| Purpose : By detecting the changes of p-PERK,p-e IF2α,ATF4 and Atg12 protein expression levels in brain tissues of each group,we explored whether eye acupuncture can affect the expression of Atg12 in brain tissues of rats with cerebral ischemia-reperfusion injury by regulating endoplasmic reticulum stress signaling pathway PERK-e IF2α-ATF4.Material and method:Seventy-five SD rats were randomly divided into three groups :control group(n = 18),sham operation group(n = 18)and model group(n = 39).The CIRI rat model was replicated by modified suture method.The rats in the control group were not treated,and the rats in the sham operation group were the same as the model group,but the fishing line was not inserted.After the model evaluation,all the rats that met the experimental requirements were divided into four groups : control group,sham operation group,model group and eye acupuncture group.The rats in the control group,sham operation group and model group were routinely fed until the end of the experiment.After reperfusion,the rats in the eye acupuncture group were treated with 31 #,0.5 inch disposable filiform needles at the time of reperfusion,12 h and 24 h(30 min before sampling),and the hepatobiliary area,renal bladder area,upper focal area and lower focal area were intervened.After the last intervention,the neurological deficit scores of each group were scored and recorded.The rats were sacrificed by intraperitoneal overdose anesthesia,and the pathological changes of brain tissue in each group were observed by HE staining.The formation of autophagosomes in brain tissue of rats in each group was observed by transmission electron microscopy.The expression level of ATF4 in brain tissue of rats in each group was observed by immunohistochemistry.The protein expression of p-PERK,p-e IF2α and Atg12 in brain tissue of rats in each group was detected by Western blot.Results:1.Changes of neurological deficit scores in rats of each groupThere was no neurological deficit symptom in the control group and the sham operation group,and the score was 0.The rats in the model group showed different degrees of neurological dysfunction,such as turning or dumping to the opposite side when walking;the contralateral forepaw could not be fully extended,indicating that the CIRI rat model was successfully replicated.Compared with the control group,the neurological deficit score of the sham operation group did not change,and the difference was not statistically significant(P > 0.05).Compared with the sham operation group,the neurological deficit score of the model group was significantly increased(P < 0.01).Compared with the model group,the neurological deficit score of the eye acupuncture group was significantly lower,and the difference was statistically significant(P < 0.01).2.Pathological changes in brain tissue of rats in each groupHE staining showed that the boundaries of gray matter and cortex in the brain tissue of rats in the control group and the sham operation group were clear,the nerve cells had a complete structure and regular arrangement,the nerve cells had clear levels,no necrosis or edema,and the distribution was relatively uniform.The neurons had rich cytoplasm,complete and clear cell membrane and nucleus.In the brain tissue of the rats in the model group,there were significant changes in the cribriform necrotic lesions,and there was no clear edge of the necrotic lesions.In the core area of the infarction,there was confusion at the level of neuronal cells,and there could be nuclear fragmentation and pyknosis.The cytoplasm of neurons showed concentrated staining atrophy,common inflammatory cell infiltration,dilated capillary inflammation,interstitial edema,light staining and loose cytoplasm.The rats in the eye acupuncture group were less likely to have reticular necrosis of the brain tissue of the rats,and there was a clear edge of the necrotic tissue.The proliferation of glial cells,inflammatory cell infiltration and telangiectasia were observed.3.The formation of autophagosomes was observed by transmission electron microscopy In the model group,neuronal cells showed different degrees of swelling,cell membrane rupture disappeared,swollen mitochondria and a small number of other organelles appeared in the cytoplasm,nuclear chromatin increased,heterochromatin was agglomerated,and autophagosomes could be observed.Among the neurons in the rat brain tissue of the eye acupuncture group,there is a high membrane permeability,the content is very complete,and the damage of organelles can be seen.Compared with the model group,there were fewer autophagosomes in the brain tissue of the eye acupuncture group,and the swelling of the rough endoplasmic reticulum and mitochondrial organelles was significantly reduced,suggesting that eye acupuncture may improve the symptoms of cerebral ischemia-reperfusion injury by reducing the level of autophagy in brain tissue.4.The expression of ATF4 protein in brain tissue of rats in each group was detected by immunohistochemistryCompared with the control group,the expression level of ATF4 protein in the brain tissue of the sham operation group was analyzed,and the difference was not statistically significant(P > 0.05).Compared with the sham operation group,the expression of ATF4 protein in the brain tissue of the model group was significantly increased,and the difference was statistically significant(P < 0.01).Compared with the model group,the expression of ATF4 protein in rat brain tissue was lower in the eye acupuncture group,and the difference was statistically significant(P < 0.01).5.Western blot was used to detect the expression of Atg12 protein in brain tissue of rats in each groupCompared with the control group,there was no significant difference in Atg12 protein in the brain tissue of the sham operation group(P > 0.05).Compared with the sham operation group,the expression of Atg12 protein in the model group was increased,and the difference was significant(P < 0.01).Compared with the model group,the Atg12 protein content in the cerebral ischemic tissue of the eye acupuncture group was significantly decreased,and the difference was statistically significant(P < 0.01).6.Western blot was used to detect the expression of p-PERK and p-e IF2α protein in brain tissue of rats in each groupThere was no significant difference between the sham operation group and the control group(P > 0.05).Compared with the sham operation group,the expression of p-PERK and p-e IF2α protein in the model group increased,and the difference was statistically significant(P < 0.01).Compared with the model group,the expression of p-PERK and p-e IF2α protein in the cerebral ischemic tissue of the eye acupuncture group was significantly decreased,and the difference was statistically significant(P < 0.01).Conclusions:1.Ocular acupuncture may regulate excessive autophagy of nerve cells by reducing the expression of Atg12 protein in brain tissue of rats with cerebral ischemia-reperfusion injury;2.Ocular acupuncture may affect the expression of Atg12 in brain tissue by regulating the endoplasmic reticulum stress PERK-e IF2α-ATF4 signaling pathway,thereby improving cerebral ischemia-reperfusion injury in rats. |
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