| ObjectiveThe pathogenesis of cerebral ischemia-reperfusion injury is very complicated.Apoptosis is one of the important causes of ischemic brain injury.Mitochondrial pathway,death receptor pathway and endoplasmic reticulum pathway are three main ways to regulate apoptosis.In recent years,the role of endoplasmic reticulum stress-induced apoptosis in cerebral ischemia-reperfusion injury has become a hotspot.In this study,rat model of middle cerebral artery occlusion/reperfusion(MCAO/R)was established by using suture method,and therapeutic effect of electro-acupuncture at GV 20 and ST 36 on cerebral ischemia-reperfusion injury rats was studied.The aim is to explore whether the protective mechanism is related to the apoptosis caused by endoplasmic reticulum,and to provide the scientific basis for acupuncture treatment on ischemic cerebrovascular disease.MethodsSixty-three adult Sprague-Dawley rats were randomly divided into the three groups:the sham(S)group,the model(M)group and the electro-acupuncture(EA)group,and then divided into 24 h and 72 h groups according to the reperfusion time.The model was established by placement of a suture to block the middle carotid artery,and reperfusion was triggered by suture removal in all groups except group S.The sham operation group only separated the right common carotid artery,internal carotid artery and external carotid artery,and no plug was inserted.Rats in the group S and the group M were not treated with only the same grabbing stimulus.The rats in EA groups received electro-acupuncture treatment by needling at GV 20 and left ST 36 every day.The neurological deficits of the rats were evaluated at the prescribed time points.The infarct volume of the rats was assessed by TTC method.The apoptosis of the brain tissue was detected by Tunel method.NeuN and GFAP were observed by immunofluorescence staining.glucose-regulated protein 78(GRP78),C/EBP homologous protein(CHOP),phosphorylated eukaryotic translation initiation complex 2a(p-eIF2a),Caspasel2 and Bcl-2/Bax protein and mRNA expression were evaluated by immunohistochemistry,western blots and Real-time PCR.Results1.Neurological deficits:Rats in three groups behaved in the same way before modeling,Longa score was 0 points,and can removed the forelimb adhesives within 10s.The Longa score and the removal time of the group M were significantly different from those in the group S(P<0.05).The Longa score in EA group significantly decreased than in M group at the same timepoint(P<0.05).At 24 h after reperfusion,the adherence and removal time of the rats in the M group were not significantly different from those in the EA group,and at 72 h after reperfusion the time in EA group was significantly shorter than that in the M group(P<0.05).2.Cerebral infarction:All brain slices were stained red in group S with no cerebral infarction area.The area of middle cerebral artery and blood supply in M group and EA group were in white colour,consistent with the location of arterial embolization around the optic chiasm.The infarct volume of the rats in group EA was significantly lower than that in the M group(P<0.05)3.TUNEL staining showed that the apoptosis of the M group and the EA group was significantly higher than that in S group(P<0.05).Compared with the model group,the cell apoptosis was significantly decreased in EA group(P<0.05).4.Immunofluorescence staining showed that the number of NeuN-positive cells in M group and EA group was significantly lower than that in S group at 72 h after reperfusion(P<0.05).Compared with model group,the number of NeuN-positive cells was significantly increased(P<0.05).At 72 h after reperfusion,the volume of GFAP positive cells and synapses were less in the S group,and the fluorescence intensity was weaker,suggesting that astrocytes were resting.The GFAP-positive cells in the M group and the EA group were larger in volume and had more synapses,stronger fluorescence intensity,suggesting that astrocytes were in the activated state.The GFAP-positive cells in the M group were significantly higher than those in the EA group(P<0.05)5.Indicators related to endoplasmic reticulum stress(ERS)(1)GRP78 ①immunohistochemistry:The expression of GRP78 protein in group S was lower than that in the group M and the group EA at the same time point(P<0.05).Compared with the M group,the expression of GRP78 protein in group EA was significantly increased at the same time point.②western blots:The expression of GRP78 protein in M group was significantly higher than that in S group at the same time point(P<0.05).There was no significant difference between M group and EA group at each time point.③)qPCR:The expression of GRP78 mRNA in M group and EA group were significantly higher than that in S group at the same time point.The mRNA expression of GRP78 in M group was significantly lower than that in EA group at the same time point(P<0.05).(2)CHOP ①immunohistochemistry:CHOP protein was expressed in the cytoplasm and nucleus.There were no significantly differences between M group and EA group at 24 h after reperfusion.At 72 h after reperfusion,CHOP protein in M group was significantly higher than that of EA group(P<0.05).②western blots:The expression of CHOP protein in M group and EA group was significantly higher than that in S group(P<0.05).Compared with M group at the same time point,CHOP protein was significantly decreased in EA group(P<0.05).③qPCR:The expression of CHOP mRNA in the M group and EA group was significantly higher than that in S operation group(P<0.05).The expression of CHOP mRNA in the EA group was significantly lower than that in the M group(P<0.05)(3)p-eIF2a ①immunohistochemistry:The expression of p-eIF2a protein in M and EA group was significantly increased compared with S group(P<0.05).The expression of p-eIF 2α protein in the M group was significantly higher than that in the EA group in 72h after reperfusion(P<0.05).②western blots:The expression of p-eIF2a protein in M group and EA group was significantly higher than that in S group(P<0.05),and the protein expression level of p-eIF2a in EA group was significantly lower than that in M group(P<0.05).③qPCR:The mRNA expression of p-eIF2a in M group and EA group was significantly higher than that in S group(P<0.05).The mRNA expression of p-eIF2a in EA group was significantly lower than that in M group at the same time point(P<0.05).(4)Caspase 12 ①immunohistochemistry:Caspase 12 protein expression in M group and EA group was significantly higher than that in S group(P<0.05).Compared with M group,the protein levels of Caspase 12 significantly increased at 72 h time point(P<0.05),and there were no significantly differences between EA group and the M group at 24 h time point(P>0.05).②western blots:The protein levels of Caspase 12 in S group were much lower than those in the M group and EA group(P<0.05),and the protein levels of Caspase 12 in EA groups was lower than M group obviously at 24h and 72 h time points(P<0.05).③qPCR:Compared with M groups,the protein levels of Caspase-12 in the EA group significantly decreased at the same time point(P<0.05).(5)Bcl-2/Bax ①immunohistochemistry:The protein levels of Bcl-2 in the M groups significantly decreased and protein levels of Bax in the M groups significantly increased compared with the S group(P<0.05).Compared with the M group,the protein levels of Bcl-2 in the EA groups significantly increased(P<0.05).No difference was found between M group and EA group in the expression of Bax in 24 h,and the protein levels of Bax in the EA group significantly decreased compared with the M group at 72 h time point(P<0.05)②western blots:The protein levels of Bcl-2 in the M groups significantly decreased and protein levels of Bax in the M groups significantly increased compared with the S group(P<0.05).Compared with the M group,the protein levels of Bcl-2 significantly increased and Bax significantly decreased in the EA groups(P<0.05).③qPCR:Compared with the M group at the same time point,the protein levels of Bcl-2 significantly increased and protein levels of Bax significantly decreased in the EA groups(P<0.05).Conclusion1.Electro-acupuncture at GV 20 and ST 36 can significantly improve the neurological deficit and cerebral infarct volume and reduce the proportion of apoptosis in rats with cerebral ischemia-reperfusion injury,which has a protective effect on ischemic brain at 72 h reperfusion time piont.2.Electro-acupuncture at GV 20 and ST 36 can promote the upregulation expression of NeuN and down regulation the expression of GFAP,and have a function of protecting the ischemic penumbra neurons,reducing the loss of neurons and inhibiting the activation of astrocytes at 72 h reperfusion time piont.3.At 24 h and 72 h reperfusion time pionts,the protein and mRNA expression of GRP 78，p-eIF 2α,CHOP and Caspase 12 in the endoplasmic reticulum stress-related markers were increased after cerebral ischemia-reperfusion injury in rats.Endoplasmic reticulum stress was induced by cerebral ischemia.Electro-acupuncture at GV 20 and ST 36 can participate in endoplasmic reticulum stress apoptotic pathway to reduce cerebral ischemia-reperfusion injury,play a protective role of the brain,the mechanism involved may as follows:promote the recovery of endoplasmic reticulum through UPR,up-regulate the expression of Bcl-2 and down-regulate the protein and mRNA expression of p-eIF2a,CHOP,Caspase 12,reduce the expression of apoptosis gene Bax,and to inhibit the endoplasmic reticulum stress response and apoptotic signaling pathway,thereby reducing neuronal apoptosis and maintaining nerve function stability.4.Endoplasmic reticulum stress apoptotic pathway play a protective role in pathological mechanism of cerebral ischemia-reperfusion injury.Our study confirmed that electroacupuncture can interfere with endoplasmic reticulum stress to alleviate cerebral ischemia-reperfusion injury,provides a scientific basis for clinical acupuncture treatment on cerebrovascular disease.InnovationAfter the CIRI model based on the classic improved sature occlude method were successfully established,we stratified three groups randomization according to different time points of neural function and cerebral infarct volume of rats to verify the effectiveness of acupuncture apparently.Finally,from the level of molecular biology to validation the mechanism of acupuncture of GV 20 and ST 36 in the rat with cerebral ischemia reperfusion injury through the detection of GRP78,CHOP,p-eIF2α and Caspase 12 on endoplasmic reticulum stress related pathways.The present research can provide the theory and experimental basis for acupuncture application in ischemia stroke prevention and treatment.There have not been found any researches on intervention mechanism of GRP78,CHOP,p-eIF2a and Caspase 12 in brain tissue of rat with cerebral ischemia reperfusion injury when acupuncture at GV 20 and ST 36 at the same time through the literature retrieval. |
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