The Role Of DNA Methylation In The Damage Of Hippocampal Neurons Induced By Benzo[a]Pyrene And The Intervention Of 5-Aza-2’-Deoxycytidine

Objective:1.To study the role of DNA methylation in hippocampal neuron injury induced by benzo[a]pyrene(B[a]P)in rats,and to provide a new experimental basis for the study.2.To investigate the effect of 5-aza-2’-deoxycytidine(5-Aza-cd R)on B[a]P-induced rat neuronal injury and to provide new molecular targets and measures for preventing the neurotoxicity of B[a]P.Methods:Part Ⅰ: Hippocampal neurons were extracted from SD rats born within 24 h and cultured with Neurobasal medium and B-27 serum-free medium.The morphology of well-grown neuron cells was observed by inverted microscope and identified by immunofluorescence method.The concentration gradient of B[a]P pretest was set as 0,10,20,40,80,100 μmol/L.Hippocampal neurons grew and matured and CCK-8 method was used to detect cell viability and screen subsequent B[a]P dose 24 h after exposure.Experimental set up the control group(0 μmol/L)and B [a] P infected group(10 μmol/L,20 μmol/L,40 μmol/L),which inverted microscope to observe the shape variation of hippocampal neurons.The length of cell process and the number of branches were observed by immunofluorescence method.The protein expression levels of DNA methyltransferase DNMT1,DNMT3 A and DNMT3 B were detected by Western Blot.The m RNA relative expression levels of DNA methyltransferase DNMT1,DNMT3 A and DNMT3 B were detected by q RT-PCR.5-methylcytosine(5-mc)immunofluorescence technique was used to determine the total methylation level of genome in cells.Hoechst33342 staining was used to observe cell apoptosis.SPSS 26.0 software was used to statistical analysis.The results using mean ± standard deviation(? x ± s)and the comparison between the groups using single factor analysis of variance and comparing two further use of LSD-t method.Alpha=0.05 is the inspection level.Part Ⅱ: Primary hippocampal neuron cells were cultured in vitro and treated with0,2.5,10,20,40 μmol/L 5-Aza-cd R for 24 h.The cell viability was detected by CCK-8method.Screening 10 and 20 μmol/L 5-Aza-cd R for follow-up detection.Hippocampal neurons were divided into 4 groups: control group,20 μmol/LB[a]P exposed group,20μmol/L B[a]P + 10 μmol/L 5-Aza-cd R intervention group,20 μmol/L B[a]P + 20 μmol/L5-Aza-cd R intervention group,The intervention concentration of 5-Aza-cd R was determined by CCK-8 method and cell viability was calculated.After the intervention,the test was carried out according to the above experimental methods.Results:Part Ⅰ: The primary hippocampal neurons were cultured using serum-free culture technique and the hippocampal neurons newly inoculated on the culture plate were small in volume,circular or polygonal with good light transmission,no protrusion,suspension,and uniform distribution.After 1 day,the cells had strong stereoscopic sense and refraction and halo was obviously around the cells.Cell shape can be round,fusiform or irregular shape,full cell body,protrusion growth fast and the number increases.After 4-5days,the typical morphology of neurons could be observed with the cell body in the shape of a spindle or triangle and the protrusions increased significantly,gradually forming a network of interconnected nerve fibers.The cultured cells were identified as hippocampal neurons by immunofluorescence method with purity>95%,which met the criteria.With cell survival rate>70% as the standard,B[a]P intervention concentrations were as follows: control group(0 μmol/L),low dose group(10 μmol/L),medium dose group(20 μmol/L)and high dose group(40 μmol/L)and the difference was statistically significant(P<0.05).With the increase of B[a]P dose,cell viability decreased,showing an obvious dose-effect relationship.In the control group,the hippocampal neuron cell body was clear,the refraction was strong,the axon shape was normal and the structure was complete.With the increase of B[a]P dose,the number of neuronal cell bodies decreased,the axon shrank and denaturated and the length and number of branches of hippocampal neuronal processes decreased significantly in each exposed group(P<0.05).Some cells shed and died,indicating that the function and morphology of neurons were inhibited after B[a]P induction.Compared with the control group,the protein expression levels of DNA methyltransferase DNMT1,DNMT3 A and DNMT3 B increased with the increase of B[a]P exposure dose(P<0.05),the relative expression levels of DNMT1,DNMT3 A and DNMT3 B m RNA increased(P<0.05),showed significant dose.The total methylation level of hippocampal neuron genome was increased in different B[a]P groups(P<0.05).When B[a]P was exposed for 24 h,the neuronal apoptosis rate of each dose group was significantly increased and the rising trend showed a significant doseeffect relationship(P<0.05).Under the fluorescence microscope,the hippocampal neurons in the control group showed greyish-blue fluorescence and the fluorescence intensity of apoptotic neurons in the B[a]P group was significantly enhanced.The higher the concentration of B[a]P and the brighter the fluorescence of the nucleus and the changes such as nuclear fragmentation and nuclear concentration were observed.Part Ⅱ: Through the combined effect of B[a]P and 5-Aza-cd R on hippocampal neurons,the cell viability of B[a]P+5-Aza-cd R intervention group was significantly increased(P<0.05),and 10 μmol/L 5-Aza-cd R was selected as the subsequent intervention concentration.In the control group,the morphology of neurons was normal and the structure was complete.In the B[a]P group,fewer cells were observed,neuronal processes shrank and interprocess connections were broken and in the B[a]P + 5-Azacd R group,a few atrophic cells were observed.Compared with B[a]P exposed group,the length of cell process and the number of cell branches in B[a]P + 5-Aza-cd R intervention group were increased(P<0.05),indicating that the cell function and morphology of neurons were improved after 5-Aza-cd R intervention.Compared with B[a]P + 5-Azacd R group,the protein levels of DNA methyltransferase DNMT1,DNMT3 A and DNMT3 B were decreased in B[a]P + 5-Aza-cd R intervention group and the relative m RNA expression levels of DNMT1,DNMT3 A and DNMT3 B were decreased,with statistical significance(P<0.05).The fluorescence intensity of 5-mc was decreased and the total methylation level was decreased(P<0.01).In the B[a]P group,nucleus staining,nucleus fragmentation and the number of apoptotic cells were observed.a small number of apoptotic cells could be observed in B[a]P + 5-Aza-cd R intervention group.Compared with B[a]P exposed group,the apoptosis rate of hippocampal neurons in B[a]P + 5-Azacd R intervention group was significantly decreased(P<0.01)..Conclusions:1.Exposure to B[a]P can reduce the viability of hippocampal neurons in SD rats,which change the normal morphological structure and induce apoptosis and increase the expression levels of DNMTs protein and DNMTs m RNA and increase the total methylation level of cell genome.These results suggest that DNA methylation may play an important role in neuron damage caused by B[a]P.2.5-Aza-cd R has a protective effect on B[a]P-induced hippocampal neuronal cell damage.The possible mechanism is that 5-Aza-cd R can reduce the expression level of DNA methyltransferase protein and gene and reduce the total DNA methylation level of cell genome.