The Role And Mechanism Of Prdx1 In Secondary Injury Induced Intracerebral Hemorrhage

Background and objective:Stroke is currently the largest cause of death in China.Among them,intracerebral hemorrhage accounts for about 15%.The incidence of intracerebral hemorrhage in China is higher than that in developed countries such as Europe and America.Intracerebral hemorrhage has the characteristics of high disability,high mortality,and high morbidity.The damage caused by intracerebral hemorrhage mainly includes two aspects:the mechanical compression of hematoma and the secondary injury caused by the substances released by blood.The mechanical compression of hematoma is related to the volume and the location of hematoma and it is difficult to intervene in clinic treatment.Many clinical studies have shown that evacuation of hematoma does not improve the prognosis and reduce the mortality of patients.The secondary injury mainly comes from inflammatory response,oxidative stress and toxic effects of blood components after intracerebral hemorrhage.Recent researches have shown that reducing secondary injury after intracerebral hemorrhage can significantly reduce neurological deficits and mortality in animals.Therefore,the intervention of secondary injury is the main direction of current research on intracerebral hemorrhage.There are increasing evidences showed that post-transcriptional regulation was involved in the development of stroke.Dykstra-Aiello C et al.collected peripheral blood samples from patients with acute intracerebral hemorrhage and cerebral infarction and normal people to perform RNA high-throughput sequencing.The RNA of cerebral infarction patients and intracerebral hemorrhage patients has occurred significantly alternative splicing compare with normal people,and these differential transcriptomes and alternative splicing were related to inflammatory response,oxidative stress,cytokines and other related pathways,suggesting that post-transcriptional regulation may be closely related to secondary injury after intracerebral hemorrhage.Post-transcriptional regulation is a variety changes that occur after genes transcribed into RNA,including alternative splicing,alternative polyadenylation,differential expression of genes,RNA methylation,and so on.No matter what type of post-transcriptional regulation,RNA-binding proteins are indispensable,because RNA-binding proteins are involved in the beginning and end of post-transcriptional regulation and they play central roles in the post-transcriptional regulation.Previous studies have shown that regulating the post-transcriptional regulation through RNA-binding proteins can change the development of disease.These evidences suggest that it is feasible to regulate the post-transcriptional regulation of intracerebral hemorrhage through RNA-binding proteins,thereby reducing the secondary injury of intracerebral hemorrhage.Peroxiredoxin 1?Prdx1?is a member of the Peroxiredoxin family and mainly involved in redox reactions in creature,and play a protective role by reducing oxidized proteins,oxygen free radicals,etc.Prdx1 has various functions that are involved in tumor invasion and metastasis;it also plays a very important role in inflammatory response and apoptosis.In addition,Prdx1 is an RNA-binding protein,however,as an RNA-binding protein,how Prdx1 participates in post-transcriptional regulation is unclear.Moreover,previous studies have shown that the expression of Prdx1 is significantly increased after intracerebral hemorrhage,and how the increased Prdx1 is involved in the secondary injury of intracerebral hemorrhage is largely unclear.To this end,we over-expressed Prdx1 by injecting adeno-associated virus in intracerebral hemorrhage rats,combined with RNA Immunoprecipitation and high-throughput sequencing in vitro to explore the effects and mechanisms of Prdx1 on the secondary injury of intracerebral hemorrhage.Method:Part?:Role of Prdx1 on secondary injury after intracerebral hemorrhageChanges of Prdx1 expression and cell localization in the perihematomal brain tissues after intracerebral hemorrhage.1.Detection of Prdx1 expression changes in the perihematomal brain tissues after intracerebral hemorrhage.The sham model and intracerebral hemorrhage model were established and sacrificed at3 d after intracerebral hemorrhage,the perihematomal brain tissue were used to RNA and protein extraction.The expression of Prdx1 mRNA level was detected by qRT-PCR,and the protein level of Prdx1 was detected by Western-blotting method.Rats were sacrificed at 3 d after intracerebral hemorrhage,the brain tissues were sectioned and the number of Prdx1-positive cells in the perihematomal area was detected by immunohistochemical staining.2.Detection cell localization of Prdx1 in the perihematomal area after intracerebral hemorrhage.The immunofluorescence staining was used to detect the cell localization of Prdx1 in neurons,astrocytes and microglia in the perihematomal area.To study the effect of Prdx1 on secondary injury of intracerebral hemorrhage.1.Prdx1 overexpressing adeno-associated virus or empty vector were injected into striatum of rats,and then the intracerebral hemorrhage models were established 3 weeks later,compared mortality in the sham group,WT intracerebral hemorrhage group,Prdx1overexpressing group and vector group.2.Sacrificed rats at 3 d after intracerebral hemorrhage in four groups,brain tissues were taken,and the wet weights of the ipsilateral and contralateral brain tissues in each group were recorded.The brain tissues were dried at 100?overnight,and the dry weights were calculated.statistic the dry weight and calculate the brain water content in each group.3.At 3 d after intracerebral hemorrhage,the neurological function of each group was evaluated using modified neurological severity score.4.Four groups of rats were sacrificed at 3 d after intracerebral hemorrhage,brain tissues were taken,and cut into coronal sections of 1 mm.Image-Pro Plus 5.0 was used to calculate the hematoma volume of each rat.5.The perihematomal brain tissues in four groups were used to extracted RNA and protein,and the expression of apoptosis-related proteins Bcl-2 and Bax were detected by Western blot.The inflammatory factor interleukin-6,interleukin-10,and tumor necrosis factor-?expression changes were detected by qRT-PCR.6.Four group of rats were sacrificed at 3 d after intracerebral hemorrhage.The brain tissues were sectioned,Nissl and Fluoro-Jade B?FJB?staining were used to detect the number of damage cell.Part?:Mechanisms of Prdx1 regulating secondary injury in intracerebral hemorrhage.1.Screen for RNAs that Prdx1 bind toHeLa cells were used to screen all RNAs that bound to Prdx1 by RNA Immunoprecipitation and high-throughput sequencing,and perform GO enrichment analysis on these RNAs.2.Screen Prdx1-dependent differentially expressed genesPrdx1 overexpressing plasmid was transfected in HeLa cells,and a HeLa cell line overexpressing Prdx1 was established.RNA high-throughput sequencing was used to screen out differentially expressed genes caused by Prdx1,and perform GO enrichment analysis on these differential genes.3.Verification of Prdx1 mechanism through in vivo and in vitro experimentsPrimary astrocytes were extracted from rat brain,and RNA immunoprecipitation combined with qRT-PCR was used to verify RIP-seq data.RNA was extracted from the brain tissue of Prdx1 overexpression group,empty vector group,WT intracerebral hemorrhage group and sham group,and using qRT-PCR to verify RNA-seq data.Results:Part?:1.Compared with the sham group,Prdx1 mRNA level?F=8.421,t=-19.474,**P<0.01 vs sham,n=3?and protein level?F=2.014,t=-3.432,**P<0.01 vs sham,n=3?in the perihematomal brain tissues were significantly increased,and the number of Prdx1positive cells in the perihematomal area were significantly increased compared with the sham group?100?m,F=1.225,t=-7.288,**P<0.01 vs sham,n=3?.2.Immunofluorescence staining showed that after intracerebral hemorrhage,elevated-Prdx1 in the striatum was mainly expressed in astrocytes and microglia,but not in neurons(25?m,F=10.964,t=-11.790,^##P<0.01 vs Prdx1/NeuN.F=12.000,t=-60.228,**P<0.01 vs Prdx1/NeuN,n=3).3.Overexpression of Prdx1 in rat brain tissue can significantly reduce mortality after intracerebral hemorrhage??2=14.310,df=3,*P<0.05 vs Vector,^#P<0.05 vs WT,n=20?,reduce neurological deficits(df=3,F=75.196,**P<0.01 versus Vector,^##P<0.01 versus WT,n=5),inhibit cerebral edema(df=3,F=9.324,*P<0.05 vs Vector,^##P<0.01 vs WT,n=6),and reduce hematoma volume?df=3,F=151.467,*P<0.05 vs Vector,^#P<0.05 vs WT,n=6?.4.The number of FJB-positive cells around the perihematomal area in the Prdx1overexpression group were significantly reduced compared with the WT intracerebral hemorrhage group and the empty vector group(50?m,df=3,F=435.050,**P<0.01 vs Vector;^##P<0.01 vs WT,n=3),while the positive cells of Nissl staining were significantly increased compared with the other two groups(50?m,df=3,F=174.206,**P<0.01 vs Vector;^##P<0.01 vs WT,n=3).5.After over expressing of Prdx1,the levels of inflammatory factors IL-6(df=3,F=27.046,*P<0.05 vs Vector;^##P<0.01 vs WT,n=3),IL-10(df=3,F=79.041,**P<0.01vs Vector,^##P<0.01 vs WT,n=3)and TNF-?mRNA?df=3,F=10.274,**P<0.01 versus Vector;^#P<0.05 versus WT,n=3?around the hematoma were lower than those in the WT intracerebral hemorrhage group and the empty vector group,and the protein levels of Bcl-2/Bax were significantly higher than those in the other two groups?df=3,F=32.759,*P<0.05 vs Vector;^#P<0.05 vs WT,n=3?.Part?:1.RIP-seq results showed that Prdx1 binds a total of 10466 RNAs,mainly in the CDS,5?UTR and 3?UTR of these RNAs.GO analysis results show that these RNAs are mainly concentrated in RNA/m RNA processing and splicing.2.RNA-seq data showed that after Prdx1 overexpression,a total of 863 genes were differentially expressed,of which 392 genes were down-regulated and 471 genes were up-regulated.GO analysis results show that these RNAs are mainly related to redox reactions,cytokine pathways,inflammatory responses,apoptosis and DNA repair.3.In vivo and in vitro results show that Prdx1 can bind to RNAs related to inflammatory response and apoptosis,such as ANGPTL4,GADD45A,THBS1,and cause differential expression of these RNAs,thereby producing the effect of inhibiting inflammatory response and apoptosis.Conclusion:The above results have shown that after intracerebral hemorrhage,Prdx1 expression in the perihematomal area is significantly increased,and the overexpressed Prdx1 is mainly concentrated in astrocytes and microglia.By overexpressing Prdx1 in vivo,we found that Prdx1 could significantly reduce the mortality of rats after intracerebral hemorrhage,improve neurological deficits,reduce cerebral edema and hematoma volume,inhibit degeneration of neurons,and alleviate inflammation and apoptosis after intracerebral hemorrhage.Prdx1 could combines the inflammation-and apoptosis-related RNAs,and change the stability of these RNAs,causing the differential expression of inflammation-and apoptosis-related RNAs,thereby producing anti-inflammation and apoptosis effects.