吡咯喹啉醌对神经细胞氧糖剥夺损伤的保护作用及机制研究
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摘要
目的:脑缺血后的病理生理过程复杂,脑缺血再灌注损伤级联反应其中一环是细胞凋亡,抑制神经细胞凋亡有利于缺血性脑血管病的治疗和康复。近期国内外研究表明,吡咯喹啉醌(Pyrroloquinoline quinone ,PQQ)具有较强的抗氧化作用,可能通过抗氧化应激对脑缺血有保护作用。而氧化应激反应被认为是导致凋亡的最重要途径之一,因此本文研究吡咯喹啉醌(PQQ)是否具有抗凋亡作用及其可能机制。研究吡咯喹啉醌(PQQ)对体外培养小鼠神经母细胞瘤Neuro2A细胞株氧糖剥夺/复氧模型(OGD/R)的影响,在细胞水平探讨PQQ神经保护作用的可能机制。
方法:利用不同浓度PQQ预处理Neuro2A细胞后,通过无氧密闭盒和无糖培养液,建立氧糖剥夺模型。取复氧后6h这一时间点,观察PQQ干预对Neuro2A细胞的细胞形态、存活率、凋亡率以及氧化应激检测指标活性氧(ROS)和还原型谷胱甘肽(GSH)的影响。细胞形态学采用倒置相差显微镜观察;细胞存活率采用四唑盐(MTT)法检测;细胞凋亡率采用荧光显微镜观察;ROS和GSH采用荧光分光光度计检测。
结果:一.倒置相差显微镜观察细胞形态:OGD 2h复氧6h后Neuro2A细胞形态出现严重损伤,6.4μM和12.8μM的PQQ预处理有减轻神经细胞损伤的作用;二.MTT法检测细胞存活率:OGD 2h复氧6h后,0‐12.8μM的PQQ预处理组细胞的存活率呈逐渐增高趋势(P<0.05)。三.荧光显微镜下计数细胞凋亡率:OGD 2h复氧6h后,6.4μM、12.8μM的PQQ预处理组和OGD无保护组相比细胞凋亡数逐渐减少(P<0.05)。四.PQQ预处理后细胞内ROS生成减少(P<0.01),GSH水平增高(P<0.05),与氧糖剥夺/复氧组相比有统计学差异。
结论:一.PQQ对神经细胞氧糖剥夺损伤有一定的保护作用。二.PQQ对氧糖剥夺损伤的Neuro2A细胞的神经保护作用可能是通过减轻氧化应激反应,降低细胞凋亡率来实现的。
Objective: Stroke is a life-threatening disease characterized by rapidly developing clinical signs of focal or global disturbance of cerebral function due to cerebral ischemia. The physiopathologic mechanisms of cerebral ischemia reperfusion injury were extremely complex. Multiple pathways were involved in the ischemic process that ultimately leads to cell death. In particular, the neuron apoptosis after ischemia/reperfu- sion is one of the major pathways that leads to the process of cell death .Therefore ,prot- ectting neurons from apoptosis may be beneficial to the therapy of ischemic disease. Pyrroloquinoline quinine(PQQ),which is an essential nutrient, has been demonstrated to act as a strong antioxidant , and thought to be have the function against cerebral ischemia by inhibting the oxidative stress. For thus ,we hypothesized that PQQ might have a neuroprotective effect via modulation of multiple pathways associated with apoptosis.
To investigate the neuroprotective effects of PQQ against oxygen and glucose deprivation (OGD) in cultured rat neuroblastoma cells Neuro2A and the possible mechanisms involved.
Methods: Cultured rat neuroblastoma cells Neuro2A were pretreated or not pretreated with increasingly concentrations of PQQ , exposed to 2 hours combined OGD in an anaerobic chamber followed by reoxygenation of 6 hours .Cellular morphology was observed by inverted phase contract microscope. Assessment of cell viability was quantitatively performed by the reduction of 3-(4,5-Dimethylthialzol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT) at reoxygenation 6 hours after OGD, and the percentage of apoptotic cells was tested by flow cytometry and Hoechst 33342 staining. The contents of intracellular ROS and GSH level were measured with fluorospectrophotometer .
Results: 1. After OGD treating in cortical neurons for 2 hours and reoxygenation treating for 6 hours the swelling of neuronal body were significant ,while PQQ could lessen the swelling of neuronal body. 2. PQQ(0.4,0.8,1.6,3.2,6.4,12.8μM)group increased the cell viability following OGD/reoxygenation, and, the maximal neuroprotection was afforded by 12.8μM PQQ ,while the toxic dose of PQQ appeared at 25.6μM. 3. After OGD treating in Neuro2A cells for 2 hours and reoxygenation treating for 6 hours the rate of apoptotic neurons increased significantly. PQQ(6.4,12.8μM) could significantly inhibited OGD/reoxygenation-induced apoptosis of cultured Neuro2A cells (P<0.01). 4. The content of ROS was increased and the level of GSH was decreased at 6h after OGD, we observed that PQQ (6.4,12.8μM) decreeased the content of ROS and increased the GSH level, significantly.
Conclusion: 1. PQQ exhibits remarkable protection against hypoxia/Reoxygenat- ion injury in Neuro2A cells. 2. The mechanism of PQQ neuroprotection may related to its ability of inhibiting the oxidative stress and subsequently suppressing the neurons apoptosis.
方法:利用不同浓度PQQ预处理Neuro2A细胞后,通过无氧密闭盒和无糖培养液,建立氧糖剥夺模型。取复氧后6h这一时间点,观察PQQ干预对Neuro2A细胞的细胞形态、存活率、凋亡率以及氧化应激检测指标活性氧(ROS)和还原型谷胱甘肽(GSH)的影响。细胞形态学采用倒置相差显微镜观察;细胞存活率采用四唑盐(MTT)法检测;细胞凋亡率采用荧光显微镜观察;ROS和GSH采用荧光分光光度计检测。
结果:一.倒置相差显微镜观察细胞形态:OGD 2h复氧6h后Neuro2A细胞形态出现严重损伤,6.4μM和12.8μM的PQQ预处理有减轻神经细胞损伤的作用;二.MTT法检测细胞存活率:OGD 2h复氧6h后,0‐12.8μM的PQQ预处理组细胞的存活率呈逐渐增高趋势(P<0.05)。三.荧光显微镜下计数细胞凋亡率:OGD 2h复氧6h后,6.4μM、12.8μM的PQQ预处理组和OGD无保护组相比细胞凋亡数逐渐减少(P<0.05)。四.PQQ预处理后细胞内ROS生成减少(P<0.01),GSH水平增高(P<0.05),与氧糖剥夺/复氧组相比有统计学差异。
结论:一.PQQ对神经细胞氧糖剥夺损伤有一定的保护作用。二.PQQ对氧糖剥夺损伤的Neuro2A细胞的神经保护作用可能是通过减轻氧化应激反应,降低细胞凋亡率来实现的。
Objective: Stroke is a life-threatening disease characterized by rapidly developing clinical signs of focal or global disturbance of cerebral function due to cerebral ischemia. The physiopathologic mechanisms of cerebral ischemia reperfusion injury were extremely complex. Multiple pathways were involved in the ischemic process that ultimately leads to cell death. In particular, the neuron apoptosis after ischemia/reperfu- sion is one of the major pathways that leads to the process of cell death .Therefore ,prot- ectting neurons from apoptosis may be beneficial to the therapy of ischemic disease. Pyrroloquinoline quinine(PQQ),which is an essential nutrient, has been demonstrated to act as a strong antioxidant , and thought to be have the function against cerebral ischemia by inhibting the oxidative stress. For thus ,we hypothesized that PQQ might have a neuroprotective effect via modulation of multiple pathways associated with apoptosis.
To investigate the neuroprotective effects of PQQ against oxygen and glucose deprivation (OGD) in cultured rat neuroblastoma cells Neuro2A and the possible mechanisms involved.
Methods: Cultured rat neuroblastoma cells Neuro2A were pretreated or not pretreated with increasingly concentrations of PQQ , exposed to 2 hours combined OGD in an anaerobic chamber followed by reoxygenation of 6 hours .Cellular morphology was observed by inverted phase contract microscope. Assessment of cell viability was quantitatively performed by the reduction of 3-(4,5-Dimethylthialzol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT) at reoxygenation 6 hours after OGD, and the percentage of apoptotic cells was tested by flow cytometry and Hoechst 33342 staining. The contents of intracellular ROS and GSH level were measured with fluorospectrophotometer .
Results: 1. After OGD treating in cortical neurons for 2 hours and reoxygenation treating for 6 hours the swelling of neuronal body were significant ,while PQQ could lessen the swelling of neuronal body. 2. PQQ(0.4,0.8,1.6,3.2,6.4,12.8μM)group increased the cell viability following OGD/reoxygenation, and, the maximal neuroprotection was afforded by 12.8μM PQQ ,while the toxic dose of PQQ appeared at 25.6μM. 3. After OGD treating in Neuro2A cells for 2 hours and reoxygenation treating for 6 hours the rate of apoptotic neurons increased significantly. PQQ(6.4,12.8μM) could significantly inhibited OGD/reoxygenation-induced apoptosis of cultured Neuro2A cells (P<0.01). 4. The content of ROS was increased and the level of GSH was decreased at 6h after OGD, we observed that PQQ (6.4,12.8μM) decreeased the content of ROS and increased the GSH level, significantly.
Conclusion: 1. PQQ exhibits remarkable protection against hypoxia/Reoxygenat- ion injury in Neuro2A cells. 2. The mechanism of PQQ neuroprotection may related to its ability of inhibiting the oxidative stress and subsequently suppressing the neurons apoptosis.
引文
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8.Nunome K, Miyazaki S, Nakano M, et al. Pyrroloquinoline quinone prevents oxidative stress-induced neuronal death probably through changes in oxidative status of DJ-1. Biol Pharm Bull. 2008 Jul;31(7):1321-6.
9.Scanlon JM, Aizenman E, Reynolds IJ. Effects of pyrroloquinoline quinone on glutamate-induced production of reactive oxygen species in neurons. Eur J Pharmacol. 1997 May 12;326(1):67-74.
10.Zhang Y, Feustel PJ, Kimelberg HK.Neuroprotection by pyrroloquinoline quinone (PQQ) in reversible middle cerebral artery occlusion in the adult rat. Brain Res. 2006 Jun 13;1094(1):200-6. Epub 2006 May 18.
11.Sanchez RM, Wang C, Gardner G, et al. Novel role for the NMDA receptor redox modulatory site in the pathophysiology of seizures. J Neurosci. 2000 Mar 15;20(6):2409-17.
12.Aizenman E, Hartnett KA, Zhong C, et al. Interaction of the putative essential nutrient pyrroloquinoline quinone with the N-methyl-D-aspartate receptor redox modulatory site. J Neurosci. 1992 Jun;12(6):2362-9.
13.Zhang P, Xu Y, Sun J,et al. Protection of pyrroloquinoline quinone against methylmercury- induced neurotoxicity via reducing oxidative stress. Free Radic Res. 2009 Mar;43(3):224-33.
14.Alexandrova ML, Bochev PG. Oxidative stress during the chronic phase after str-oke. Free Radic Biol Med. 2005 Aug 1;39(3):297-316.
15.Jensen FE, Gardner GJ, Williams AP, et al. The putative essential nutrient pyrroloquinoline quinone is neuroprotective in a rodent model of hypoxic/ischemic b- rain injury. Neuroscience. 1994 Sep;62(2):399-406.
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