The Role Of The DNA Methylation In Bilirubin-induced Neurotoxicity In Cortical Neurons Of Rats In Vitro

Objective To explore the role of DNA methylation in bilirubin-induced neurotoxicity in vitro so as to provide a theoretical basis for the development of small molecule "target drug" aiming to DNA methylation as a target,and to provide new ideas for the prevention and treatment for kernicterus.Methods(1)Establishment of primary nerve cell culture system:SD rats were sacrificed within 24 hours after birth,and cerebral cortical cells were extracted and incubated in Neurobasal serum-free medium on culture plates precoated with polylysine for 72 hours in 37℃,5%CO2 incubator.The immumofluorescence method of NSE was used to identify nerve cells.(2)The experimental grouping and bilirubin exposure:wells in which nerve cells were incubated were divided into four groups:control group(0.0μmol/L),low dose group(25μmol/L),middle dose group(50μmol/L)and high dose group(100μmol/L).The bilirubin in Nurobasal solution was added to the medium under dark conditions and made the final concentration to the given concentration,and the same amount of Neurobasal solution was added to the control group.The cells were harvested at 24hours,and some endpoints were subsequently detected.(3)Cell viability was determined by MTT assay.(4)Apoptosis of neurons was measured by Hoechst 33342 fluorescent staining.(5)Immunofluorescence assay was used to detect the methylation level of whole genome DNA by taking Mouse anti-5mc antibody as a primary antibody,FITC as a marker of sheep anti-mouse IgG as secondary antibody.(6)The expression of DNMTs protein were detected by Western blot.(7)Determination of mRNA expression of DNA methyltransferase(DNMTs):The mRNA levels of DNMT1,DNMT3A and DNMT3B in cultured neurons were detected by real-time fluorescence quantitative PCR.(8)Statistical analysis:4-8 parallel samples were set for each group.The results were expressed as (?)±S.The significant difference among different dose groups and between two groups were analyzed by ANOVA and SNK-q test,respectively.Results(1)In vitro model of hyperbilirubinemia was established by using serum-free primary culture technique.The density of cells was moderate,the morphology was good and the growth was vigorous.The cultured cells were identified as neurons.(2)Compared with the control group,the cell viability in the middle and high exposed group decreased significantly with an obvious dose-effect relationship(P<0.001).(3)Immunofluorescence assay showed that the morphological changes of nerve cell nucleus pycnosis and fracture were observed in the bilirubin exposed group.With the increase of the dose,the apoptosis rate increased gradually,showing obvious dose-effect(P<0.001).(4)Bilirubin could lead to elevation of whole genomic DNA methylation of neurol cells.With the increase of bilirubin dose,the gray value of cell staining increased gradually,indicating that the level of methylation of whole genome DNA gradually increased,and a significant difference between each bilirubin exposed group and control group was observed(P<0.05).The methylation level in 50μmol/L group was the highest,which was about twice as high as that of the control group.The methylation level of the 100μmol/L group increase slightly but significantly compared with the control group.(5)Western-blot results showed that the protein expression of three enzymes elevated to the some extent.The expression level of DNMT3A significantly imcreased in 25μmol/L and 50μmol/L bilirubin dosage groups(P<0.05),and the expression level of DNMT3B in 50μmol/L was significantly higher than that of the control group(P<0.05).The expression of DNMT1 protein was significantly higher in 20μmol/L,50μmol/L and 100μmol/L groups than that of control group(P<0.05).In the 100μmol/L dose group,the induction of bilirubin on the expression of three enzyme proteins was weakened to some extent.(6)Real-time quantitative PCR results showed that the transcription level of several DNA methyltransferase genes showed an increasing trend with the increase of bilirubin exposure(P<0.05).In the 100μmol/L group,mRNA levels of DNMT3A and DNMT3B increased obviously(P<0.05).The mRNA level of DNMT1 significantly elevated both in the 50μmol/L and the 100μmol/L groups compared with the control group.Conclusion Under this experimental condition,bilirubin exposure induces the expression of DNA methyltransferase genes in the cerebral cortex cells,and leads to elevation of the level of DNA methylation in global genome in vitro.The results suggested that DNA methylation modifation plays an important role in bilirubin-induced neuronal damage.