Functional Study Of SNAP25 On Cortical Neuron Protection In Neonatal Hypoxic-ischemic Rats

Objective:Neonatal hypoxic ischemic encephalopathy(NHIE)induces irreversible nerve damage in brain cells,leading to severe sequelae and unpredictable effects.SNAP25 plays an important role in the coordination of neuronal synaptic transmission,and the lack of SNAP25 leads to memory impairment and neurological disfunction.Therefore,this article will be divided into two parts in vivo and in vitro to study whether SNAP25 plays a neuroprotective role in neonatal cerebral hypoxia-ischemia(HI)rats,and to explain the possible mechanism of SNAP25 in neuronal protective.Methods:The experiment in this paper is divided into vivo and vitro.In vivo:7 days SD rats were disconnected from the right carotid artery and hypoxia for 2 h to establish HI model.After 24h,TTC staining was used to observe the acute cerebral infarction.Zea-longa score was used to evaluate the success of HI model.NEUN immunofluorescence staining was used to detect the number of NEUN positive cells in acute phase after HI.HE staining was used to observe the death of 24 h and 30 d cerebral cortical cells after HI.Water maze test and Y maze detect 30 d memory impairment after HI,the open field experiment was used to evaluate the anxiety state of rats,the NSS score was used to evaluate the neurological function,and the rotating rod experiment was used to evaluate the movement after HI.Finally,the expression of SNAP25 in the 24 h left and right cortex and the left and right hippocampus was detected by qRT-PCR.In vitro:After establishing an oxygen glucose deprivation(OGD)model,qRT-PCR was used to observe the expression of SNAP25-mRNA at 2 h and 3 h,which to determine the OGD model time.NCBI searched for the double-stranded RNA(dsRNA)sequence of SNAP25,and designed and prepared three RNA fragments and a nonsense fragment(NC)by Guangzhou Ruibo.Neonatal SD rats were used to culture primary cortical neurons,then we resuscitated PC 12 cells and transfected with SNAP25-siRNA fragment positive control(CY3)in primary cortical neurons and PC 12 cells to observe transfection efficiency.Three SNAP25-siRNA fragments were transfected into primary cortical neurons,and the most efficient siRNA fragments were screened by qRT-PCR.Then the most effective SNAP25-siRNA fragment was transfected into primary cortical neurons.After the neurons were subjected to OGD,Tuj 1 immunofluorescence was used to detect cell size,axon length and number of neurons.Finally,SNAP25-associated molecules were searched by GENEMANIA software,and qRT-PCR was used to detect these associated molecules.Results:In vivo:We found a significant infarct area in the right cortex 24 h after TTC staining for HI group compared with the sham group(P<0.05).The Zea-longa score of the HI group was significantly increased(P<0.05),and the number of NEUN positive cells was also decreased(P<0.05).HE staining showed that neurons died in the cerebral cortex 24 h after HI,the cell cytoplasm was loosely stained,and the neurons were disorderly arranged.A large piece of death and nuclear pyknosis occurred in the cortical neurons of rats 30 days after HI.The water maze test showed that the escape latency of the HI group was higher than sham group 30 d after HI(P<0.05),but the number of target crossings,the time of target zone(%)and the distance of target zone(%)were lower than sham group(P<0.05).Y maze test found that the error rate and the time of false arm in the HI group increased compared with the sham group(P<0.05),and the correct rate and the food arm stay time decreased 30 d after HI(P<0.05).In the open field experiment,the standing time and grooming time of rats 30 d after HI were decreased(P<0.05),and the NSS score showed that the HI group was higher than sham group(P<0.05).The rotating rod experiment showed that the time of the rats in the HI group on the rotating rod was lower than sham group 30 d after HI(P<0.05).The qRT-PCR results showed that the mRNA expression of SNAP25 was increased in the right cortex compared with sham group(P<0.05),and the SNAP25 in the left cortex,left hippocampus and right hippocampus was increased,but there was no statistical significance.In vitro:qRT-PCR analysis showed that the expression of SNAP25-mRNA was higher in OGD 2 h group than in hypoxia group(P<0.05),and the expression of SNAP25-mRNA was decreased in OGD 3h compared with OGD 2 h(P<0.05).After transfecting three fragments of SNAP25-siRNA,the expression of SNAP25-mRNA in F1 fragment was significantly lower than normal group(P<0.05).Therefore,F1 fragment was selected as the most effective interference fragment.After transfection of effective interference fragment(F1),it was found that the expression of SNAP25-mRNA in SNAP25-si group decreased under normal condition and OGD condition(P<0.05),and the transfection efficiency of CY3 reached 80%,it indicated that the successful silencing of SNAP25.Immunofluorescence staining showed that axon length,cell body area and cell number were smaller than sham group in HI group under normal condition and OGD condition(P<0.05).The axon length,cell body area and cell number of the SNAP25-si group were also reduced compared with the NC group(P<0.05).Finally,we screened 11 molecules associated with SNAP25,namely Bcl-2,NGF,VEGF,CREB,SNCB,SYP,STAT3,ERK1,AKT,JAK1,and PI3K.It was found that compared with the NC group,the expression of Bcl-2,NGF and VEGF was increased and the expression of CREB and SYP was decreased in the SNAP25-si group under normal condition(P<0.05).Under OGD condition,NGF expression increased,CREB,SNCB and SYP expression decreased in SNAP25-si group(P<0.05),and STAT3 and ERK1 expression increased after SNAP25 interference under normal condition(P<0.05).Conclusions:We successfully constructed an in vivo HI model and an in vitro OGD model,and verified that silencing SNAP25 aggravated neuronal damage,At the same time,it suggests that SNAP25 may have the function of protecting neurons.We considered that the function of SNAP25 in neonatal HIE rats may be related to down-regulation of STAT3 and ERK1 and up-regulation of CREB.This study provides a new idea for the functional of SNAP25 in neonatal hypoxic ischemic encephalopathy.