| Objective:To investigate the effect of enriched environment(EE)combined with recombinant adenovirus-mediated hypoxia-inducible factor-1α(Ad HIF-1α)gene on pyroptosis in focal cerebral ischemia-reperfusion injury(CIRI)rats and the possible mechanism.Methods:132 male Sprague-Dawley rats were divided into 6 groups using the random number table method.These included Sham group,CIR group,recombinnant adenovirus empty vector(Ad)group,Ad HIF-1α group,EE group,and EE+Ad HIF-1α group.There were 24 rats in Ad and Ad HIF-1α groups respectively,and each of the other four groups had 21 rats.The models of right middle cerebral artery occlusion(MCAO)were established in all groups except Sham group.After modeling,different solutions were injected into the right ventricle of rats.Sham,CIR and EE groups were injected with sterile saline.Ad group was injected with Ad solution.Ad HIF-1α and EE+Ad HIF-1α groups were injected with Ad HIF-1α solution.Ad and the vector of Ad HIF-1α gene carry independently expressed green fluorescent protein(GFP)gene as tracer marker.After operation,EE and EE+Ad HIF-1α groups were fed in EE,and the other groups were fed in standard environment(SE).The modified neurological severity score(m NSS)was performed on each group of rats before modeling and on the 1st day,7th day and 14 th day after operation.The expression of hypoxia-inducible factor-1α(HIF-1α),nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)and cysteinyl aspartate specific proteinase-1(caspase-1)proteins in hippocampal tissue on the ischemic side of the rats was detected by western blot(WB)on the 1st day and 14 th day after operation.On the 14 th day after operation,fluorescence microscope was used to observe the expression of GFP in hippocampal tissue on the ischemic side of rats in Ad and Ad HIF-1α groups,hematoxylin and eosin(HE)staining was used to observe the pathomorphological changes of the hippocampal CA1 region on the ischemic side of the rats in each group,immunohistochemical method was applied to detect the expression of NLRP3 and caspase-1 proteins in the hippocampal CA1 region tissue on the ischemic side,and transmission electron microscopy was used to observe the ultrastructural changes of neurons in the hippocampal CA1 region on the ischemic side.Results:1.The expression of GFP observed under fluorescence microscope: On the 14 th day after operation,the expression of GFP was observed in hippocampal tissue on the ischemic side of rats in Ad and Ad HIF-1α groups.2.m NSS: The score of rats was 0 in each group before modeling and Sham group.On the 1st day and 7th day after operation,the scores of rats in CIR,Ad,Ad HIF-1α,EE and EE+Ad HIF-1α groups were higher than those in Sham group(P<0.05),but there was no significant difference in the scores among these five groups(P>0.05).On the 14 th day after operation,rats in CIR,Ad,Ad HIF-1α,EE and EE+Ad HIF-1α groups all scored higher than Sham group(P<0.05),but there was no significant difference in the scores between CIR and Ad groups(P>0.05);compared with CIR and Ad groups,the scores in Ad HIF-1α and EE groups were lower(P<0.05),but there was no significant difference in the scores between these two groups(P>0.05);compared with Ad HIF-1α and EE groups,the scores in the EE+Ad HIF-1α group were reduced(P<0.05).3.HE staining: On the 14 th day after operation,in Sham group,the structure of right hippocampal tissue was intact and clear,the morphology of pyramidal cells in CA1 region was normal,the nuclei were round or oval,the nucleus was full,the nucleoli were obvious,and the cells were arranged regularly and compactly.The hippocampal tissue in the ischemic side of the rats in CIR and Ad groups had loose structure and interstitial edema,a large number of pyramidal cells in the CA1 region had irregular morphology,the nuclei pyknosis was deeply stained and surrounded by vacuoles,the nucleoli disappeared,the pyramidal cells were disordered,and the density of neurons was reduced.The above pathological changes in Ad HIF-1α,EE and EE+Ad HIF-1α groups were lighter than those in CIR and Ad groups,and those in EE+Ad HIF-1α group were the lightest.4.Immunohistochemistry: On the 14 th day after operation,NLRP3 and caspase-1 proteins were seen to express in small amounts in the CA1 region of the right hippocampus in Sham group rats.The expression levels of NLRP3 and caspase-1 proteins in the hippocampal CA1 region on the ischemic side of rats in CIR,Ad,Ad HIF-1α,EE and EE+Ad HIF-1α groups were all increased compared with Sham group(P<0.05),with no significant difference between groups CIR and Ad(P>0.05),Ad HIF-1α and EE(P>0.05).Compared with CIR and Ad groups,the expression levels of NLRP3 and caspase-1 proteins in Ad HIF-1α,EE and EE+Ad HIF-1α groups were all decreased(P<0.05).And the expression levels of NLRP3 and caspase-1 proteins in EE+Ad HIF-1α group were lower than those in Ad HIF-1α and EE groups(P<0.05).5.WB: The expression levels of HIF-1α,NLRP3 and caspase-1 proteins were low in the right hippocampal tissue of rats in Sham group.On the 1st day after operation,compared with Sham group,the expression levels of HIF-1α,NLRP3 and caspase-1 proteins were higher in the hippocampal tissues on the ischemic side of rats in CIR,Ad,Ad HIF-1α,EE and EE+Ad HIF-1α groups(P<0.05),but there was no significant difference in the expression levels of each protein among the five groups(P>0.05).On the 14 th day after operation,the expression levels of HIF-1α,NLRP3 and caspase-1 proteins in the hippocampal tissues on the ischemic side of rats in CIR,Ad,Ad HIF-1α,EE and EE+Ad HIF-1α groups were all increased compared with Sham group(P<0.05),but there was no significant difference in the expression levels of each protein between CIR and Ad groups(P>0.05);compared with CIR and Ad groups,the expression levels of HIF-1α protein were increased in both Ad HIF-1α and EE groups(P<0.05),and the expression levels of NLRP3 and caspase-1 proteins were decreased(P<0.05),but there was no significant difference in the expression levels of each protein between these two groups(P>0.05);compared with Ad HIF-1α and EE groups,the expression levels of HIF-1α protein were increased in EE+Ad HIF-1α group(P<0.05),and the expression levels of NLRP3 and caspase-1 proteins were decreased(P<0.05).6.Transmission electron microscopy: On the 14 th day after operation,in Sham group,the ultrastructure of neurons in the right hippocampal CA1 region was normal,the structure of cell membrane was intact,the organelles in the cytoplasm were abundant and evenly distributed,the structures of mitochondria and rough endoplasmic reticulum were clear and normal,and the chromatin was evenly distributed in the nucleus.The ultrastructural changes of neurons in hippocampal CA1 region of ischemic side were similar in CIR and Ad groups,both of which showed obvious pathological changes of pyroptosis,with many holes formed in cell membrane,cell edema,disappearance of most of organelles in cytoplasm,significant reduction of contents,swelling of mitochondria,disappearance of mitochondrial cristae to vacuolization,rupture of the outer membrane of some mitochondrias to unclear structure,pyknosis of nucleus,increase of heterochromatin which gather around the nuclear membrane resulting in dense masses.In Ad HIF-1α and EE groups,the pathological changes of neuronal pyroptosis were significantly reduced compared with those in CIR and Ad groups,a few holes were occasionally seen on the cell membrane,cell edema was alleviated,there was no significant reduction of organelles and contents in the cytoplasm,a small amount of mitochondrial damage was observed,the nucleus was not significantly pyknotic,and the heterochromatin in the nucleus was reduced.The ultrastructure of neurons in EE+Ad HIF-1α group tended to be normal,with an intact cell membrane structure,many intracytoplasmic organelles,occasional disappearance of a small amount of mitochondrial cristae,and no obvious abnormality in the nucleus.Conclusions:1.EE may inhibit the NLRP3/caspase-1-mediated classical pyroptosis pathway by up-regulating the expression of HIF-1α,thus promoting the recovery of neurological function in CIRI rats.2.Ad HIF-1α gene intervention may down-regulate the expression of pyroptosisrelated proteins NLRP3 and caspase-1 by up-regulating the expression of HIF-1α,inhibit pyroptosis,and then improve CIRI and play a protective role in brain.3.The combination of EE and Ad HIF-1α gene can play a better role in brain protection than single therapy,which may be related to the fact that HIF-1α is the common pathway for both to play a role. |
