白藜芦醇通过激活mTOR/自噬抑制缺氧缺糖/复氧复糖诱导的PC12细胞损伤及凋亡
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摘要
目的:白藜芦醇对缺氧缺糖/复氧复糖(OGD/R)诱导的PC12细胞损伤及凋亡的影响及作用机制。方法:以PC12细胞建立OGD/R自噬性损伤模型,设置空白对照组、模型组、不同浓度(5、10μmol/L)白藜芦醇组,自噬抑制剂(3-MA)组。使用CCK-8试剂盒检测PC12的增殖能力;流式细胞术检测各组细胞凋亡率;ELISA检测各组细胞培养液中TNF-α和IL-6的浓度变化;DHE荧光探针检测ROS的变化;特定试剂盒检测乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH-PX)含量;Westernblot法检测各组mTOR的磷酸化情况及LC3-Ⅱ、Beclin-1蛋白表达。结果:与空白对照组相比模型组能够显著降低PC12的增殖能力,增加细胞中ROS的含量和凋亡率,诱导TNF-α和IL-6分泌,LDH、MDA含量显著增加,SOD、GSH-PX活性显著下降,而P-mTOR/mTOR、LC3-Ⅱ、Beclin-1表达增加(P<0.01)。与模型组比较,不同浓度白藜芦醇作用PC12时,细胞存活率逐渐上升,ROS含量及凋亡率逐渐下降,LDH、MDA含量减少,SOD、GSH-PX活性随之上升,TNF-α和IL-6分泌显著下降,P-mTOR/mTOR、LC3-Ⅱ、Beclin-1表达进一步增加(P<0.01)。自噬抑制剂(3-MA)组上述指标的检测结果与白藜芦醇组相反。结论:白藜芦醇可能部分通过激活mTOR/自噬通路对神经细胞发挥保护作用。
Objective: To investigate the effect of Resveratrol on injury and apoptosis of PC12 cells induced by OGD/R and explore the possible mechanism. Methods: PC12 cells were cultured in vitro. OGD/R acted on PC12 cells to induce the injury and apoptosis.The cell proliferation ability of each group was detected by CCK-8 assay.The apoptosis rate of cells in each group was detected by flow cytometry.The concentration of nitric oxide( NO),TNF-α and IL-6 in the culture medium of each group was detected by ELISA. LDH、MDA、SOD and GSH-PX were detected by specific kits.Detection of ROS changes in Cerebral cortical nerve cells by DHE fluorescence probe.Western blot was used to detect the expression of protein( P-mTOR/mTOR、LC3-Ⅱ、Beclin-1) in each group.Results: Resveratrol significantly enhanced PC12 cells proliferation ability and increased the activities of antioxidant enzymes(superoxide dismutase,catalase),scavenged reactive oxygen species(ROS),inhibited TNF-α,IL-6 secretion.Resveratrol also reduced the rate of apoptosis of cells.Resveratrol upregulated protein( P-mTOR/mTOR、LC3-Ⅱ、Beclin-1) expression.3-MA( inhibitor of autophagy) could prevent this protection.Conclusion: The protective effect of Resveratrol on PC12 cells injury and apoptosis induced by OGD/R via activating mTOR/autophagy.
Objective: To investigate the effect of Resveratrol on injury and apoptosis of PC12 cells induced by OGD/R and explore the possible mechanism. Methods: PC12 cells were cultured in vitro. OGD/R acted on PC12 cells to induce the injury and apoptosis.The cell proliferation ability of each group was detected by CCK-8 assay.The apoptosis rate of cells in each group was detected by flow cytometry.The concentration of nitric oxide( NO),TNF-α and IL-6 in the culture medium of each group was detected by ELISA. LDH、MDA、SOD and GSH-PX were detected by specific kits.Detection of ROS changes in Cerebral cortical nerve cells by DHE fluorescence probe.Western blot was used to detect the expression of protein( P-mTOR/mTOR、LC3-Ⅱ、Beclin-1) in each group.Results: Resveratrol significantly enhanced PC12 cells proliferation ability and increased the activities of antioxidant enzymes(superoxide dismutase,catalase),scavenged reactive oxygen species(ROS),inhibited TNF-α,IL-6 secretion.Resveratrol also reduced the rate of apoptosis of cells.Resveratrol upregulated protein( P-mTOR/mTOR、LC3-Ⅱ、Beclin-1) expression.3-MA( inhibitor of autophagy) could prevent this protection.Conclusion: The protective effect of Resveratrol on PC12 cells injury and apoptosis induced by OGD/R via activating mTOR/autophagy.
引文
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[3]卢娜,魏林郁,王宝英,等.自噬参与低氧预处理抗PC12细胞糖氧剥夺损伤过程[J].中国病理生理杂志,2015,31(9):1627-1632.
[4]ALIREZAEI M,KEMBALL CC,WHITTON JL.Autophagy,inflammation and neurodegenerative disease[J].European Journal of Neuroscience,2011,33(2):197-204.
[5]Pervaiz S,Holme AL.Resveratrol:its biologic targets and functional activity[J].Antioxid Redox Signal,2009,11:2851-2897.
[6]Albani D,Polito L,Signorini A,et al.Neuroprotective properties of resveratrol in different neurodegenerative disorders[J].Biofactors,2010,36:370-376.
[7]Foti CV,Ciurleo R,Giacoppo S,et al.Role of resveratrol and its analogues in the treatment of neurodegenerative diseases:focus on recent discoveries[J].CNS Neurol Disord Drug Targets,2011,10:849-862.
[8]PENG J,MAO XO,STEVENSON FF,et al.The herbicide paraquat induces dopaminergic nigral apoptosis through sustained activation of the JNK pathway[J].Journal of Biological Chemistry,2004,279(31):32626-32632.
[9]ADHAMI F,LIAO G,MOROZOV YM,et al.Cerebral ischemiahypoxia induces intravascular coagulation and autophagy[J].Am JPathol,2006,169(2):566-583.
[10]QIN AP,ZHANG HL.Mechanisms of lysosomal proteases participating in cerebral ischemia-induced neuronal death[J].Neuroscience Bulletin,2008,24(2):117-123.
[11]MEIJER AJ,CODOGNO P.Regulation and role of autophagy in mam-malian cells[J].Int J Biochem Cell Biol,2004,36(12):2445-2462.
[12]C Barbara,L Maria Teresa,L Gloria,et al.Autophagy-related protein LC3 and Beclin-1 in the first trimester of pregnancy[J].Clin Exp Reprod Med,2013,40(1):33-37.
[13]BARTH S,GLICK D,MACLEOD KF.Autophagy:assays and artifacts[J].Journal of Pathology,2010,221(2):117-124.
[14]PARK HK,CHU K,JUNG KH,et al.Autophagy is involved in the ischemic preconditioning[J].Neuroscience Letters,2009,451(1):16-19.
[15]SHENG R,ZHANG LS,HAN R,et al.Autophagy activation is associated with neuroprotection in a rat model of focal cerebral ischemic preconditioning[J].Autophagy,2010,6(4):482-494.
[16]C Dai,GD Ciccotosto,R Cappai,et al.Rapamycin Confers Neuroprotection against Colistin-Induced Oxidative Stress,Mitochondria Dysfunction,and Apoptosis through the Activation of Autophagy and mTOR/Akt/CREB Signaling Pathways[J].Acs Chemical Neuroscience,2018,9(4):824-837.
[17]M Wei,L Gan,Z Liu,et al.MiR125b-5p protects endothelial cells from apoptosis under oxidative stress[J].Biomed Pharmacother,2017,95:453-460.
[18]郭思琪,张明月,张琳,等.血清炎症因子和氧化应激产物的动态监测在急性脑出血患者中的应用[J].黑龙江医药科学,2018(04):1.
[19]师宁宁,徐俊华,张红.阿托伐他汀联合依折麦布治疗冠状动脉粥样硬化性心脏病对患者血脂、心功能、氧化应激反应的影响[J].中国药物经济学,2018(07):1.
[20]彭安洋.探讨综合疗法与神经节苷脂钠治疗中老年重症脑卒[J].安徽卫生职业技术学院学报,2016(03):1.
[21]顾民华,洪文,唐传其,等.芒柄花素保护前脑缺血再灌注损伤中的血脑屏障并抑制神经炎症[J].暨南大学学报(自然科学与医学版),2015,36(1):34-39.