Study On The Association Between Changes In Gene Methylation Levels Induced By PM2.5 Exposure And Ischemic Stroke

Purpose:More and more evidence shows that PM2.5 exposure is significantly associated with the increased risk and severity of cerebrovascular diseases,especially ischemic stroke,but the relevant mechanism is still unclear.In this study,rats were exposed to PM2.5 and the MCAO model of rats was established to observe the effects of PM2.5 exposure on cerebral infarction volume,nerve function defect,inflammatory reaction,oxidative stress and blood lipid level in MCAO rats.Using the methods of RRBS and TBS methylation sequencing,we preliminarily explored the relationship between the changes of DNA methylation level of related genes induced by PM2.5 exposure and ischemic stroke,and provided relevant theoretical basis for further study of the mechanism of DNA methylation in the aggravation of cerebral ischemia injury by PM2.5 exposure.Method:In this study,male SD rats were randomly divided into five groups after being raised in the SPF level laboratory for one week:sham operation group(share group),normal saline treated MCAO model control group(tMCAO+NS group),low concentration PM2.5 exposed MCAO model group(tMCAO+Low PM2.5),medium concentration PM2.5 exposed MCAO model group(tMCAO+Mid PM2.5),high concentration PM2.5 exposed MCAO model group(tMCAO+High PM2.5).Nerve function defects were scored at 24h and 48h after PM2.5 poisoning and modeling,and then cardiac perfusion and blood,artery and brain tissue samples were taken.The volume of cerebral infarction in rats was measured by TTC method,the thickness of carotid and aortic intima was evaluated by HE staining method,the levels of TC,LDL and HDL were measured by automatic biochemical analyzer(AU 480),and the inflammatory cytokines IL-6,IL-8 and IL were measured by ELISA method-β、TNF-α、INF-γ The activity of SOD,an indicator of oxidative stress,was measured by WST-1 method.The differential methylation genes between tMCAO+NS group and tMCAO+High-PM2.5 group were detected by RRBS methylation sequencing method,and the differential methylation genes related to IS were screened by GO and KEGG enrichment analysis,and the methylation changes of the selected genes were verified by TBS methylation sequencing method.Finally,RT-PCR and WB methods were used to detect the mRNA and protein expression levels of PM-induced IS-related methylation change genes(MTHFR,FOXO1,LCAT,DAPK1,TRAF3)in the brain tissues of rats in each group.Result:1.Neurological function defect score and TTC staining results of brain tissue infarction area.Compared with tMCAO+NS group,there was no statistical difference in the neurological function defect score and cerebral infarction volume at 24h and 48h in tMCAO+Low-PM2.5 group;While tMCAO+Mid-PM2.5 group and tMCAO+High-PM2.5 group increased significantly,with statistical significance.2.HE staining of main and carotid artery pathological changes and blood lipid level detection results.The arterial tissue structure of rats in the same group and tMCAO+NS group had no obvious pathological damage,and the blood lipid level had no significant change.In the tMCAO+High PM2.5 group,there were slight atherosclerosis and venous fibrous thickening;The pathological damage of tMCAO+Low-PM2.5 group and tMCAO+Low-PM2.5 group was more slight than that of tMCAO+High-PM2.5 group,with smooth muscle cell deformation and a small amount of fibrous lesions.Compared with tMCAO+NS group,the levels of LDL-C and TG in tMCAO+Low-PM2.5 group,tMCAO+Mid-PM2.5 group and tMCAO+High-PM2.5 group increased,while the levels of HDL-C decreased.3.Test results of inflammatory cytokines and oxidative stress indicators.The levels of inflammatory factors in the same group were significantly lower than those in other groups,and the activity of SOD was significantly higher than that in other groups;Compared with tMCAO+NS group,the levels of IL-6,IL-8 and ILβ in tMCAO+Low-PM2.5 group was no significant statistical difference in the levels,but the levels of tMCAO+Mid-PM2.5 group and tMCAO+High-PM2.5 group were significantly higher;Compared with tMCAO+NS group,The level TNF-α、INF-γ of low,medium and high PM2.5 exposure groups showed a significant upward trend,and the activity of SOD showed a significant downward trend.4.RRBS and TBS methylation sequencing results.A total of 8873 differential methylation genes were detected between tMCAO+NS group and tMCAO+HighPM2.5 group,of which hypermethylation genes accounted for 55.19%and hypomethylation genes accounted for 44.81%.After enrichment and analysis of GO and KEGG databases,important IS-related hypermethylation genes MTHFR,FOXO1,LCAT,and hypomethylation genes DAPK1,TRAF3 were selected.TBS methylation sequencing confirmed this result.Compared with tMCAO+NS group,the methylation level of MTHFR,FOXO1,LCAT genes in low,medium and high PM2.5 exposure groups showed a significant upward trend,while the methylation level of DAPK1,TRAF3 genes showed a significant downward trend.5.Detection of MTHFR,FOXO1,LCAT,DAPK1,TRAF3 gene mRNA and protein expression level.The mRNA and protein expression levels of MTHFR,FOXO1 and LCAT genes in the share group were significantly higher than those in the other four groups,while the expression levels of DAPK1 and TRAF3 genes were significantly lower than those in the other four groups;Compared with tMCAO+NS group,the mRNA and protein expression levels of MTHFR,FOXO1 and LCAT genes in low,medium and high PM2.5 exposure groups decreased significantly,while the expression levels of DAPK1 and TRAF3 genes increased significantly.Conclusion:1.PM2.5 exposure can lead to a larger range of cerebral infarction and higher neurological deficit score in MCAO rats;More serious pathological damage of the intima-media of the main and carotid arteries;At the same time,it can induce changes in blood lipid level,inflammatory cascade reaction and oxidative stress reaction.2.PM2.5 exposure changes the DNA methylation pattern of rat brain tissue,and increases the methylation level of MTHFR,FOXO1 and LCAT genes,and decreases the mRNA and protein expression level;The methylation level of DAPK1 and TRAF3 genes decreased and the gene expression level increased.3.The IS-related genes induced by PM2.5 exposure:MTHFR,FOXO1,LCAT gene hypermethylation,DAPK1,TRAF3 gene hypomethylation,which may increase the damage of rat brain tissue and nerve element by changing the level of blood lipids,inducing the injury of arterial intima-media membrane,inflammatory cascade reaction,oxidative stress reaction,etc.4.The change of DNA methylation level of related genes induced by PM2.5 exposure is associated with ischemic stroke.