Effects Of Hsp70-hom+2437 Gene Polymorphism On Rat MCAO Model And Related Studies

Objectives:1.To explore whether the HSP70-hom+2437 C and T alleles have a protective effect on the rat MCAO model.2.To investigate whether there is a difference in the protective effect of HSP70-hom+2437 C and T alleles on rat MCAO model.3.To explore the possible mechanism of protection and differential protection of HSP70-hom+2437 C and T alleles on the rat MCAO model by detecting the apoptotic rate and the expression levels of HSPAIL,PI3K,p-AKT,Bax,Bcl-2,cleaved-caspase3,and BDNF in each group.Methods:1.Lateral ventricle injection:65 male SD rats,weighing 220-240 g were randomly divided into 5 groups:model group,saline group,negative control virus group,C allele group,T allele group(n=13).The model group was not performed the lateral injection.The normal saline group,the negative control virus group,the C allele group,and the T allele group were injected with the same amount of saline or equal viral particles,the control negative lentivirus,the C allele lentivirus and the T allele lentivirus,respectively.On the 7th day of lentivirus injection,3 rats in each group were sacrificed to take brain tissue for detecting the level of expression of HSPA1L by western blot and qPCR.This step is for ensuring the successful preparation of the overexpressed animal model.2.the rat MCAO model:7 days after lateral ventricle injection,rats in each group were performed MCAO model.The lysine coated nylon thread was inserted from the common carotid artery(CCA)near the bifurcation into the internal carotid artery(ICA),until entering the initial segment of the middle cerebral artery(MCA),blocking the blood flow of the MCA,ligating CCA and nylon thread,forming a permanent model of middle cerebral artery infarction.3.Neurological deficit score:The severity of neurological deficit was assessed by Longa five-point method 24 hours after MCAO model surgery.4.TTC staining for infarct volume:Rats were sacrificed at 24 h after MCAO model in each group.A part of rats were sacrificed,taking brain tissues for TTC staining.The tissue was cut into 2mm thick slices with brain trough.Brain slices were stained in 1%TTC at 37? for 15 min,then fixed with 4%paraformaldehyde for 6 h.The fixed slices were photographed by camera and image J software was used to measure infarct volume in each group.5.Tunnel staining for apoptosis rates:The 3rd tissue sections of each group were fixed with 4%paraformaldehyde for 24h,then embedded in paraffin and sliced.for dewaxing and repair.Dyeing,DAPI staining.The anti-fluorescent quencher was photographed and photographed under a:fluorescence microscope.Image J software was used to count the apoptosis rate of brain tissue cells of each group.6.Western blot for detecting the expression of HSPAIL,PI3K,p-AKT,Bax,Bcl-2,BDNF and cleaved-caspase3:The total protein was extracted from brain tissue in peripheral region of cerebral infarction and then several of apoptosis-related proteins were detected by western blot.The relative expression of each protein was measured by grayscale statistical analysis using Image J software.Results:1.the expression of HSP70-hom gene carried in lentivirus:on the 7th day after injection of lentivirus in the lateral ventricle,there was no significant difference in mRNA and protein levels between the model group,the saline group and the negative control lentivirus group.Compared to the three groups,the transcription levels of the HSP70.hom were significantly increased in C and T alleles groups(p<0.05).Compared to the model group,the content of HSPAIL protein in C and T alleles groups were increased by 60%and 62%,respectively(P<0.05).There was no significant difference of he mRNA and protein levels between C and T alleles groups(P>0.05).2.Neurological deficit score:After 24 hours of rat MCAO model surgery,there was no significant difference in neurological deficit scores between model group,saline group and negative control virus group.Compared to the former three groups,C and T alleles groups significantly reduced the neurological deficit score(P<0.05).There was no significant difference between the the two groups(P>0.05).3.TTC staining for cerebral infarct size:There was no significant difference in the volume of cerebral infarction between the three control groups,model group,saline group and negative control virus group(P>0.05).Compared to these three groups,the infarct size of the C and T alleles decreased(P<0.05),and the infarct volume of the T allele group was less more than the C allele group(P<0.05),addicating the the stronger protection of T allele.4.Apoptotic rates measured by tunnel staining:There was no significant difference in apoptotic rates between model group,saline group and negative control virus group.C and T allele groups had more lower rates than these three control groups.The apoptotic rate of allele groups decreased(P<0.05).Compared to the C allele group,the T allele group had a lower apoptotic rate(P<0.05),stronger inhibitory effect on apoptosis5.The expression of apoptosis-related proteins:Compared with the model group,saline group and negative control virus group,overexpressing C and T alleles increased the expression of HSPAIL and pAKT,and inhibited the expression of the pro-apoptotic protein Bax.Compared with the C allele group,the level of Bax in the T allele was significantly lower(P<0.05).Conclusions:1.Overexpression of HSP70-hom gene decreased the neurological deficit score in the rats MCAO model.2.Overexpression of HSP70-hom gene reduced the infarct volume of the rats MCAO model rats,cerebral infarct volume of T allele group was smaller than C allele group,indicating T allele The gene has a stronger protective effect on the rat MCAO model.3.HSP70-hom gene decreased the apoptosis rate of ischemic brain tissue in MCAO rats,and the apoptosis rate of T allele is lower.The protection of HSP70-hom on MCAO model may related to the inhibition of apoptosis.The T allele inhibits apoptosis more strongly than the C allele.4.Overexpression of HSP70-hom increased the expression of HSPA1L and pAKT proteins in the brain tissue,and the expression level of proapoptotic protein Bax was significantly decreased,indicating that HSP70-hom protected rat MCAO model related to the expression of HSPA1Land pAKT,inhibition of Bax.Moreover,the differential protection of the C and T alleles of the HSP70-hom gene to the MCAO model was related to the inhibition level of Bax protein.