| Aims:To study the mechanism of Neuromodulin 1β(NRG1β)improving Middle cerebral artery occlusion reperfusion(MCAO/R)injury in rats.And its effect on SIRT1-FOXO1 signaling pathway.Methods:210 healthy male SD rats were randomly divided into Sham group(Sham group),model group(MCAO/R group),treatment group(NRG1β group),agonist group(SRT501group),agonist + treatment group(SRT501+ NRG1β group),inhibitor group(EX527group)and inhibitor + treatment group(EX527+NRG1β group),with 30 rats in each group.The modified thread plug method was used to establish a rat model of MCAO/R.The thread plug was inserted into the internal carotid artery about 18~22 mm from the external carotid artery to the left anterior cerebral artery.After 2 hours of ischemia,the thrombus was slowly pulled out and cerebral blood flow was restored for 22 h.EX527(5mg/kg)and SRT501(100 mg/kg)were injected intraperitoneally 30 min before surgery,and NRG1β 2 μg/kg were injected into the internal carotid artery by microsyringe after the suture plug was pulled out.The neurobehavioral function of the seven groups of rats was evaluated by modified neurological severity score(m NSS)after 2 h ischemia and 22 h reperfusion.Triphenyltetrazole chloride(TTC)staining was used to calculate the percentage of cerebral infarction volume in rats.Hematoxylin-eosin(HE)and toluidine blue(Nissl)staining were used to observe morphological changes of neurons.Western Blotting(WB)and immunofluorescence(IF)were used to detect ischemic penumbra in the ischemic frontal cortex,SIRT1,FOXO1,LC3 and P62 were expressed.The ultrastructure of neurons and the number of autophagosomes and autophagolysosomes were observed by transmission electron microscopy(TEM).Results:NRG1β can significantly improve the neurological function defect,cerebral infarction volume and morphological and structural damage of neurons in rats.Enhance the expression of SIRT1,FOXO1,P62,and inhibit LC3;Reduce the number of autophagosomes and autophagolysosomes.m NSS and TTC showed that there was no neurobehavioral function injury and infarct area in Sham group.m NSS and infarct volume were higher in MCAO/R group,EX527 group and EX527+NRG1β group.The m NSS and infarct volume of NRG1β group,SRT501 group and SRT501+NRG1β group were significantly reduced compared with the previous groups.The results of HE and Nissl staining showed that the morphology of neurons in Sham group was nearly normal, and the damage of neurons in MCAO/R group,EX527 group and EX527+NRG1β group was more serious,while the damage of neurons in NRG1β group,SRT501 group and SRT501+NRG1β group was significantly reduced.The results of WB and IF showed that the expressions of SIRT1,FOXO1 and P62 were consistent,and the expressions of MCAO/R group were decreased compared with Sham group.The expression of NRG1βgroup,SRT501 group and SRT501+NRG1β group was enhanced compared with MCAO/R group,and the expression of SRT501+NRG1β group was the strongest.The expression of MCAO/R group,EX527 group and EX527+ NRG1β group was weak,and the expression of EX527 group was the weakest.The expression trend of LC3 protein was opposite to that of P62 protein.TEM showed that the morphology and structure of neurons in the Sham group were intact,and the nuclei and mitochondria were normal.In MCAO/R group,EX527 group and EX527+NRG1β group,autophagosomes and autophagolysosomes increased,nuclear atrophy and vacuole were observed,and some mitochondrial structures were seriously damaged,especially in EX527 group.The number of autophagosomes and autophagolysosomes decreased in NRG1β group,SRT501 group and SRT501+NRG1β group,and the morphological structure of neurons was relatively complete and the damage was light,which was significantly improved compared with MCAO/R group and EX527 group,especially SRT501+NRG1β group.Conclusion:1.The improvement of NRG1β can alleviate the nerve function injury in MCAO/R rats;2.NRG1β can inhibit autophagy by activating SIRT1-FOXO1 signaling pathway,thus reducing cerebral ischemic reperfusion injury in MCAO/R rats. |
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