Preliminary Study On The Role Of Global Genome DNA Methylation Modification In Brain Injury Induced By Hyperbilirubinemia In Mice

Objective In this study,we established a mouse model of hyperbilirubinemia to observe the changes in the global genome methylation and expression levels of related methyltransferase genes in nerve cells of mice brain tissue so as to initially explore the role of global genome methylation in hyperbilirubinemia-induced nerve injury and provide new prevention strategies and methods for hyperbilirubinemia-induced brain nerve injury.Methods Forty-eight 7-day-old newborn 129 s mice were randomly divided into four groups including control group(normal saline),low-dose group(25 mg/kg),medium-dose group(50 mg/kg),and high-dose group(100 mg/kg).There were 12 animals in each group.The method of intraperitoneal injection was used to inject Phenylhydrazine hydrochloride solution according to grouped doses for exposure.The mice were sacrificed 24 hours after exposure,and blood and brain tissues were collected.1.HE stained slices of brain tissue were prepared and the pathological changes of brain tissue were observed under light microscope.2.Spectrophotometry and microplate reader were used to detect total bilirubin levels in blood samples and brain tissue,catalase(CAT),superoxide dismutase(SOD),malondialdehyde(MDA),glutathione peroxidation enzyme activity(GSH-Px)in brain tissues.Enzyme-linked immunosorbent assay was used to determine the concentrations of inflammatory cytokines interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),neuron specific enolase(NSE)and S-100,calcium-binding protein β(S-100β).3.Immunofluorescence method was used to detect the global genome methylation in cerebral cortical nerve tissue.4.The mRNA expressions of Dnmt1,Dnmt3 a and Dnmt3 b genes in cerebral cortical nerve tissue were conducted by qRT-PCR.5.The protein expression levels of DNA methyltransferase genes(Dnmt1,Dnmt3 a andDnmt3b)in cerebral cortical nerve tissue were conducted by Western Blot.Results 1.The model of hemolytic hyperbilirubinemia was successfully established by using phenylhydrazine hydrochloride solution.The evaluation indicators are:(1)Pathological section:In the control group,the brain pyramidal cell structure of the mice was normal,and the cell layers in the hippocampal CA1 area were arranged neatly.In the low,medium,and high dose groups,the pyramidal cell nucleus and cytoplasm of the brain tissue were lightly stained,and the structure was missing.The number of cells in CA1 area decreased,the arrangement was scattered,and the high-dose group was particularly prominent.(2)Oxidative stress factors:The results showed the SOD,CAT,and GSH-Px activity in the high-dose group were significantly reduced compared with the control group(P < 0.05).The level of malondialdehyde(MDA)in the brain tissue of the high-dose group increased compared with the control group(P < 0.05);(3)Inflammatory factors and markers of nervous system damage:The concentrations of IL-1β,TNF-α,NSE and S-100β in the brain tissue of the high-dose group increased significantly compared with the control group(P < 0.05);(4)the total bilirubin(TBIL)in the brain and plasma showed an increasing trend.Compared with the control group,the blood and brain TBIL in the high-dose group increased significantly(P < 0.05).2.Global genome DNA methylation modification level and related DNA methyltransferase changes:(1)Compared with the control group,the DNA methylation level of cortical neurons in the low,middle,and high groups was increased;the high-dose group had the highest DNA methylation level(P < 0.05).(2)The mRNA levels of the three types of DNMTs in the low,medium and high dose groups increased compared with the control group.Among them,Dnmt3 b was significantly expressed in the medium and high dose groups(P < 0.05),Dnmt1 and Dnmt3 a were significantly expressed in the high dose group(P < 0.05);the protein expression levels of Dnmt1,Dnmt3 a,and Dnmt3 b all increased to varying degrees,of which Dnmt1,Dnmt3 a and Dnmt3 b were significantly increased in the high-dose group(P < 0.05).Conclusion 1.Intraperitoneal injection of phenylhydrazine hydrochloride solution at a certain concentration can successfully establish a mouse model of hyperbilirubinemia.The main manifestations are: pathological changes of mouse brain tissue cells,oxidative stress,changes in neuronal specific damage indicators and inflammatory response.2.The Global genome methylation of brain tissue cells in mice with hyperbilirubinemia increases,Three Methyltransferase(Dnmt1,Dnmt3 a,and Dnmt3b)gene expression levels were significantly increased at the mRNA and protein levels,it is suggested that the increase in the global genome methylation may play a role in the mechanism of neuronal damage caused by hyperbilirubinemia.