Potential Mechanism Study Of Benzo[a]Pyrene-downregulaated MVP And Benzo[a]Pyrene-induced Neurotoxicity

Objective: To study the effect of major vault protein(MVP)expression on the physiological functions of neurons,and to investigate the possible reasons for B[a]P to inhibit MVP expression.Then,to investigate the possible reasons for MVP protein inhibition from the perspective of DNA methylation.It also provides a new idea for the mechanism of B[a]P-induced nervous system damage in terms of DNA methylation.Methods: PC12 cells were first transfected with MVP-silencing virus and over-expressing plasmid to construct MVP over-expression and silencing models,and verified by Western blot and Real-time PCR.Subsequently,CCK8 experiments and Hoechst apoptotic staining were used to explore the role of MVP in PC12 cell viability and apoptosis.The same experimental method was used to test the effect of B[a]P exposure on the viability and apoptosis of PC12 cells after MVP silencing and overexpression plasmids.Twenty male 3-week-old male SD rats live in a lab for one week before B[a]P exposure,and then randomly divided into a control group and a B[a]P-exposed group(2.0mg/kg)for 7-week intragastric administration.After the exposure,Morris water maze was used to determine the spatial memory and learning of rats,followed by HE staining and RBSS sequencing.The sequencing results were analyzed by GO and KEGG.The mRNA expression of Tnr gene and Pla2g2 a gene was detected by Real-time PCR.Result:1.The overexpression and silencing model of PC12 cells was successfully constructed.Compared with the empty plasmid group,the relative expression of MVP protein and mRNA increased in PC12 cells transfected with the overexpressing plasmid by WB and Real-time PCR,and the difference was statistically significant(P <0.05).Compared with the empty virus group,the relative expression of MVP protein and mRNA were reduced in PC12 cells transfected with MVP-silent virus,and the difference was statistically significant(P <0.05).2.Overexpression and silencing of MVP had no effect on the physiological functions of PC12 cell viability and apoptosis,and there was no statistical difference in positive cell counts between groups in each experiment(P <0.05).3.PC12 cells exposed to B[a]P after MVP overexpression and silencing had no change in physiological functions such as viability and apoptosis,and there was no statistical difference in positive cell counts between groups in each experiment(P <0.05).4.Morris water maze results showed that B[a]P exposure impairing rats spatial learning and memory.5 HE staining tissue morphology observation hippocampal tissue found that the exposure of B [a] P caused the disorder of the arrangement of granule cells and pyramidal cells in the DG and CA1 areas of the hippocampus.Both of these cells showed even red staining and atrophy.6.DNA methylation sequencing showed 32 differentially methylated regions and no DNA methylation change was found in MVP gene sequence.GO analysis enriched 871 GO entries.KEGG analysis yielded 52 pathways,of which 145 GO entries and 11 pathways were significantly enriched(P < 0.05).Compared with the control group,the relative expression of Tnr was reduced and the relative expression of Pla2g2 a was increased in the B[a]Ptreated group.Conclusion:1.MVP is not a key functional protein for proliferation and apoptosis in PC12 cells exposed to B[a]P.2.In vivo model of rats exposed to B[a]P,DNA methylation did not inhibit transcription of the MVP.The down-regulation MVP may be a result due to the failure of neuronal homeostasis after B[a]P exposure.3.The genes and their related pathways changed by DNA methylation might be potential mechanisms of B[a]P neurotoxicity.