| At present,studies on collateral circulation mostly focus on acute ischemia.However,there is less research on the relationship between chronic ischemia and collateral circulation.The genetic regulation of collateral-circulation establishment is currently not fully understood,and most of the research has focused on mRNA?in recent years,With the development in gene technology,molecular biology,and gene sequencing technologies,it has been found that long-chain non-coding RNAs are closely related to cell proliferation,differentiation,and metabolic apoptosis.The occurrence and development of human diseases are also closely related to it.In particular,studies on vascular endothelial cells and vascular smooth muscle have found that lncRNA regulates it in many ways,but Cerebrovascular disease research in this area is rarely reported.Therefore,we performed the acute and chronic ischemia models of rat,and further observed the changes of blood vessels in the brain of rat under acute or chronic ischemia conditiongs to analysis the possible influencing factors.A new generation of RNA-seq sequencing technology is applied to RNA-seq sequencing of cerebral vessels in the ischemic area.We attempt to find out the similarities and differences between angiogenesis and collateral circulation in the chronic ischemia and acute ischemia in rats,providing some evidence for possible gene regulation targets.Establishment of acute and chronic ischemia model in rats and assessment of collateral circulation.Method:To Establish the model of acute and chronic ischemia in rats.Adult male Wistar healthy rats were randomly divided into three groups: A group(sham operation group,B group(MCAO model group),and C group(chronic ischemia group).All rats were subjected to laser speckle imaging after modeling.At 2 hours,12 hours,24 hours,48 hours,72 hours,and 1 week,neurological scores were sacrificed in each group at each time period.TTC,HE,and CD105 staining were performed to the establishment of collateral circulation and there is a small amount of angiogenesis.Results:TTC staining : the percentage of infarcted area in group B was 20.30±4.62%,and the percentage of infarcted area in group C was 3.02±1.26%.Laser speckle blood flow diagram: in A group,Cerebral blood perfusion did not change significantly after establishment of the model and before.In B group,cerebral perfusion of nfarcted cerebral hemispheres was significantly reduced compared with that before,with a decrease of more than 60%.the pial collateral arteries were opened to the ischemic area following by occlusion in rats.In C group,the total perfusion of cerebral reduced significantly more than 50% compared with preoperative.there was still blood flow in the middle and front artery of the brain,which was significantly lower than before.CD105 staining : In B group,neovascularization in both the infarct core and infarct around the Infarct area.There was also neovascularization in C group.Differential Expression of RNA in Cerebral Vascular of Rat with Cerebral infarctionMethod:The MCAO model and chronic ischemia model of adult male Wistar healthy rats were established,and then randomly divided into three groups: A group(sham operation group,B group(MCAO model group),and C group(chronic ischemia group).After 48 hours,the arteris of right cerebral ischemic region was immediately taken to extract RNA,following the rats were sacrificed.and then the next-generation technology high-throughput RNA-seq sequence was performed.Ballgown was used to compare the transcripts of the two groups to find differential genes.Then the KEGG database was used to perform GO.Analysis,pathway analysis,signal-net analysis and lncRNA and mRNA co-expression network(lncRNA-mRNA-net)analysis.Differential gene real-time quantitative PCR(qRT-PCR)validationResults:1.Analysis of lncRNA and mRNA in chronic ischemic group and control group: 276 differently expressed lncRNAs were screened,of which 66 were up-regulated and 160 were down-regulated.There were 3289 differently expressed mRNAs,of which 1776 were up-regulated and 1513 were down-regulated.In the mRNA-interactive network,Mapk1,Prkca,Nras and Kras constitute the main interaction network for angiogenesis.Nras,Kras and Mapk1 may interact more closely and can regulate Mapk8 and Mapk9.We speculate that the generation of blood vessels during chronic ischemia is mainly regulated by Mapks pathway.By analyzing the co-expression network of lncRNA and mRNA,we found that ENSRNOT00000092872 negatively regulates Kras and ENSRNOT00000076004 Positively controls Nras.The strongest of the lncRNAs regulating Mapk1 are ENSRNOT00000090397 and ENSRNOT00000076495.ENSRNOT00000090397 is negatively regulated Mapk1.ENSRNOT00000092832 Positively regulates Prkca.2.The analysis of lncRNA and mRNA in acute ischemic group and control group: The analysis of lncRNA and mRNA in acute ischemic group and control group showed that there were 71 d differently expressed lncRNAs,of which 34 were up-regulated and 37 were down-regulated.There were 1963 differently expressed mRNAs,of which 1142 were up-regulated and 821 were down-regulated.In the mRNA interaction network,Tek,Kdr,Plcb4,and Plcg1 play a very important role in the entire regulatory network,related to angiogenesis.Tek and Kdr can control this network,which consisting of Prkce,Prkcb,Prkca,Itpr1,Pla2g4 a,Mapk13,and Mapk11,by activating Plcb4.Kdr can also regulate the network by activating Plcg1.Itgb2 can influence Adcy5 and then affect the regulatory network by regulating Fgf2.ENSRNOT00000036680,ENSRNOT00000084727 regulate Plcg1 at the same time,the former is positive regulation and the latter is negative regulation.lncRNA was not find to regulate Tek.there were three lncRNAs to regulate Kdr.In which,ENSRNOT00000077132 had the strongest negative regulation ability and ENSRNOT00000092096 had positive regulation ability.ENSRNOT00000073655 negatively regulates Plcb4.ENSRNOT00000087051 positively regulates Adcy5.3.Analysis of lncRNA and mRNA in Chronic Ischemic Group and Acute Ischemic Group: Comparison of RNA data analysis between acute and chronic ischemia groups: Among the angiogenesis-related mRNAs,330 RNA were expressed in both groups.There are 21 differentials expressed more than 4 times in them.Kcnab2,Atp6v1g2,Stmn4,Stim1,Cyp1b1,and Fgf2 were opposite Regulated between the two groups,and Kng2 was up-regulated in the two groups but the difference in the B group was more significant.Compared differences between two groups by the way of GO analysis and pathway analysis,Kcnab2 and Stmn4 had significant differences only in group C.Stim1 and Cyp1b1 were only significantly different in group C.Atp6v1g2 was down-regulated in group B,and was up-regulated in group C.Atp6v1g2 was down-regulated in group B and up-regulated in group C in The metabolic pathway.From the above data,we can see that Kng2,Fgf2,and Atp6v1g2 have the same functions in both groups B and groups C,and in the same regulatory pathway.ENSRNOT00000087079 has the strongest regulatory ability to regulate Stmn4.ENSRNOT00000002990 negatively regulates Serpine1,but ENSRNOT00000076446 is more regulative than ENSRNOT00000002990 in the C group.There were difference between the two groups in Regulation of Kng2.In both groups,ENSRNOT00000079300 negatively regulates lgf1.ENSRNOT00000092605 positively regulates Atp6v1g2 in both groups,but ENSRNOT00000089288 has stronger regulation than ENSRNOT00000092605 in group C.Conclusion1.The establishment of ultra-early collateral circulation in acute infarction is based on existing arteries,such as the Willis artery ring and the pial artery.More angiogenesises occurred within 24 hours following Cerebral infarction.In the case of chronic vascular stenosis,the establishment of medial cranial circulatory circulation is mainly based on existing arteries and angiogenesis is not obvious.2.In acute ischemia model,Tek,Kdr,Plcb4 and Plcg1 play important roles in the angiogenesis.Which may be regulated angiogenesis by VEGF signaling pathway,PI3K-Akt signaling pathway,HIF-1 signaling pathway,and Mapks signaling pathway.Mapks signaling pathway may be more obvious than other pathways.ENSRNOT00000077132,ENSRNOT00000092096,and ENSRNOT000000-73655 regulate above mRNAs.3.In the chronic ischemia model,Mapk1,Prkca,Nras and Kras genes play an important role in the regulation of these genes.The PI3K-Akt signaling pathway and Mapk signaling pathway contribute more than Others.LncRNAs such as ENSRNOT00000092872,ENSRNOT00000076004,and ENSRNOT00000092832 have strong regulatory effects on these above mRNAs.4.Kng2,Fgf2 and Atp6v1g2 have same functions and regulatory pathways in both groups B and C.In both groups,ENSRNOT00000079300 negatively regulates lgf1.ENSRNOT00000092605 positively regulates Atp6v1g2 in both groups.In C group,ENSRNOT00000089288 has the stronger regulation than ENSRNOT00000092605. |
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