The Protective Effects And Mechanism Of Overexpression Of Egr2 And Egr4 In Ischemic Stroke

Background:Ischemic stroke is one of the most common types of neurological diseases in the brain.It is usually caused by the decrease of intracranial blood supply induced by vascular lesions,which leads to the damage of intracranial central nervous system,and then leads to the necrosis and functional loss of cerebral nerve tissue in patients.Nerve cell survival is a key factor in the pathology and prognosis of ischemic stroke.Among them,the level of neuronal apoptosis and neuronal inflammation induced by ischemia is one of the core links affecting the survival of nerve cells.Therefore,based on the clinical basic research of neuroinflammation and neuronal apoptosis,screening the clinical treatment and prognosis targets of ischemic stroke has become one of the mainstream directions of ischemic stroke research.Transcription factor is the key hub of intracellular signal transducation.After receiving intracellular and intracellular signals,it enters the nucleus from the cytoplasm to participate in the transcriptional regulation of target genes,so as to initiate the response of intracellular signals.Therefore,it participates in a variety of life activities such as cell apoptosis and inflammation.Transcription factors Egr2 and Egr4 have become one of the potential clinical therapeutic targets in the pathological process of ischemic stroke.However,the roles and mechanisms of transcription factors Egr2 and Egr4 in ischemic stroke remain unclear.In particular,whether Egr2 and Egr4 participate in the survival of nerve cells in ischemic stroke by regulating neuroinflammation and neuronal apoptosis has become a key issue for further analysis of the role of Egr2 and Egr4 in ischemic stroke lesions.Therefore,this study analyzed the correlation between Egr2 and Egr4 and ischemic stroke through database,constructed rat ischemic stroke model(MCAO),Adenovirus was used to construct Egr2 overexpressing MCAO rats,Egr4 overexpressing MCAO rats,and Egr2+Egr4 overexpressing MCAO rat models.TTC staining,neurological impairment score,Nissl staining,The role and mechanism of Egr2 and Egr4 in the pathological link of ischemic stroke were analyzed by qRT-PCR analysis,WB detection and ELSIA detection,which provided a further basis and new direction for the study of ischemic stroke.The first partObjective:To screen the clinical therapeutic targets for ischemic stroke and analyze the changes of Egr2 and Egr4 expression in cerebral tissue of ischemic stroke rats.Methods:Bioinformatics method was used to analyze the differentially expressed genes in pathological links of ischemic stroke.The experimental animals were rats(Wistar,male,SPF grade),and the modeling of ischemic stroke rats(MCAO)was carried out in the Animal Experimental Center.Rats were selected to undergo pre-operative treatment,carotid artery separation operation,carotid artery operation and postoperative treatment after anesthesia,and the ischemic stroke rat model(MCAO)was established.Neurological impairment score(NSS score)was used to analyze the neurological impairment of rats in different groups.The changes of cerebral infarction in different groups were analyzed by TTC staining.Nissl staining was used to analyze the survival changes of nerve cells in different groups of rats.Western blot was used to detect the expression of Egr2 and Egr4 proteins in different groups of rats.The gene expression of Egr2 and Egr4 in rat brain was detected by qRT-PCR.Results:1.Bioinformatics analysis showed that Egr2 and Egr4 were differentially expressed genes in ischemic stroke and had potential for targeted gene analysis.2.Neurological impairment score(NSS score)results:The cerebral neurological impairment score(NSS score)of ischemic stroke rats(MCAO)was significantly higher than that of sham operation group and blank control group,P<0.01.3.Nissl staining analysis showed that the volume of cerebral infarction in ischemic stroke rats(MCAO)was significantly higher than that in sham operation group and blank control group,P<0.01.4.Nissl staining analysis showed that the number of Nissl staining positive cells in MCAO brain tissue was significantly lower than that in sham operation group and blank control group,P<0.01.5.The protein and gene expression levels of Egr2 and Egr4 in ischemic stroke group were significantly lower than those in blank group and sham group,P<0.01.Conclusion:Egr2 and Egr4 are targets for clinical diagnosis and treatment of ischemic stroke,and they are abnormally low expressed in the brain tissue of ischemic stroke rats.The second partObjective:To explore the mechanisms of neuroprotection of overexpression of Egr2 and Egr4 in ischemic strokeMethods:After the establishment of ischemic stroke rats(MCAO),Adenovirus was used to construct Egr2 overexpressing MCAO rats,Egr4 overexpressing MCAO rats,and Egr2+Egr4 overexpressing MCAO rat models.After the establishment of the animal model,neurological impairment score(NSS score)was used to analyze the neurological impairment of rats in different groups.The changes of cerebral infarction in different groups were analyzed by TTC staining.Nissl staining was used to analyze the survival changes of nerve cells in different groups of rats.Western blot was used to detect the expression of Egr2 and Egr4 proteins in different groups of rats.The gene expression of Egr2 and Egr4 in rat brain was detected by qRT-PCR.Results:1.The expression of Egr2 protein and gene in the brain of Egr2 overexpressing MCAO rats and Egr2+Egr4 overexpressing MCAO rats was significantly higher than that of ischemic stroke rats(MCAO).The expression of Egr4 protein and gene in the brain of Egr4 overexpressed MCAO rats and Egr2+Egr4 overexpressed MCAO rats was significantly higher than that of ischemic stroke rats(MCAO),P<0.01,the difference was extremely significant.2.Neurological impairment score(NSS score):Cerebral neurological impairment score(NSS score)of ischemic stroke rats(MCAO),Egr2-overexpressing MCAO rats,Egr4-overexpressing MCAO rats,and Egr2+Egr4-overexpressing MCAO rats were significantly higher than those of sham operation group and blank group.The cerebral nerve function injury score(NSS score)of Egr2 overexpressing MCAO rats,Egr4 overexpressing MCAO rats and Egr2+Egr4 overexpressing MCAO rats was significantly lower than that of ischemic stroke rats(MCAO),P<0.01.3.TTC staining analysis showed that the cerebral infarction volume of ischemic stroke rats(MCAO),Egr2 overexpressed MCAO rats,Egr4 overexpressed MCAO rats,and Egr2+Egr4 overexpressed MCAO rats were significantly higher than that of sham operation group.The cerebral infarction volume of Egr2 overexpressed MCAO rats,Egr4 overexpressed MCAO rats and Egr2+Egr4 overexpressed MCAO rats was significantly lower than that of ischemic stroke rats(MCAO),P<0.01.4.Nissl staining analysis showed that the number of Nissl staining positive cells in ischemic stroke rats(MCAO),Egr2 overexpressing MCAO,Egr4 overexpressing MCAO and Egr2+Egr4 overexpressing MCAO were significantly lower than those in sham operation group.The number of Nissl staining positive cells in Egr2 overexpressing MCAO rats,Egr4 overexpressing MCAO rats and Egr2+Egr4 overexpressing MCAO rats was significantly higher than that in ischemic stroke rats(MCAO),P<0.01.Conclusion:Rat models of overexpressing Egr2,overexpressing Egr4,and overexpressing Egr2+Egr4 MCAO were constructed.overexpression of Egr2 and Egr4 can inhibit cerebral infarction and promote the survival of nerve cells in ischemic stroke.The third partObjective:To investigate the mechanism of Egr2 and Egr4 promoting nerve survival in ischemic stroke.Methods:The model building of ischemic stroke rats(MCAO)was carried out in the Animal Experimental Center.Ischemic stroke rats(MCAO),Egr2 overexpressed MCAO rats,Egr4 overexpressed MCAO rats and Egr2+Egr4 overexpressed MCAO rats were successfully constructed.After the establishment of the animal model,the expression levels of p-JNK and p-c-JUN related proteins in the brain tissues of different groups of rats were detected by Western blot.The expression of inflammatory factors IL-1β,IL-6 and TNF-α in serum and brain tissue of different groups of rats was detected by ELISA.Results:1.The expression levels of JNK signaling pathway related proteins p-JNK and p-c-JUN in cerebral tissue of rats in ischemic stroke group were significantly higher than those in blank group and sham group,P<0.01;2.The expression levels of p-JNK and p-c-JUN in brain tissue of Egr2 overexpressed MCAO rats,Egr4 overexpressed MCAO rats and Egr2+Egr4 overexpressed MCAO rats were significantly lower than those in ischemic stroke rats(MCAO),P<0.05.3.The expression levels of inflammatory cytokines IL-6,IL-1β and TNF-α in brain tissue and serum of rats in ischemic stroke group were significantly higher than those in blank group and sham group,P<0.01;The expression levels of inflammatory cytokines IL-6,IL-1β and TNF-α in brain tissue and serum of Egr2 overexpressed MCAO rats,Egr4 overexpressed MCAO rats and Egr2+Egr4 overexpressed MCAO rats were significantly lower than those in ischemic stroke rats(MCAO),P<0.05.Conclusion:Reduced Egr2 and Egr4 prompted the expression of JNK signaling-related proteins p-JNK and p-c-JUN and inflammation in ischemic stroke rats,indicating the activation of the JNK/c-JUN pathway.Upregulation of Egr2 and Egr4 inhibited the expression of p-JNK,p-c-JUN,and neuroinflammation in MCAO rats.Thus,the reduces the damage to the brain tissue and neurons.