羟氯喹促进ERK1/2磷酸化减轻大鼠脑缺血再灌注损伤
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摘要
目的:探讨羟氯喹(hydroxychloroquine,HCQ)对大鼠脑缺血再灌注损伤(ischemia/reperfusion,I/R)的影响。方法:采用随机数字表法将健康雄性SD大鼠72只分为6组:对照组、缺血再灌注组(I/R组)、低剂量羟氯喹预处理组(HCQ250组)、中剂量羟氯喹预处理组(HCQ500组)、高剂量羟氯喹预处理组(HCQ1000组)和细胞外信号调节激酶1/2(extracellular regulated protein kinases 1/2,ERK1/2)抑制剂U0126预处理组(U0126组),每组均为12只。采用线栓法行短暂性大鼠大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)建立大鼠缺血性脑卒中模型。I/R组行MCAO后2 h再灌注,HCQ250组、HCQ500组、HCQ1000组和U0126组分别在MCAO前72 h、48 h和24 h经侧脑室注射8μL 250、500、1 000和1 000μmol/L HCQ,其中U0126组在每次注射1 000μmol/L HCQ后12 h再经侧脑室注射8μL 1 000μmol/L U0126;对照组不栓塞大脑中动脉,其余处理同I/R组。再灌注6 h时将每组一半大鼠断头取脑,采用蛋白质印迹法检测缺血半暗带脑组织ERK1/2、p-ERK1/2、抗凋亡因子Bcl-2和促凋亡因子cleaved caspase-3、环磷腺苷效应元件结合蛋白(c AMP-response element binding protein,CREB)、磷酸化CREB(p-CREB)、蛋白激酶B(Akt)和磷酸化Akt(p-Akt)的表达;再灌注24 h时采用神经功能缺陷评分量表检测剩余大鼠的神经功能,再断头取脑后采用2%TTC染色测量脑梗死体积。结果:与对照组比较,其余5组神经功能缺陷评分显著降低,脑梗死体积明显增大,p-ERK1/2、cleaved caspase-3、p-CREB和p-Akt表达显著增高,Bcl-2表达降明显低(P均<0. 05);与I/R组比较,HCQ250组、HCQ500组、HCQ1000组神经功能缺陷评分明显升高,脑梗死体积显著减少,pERK1/2、Bcl-2、p-CREB、p-Akt表达明显增高,cleaved caspase-3表达降低明显(P均<0. 05);与HCQ1000组比较,U0126组神经功能缺陷评分明显降低,脑梗死体积显著增大,p-ERK1/2、Bcl-2、p-CREB和p-Akt表达降低显著,cleaved caspase-3表达明显增高(P均<0. 05)。结论:羟氯喹可能通过CREB/Akt信号通路促进p-ERK1/2表达,参与大鼠脑缺血再灌注损伤时的内源性保护机制。
Objective: To evaluate the attenuation of hydroxychloroquine( HCQ) for cerebral ischemia reperfusion( I/R) injury induced by transient middle cerebral artery occlusion( MCAO) in rats and potential mechanisms. Methods: A total of 72 healthy male SD rats were randomly divided into 6 groups( n =12) : control group,ischemia-reperfusion group( group I/R),low dose HCQ pretreatment group( group HCQ250),medium dose HCQ pretreatment group( group HCQ500),high dose HCQ pretreatment group( group HCQ1000) and extracellular regulated protein kinase 1/2( ERK1/2) inhibitor U0126 pretreatmentgroup( group U0126). MCAO was used to establish rat ischemic stroke model. Control group was the same as group I/R,but not occluded the middle cerebral artery; group I/R was reperfused after MCAO for 2 h; group HCQ250,group HCQ500,group HCQ1000 and group U0126 were injected with 8 μL 250μmol/L,500 μmol/L,1 000 μmol/L and 1 000 μmol/L HCQ by lateral ventricle( LV) at 24 h,48 h and 72 h,respectively,before MCAO,and U0126 group was injected with 8 μL of 1 000 μmol/L U0126 after 12 h each injection of HCQ. Brain tissues were collected by decollation after reperfusion 6 h,the expression of ERK1/2,p-ERK1/2,anti-apoptotic factor Bcl-2,pro-apoptotic factor cleaved caspase-3,pCREB and p-Akt was detected by Western blotting in ischemic penumbra. Neurological deficit score was measured and followed infarct volume was detection by TTC staining 24 h after reperfusion. Results:Compared with control group,neurological deficit score was reduced,infarct volume was magnified,the expression of p-ERK1/2,cleaved caspase-3,pCREB,pAkt was increased,Bcl-2 expression was decreased in group I/R,group HCQ250,group HCQ500,group HCQ1000 and group U0126( P < 0. 05).Compared with group I/R,neurological deficit score was enlarged,infarct volume was reduced,the expression of p-ERK1/2,Bcl-2,pCREB,pAkt was increased,the expression of cleaved caspase-3 was decreased in group HCQ1000( P < 0. 05). However,compared with group HCQ1000,neurological deficit score was reduced,infarct volume was magnified,the expression of p-ERK1/2,Bcl-2,p CREB,pAkt was decreased,the expression of cleaved caspase-3 was increased in group U0126( P < 0. 05). Conclusion:Hydroxychloroquine may promote the expression of p-ERK1/2 and protects agaist cerebral ischemia through reperfusion injury through CREB/Akt signaling way in rats.
Objective: To evaluate the attenuation of hydroxychloroquine( HCQ) for cerebral ischemia reperfusion( I/R) injury induced by transient middle cerebral artery occlusion( MCAO) in rats and potential mechanisms. Methods: A total of 72 healthy male SD rats were randomly divided into 6 groups( n =12) : control group,ischemia-reperfusion group( group I/R),low dose HCQ pretreatment group( group HCQ250),medium dose HCQ pretreatment group( group HCQ500),high dose HCQ pretreatment group( group HCQ1000) and extracellular regulated protein kinase 1/2( ERK1/2) inhibitor U0126 pretreatmentgroup( group U0126). MCAO was used to establish rat ischemic stroke model. Control group was the same as group I/R,but not occluded the middle cerebral artery; group I/R was reperfused after MCAO for 2 h; group HCQ250,group HCQ500,group HCQ1000 and group U0126 were injected with 8 μL 250μmol/L,500 μmol/L,1 000 μmol/L and 1 000 μmol/L HCQ by lateral ventricle( LV) at 24 h,48 h and 72 h,respectively,before MCAO,and U0126 group was injected with 8 μL of 1 000 μmol/L U0126 after 12 h each injection of HCQ. Brain tissues were collected by decollation after reperfusion 6 h,the expression of ERK1/2,p-ERK1/2,anti-apoptotic factor Bcl-2,pro-apoptotic factor cleaved caspase-3,pCREB and p-Akt was detected by Western blotting in ischemic penumbra. Neurological deficit score was measured and followed infarct volume was detection by TTC staining 24 h after reperfusion. Results:Compared with control group,neurological deficit score was reduced,infarct volume was magnified,the expression of p-ERK1/2,cleaved caspase-3,pCREB,pAkt was increased,Bcl-2 expression was decreased in group I/R,group HCQ250,group HCQ500,group HCQ1000 and group U0126( P < 0. 05).Compared with group I/R,neurological deficit score was enlarged,infarct volume was reduced,the expression of p-ERK1/2,Bcl-2,pCREB,pAkt was increased,the expression of cleaved caspase-3 was decreased in group HCQ1000( P < 0. 05). However,compared with group HCQ1000,neurological deficit score was reduced,infarct volume was magnified,the expression of p-ERK1/2,Bcl-2,p CREB,pAkt was decreased,the expression of cleaved caspase-3 was increased in group U0126( P < 0. 05). Conclusion:Hydroxychloroquine may promote the expression of p-ERK1/2 and protects agaist cerebral ischemia through reperfusion injury through CREB/Akt signaling way in rats.
引文
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[2]Li M,Feng B,Wang L,et al.Tollip is a critical mediator of cerebral ischaemia-reperfusion injury[J].J Pathol,2015,237(2):249-262.
[3]Martinez-Lopez N,Athonvarangkul D,Mishall P,et al.Autophagy proteins regulate ERK phosphorylation[J].Nat Commun,2013,4:2799.
[4]Li L,Xiong Y,Qu Y,et al.The requirement of extracellular signal-related protein kinase pathway in the activation of hypoxia inducible factor 1αin the developing rat brain after hypoxia-ischemia[J].Acta Neuropathol,2008,115(3):297-303.
[5]Liu L,Zhang R,Liu K,et al.Tissue kallikrein protects cortical neurons against in vitro ischemia-acidosis/reperfusion-induced injury through the ERK1/2 pathway[J].Exp Neurol,2009,219(2):453-465.
[6]Bourke L,Mccormick J,Taylor V,et al.Hydroxychloroquine protects against cardiac ischaemia/reperfusion injury in vivo via enhancement of ERK1/2 phosphorylation[J].PLo S One,2015,10(12):e143771.
[7]Liang X,Hu Q,Li B,et al.Follistatin-like 1 attenuates apoptosis via disco-interacting protein 2 homolog A/Akt pathway after middle cerebral artery occlusion in rats[J].Stroke,2014,45(10):3048-3054.
[8]Hommel JD,Sears RM,Georgescu D,et al.Local gene knockdown in the brain using viral-mediated RNA interference[J].Nat Med,2003,9(12):1539-1544.
[9]Mebratu Y,Tesfaigzi Y.How ERK1/2 activation controls cell proliferation and cell death:Is subcellular localization the answer?[J].Cell Cycle,2009,8(8):1168-1175.
[10]Wang Y,Han R,Zuo Z.Dexmedetomidine post-treatment induces neuroprotection via activation of extracellular signal regulated kinase in rats with subarachnoid haemorrhage[J].Br J Anaesth,2016,116(3):384-392.
[11]Lee S-J,Silverman E,Bargman JM,et al.The role of antimalarial agents in the treatment of SLE and lupus nephritis[J].Nat Rev Nephrol,2011,7:718-729.
[12]Barth S,Glick D,Macleod KF,et al.Autophagy:assays and artifacts[J].J Pathol,2010,221:117-124.
[13]Johnson R,Charnley J.Hydroxychloroquine in prophylaxis of pulmonary embolism following hip arthroplasty[J].Clin Orthop Relat Res,1979(144):174-177.
[14]Dworkin S,Mantamadiotis T.Targeting CREB signaling in neurogenesis[J].Expert Opin Ther Targets,2010,14(8):869-879.