| ObjectiveStroke is the second leading cause of death in the world,of which ischemic stroke(IS)accounts for 85%.Remote ischemic preconditioning(RIPC)induces endogenous protection by stimulating nonlethal ischemia of nonvital organs(usually the upper or lower extremities),which is a promising approach for stroke.However,the mechanism of RIPC is still in infancy,and the key molecules involved in neuroprotection induced by RIPC needed to be found.Therefore,this study aims to find the key gene and functions of differently expressed genes involved in RIPC in the early and late stages exerting neuroprotection through microarray.MethodsTwenty-seven healthy male SD(Sprague-Dawley)rats(11-12 weeks,weight 270-320g)were randomly divided into shame group(shame,n=3),focal cerebral ischemia group(IS,n=12)and remote ischemic preconditioning group(RIPC,n=12).The groups of IS and RIPC were divided into four subgroups: 1h,3h,6h and 24 h,respectively.In IS group,we established distal middle cerebral artery infarction occlusion(dMCAO)model.The dMCAO model was successfully made by permanent left middle cerebral artery infarction occlusion and bilateral common carotid artery occlusion(CCAO)for 30 minutes.In RIPC group,the left femoral artery was clipped for 15 min/reperfused for 15 min.which was repeating three cycles,then focal cerebral ischemia was performed immediately.The shame group only performed craniotomy.Total RNA was extracted from brain tissue,and mRNA expression was then measured by microarray.Bioinformatics methods were used to identify the differentially expressed genes(DEGs)that play a key role in RIPC,and RT-q PCR was performed to verify some of them.Finally,the DEGs screened above were studied at the cellular level.ResultsCompared with the shame group,the number of DEGs in the RIPC group at1 h,3 h,6 h and 24 h were 623,854,1019 and 2915,respectively.The number of DEGs in the IS group was 354,628,923 and 2268,respectively.First,we investigated the DEGs in early(<4h)stage of RIPC.The DEGs were intersected into three parts by venny: DEGs related to RIPC(RIPC＿DEGs),DEGs related to IS(IS＿DEGs),DEGs co-expressed in group of RIPC and IS(co-expressed＿DEGs).Then,GO and KEGG enrichment analysis showed that DEGs of RIPC＿DEGs significantly enriched the G protein-coupled signaling pathway and olfactory transduction pathway.However,the DEGs of IS＿DEGs and co-expressed＿DEGs significantly enriched the inflammatory response.Furthermore,the top 60 DEGs were further analyzed.It was found that RIPC still significantly enriched the G protein-coupled signaling pathway and olfactory transduction pathway,and Rreb1,Cxcl11,Ptgs2 and Olr1356 may be key genes in the early stage of RIPC.Secondly,DEGs related to RIPC24h(RIPC24h＿DEGs)can significantly enrich the neural active receptor ligand pathway.On this basis,we found that there were 28 immune-related genes in RIPC24h＿DEGs.Finally,the gene sets including interference on Gamma response,TNFa signaling via NFkB,Inflammtory response,Interferon gamma response,Allograft rejection,IL6 JAK STAT3 signaling were activated at 1h and reached the highest at 24 h during RIPC.Based on the DEGs screened by the above analysis,we successfully established a ischemic preconditioning model at the cells of SH-SY5 Y and found that CXCL11 and MST1 were significantly upregulated in group of ischemic preconditioning.ConclusionIn this study,we found that most enriched gene sets in RIPC were gradually activated at 1h,3h,6h and 24 h.At 1 h and 3 h,RIPC play a role mainly through G protein-coupled signaling pathways and olfactory transduction pathways.At24 h,RIPC induced cell signal transduction,ERK1 and ERK2 cascade activation,chemical neurotransmitter transmission,angiogenesis and other biological processes,but mainly through the neuroactive ligand-receptor interaction.Among them,Bmp2、Ccn1、Inhba、Npy may play important roles in RIPC24 h.Based on the DEGs which were screened,this study further found that CXCL11 and MST1 were involved in the regulation of preconditioning at the cellular level. |
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