The Role And Mechanism Of Histone Modifications In Aging-induced Aggravation Of Intracerebral Hemorrhage

Background and objectsIntracerebral hemorrhage(ICH)refers to intracranial hemorrhage caused by vascular rupture in cerebral parenchyma,including traumatic and spontaneous ICH.Among them,spontaneous ICH is the second most common type of stroke,with high morbidity,high mortality and high disability rate.Study found that aging is an important promoting factor for the occurrence and development of spontaneous ICH,but its role and mechanism in ICH induced cerebral injury are still unclear.Aging is characterized by a variety of hallmarks.among them,epigenetic abnormalities can regulate gene transcription without changing gene sequences,attracting much attention in aging research.The modification of histone methylation and acetylation is one of the mainly regulatory mechanisms of epigenetics.Studies have shown that H3K27me3(inhibition),H3K4me3(activation)and H3K9ac(activation)modifications are not only important markers of brain aging,but also closely related to the progression of neurodegenerative diseases,suggesting that the a bove three histone modifications may play an important role in aging-induced aggravation of ICH injury.Recent studies have found that young plasma,taken from healthy young populations,is an effective treatment for aging and aging related diseases.Young plasma is rich in a variety of secreted proteins,hormones,metabolites or lipids and other young factors.They can affect the aging process of various tissues and organs through local and long-range action after secreted into blood.Moreover,studies have discovered that young plasma can delay aging,AD and ischemic stroke in the elderly,suggesting that young plasma may be able to relieve the ICH injury caused by aging.However,the effects and mechanisms of young plasma on aging-induced aggravation of ICH injury are still not clear.Above all,this study focused on the role and mechanism of aging in ICH induced brain injury.To achieve this,we adopted ICH model with different ages to evaluate the effects of H3K27me3,H3K4me3 and H3K9 ac histone modification in aging ICH by Ch IP-seq,RNA-seq techniques.In addition,the role of young plasma in the treatment of aging-induced aggravation of ICH injury,its relationship with histone modifications and the possible key factors were also clarified.This study was expected to reveal the internal molecular mechanism of aging aggravating ICH induced brain injury,and lay ed a solid theoretical and experimental basis for the treatment of elderly patients with ICH.Materials and methods1.Construction of ICH model: ICH model was established in rats aged 13 months(middle age)and 22 months(old age).Mortality at 7 days after ICH was recorded in both groups,and brain tissue surrounding the hematoma was collected at 3 days after ICH.All specimens were frozen at-80? or liquid nitrogen immediately after collection.2.Distribution of three histone modifications after ICH: WB and immunohistochemistry were used to detect the changes and cell localization of three histone modifications at the 3 day after ICH with different ages.The differentially modified genes of three histones in the whole genome were detected by Ch IP-seq.3.Transcriptome detection of brain tissue after ICH: RNA-seq was used to detect transcriptome changes at 13 and 22 months old ICH rats,and correlation analysis was performed with Ch IP-seq,and q PCR was performed to verify the results.4.Young plasma intervention: The construction of parabiosis model: young-old,old-old.The parabiosis model was removed after 50 days,and the ICH model was constructed in elderly rats to detect brain injury and histone modifications of H3K27me3 and H3K9ac.At the same time,plasma was collected from young and old rats and infused through tail vein 30 min after ICH to observe the changes of brain injury in old rats.5.Detection of plasma compositions: The plasma of young,middle-aged and elderly healthy people were collected,and the protein changes in plasma were detected by LC-MS/MS.The "youth factor" was screened and verified by ELISA and q PCR in plasma and brain tissues of ICH rats with different ages.6.Detection of brain injury: The mortality rate,survival curve and modified neurological defense scores(m NDS)were measured within 7 days after ICH.Brain water content(BWC)on the 3 day after ICH was assessed and the neuronal damage around the hematoma was detected by TUNEL,FJB and Nissl staining on 3 day after ICH.7.Others: Interferon-?(IFN-?)antibody or insulin-like growth factor(IGF-1)protein was applied to ICH rats to detect the key effector pathway of young plasma in the treatment of aging-induced aggravation of ICH.Results1.Aging could aggravate brain damage caused by ICH: Compared with 13 months old(middle age),the 7-day mortality and m NDS of 22 month old(old age)ICH rats were higher,and the number of BWC and TUNEL,FJB,Nissl staining and other neuronal damage on the 3rd day of ICH were significantly increased.BWC and TUNEL and FJB positive injured neurons at 3 day of ICH were significantly increased.2.H3K27me3 and H3K9 ac histone modifications were redistributed in the genomes of aging rats with ICH : The levels of H3K4Me3,H3K27Me3 and H3K9 Ac changed after ICH.Among them,H3K27Me3 was significantly decreased in 13-and22-month-old groups.The modification level of H3K4ME3 showed a decreasing trend in the two groups,but the difference was not statistically significant.H3K9 AC modification was significantly increased only in 22 months old ICH rats.The results of Ch IP-Seq analysis indicated that the three histone modifications were mainly distributed in the TSS region of the genome,suggesting that they were mainly involved in the regulation of gene transcription.Further GO analysis revealed that the three histone modifications involved in the regulation of distinct molecular pathways at different age stages.3.H3K27me3 and H3K9 ac histone modification redistribution leaded to the upregulation of inflammatory and immune response related factors after ICH in the elderly: RNA-seq suggested that IFN-?,oxylipid-related enzymes and other factors related to inflammation and immune response could be up-regulated after ICH.However,the expression levels of these molecules were different between 13 months and 22 months old ICH rats,which might be caused by brain aging.Further correlation analysis with Ch IP-seq showed that the differentially modified genes by H3K27me3 and H3K9 ac histones were substantially coincident with the upregulated expressed genes by RNA-seq in the22-month-old rats with ICH.The corresponding genes of H3K4me3 and H3K9 ac were dominant in rats with ICH at 13 months of age.GO analysis showed that these overlapping genes were mainly involved in inflammation and immune response after ICH.4.The application of young plasma could alleviate brain injury caused by aging-associated ICH,and IGF-1 was the possible important effector: Using the parallel model of young and old mice,it was found that the parallel model of young mice could significantly reduce the mortality,m NDS and BWC of the aged rats with ICH.Meanwhile,TUNEL,FJB and Nissl staining indicated that the number of damaged or apoptotic neurons around the hematoma was significantly reduced in the parallel group of young mice.Through caudal vein infusion of young plasma,it was found that the use of young plasma could also reduce the mortality,m NDS,BWC and neuronal damage of the aged rats with ICH.All these results suggested that the application of young plasma could reduce aging-associated brain injury induced by ICH.LC-MS/MS analysis of the changes in human plasma at different ages showed that plasma IGF-1 decreased significantly with age.It was found that IGF-1 decreased with the increased age,and aging could inhibit the expression of IGF-1 after ICH.The infusion of young plasma could increase the IGF-1 content in the brain tissue of ICH.Further research found that these increased IGF-1 was not from the expression of the brain tissue itself,but came from the IGF-1 in young plasma.Further,IGF-1 recombinant protein was infused into the aged ICH rats,and it was found that IGF-1 could significantly reduce aging-associated ICH injury.5.H3K27me3 and H3K9 ac histone modifications were involved in the regulation of inflammation and immune response in young plasma in aging-induced aggravation of ICH injury: WB and histochemistry suggested that the treatment of young plasma could reverse the distribution of H3K27me3 and H3K9 Ac histone modifications in the elderly ICH to some extent.Ch IP-seq results showed that IFN-?,Lrg1 and other inflammatory and immune molecules modified by H3K27me3 or H3K9 ac histones could be altered by young plasma,thereby inhibiting their transcriptional expression.Further,it was found that the mortality,m NDS,BWC and neuronal damage of the elderly ICH rats were significantly decreased after the application of IFN-? antibody,suggesting that the inhibition of IFN-?might be an important way for young plasma to cure the aging-induced aggravation of ICH injury.ConclusionsAs an important risk factor for ICH,aging can significantly aggravate ICH induced brain injury.The redistribution of H3K27me3,H3K4me3 and H3K9 ac histone modifications plays an important role in aging-induced aggravation of ICH injury.Among them,aging mainly mediates the reduction of H3K27me3 and the increase of H3K9 ac in ICH,and the redistribution of H3K27me3 and H3K9 ac modifications can promote the up-regulation of the expression of factors related to inflammation and immune response after ICH.Young plasma can reverse the redistribution of H3K27me3 and H3K9 Ac histone modifications in elderly ICH to a certain extent,and thus inhibit the expression of pro-inflammatory factors such as IFN-?,so as to reduce the brain injury of aging-associated ICH.