褪黑素通过沉默信息调节因子1减轻线粒体氧化应激对脑缺血-再灌注小鼠的作用
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摘要
目的探讨褪黑素通过沉默信息调节因子1(SIRT1)减轻线粒体氧化应激机制对脑缺血-再灌注(IR)小鼠的作用。方法通过线栓法建立小鼠短暂性大脑中动脉阻塞(MCAO)IR模型,对190只小鼠经腹腔注射褪黑素和侧脑室注射SIRT1抑制剂EX527,剔除死亡小鼠30只,按随机数字表法分为IR组、褪黑素组、褪黑素+EX527组、EX527组各40只,通过2,3,5-氯化三苯基四氮唑(TTC)法测定脑梗死体积、干湿比重法测定脑水肿并进行神经功能缺损评分。通过Western blot检测线粒体和胞质SIRT1、Ac-P53、Ac-核因子κB(Ac-NF-κB)、BCL2、BAX蛋白以及细胞色素C蛋白表达。多组间比较采用单因素方差分析。结果 (1)4组间脑梗死体积、神经功能障碍评分和脑水肿的差异均有统计学意义(F值分别为16.452、23.622和18.786,均P<0.05)。4组SITR1、Ac-P53、Ac-NF-κB、BCL2、BAX表达差异均有统计学意义(F值分别为2 348.158、1 434.841、7 042.563、14 627.128和691.475,均P<0.05)。4组小鼠线粒体膜电位、线粒体活性氧和复合体Ⅰ活性差异有统计学意义(F值分别为28.454、33.728和29.716,均P<0.05)。(2)与IR组相比,褪黑素组的脑梗死体积缩小(32±5)mm~3比(57±5)mm~3,P<0.05);神经功能障碍评分降低(2.4±0.3比3.5±0.3,P<0.05);脑水肿减轻(80.2±0.9)%比(83.9±1.2)%,P<0.05);SIRT1和抗凋亡蛋白BCL2的表达增加(P<0.05);促凋亡蛋白BAX和Ac-P53、Ac-NF-κB的表达减少(P<0.05);线粒体膜电位、线粒体复合体Ⅰ活性和细胞色素C水平提高(P<0.05);胞质活性氧产物和细胞色素C水平降低(P<0.05)。(3)与褪黑素组相比,褪黑素+EX527组的脑梗死体积增大[(42±5)mm~3比(32±5)mm~3,P<0.05];神经功能障碍评分增高(3.2±0.3比2.4±0.3,P<0.05);脑水肿加重[(83.4±0.8)%比(80.2±0.9)%,P<0.05];SIRT1和抗凋亡蛋白BCL2的表达减少(P<0.05);促凋亡蛋白BAX和Ac-P53、Ac-NF-κB的表达增加(P<0.05);线粒体膜电位、线粒体复合体Ⅰ活性和细胞色素C水平降低(P<0.05);胞质活性氧产物和胞质细胞色素C水平提高(P<0.05)。结论在缺血性卒中模型小鼠中,褪黑素通过激活SIRT1信号通路,减轻线粒体氧化应激损伤和细胞死亡,从而发挥脑保护作用。
Objective To investigate the effect of melatonin on mice with ischemia-reperfusion via a silent information regulator 1( SIRT1) reducing mitochondrial oxidative stress mechanism. Methods A transient middle cerebral artery occlusion( MCAO) cerebral ischemia-reperfusion( IR) model in mice was established by the suture-occluded method. One hundred and ninety mice were injected with melatonin intraperitoneally or the SIRT1 inhibitor( EX527) intracerebroventricularly,30 dead and model failure mice were excluded. They were divided into IR,melatonin,melatonin + EX527,and EX527 groups( n = 40 in each group) according to the random number table. The cerebral infarct volume was detected by the triphenyltetrazolium chloride( TTC) method,the brain edema was measured by the wet and dry weight method and the neurological deficit scores were measured. Western blot was used to detect SIRT1,Ac-P53,acetylated-nuclear factor κB( Ac-NF-κB),BCl2,Bax proteins in the mitochondria and cytoplasm,as well as the cytochrome C protein expression. A single factor analysis of variance was used for comparison among the groups. Results( 1) There were significant differences in cerebral infarction volume,neurological dysfunction scores and cerebral edema among the four groups( F values,16. 452,23. 622,and 18. 786,respectively( all P < 0. 05). There were significant differences in the expression levels of SIRT1,Ac-P53,Ac-NF-κB,BCl2,and Bax among the four groups( F values,2 348. 158,1 434. 841,7 042. 563,14 627. 128,and 691. 475,respectively,all P < 0. 05). There were significant differences in mitochondrial membrane potential,mitochondrial reactive oxygen species,and complex I activity in mice among the four groups( F value,28. 454,33. 728 and 29. 716,respectively,all P < 0. 05).( 2) Compared with the IR group,the infarct volume was reduced( 32 ± 5 mm~3 vs. 57 ± 5 mm~3,P < 0. 05),neurological deficit scores were decreased( 2. 4 ± 0. 3 vs. 3. 5 ± 0. 3,P < 0. 05); brain edema was reduced( 80. 2 ± 0. 9% vs. 83. 9 ±1. 2%,P < 0. 05); the expression levels of SIRT1 and anti-apoptosis protein BCL2 were increased in the melatonin group( P < 0. 05); the expression levels of pro-apoptotic protein BAX and Ac-P53,Ac-NF-κB were reduced( P < 0. 05); the mitochondrial membrane potential,mitochondrial complex I activity and cytochrome C level were increased( P < 0. 05); and the cytoplasmic reactive oxygen species and cytochrome C level were decreased( P < 0. 05).( 3) Compared with the melatonin group,cerebral infarction volume were increased( 42 ± 5 mm~3 vs. 32 ± 5 mm~3,P < 0. 05); nerve dysfunction scores were increased( 3. 2 ± 0. 3 vs. 2. 4 ± 0. 3,P < 0. 05); cerebral edema was aggravated( 83. 4 ± 0. 8 % vs.80. 2 ± 0. 9 %,P < 0. 05); the expression levels of SIRT1 and anti-apoptotic protein BCL2 were reduced( P < 0. 05); the pro-apoptotic protein BAX,Ac-P53,and Ac-NF-κB expression levels were increased( P < 0. 05); the mitochondrial membrane potential and mitochondrial complex I activity and cytochrome C level were decreased( P < 0. 05); and the cytoplasmic reactive oxygen species and cytoplasmic cytochrome C level were increased in the melatonin + EX527 group( P < 0. 05). Conclusion In ischemic stroke model mice,melatonin plays a neuroprotective role by activating the SIRT1 signaling pathway and reducing oxidative stress injury and cell death in mitochondria,thus plays a role in cerebral protection.
Objective To investigate the effect of melatonin on mice with ischemia-reperfusion via a silent information regulator 1( SIRT1) reducing mitochondrial oxidative stress mechanism. Methods A transient middle cerebral artery occlusion( MCAO) cerebral ischemia-reperfusion( IR) model in mice was established by the suture-occluded method. One hundred and ninety mice were injected with melatonin intraperitoneally or the SIRT1 inhibitor( EX527) intracerebroventricularly,30 dead and model failure mice were excluded. They were divided into IR,melatonin,melatonin + EX527,and EX527 groups( n = 40 in each group) according to the random number table. The cerebral infarct volume was detected by the triphenyltetrazolium chloride( TTC) method,the brain edema was measured by the wet and dry weight method and the neurological deficit scores were measured. Western blot was used to detect SIRT1,Ac-P53,acetylated-nuclear factor κB( Ac-NF-κB),BCl2,Bax proteins in the mitochondria and cytoplasm,as well as the cytochrome C protein expression. A single factor analysis of variance was used for comparison among the groups. Results( 1) There were significant differences in cerebral infarction volume,neurological dysfunction scores and cerebral edema among the four groups( F values,16. 452,23. 622,and 18. 786,respectively( all P < 0. 05). There were significant differences in the expression levels of SIRT1,Ac-P53,Ac-NF-κB,BCl2,and Bax among the four groups( F values,2 348. 158,1 434. 841,7 042. 563,14 627. 128,and 691. 475,respectively,all P < 0. 05). There were significant differences in mitochondrial membrane potential,mitochondrial reactive oxygen species,and complex I activity in mice among the four groups( F value,28. 454,33. 728 and 29. 716,respectively,all P < 0. 05).( 2) Compared with the IR group,the infarct volume was reduced( 32 ± 5 mm~3 vs. 57 ± 5 mm~3,P < 0. 05),neurological deficit scores were decreased( 2. 4 ± 0. 3 vs. 3. 5 ± 0. 3,P < 0. 05); brain edema was reduced( 80. 2 ± 0. 9% vs. 83. 9 ±1. 2%,P < 0. 05); the expression levels of SIRT1 and anti-apoptosis protein BCL2 were increased in the melatonin group( P < 0. 05); the expression levels of pro-apoptotic protein BAX and Ac-P53,Ac-NF-κB were reduced( P < 0. 05); the mitochondrial membrane potential,mitochondrial complex I activity and cytochrome C level were increased( P < 0. 05); and the cytoplasmic reactive oxygen species and cytochrome C level were decreased( P < 0. 05).( 3) Compared with the melatonin group,cerebral infarction volume were increased( 42 ± 5 mm~3 vs. 32 ± 5 mm~3,P < 0. 05); nerve dysfunction scores were increased( 3. 2 ± 0. 3 vs. 2. 4 ± 0. 3,P < 0. 05); cerebral edema was aggravated( 83. 4 ± 0. 8 % vs.80. 2 ± 0. 9 %,P < 0. 05); the expression levels of SIRT1 and anti-apoptotic protein BCL2 were reduced( P < 0. 05); the pro-apoptotic protein BAX,Ac-P53,and Ac-NF-κB expression levels were increased( P < 0. 05); the mitochondrial membrane potential and mitochondrial complex I activity and cytochrome C level were decreased( P < 0. 05); and the cytoplasmic reactive oxygen species and cytoplasmic cytochrome C level were increased in the melatonin + EX527 group( P < 0. 05). Conclusion In ischemic stroke model mice,melatonin plays a neuroprotective role by activating the SIRT1 signaling pathway and reducing oxidative stress injury and cell death in mitochondria,thus plays a role in cerebral protection.
引文
[1]刘玲,杨昉,刘海波,等.组织激肽释放酶对酸敏感离子通道1介导的大鼠酸中毒神经元氧化应激的影响[J].中国脑血管病杂志,2012,9(4):200-205.
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[3]陈路佳,蔡军,赵京晶,等.应用同步辐射血管造影观察褪黑素对大鼠蛛网膜下腔出血致血管痉挛的影响[J].中国脑血管病杂志,2012,9(5):259-264.
[4]谭凌菁,王金凤,梅志刚.沉默信息调节因子1信号通路在脑缺血-再灌注损伤中的保护作用[J].中国脑血管病杂志,2016,13(8):442-445.
[5]Hernández-Jiménez M,Hurtado O,Cuartero MI,et al.Silent information regulator 1 protects the brain against cerebral ischemic damage[J].Stroke,2 0 1 3,4 4(8):2333-2337.
[6]Carloni S,Albertini MC,Galluzzi L,et al.Melatonin reduces endoplasmic reticulum stress and preserves sirtuin 1 expression in neuronal cells of newborn rats after hypoxia-ischemia[J].J Pineal Res,2014,57(2):192-199.
[7]Longa EZ,Weinstein PR,Carlson S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20(1):84-91.
[8]Chern CM,Liao JF,Wang YH,et al.Melatonin ameliorates neural function by promoting endogenous neurogenesis through the MT2 melatonin receptor in ischemic-stroke mice[J].Free Radic Biol Med,2012,52(9):1634-1647.
[9]Reiter RJ,Cabrera J,Sainz RM,et al.Melatonin as a pharmacological agent against neuronal loss in experimental models of H untington's disease,A lzheim er's disease and Parkinsonism[J].Ann N Y Acad Sci,1999,890(10):471-485.
[10]Reiter RJ,Paredes SD,Manchester LC.et al.Reducing oxidative/nitrosative stress:a newly-discovered genre for melatonin[J].Crit Rev Biochem Mol Biol,2009,44(4):175-200.
[11]Zhao W,Kruse JP,Tang Y,et al.Negative regulation of the deacetylase SIRT1 by DBC1[J].Nature,2008,451(7178):587-590.
[12]Yang Y,Duan W,Li Y,et al.Novel role of silent information regulator 1 in myocardial ischemia[J].Circulation,2013,128(20):2232-2240.
[13]Carloni S,Albertini MC,Galluzzi L,et al.Melatonin reduces endoplasm ic reticulum stress and preserves sirtuin 1 expression in neuronal cells of newborn rats after hypoxiaischemia[J].J Pineal Res,2014,57(2):192-199.
[14]Khader A,Yang WL,Kuncewitch M,et al.Sirtuin 1 activation stim ulates m itochondrial biogenesis and attenuates renal injury after ischemia-reperfusion[J].Transplantation,2014,98(2):148-156.
[15]Herskovits AZ,Guarente L.SIRT1 in neurodevelopment and brain senescence[J].Neuron,2014,81(3):471-483.
[16]Patki G,Lau YS.Melatonin protects against neurobehavioral and mitochondrial deficits in a chronic mouse model of Parkinson's disease[J].Pharmacol Biochem Behav,2011,99(4):704-711.
[17]Garcia-Macia M,Vege-Naredo I,De Gonzalo-Calvo D,et al.Melatonin induces neural SOD2 expression independent of the NF-kappa B pathway and improves the mitochondrial population and function in old mice[J].J Pineal Res,2011,50(1):54-63.
[2]Mauriz JL,Collado PS,Veneroso C,et al.A review of the m olecular aspects of m elatonin's anti-inflam m atory action s:recent in sights and new perspectives[J].J Pineal Res,2013,54(1):1-14.
[3]陈路佳,蔡军,赵京晶,等.应用同步辐射血管造影观察褪黑素对大鼠蛛网膜下腔出血致血管痉挛的影响[J].中国脑血管病杂志,2012,9(5):259-264.
[4]谭凌菁,王金凤,梅志刚.沉默信息调节因子1信号通路在脑缺血-再灌注损伤中的保护作用[J].中国脑血管病杂志,2016,13(8):442-445.
[5]Hernández-Jiménez M,Hurtado O,Cuartero MI,et al.Silent information regulator 1 protects the brain against cerebral ischemic damage[J].Stroke,2 0 1 3,4 4(8):2333-2337.
[6]Carloni S,Albertini MC,Galluzzi L,et al.Melatonin reduces endoplasmic reticulum stress and preserves sirtuin 1 expression in neuronal cells of newborn rats after hypoxia-ischemia[J].J Pineal Res,2014,57(2):192-199.
[7]Longa EZ,Weinstein PR,Carlson S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20(1):84-91.
[8]Chern CM,Liao JF,Wang YH,et al.Melatonin ameliorates neural function by promoting endogenous neurogenesis through the MT2 melatonin receptor in ischemic-stroke mice[J].Free Radic Biol Med,2012,52(9):1634-1647.
[9]Reiter RJ,Cabrera J,Sainz RM,et al.Melatonin as a pharmacological agent against neuronal loss in experimental models of H untington's disease,A lzheim er's disease and Parkinsonism[J].Ann N Y Acad Sci,1999,890(10):471-485.
[10]Reiter RJ,Paredes SD,Manchester LC.et al.Reducing oxidative/nitrosative stress:a newly-discovered genre for melatonin[J].Crit Rev Biochem Mol Biol,2009,44(4):175-200.
[11]Zhao W,Kruse JP,Tang Y,et al.Negative regulation of the deacetylase SIRT1 by DBC1[J].Nature,2008,451(7178):587-590.
[12]Yang Y,Duan W,Li Y,et al.Novel role of silent information regulator 1 in myocardial ischemia[J].Circulation,2013,128(20):2232-2240.
[13]Carloni S,Albertini MC,Galluzzi L,et al.Melatonin reduces endoplasm ic reticulum stress and preserves sirtuin 1 expression in neuronal cells of newborn rats after hypoxiaischemia[J].J Pineal Res,2014,57(2):192-199.
[14]Khader A,Yang WL,Kuncewitch M,et al.Sirtuin 1 activation stim ulates m itochondrial biogenesis and attenuates renal injury after ischemia-reperfusion[J].Transplantation,2014,98(2):148-156.
[15]Herskovits AZ,Guarente L.SIRT1 in neurodevelopment and brain senescence[J].Neuron,2014,81(3):471-483.
[16]Patki G,Lau YS.Melatonin protects against neurobehavioral and mitochondrial deficits in a chronic mouse model of Parkinson's disease[J].Pharmacol Biochem Behav,2011,99(4):704-711.
[17]Garcia-Macia M,Vege-Naredo I,De Gonzalo-Calvo D,et al.Melatonin induces neural SOD2 expression independent of the NF-kappa B pathway and improves the mitochondrial population and function in old mice[J].J Pineal Res,2011,50(1):54-63.