Role Of Methylation Of BDNF Gene Methylation In Benzo[a]pyrene-induced Cognitive Impairment And Its Intervention Of Aspirin In Mice

Objective:1.To explore the role of brain-derived neurotrophic factor(BDNF)gene methylation in benzo[a]pyrene(B[a]P)-induced cognitive impairment in mice;2.The interventional study of B[a]P-induced cognitive impairment in mice with low-dose aspirin.Methods:Part 1:Forty-eight adult male ICR mice were randomly divided into 4 groups according to their body weights,a control group and B[a]P-treated groups at three doses of 0.5 mg/kg(low-dose),2.0 mg/kg(mid-dose),and 10.0 mg/kg(high-dose).Mice were administered intraperitoneally with B[a]P at doses of 0.5,2.0,and 10.0 mg/kg body weight once every two days,continuously for 30 times of administration.At the end of 30 th administration,mice were tested spatial learning and memory using morris water maze test,the ability of the space exploration of the mice was detected by the open field test,and the pathological changes of the cerebral cortex of the mice were observed by the hematoxylin-eosin staining(HE staining).Transmission electron microscopy was used to observe the change of synaptic connections in hippocampus of mice.Flow cytometry(Annexin V-FITC/PI method)was used to detect the neuronal apoptosis in the cerebral cortex of each exposed group.Real-time fluorescence quantitative nucleic acid amplification(QPCR)detection of BDNF,DNMT1,DNMT3 a and DNMT3 b gene expression levels in mice cerebral cortex;enzyme-linked immunosorbent assay(ELISA)detection of BDNF,DNMT1,DNMT3 a and DNMT3 b protein expression levels;the methylation-specific PCR(MSP)method was used to detect the gene methylation level of exon IV promoter region of BDNF gene.Part 2: Thirty-six adult SPF grade male ICR mice were randomly divided into 3 groups according to body weight,12 in each group,which were peanut oil control group,10.0 mg/kg(high-dose)and the aspirin intervention group(10.0 mg/kg B[a]P+10.0 mg/kg aspirin).The high-dose treatment group and aspirin intervention group were intraperitoneally injected with 10.0 mg/kg B[a]P every other day,the control group was injected with equal volume peanut oil was continuously intoxicated 30 times.The aspirin intervention group drank drinking water containing aspirin(0.05 mg/m L)every day.After the end of the exposure,the test was performed according to the part 1.Results:Part 1:Morris water maze test showed that the mice's average escape latency times were increased with the dose of B[a]P,which was significantly higher in the high-dose group than in the control group(P<0.05).The frequency of crossing platform and the swimming distance in the target quadrant were gradually decreased with the B[a]P doses,which were both significantly increased in the high-dose group as compared to the solvent control group(P<0.05).The open field test results showed that: the total activity of mice in the high-dose group,activity distance in the surrounding area,and activity distance in the central area were all significantly lower than those in other groups as compared to the solvent control group(P<0.05).The number of standing times and stool grains in the high-dose group was lower than other groups,but the difference was not statistically significant(P>0.05).The results of HE staining showed that with the increase in the dose,various degrees of nerve cell damage occurred in mice in each dose group,especially in the high-dose group.A large number of edema nerves appeared in the cerebral cortex of the high-dose group.A large number of lattice cells can be seen;transmission electron microscopy results show that with the increase of the dose,nerve cell nucleus,mitochondrial damage gradually increased,mainly for the condensation of chromatin condensation,nuclear membrane rupture,mitochondrial swelling and reduced the number.The destruction of mitochondrial cristae gradually aggravated,and the dendritic dissolution and vacuolar degeneration gradually increased,and the high-dose group suffered the most serious damage.The QPCR and ELISA results of BDNF showed that as the dose increased,the expression of BDNF promoter mRNA and BDNF mRNA and protein in the cerebral cortex of mice with B[a]P poisoning dose gradually decreased,compared with the solvent control group.The difference was statistically significant(P<0.05).QPCR and ELISA results of DNA methyltransferases DNMT1,DNMT3 a,and DNMT3 b showed that as the dose increased,mRNA and protein expression of DNMT1,DNMT3 a,and DNMT3 b in cerebral cortex of mice exposed to B[a]P in each dose.With the doses gradually increased,with a significant dose-dependent,compared with the control group,the difference was statistically significant(P<0.05).The results of MSP method in exon IV promoter region of BDNF gene showed that methylation levels of exon IV promoter region of BDNF gene in cerebral cortex of B[a]P poisoned mice at each dose increased with increasing dose gradually increased,compared with the solvent control group,the difference was statistically significant(P<0.05).Part 2: Compared with the B[a]P poisoned group(10.0 mg/kg B[a]P),the spatial learning and memory abilities of the aspirin-treated mice were significantly improved,showing a shortened latency to reach the platform and a total of the target quadrants.Both the number of times and swimming distance in the target quadrant increased,and the difference was statistically significant(P<0.05).However,there was no significant improvement in self-issued ability(P>0.05).HE staining showed that the degree of edema was significantly lower than that of the high-dose group.The number of edema cells was also significantly less than that of B[a]P-infected group alone;transmission electron microscopy revealed that nuclear cells in the aspirin-treated group were less nucleus-distorted and chromatin-rimmed than those in the B[a]P-poisoned group alone.The nuclear membrane was intact,the mitochondrial edema was mild,the gingival rupture was mild,and the dendritic dissolution was improved.The flow cytometry results showed that the early apoptosis mice of the cells in the mice cerebral cortex was significantly decreased,and the difference was statistically significant(P<0.05).Compared with pure B[a]P exposure group,mRNA expression of BDNF promoter IV region and BDNF total mRNA and protein expression in mice cerebral cortex increased significantly(P<0.05),mRNA and protein of DNMT1,DNMT3 a and DNMT3 b.Expressed Decreased(P<0.05),cortex of BDNF gene exon IV promoter methylation level of the promoter region significantly lower than B[a]P exposure groups was statistically significant(P<0.05)difference.Conclusion:1.Intraperitoneal injection of B[a]P can cause a decline in cognitive function and self-issuing ability in mice,which may be associated with increased activity of cortical DNMTs and methylation of BDNF exon IV promoter region,BDNF exon IV promoter mRNA,BDNF total mRNA and BDNF protein expression levels were related to the decrease.2.Aspirin intervention has a protective effect on B[a]P-induced neuronal damage and cognitive impairment in mice.