高糖通过Nox4型NADPH氧化酶影响施旺细胞凋亡的机制研究
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摘要
目的:探讨高糖通过Nox4型NADPH氧化酶影响施旺细胞凋亡的机制。方法:提取Wistar大鼠新生鼠的施旺细胞体外培养。分为对照组、高糖组、NOX4 siRNA组及对照siRNA组(n=10)。采用WST-1法检测细胞活力,DCFH-DA法检测细胞内活性氧自由基(ROS)含量,荧光实时定量RT-PCR检测Nox4和Caspase3 mRNA表达,蛋白印迹法检测Nox4和Caspase3蛋白表达。结果:高糖培养上调施旺细胞Nox4 mRNA及蛋白表达,降低施旺细胞活性,增加细胞内ROS含量,通过增加Caspase3 mRNA及蛋白表达促进细胞凋亡。NOX4 siRNA通过抑制Nox4基因表达,阻止高糖培养的施旺细胞内ROS蓄积,降低高糖对施旺细胞的活性损害,通过下调Caspase3 mRNA及蛋白表达减少细胞凋亡。结论:Nox4参与高糖引起的施旺细胞凋亡,针对Nox4表达或功能的调控方式可能成为治疗糖尿病周围神经病变的新途径。
Objective: To investigate the mechanism of high glucose affecting the apoptosis of schwann cells through Nox4 NADPH oxidase. Methods: The schwann cells of newborn Wistar rats were cultured in vitro. The cultured cells were divided into four groups: control group,high-glucose group,NOX4 siRNA group and control siRNA group( n = 10). The WST-1 method was used to detect the cell vitality,and the DCFH-DA method was used to detect the contents of intracellular reactive oxygen free radicals( ROS).Nox4 and Caspase3 mRNA expressions were detected by real-time fluorescence quantitative RT-PCR. Nox4 and Caspase3 protein expressions were determined by Western blot. Results: High glucose culture up-regulated Nox4 mRNA and protein expressions of schwann cells,decreased activity of schwann cells,increased intracellular ROS content,and promoted apoptosis by increasing Caspase3 mRNA and protein expressions. NOX4 siRNA blocked the accumulation of ROS in the high glucose cultured schwann cells,and reduced the damage of glucose on cell viability,by inhibiting NOX4 gene expression. NOX4 siRNA also reduced cell apoptosis by down-regulating Caspase3 mRNA and protein expressions. Conclusion: Nox4 was involved in the hyperglycemic-induced apoptosis of schwann cells through ROS. The regulation of Nox4 expression or function might be a new way to treat diabetic peripheral neuropathy.
Objective: To investigate the mechanism of high glucose affecting the apoptosis of schwann cells through Nox4 NADPH oxidase. Methods: The schwann cells of newborn Wistar rats were cultured in vitro. The cultured cells were divided into four groups: control group,high-glucose group,NOX4 siRNA group and control siRNA group( n = 10). The WST-1 method was used to detect the cell vitality,and the DCFH-DA method was used to detect the contents of intracellular reactive oxygen free radicals( ROS).Nox4 and Caspase3 mRNA expressions were detected by real-time fluorescence quantitative RT-PCR. Nox4 and Caspase3 protein expressions were determined by Western blot. Results: High glucose culture up-regulated Nox4 mRNA and protein expressions of schwann cells,decreased activity of schwann cells,increased intracellular ROS content,and promoted apoptosis by increasing Caspase3 mRNA and protein expressions. NOX4 siRNA blocked the accumulation of ROS in the high glucose cultured schwann cells,and reduced the damage of glucose on cell viability,by inhibiting NOX4 gene expression. NOX4 siRNA also reduced cell apoptosis by down-regulating Caspase3 mRNA and protein expressions. Conclusion: Nox4 was involved in the hyperglycemic-induced apoptosis of schwann cells through ROS. The regulation of Nox4 expression or function might be a new way to treat diabetic peripheral neuropathy.
引文
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[13]Yao M,Cao F,Wang X,et al.Nox4 is involved in high glucose-induced apoptosis in renal tubular epithelial cells via Notch pathway[J].Mol Med Rep,2017,15(6):4319-4325.
[14]Hou Q,Lei M,Hu K,et al.The effects of high glucose levels on reactive oxygen species-induced apoptosis and involved signaling in human vascular endothelial cells[J].Cardiovasc Toxicol,2015,15(2):140-146.
[15]Jha JC,Gray SP,Barit D,et al.Genetic targeting or pharmacologic inhibition of NADPH oxidase nox4 provides renoprotection in long-term diabetic nephropathy[J].J Am Soc Nephrol,2014,25(6):1237-1254.
[2]Yu T,Li L,Bi Y,et al.Erythropoietin attenuates oxidative stress and apoptosis in Schwann cells isolated from streptozotocin-induced diabetic rats[J].J Pharm Pharmacol,2014,66(8):1150-1160.
[3]陈娟,崔节达,郭晓桐,等.慢性阻塞性肺疾病小气道NOX4、TGF-β的表达与气道重塑的关系[J].中国应用生理学杂志,2017,33(6):481-487.
[4]Nisimoto Y,Diebold BA,Cosentino-Gomes D,et al.Nox4:a hydrogen peroxide-generating oxygen sensor[J].Biochemistry,2014,53(31):5111-5120.
[5]Gon9alves NP,Vgter CB,Pallesen LT.Peripheral glial cells in the development of diabetic neuropathy[J].Front Neurol,2018,9:268.
[6]马益梅,李传达,朱雅冰,等.Rv D1对大鼠2型糖尿病神经病理性痛的作用及机制研究[J].中国应用生理学杂志,2017,33(3),277-281.
[7]Lennertz RC,Medler KA,Bain JL,et al.Impaired sensory nerve function and axon morphology in mice with diabetic neuropathy[J].J Neurophysiol,2011,106(2):905-914.
[8]Volpe CMO,Villar-Delfino PH,Dos Anjos PMF,et al.Cellular death,reactive oxygen species(ROS)and diabetic complications[J].Cell Death Dis,2018,9(2):119.
[9]Zhang L,Pang S,Deng B,et al.High glucose induces renal mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway,which is inhibited by resveratrol[J].Int J Biochem Cell Biol,2012,44(4):629-638.
[10]何婷.白藜芦醇抑制糖尿病肾病肾间质纤维化的作用和机制研究[D].重庆:第三军医大学,2015:75-76.
[11]Rozentsvit A,Vinokur K,Samuel S,et al.Ellagic acid reduces high glucose-induced vascular oxidative stress through ERK1/2/NOX4 signaling pathway[J].Cell Physiol Biochem,2017,44(3):1174-1187.
[12]Li Q,Lin Y,Wang S,et al.GLP-1 inhibits high-glucose-induced oxidative injury of vascular endothelial cells[J].Sci Rep,2017,7(1):8008.
[13]Yao M,Cao F,Wang X,et al.Nox4 is involved in high glucose-induced apoptosis in renal tubular epithelial cells via Notch pathway[J].Mol Med Rep,2017,15(6):4319-4325.
[14]Hou Q,Lei M,Hu K,et al.The effects of high glucose levels on reactive oxygen species-induced apoptosis and involved signaling in human vascular endothelial cells[J].Cardiovasc Toxicol,2015,15(2):140-146.
[15]Jha JC,Gray SP,Barit D,et al.Genetic targeting or pharmacologic inhibition of NADPH oxidase nox4 provides renoprotection in long-term diabetic nephropathy[J].J Am Soc Nephrol,2014,25(6):1237-1254.