Protective Mechanism Of Electroacupuncture Combined With Enriched Rehabilitative Training On Hippocampal Neurons In Rats With Cerebral Ischemia-Reperfusion Injury Based On Endoplasmic Reticulum Stress Response

Objective:To observe the effects of electroacupuncture combined with enriched rehabilitative training on endoplasmic reticulum stress(ERS)-associated proteins,i.e.,protein kinase R–like endoplasmic reticulum kinase(PERK),CCAAT/enhancer binding protein homologous protein(CHOP),B-cell lymphoma-2(Bcl-2),and Bcl-2 associated X protein(Bax),in rats with cerebral ischemia-reperfusion injury,and to investigate the protective mechanism of electroacupuncture combined with enriched rehabilitative training in relation to the hippocampal neurons in rats with cerebral ischemia-reperfusion injury.Methods:The enrolled SD rats were randomly divided into a normal group(Normal),sham surgery group(Sham),model group(Model),electroacupuncture group(EA),enriched rehabilitative training group(RT),and electroacupuncture + rehabilitative training group(EA + RT),with 18 per group.The ischemia-reperfusion injury model was induced by modified occlusion of the middle cerebral artery using a suture technique.At 24 hours after the successful modeling in the EA group and EA + RT group,electroacupuncture was performed at the Baihui and Shenting acupoints;for the rats in the RT and EA + RT groups,an enriched environment was set up and they were involved in many rehabilitative activities.Electroacupuncture and rehabilitative training were performed once per day,with each session lasting 30 minutes,for 7 days.The rats' learning and memory function was evaluated by a water maze test.The cerebral infarction volume was observed via TTC staining.The histomorphological changes in the hippocampal CA1 area of the rats were observed by HE staining.The neuronal apoptotic index in each group was determined using the TUNEL method.The expression of ERS-associated proteins,i.e.,ERSK,CHOP,Bcl-2,and Bax,in cerebral infarction areas was detected by immunohistochemistry and Western blot.The expression of PERK,CHOP,Bcl-2,and Bax mRNA in cerebral infarction areas was measured by RT-qPCR.Results:1.Cerebral infarction volume measurementAfter TTC staining,the normal tissue was red and the ischemic tissue was white.The staining results showed that there was no white area in Normal group and Sham-operation group,but the white area was more obvious in Model group.Compared with the Model group,the volume of cerebral infarction was significantly reduced after electroacupuncture,rich rehabilitation training and intervention with combined therapy(P<0.01).Compared with the EA group and the RT group,the volume of cerebral infarction in the EA+RT group decreased more obviously,and the difference was statistically significant(P<0.01).There was no significant difference between the EA group and the RT group(P>0.05).2.Learning and memory testingCompared with the Normal group and the Sham operation group,the average escape latency time in the Model group was significantly increased,and the number of times of crossing the platform was decreased,and the difference was statistically significant(P<0.01).After electroacupuncture,rich rehabilitation training and intervention of combined treatment,the three treatment groups compared with the Model group,the average escape latency time was significantly shortened,the number of times of crossing the platform increased,and the difference was statistically significant(P<0.01).Compared with the EA group and the RT group,the average escape latency time of the EA+RT was shorter,the times of crossing the platform increased,the difference was statistically significant(P<0.05).There was no significant difference between the EA group and the RT group(P>0.05).3.Pathomorphological observationIn the Normal group and the Sham group,the brain tissues were H&E-stained uniformly,the number of neurons in the hippocampal area CA1 was Normal with a clear tissue hierarchy,the neurocytes were normally structured,the capsules were intact,the nucleoli were clear,and there was no neurocyte interstitial edema.For the rats in the Model group,the edema at the hippocampal area was obvious,the cell membrane and nuclear membrane were poorly demarcated,the nuclei were deeply stained,the intercellular space was widened and in disorderly arrangement,and the nucleoli disappeared.For the rats in the three treatment groups,their brain tissues showed reduced neuroedema to varying degrees,an elevated number of neurons,and improved cell morphology;the brain tissues of the rats in the EA + RT group had more obvious improvement than those in the other treatment groups,the cell arrangement was in good order,the cytoplasm was abundant and deformed,and the number of necrotic neurons was significantly decreased.4.Detection of apoptosis by TUNElNuclear staining in normal brain cells appeared blue,after cerebral ischemia-reperfusion injury,the nuclei of apoptotic cell appeared brownish yellow,and chromatin was condensed and concentrated.The rats in the Normal group and the Sham group had a low apoptotic rate,and the apoptosis of the Model group was significantly higher than that of the Normal group and the Sham-operation group(P<0.01).After electroacupuncture,rich rehabilitation training and intervention of combined therapy,compared with the model group,the apoptosis of the three treatment groups decreased,the difference was statistically significant(P<0.01).Compared with the EA group and the RT group,the apoptosis of the EA+RT group was less,the difference was statistically significant(P<0.05),and there was no significant difference between the EA group and the RT group(P>0.05).5.The expression of positive cells were detected by immunohistochemistryThere were only a few positive cells in the Normal group and the Sham-operation group.After cerebral ischemia-reperfusion,the expression of PERK?CHOP?Bcl-2 and Bax in hippocampus of each group increased,and the average optical density value increased significantly.After electroacupuncture,rehabilitation training and intervention of combined therapy,the expression of Bcl-2 in the three treatment groups was significantly higher than that of the Model group,and the expression of PERK?CHOP and Bax were lower than that of the Model group(P<0.01).The increase of Bcl-2 and the decrease of PERK?CHOP and Bax in the EA + RT group were more significant than those in the EA group and the RT group(P<0.05).There was no significant difference between EA group and RT group(P>0.05).6.Comparison of hippocampal area CA1-associated factor protein expression in each groupCompared with the Normal group and the Sham-operated group,the expression of PERK?CHOP?Bcl-2 and Bax protein in hippocampus of the Model group were increased by western blot(P<0.01).After electroacupuncture,rehabilitation training and intervention of combined therapy,the three treatment groups compared with the Model group,the Bcl-2 protein in the hippocampus of the rats was significantly increased,the other protein levels were significantly reversed,and the phosphorylation level of AKT was continuously increased.The difference was statistically significant(P<0.01 or P<0.05).The phosphorylation level of Bcl-2,AKT and the decrease of PERK?CHOP and Bax in the EA + RT group were higher than those in the EA group and the RT group(P<0.05),and there was no significant difference between the EA group and the RT group(P>0.05).7.Comparison of hippocampal area CA1-associated factor mRNA in each groupOur correlation analysis showed that the Model group had significantly elevated expressions of PERK,CHOP,Bcl-2 and Bax mRNA compared with those in the Normal group and the Sham group,showing statistically significant differences(P<0.01).Compared with the Model group,the three treatment groups revealed a downward trend in the expressions of PERK,CHOP,and Bax mRNA(P<0.01),and the EA+RT group had a more significant decrease in expression than the EA group and the RT group.The three treatment groups showed an upward trend in the expression of Bcl-2 mRNA(P<0.01),and the EA+RT group had a more significant increase in the expression than the EA group and the RT group.No significant differences in mRNA expression were found between the EA group and the RT group(P>0.05).Conclusion1.Electroacupuncture combined with rehabilitation training can reduce the volume of cerebral infarction,relieve the stress response of endoplasmic reticulum induced by cerebral ischemia-reperfusion,and improve the ability of learning and memory in rats.And the treatment effect is more obvious than the single use of electroacupuncture or rich rehabilitation training.2.Electroacupuncture combined with enriched rehabilitation training can reduce neurointerstitial edema in hippocampus of rats and improve the morphological structure of ischemic tissue cells in rats,and the therapeutic effect is more obvious than that of single use of electroacupuncture or rich rehabilitation training.3.Electroacupuncture combined with rehabilitation training could reduce apoptosis,increase the expression of Bcl-2 protein and mRNA,increase the level of phosphorylation of AKT,decrease the expression of PERK,CHOP,Bax protein and mRNA in the ischemic hippocampus of rats.4.Electroacupuncture,enriched rehabilitative training and electroacupuncture combined with rehabilitative training could be used to repair and protect injured brain tissue after cerebral ischemia-reperfusion.The possible underlying mechanism might be up-regulation of the expression of anti-apoptotic protein Bcl-2 and AKT phosphorylation,interfering with the ERS-mediated apoptosis pathway and reducing the apoptotic rate by inhibiting the release and activation of ERS-associated proteins PERK,CHOP,and Bax for the purpose of exerting neuroprotective effects.In addition,treating ischemia-reperfusion injury with electroacupuncture combined with enriched rehabilitative training is more obviously efficacious than electroacupuncture monotherapy or enriched rehabilitative training.