白藜芦醇通过促进线粒体自噬减轻小鼠脑缺血/再灌注损伤的实验研究
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摘要
目的研究白藜芦醇(resveratrol,Res)对脑缺血/再灌注(ischemia/reperfusion,I/R)损伤的保护作用及其可能作用机制。方法采用线栓法建立小鼠右侧大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)模型。各组小鼠术前连续腹腔给药5 d,每日1次。术后24 h对梗死体积和神经功能进行评价; Western blot检测沉默信息调节因子1(silent information regulator 1,SIRT1)和线粒体自噬相关蛋白p62、LC3B、PHB2、TOM20表达情况,以及细胞色素C (cytochrome C,CytC)分布情况;流式细胞术检测线粒体膜电位(mitochondrial membrane potential,MMP)变化;酶标仪检测胞内ATP水平变化;透射电镜观察线粒体形态变化。结果Res预处理减少了脑梗死体积和神经功能缺损,提高了SIRT1和线粒体自噬相关蛋白表达,减少了CytC的释放,恢复了MMP,提高了ATP水平。透射电镜结果显示,Res减轻了缺血导致的线粒体损伤。自噬抑制剂3-MA抑制了线粒体自噬,同时也逆转了Res的神经、线粒体保护作用。结论Res减轻小鼠脑组织I/R损伤可能与其对线粒体自噬的调控,最终确保线粒体质量有关。
Aim To investigate the effects of resveratrol(Res) pretreatment on focal cerebral ischemia/reperfusion(I/R) injury and the possible mechanism.Methods Transient focal cerebral ischemia was introduced into mice by right middle cerebral artery occlusion(MCAO) technique. For Res treatment,C57 BL/6 J mice were given an intraperitoneal injection with Res per day for 5 days. After 24 h of reperfusion,infarct volume, neurological function were assessed.Western blot was used to analyse the expression of silent information regulator 1(SIRT1),mitophagy-related proteins, and the distribution of cytochrome C(CytC). Flow cytometry assay was introduced to test mitochondrial membrane potential(MMP). A microplate reader was used to detect the cellular adenosine triphosphate(ATP) levels. Transmission electron microscopy(TEM) was conducted to observe mitochondrial morphology. Results Res pretreatment reduced infarct volume and neurological deficit,improved the expression of SIRT1 and mitophagy. Meanwhile,Res attenuated CytC release,recovered MMP,and improved ATP levels. TEM results showed Res could protect the mitochondria from I/R injury-induced destruction.However,as 3-MA was used to inhibit the activation of mitophagy,the protective effects of Res on neurological and mitochondrial function were reversed. Conclusions Res pretreatment could relieve cerebral I/R injury.The mechanism might be associated with the regulation of mitophagy to maintain mitochondrial structural and functional integrity.
Aim To investigate the effects of resveratrol(Res) pretreatment on focal cerebral ischemia/reperfusion(I/R) injury and the possible mechanism.Methods Transient focal cerebral ischemia was introduced into mice by right middle cerebral artery occlusion(MCAO) technique. For Res treatment,C57 BL/6 J mice were given an intraperitoneal injection with Res per day for 5 days. After 24 h of reperfusion,infarct volume, neurological function were assessed.Western blot was used to analyse the expression of silent information regulator 1(SIRT1),mitophagy-related proteins, and the distribution of cytochrome C(CytC). Flow cytometry assay was introduced to test mitochondrial membrane potential(MMP). A microplate reader was used to detect the cellular adenosine triphosphate(ATP) levels. Transmission electron microscopy(TEM) was conducted to observe mitochondrial morphology. Results Res pretreatment reduced infarct volume and neurological deficit,improved the expression of SIRT1 and mitophagy. Meanwhile,Res attenuated CytC release,recovered MMP,and improved ATP levels. TEM results showed Res could protect the mitochondria from I/R injury-induced destruction.However,as 3-MA was used to inhibit the activation of mitophagy,the protective effects of Res on neurological and mitochondrial function were reversed. Conclusions Res pretreatment could relieve cerebral I/R injury.The mechanism might be associated with the regulation of mitophagy to maintain mitochondrial structural and functional integrity.
引文
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[2]Yang J L,Mukda S,Chen S D.Diverse roles of mitochondria in ischemic stroke[J].Redox Biol,2018,16:263-75.
[3]Zhang X,Yan H,Yuan Y,et al.Cerebral ischemia-reperfusioninduced autophagy protects against neuronal injury by mitochondrial clearance[J].Autophagy,2013,9(9):1321-33.
[4]Sarubbo F,Esteban S,Miralles A,et al.Effects of resveratrol and other polyphenols on Sirt1:relevance to brain function during aging[J].Curr Neuropharmacol,2018,16(2):126-36.
[5]Yang Y,Duan W,Li Y,et al.New role of silent information regulator 1 in cerebral ischemia[J].Neurobiol Aging,2013,34(12):2879-88.
[6]Jardim F R,de Rossi F T,Nascimento M X,et al.Resveratrol and brain mitochondria:a review[J].Mol Neurobiol,2018,55(3):2085-101.
[7]Longa E Z,Weinstein P R,Carlson S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20(1):84-91.
[8]Anzell A R,Maizy R,Przyklenk K,et al.Mitochondrial quality control and disease:insights into ischemia-reperfusion injury[J].Mol Neurobiol,2018,55(3):2547-64.
[9]Pangeni R,Sahni J K,Ali J,et al.Resveratrol:review on therapeutic potential and recent advances in drug delivery[J].Expert Opin Drug Deliv,2014,11(8):1285-98.
[10]万磊,陈青松,周壮,等.丹酚酸B通过调控SIRT1/NF-κB/p53通路减轻缺氧/复氧诱导的大鼠肝细胞损伤[J].中国药理学通报,2018,34(5):680-5.[10]Wan L,Chen Q S,Zhou Z,et al.Sal B attenuates rat hepatocytes injury induced by hypoxia/reoxygenation via modulation of SIRT1/NF-κB/p53 pathway[J].Chin Pharmacol Bull,2018,34(5):680-5.
[11]Schiedel M,Robaa D,Rumpf T,et al.The current state of NAD+-dependent histone deacetylases(Sirtuins)as novel therapeutic targets[J].Med Res Rev,2018,38(1):147-200.
[12]Guo Y J,Dong S Y,Cui X X,et al.Resveratrol alleviates MPTP-induced motor impairments and pathological changes by autophagic degradation ofα-synuclein via SIRT1-deacetylated LC3[J].Mol Nutr Food Res,2016,60(10):2161-75.
[13]Wei Y,Chiang W C,Sumpter R Jr,et al.Prohibitin 2 is an inner mitochondrial membrane mitophagy receptor[J].Cell,2017,168(1-2):224-38.e10.
[14]Feng J,Chen X,Lu S,et al.Naringin attenuates cerebral ischemia-reperfusion injury through inhibiting peroxynitrite-mediated mitophagy activation[J].Mol Neurobiol,2018,55(12):9029-42.
[15]Shen Z,Zheng Y,Wu J,et al.PARK2-dependent mitophagy induced by acidic postconditioning protects against focal cerebral ischemia and extends the reperfusion window[J].Autophagy,2017,13(3):473-85.