evolving role of the NAD+/nicotinamide metabolome in skin homeostasis,cellular bioenergetics,and aging[J].DNA Repair (Amst),2014,23(11):59-63.
[16] Arunachalam G,Samuel SM,Marei I,et al.Metformin modulates hyperglycaemia-induced endothelial senescence and apoptosis through SIRT1[J].Br J Pharmacol,2014,171(2):523-535.
[17] Gong H,Pang J,Han Y,et al.Age-dependent tissue expression patterns of Sirt1 in senescence-accelerated mice[J].Mol Med Rep,2014,10(6):3296-3302.
[18] Pazienza V,Pomara C,Cappello F,et al.The TRPA1 channel is a cardiac target of mIGF-1/SIRT1 signaling[J].Am J Physiol Heart Circ Physiol,2014,307(7):H939-H944.
[19] Rong G,Liu W,Liu B,et al.SIRT1 suppresses cardiomyocyte apoptosis in diabetic cardiomyopathy:An insight into endoplasmic reticulum stress response mechanism[J].Int J Cardiol,2015,191(3):36-45.
[20] Ma S,Feng J,Zhang R,et al.SIRT1 activation by resveratrol alleviates cardiac dysfunction via mitochondrial regulation in diabetic cardiomyopathy mice[J].Oxid Med Cell Longev,2017,2017:4602715.
[21] Faria A,Persaud SJ.Cardiac oxidative stress in diabetes:mechanisms and therapeutic potential[J].Pharmacol Ther,2017,172(2):50-62.
[22] Zhang W,Zhang Y,Wang Z,et al.Tris(2-chloroethyl)phosphate-induced cell growth arrest via attenuation of SIRT1-independent PI3K/Akt/mTOR pathway[J].J Appl Toxicol,2016,36(7):914-924.
[2] Westermeier F,Riquelme JA,Pavez M,et al.New molecular insights of insulin in diabetic cardiomyopathy[J].Front Physiol,2016,7:125.
[3] Chen J,Zhang Z,Cai L.Diabetic cardiomyopathy and its prevention by Nrf2:current status[J].Diabetes Metab J,2014,38(5):337-345.
[4] Huynh K,Bernardo BC,McMullen JR,et al.Diabetic cardiomyopathy:mechanisms and new treatment strategies targeting antioxidant signaling pathways[J].Pharmacol Ther,2014,142(3):375-415.
[5] 王金鑫,段鹏,朱庆磊.一种建立小鼠2型糖尿病心肌病模型的方法[J].中国病理生理杂志,2018,34(4):764-768.
[6] Chen H,Yang X,Lu K,et al.Inhibition of high glucose-induced inflammation and fibrosis by a novel curcumin derivative prevents renal and heart injury in diabetic mice[J].Toxicol Lett,2017,278(15):48-58.
[7] 尹茂山,许淑红,王燕,等.2 型糖尿病大鼠主动脉 Wnt/β-catenin信号通路的变化及 SIRT1 的调节作用[J].中国药理学通报,2016,32(3):337-342.
[8] Muijen M.The state of psychiatry in Europe:facing the challenges,developing consensus[J].Int Rev Psychiatry,2012,24(4):274-277.
[9] 李越凡,徐丹,李婷,等.Sirt1 对糖尿病心肌病大鼠早期心肌 S-腺苷同型半胱氨酸水解酶的调节作用及其机制研究[J].国际心血管病杂志,2017,44(6):348-351.
[10] Karbasforooshan H,Karimi G.The role of SIRT1 in diabetic cardiomyopathy[J].Biomed Pharmacother,2017,90(9):386-392.
[11] Huby AC,Turdi S,James J,et al.FasL expression in cardiomyocytes activates ERK1/2 pathway,leading to dilated cardiomyopathy and advanced heart failure[J].Clin Sci (Lond),2016,130(4):289-299.
[12] Prescimone T,Masotti S,D'Amico A,et al.Cardiac molecular markers of programmed cell death are activated in end-stage heart failure patients supported by left ventricular assist device[J].Cardiovasc Pathol,2014,23(5):272-282.
[13] Wang S,Ding L,Ji H,et al.The role of p38 MAPK in the development of diabetic cardiomyopathy[J].Int J Mol Sci,2016,17(7):842-850.
[14] Delbridge LMD,Benson VL,Ritchie RH,et al.Diabetic cardiomyopathy:the case for a role of fructose in disease etiology[J].Diabetes,2016,65(12):3521-3528.
[15] Oblong JE.The evolving role of the NAD+/nicotinamide metabolome in skin homeostasis,cellular bioenergetics,and aging[J].DNA Repair (Amst),2014,23(11):59-63.
[16] Arunachalam G,Samuel SM,Marei I,et al.Metformin modulates hyperglycaemia-induced endothelial senescence and apoptosis through SIRT1[J].Br J Pharmacol,2014,171(2):523-535.
[17] Gong H,Pang J,Han Y,et al.Age-dependent tissue expression patterns of Sirt1 in senescence-accelerated mice[J].Mol Med Rep,2014,10(6):3296-3302.
[18] Pazienza V,Pomara C,Cappello F,et al.The TRPA1 channel is a cardiac target of mIGF-1/SIRT1 signaling[J].Am J Physiol Heart Circ Physiol,2014,307(7):H939-H944.
[19] Rong G,Liu W,Liu B,et al.SIRT1 suppresses cardiomyocyte apoptosis in diabetic cardiomyopathy:An insight into endoplasmic reticulum stress response mechanism[J].Int J Cardiol,2015,191(3):36-45.
[20] Ma S,Feng J,Zhang R,et al.SIRT1 activation by resveratrol alleviates cardiac dysfunction via mitochondrial regulation in diabetic cardiomyopathy mice[J].Oxid Med Cell Longev,2017,2017:4602715.
[21] Faria A,Persaud SJ.Cardiac oxidative stress in diabetes:mechanisms and therapeutic potential[J].Pharmacol Ther,2017,172(2):50-62.
[22] Zhang W,Zhang Y,Wang Z,et al.Tris(2-chloroethyl)phosphate-induced cell growth arrest via attenuation of SIRT1-independent PI3K/Akt/mTOR pathway[J].J Appl Toxicol,2016,36(7):914-924.
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