远隔缺血预适应对脑缺血再灌注损伤大鼠缺血半暗带区PERK/p-eIF2α通路及自噬的影响

Effect of remote ischemic preconditioning on PERK/p-eIF2αpathway and autophagy in the penumbra of rats with focal cerebral ischemia/reperfusion injury

目的研究远隔缺血预适应(remote ischemic preconditioning, RIPC)对局灶性脑缺血再灌注大鼠缺血半暗带区自噬相关蛋白Beclin1、LC3B以及内质网分子伴侣GRP78、内质网应激(endoplasmic reticulum stress, ERS)相关通路PERK/p-eIF2α的影响,探讨RIPC保护脑缺血损伤作用的相关机制。方法将24只健康雄性Sprague-Dawley大鼠采用抽签法随机分为3组:假手术(Sham)组、大脑中动脉梗塞(middle cerebral artery occlusion, MCAO)组和RIPC+MCAO组。每组8只大鼠。采用改良线栓法制备大鼠MCAO再灌注模型,于缺血90 min后拔出线栓再灌注24 h。其中RIPC+MCAO组在MCAO之前,给予大鼠连续3 d的RIPC(采用夹闭双侧股动脉10 min开放10 min,每天3个循环的方法)。免疫荧光双标染色法检测再灌注大鼠缺血半暗带区自噬相关蛋白Beclin1、LC3B以及GRP78、p-eIF2α的表达。结果 1)Sham组大鼠脑内偶见Beclin1阳性细胞。MCAO组大鼠再灌注24 h缺血半暗带区Beclin1表达水平比Sham组显著升高(P<0.05)。给予RIPC的脑缺血再灌注大鼠半暗带区Beclin1的表达水平比MCAO组显著减少(P<0.05)。Beclin1与神经元标志物NeuN共定位。2)MCAO组大鼠再灌注24 h,脑缺血半暗带区GRP78分别与Beclin1和LC3B免疫荧光染色共定位。3)MCAO组大鼠再灌注24 h,脑缺血半暗带区Beclin1与p-eIF2α免疫荧光染色共定位。Sham组大鼠未见Beclin1和p-eIF2α双标阳性细胞,MCAO组大鼠再灌注24 h半暗带区可见大量Beclin1和p-eIF2α双标阳性细胞。给予RIPC后,缺血大鼠半暗带区Beclin1和p-eIF2α双标阳性细胞数目比MCAO组明显减少(P<0.05)。结论 RIPC可能通过抑制ERS相关通路PERK/p-eIF2α,减少神经元中自噬相关蛋白产生,避免神经元过度激活自噬,发挥对脑缺血再灌注损伤的保护作用。

Objective To investigate the protective effect and mechanism of remote ischemic preconditioning(RIPC)on cerebral ischemia injury via studying the expressions of autophagy-related protein Beclin1,LC3B and chaperone of endoplasmic reticulum GRP78,as well as endoplasmic reticulum stress(ERS)-related pathway PERK/p-eIF2αin the penumbra of rats with focal cerebral ischemia/reperfusion.Methods A total of 24 male Sprague Dawley rats were divided randomly into 3 groups:Sham group,middle cerebral artery occlusion(MCAO)group and RIPC+MCAO group,with n=8 for each group.A model of MCAO was induced with the intraluminal suture method.The rats underwent 90 minutes of MCAO and then were reperfused for 24 h by withdrawal of the filament.Three cycles of RIPC had been given three times a day for 3 days before the MCAO surgery,induced by temporarily occluding the bilateral femoral arteries(10 minutes)prior to 10 minutes of reperfusion.The positive expressions of autophagy-related protein Beclin1 and LC3B,as well as GRP78 and p-eIF2αin the ischemic penumbra of the brain tissues sections were detected with immunofluorescence double staining.Results 1)There was little Beclin1 positive cells in the Sham group.Compared with Sham group,the Beclin1 positive cells in the penumbra of MCAO group obviously increased after 24 h reperfusion(P<0.05).Compared with MCAO group,the Beclin1 positive cells decreased significantly in the penumbra of RIPC+MCAO group.The Beclin1 positive cells were colocalized with NeuN,a general neuronal marker,in the brain ischemic penumbra.2)In the penumbra of MCAO group rats after 24 h reperfusion,GRP78 was colocalized with Beclin1 and LC3B,respectively,which was examined with immunofluorescence staining.3)In the penumbra of brain tissue of MCAO group with 24 h reperfusion,Beclin1 was colocalized with p-eIF2α.No Beclin1 and p-eIF2αdouble positive cell was observed in the Sham group.Lots of Beclin1 and p-eIF2αdouble positive cells were observed in the ischemic penumbra of MCAO group after 24 h reperfusion.After treated with RIPC,the Beclin1 and p-eIF2αdouble positive cells in the penumbra of the ischemia rats were significantly decreased compared with that in MCAO group(P<0.05).Conclusion RIPC may inhibit endoplasmic reticulum stress(ERS)-related pathway PERK/eIF2αand reduce the expression of autophagy-related protein,alleviate excessive activation of neuronal autophagy,and thus exerts neuroprotective effects on cerebral ischemia/reperfusion injury.