| ObjectiveEnriched environment(EE)is effective in preventing cerebral ischemia/reperfusion(I/R)injury.However,little is known about the mechanism underlying the neuroprotection of EE preprocessing.Numerous studies have reported that endoplasmic reticulum(ER)stress is widely present in I/R injury.Mild ER stress promotes cells to break away from danger signals and enter the adaptive procedure with the activation of pro-survival mechanism to rescue ischemic injury,while chronic ER stress generally serves as a detrimental role on nerve cells via triggering diverse pro-apoptotic mechanism.The purpose of this study was to explore the role of ER stress-mediated autophagy and apoptosis in I/R injury and their potential regulatory mechanisms,to provide a research basis for further investigation of the molecular mechanisms of neuroprotective effects of EE production,and to provide new ideas for the prevention of cerebral ischemia.MethodsMale Sprague Dawley rats were housed in Standard environment(SE)or EE for4 weeks and then underwent sham surgery or middle cerebral artery occlusion and reperfusion(MCAO/R)to establish an animal model of cerebral ischemic injury.After surgery,the rats were divided into sham,MCAO and MCAO+EE groups.Laser flow scatter imaging was applied to monitor the cerebral blood flow status before surgery,during occlusion and after reperfusion.The degree of neurological deficit was observed by Zea Longa score at 0,24 and 48 hours postoperatively.Rats were dissected and executed under general anesthesia at 48 h postoperatively,and TTC staining was performed immediately to calculate the cerebral infarct volume.Hematoxylin-eosin staining(HE)was used to observe the morphological structure of the ischemic penumbra neurons.The expression of rat cortical ER stress-related proteins(GRP78,p-PERK/PERK,p-IRE1/IRE1,ATF6,ATF4,CHOP and p-JNK/JNK),autophagy-related proteins(LC3II/I,Beclin-1 and p62)and apoptosis-related proteins(Bcl-2 and Bax)were detected by immunoblotting.TUNEL staining was also used to detect the level of apoptosis and immunofluorescence was used to detect the expression of p-PERK,p-IRE1 and LC3.Results1.Pre-ischemic EE reduced cerebral infarct size and acute neuronal damageTo evaluate the successful establishment of the MCAO/R model,we first examined the changes in cerebral blood flow(CBF)in rats before,during,and after surgery using laser scattering blood flow imaging.The results showed that CBF decreased to 22.33% and 17.57% of baseline in the MCAO group and MCAO+EE group rats,respectively.The TTC results showed that the cerebral infarct volume was significantly reduced in the MCAO+EE group compared with the MCAO group(P <0.01).Longa score was used to test neurological deficits in rats.Analysis showed that at 48 h after MCAO/R,the neurological deficit scores of rats in the MCAO+EE group were significantly less than those in the MCAO group and significantly different(P <0.01).HE staining showed that the arrangement of cortical neuronal cells in MCAO rats was disturbed,accompanied by morphological swelling,cell membrane rupture,and a large number of neuronal deaths.Compared with MCAO group,neuronal structure and integrity of MCAO+EE group were significantly improved,with a substantial reduction in neuronal death.These findings indicated that pre-ischemic treatment could relieve acute neuronal damage caused by cerebral I/R in rats.2.Pre-ischemic EE relieved I/R injury-induced ER stressRats were anesthetized and executed at 48 h after MCAO/R surgery.ER stress levels were detected by immunoblotting.The results showed that the expression of GRP78,a marker of ER stress,was lower in the MCAO+EE group than in the MCAO group,which was statistically different(P < 0.05).The expression of three ER transmembrane receptors p-PERK/PERK,p-IRE1/IRE1 and ATF6 in the unfolded protein response(UPR)was significantly higher in the MCAO group compared with the MCAO+EE group at the protein level,with significant differences(P<0.001,P<0.05,P<0.001).In addition,the fluorescence intensity of p-PERK and p-IRE1 in MCAO group was higher than that in MCAO+EE group(P<0.001,P<0.05).The above results indicated that pre-ischemic EE pretreatment memorably inhibits I/R injury-induced ER stress.3.Pre-ischemic EE inhibited autophagic activity after I/R injurySince ER stress-mediated autophagy plays a crucial role in I/R injury,we explored whether pre-EE treatment has an effect on autophagy.We detected the expression of autophagy marker LC3 by immunoblotting and immunofluorescence.The results showed that the protein expression of LC3II/I as well as the fluorescence intensity were significantly decreased in the MCAO+EE group compared to the MCAO group(P < 0.01).In addition,the protein expression of Beclin-1 in the MCAO+EE group was lower than that in the MCAO group(P < 0.01),and the protein expression of p62 was higher than that in the MCAO group,both of which were statistically different(P < 0.05).Together,these results revealed that pre-ischemic EE treatment could inhibit autophagy casued by MCAO surgery.4.Pre-ischemic EE attenuated I/R injury-induced apoptosisWe detected apoptosis in the affected cortical cells of rats by TUNEL staining.The apoptotic index of the MCAO+EE group was notably lower than that of the MCAO group(P < 0.01).In addition,we detected the expression levels of apoptosis proteins Bax and Bcl-2 by immunoblotting.The results indicated that the expression of Bcl-2 was decreased(P < 0.001)and Bax was increased(P < 0.001)in the MCAO group compared with the sham group,while the expression of Bcl-2 was added(P <0.01)and dropped(P < 0.05)in the MCAO+EE group compared with the MCAO group.5.Effect of pre-ischemic EE on autophagy and apoptosis-related signaling pathways under ER stressWe assessed the protein expression levels of ATF4 and CHOP under PERK signaling pathway and p-JNK/JNK under IRE1 signaling pathway by immunoblotting.The results showed that the expression levels of all three proteins were higher in the MCAO+EE group than in the MCAO group(P<0.05,P<0.001,P<0.01).The above data implied that EE preconditioning attenuates I/R injury-mediated autophagy and apoptosis possibly associated with activation of the PERK-ATF4-CHOP pathway as well as the IRE1-JNK pathway under prolonged ER stress.Conclusion1.Pre-ischemic EE inhibited ER stress,autophagy activity and apoptosis.2.The neuroprotective effect of pre-ischemic EE preconditioning may be related to the modulation of ER stress-mediated PERK-ATF4-CHOP pathway and IRE1-JNK pathway inhibiting the autophagy and apoptosis. |
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