Protective Effect Of Folic Acid On Benzo[a]pyrene Induced Cardiotoxicity

Objectives:Studies have shown that exposure to benzo[a]pyrene is associated with the development of cardiovascular diseases.Previous results have shown that folic acid has a protective effect against cardiotoxicity caused by a variety of environmental chemicals,but the way it works is still a mystery.In this study,we aim to investigate the molecular mechanism by which folic acid protects against BaP-induced cardiotoxicity by antagonizing the overactivation of the aryl hydrocarbon receptor signaling pathway.Methods:Rat cardiomyocyte H9C2 was selected as the study model and cultured in a proliferation system,and the cytotoxicity was detected by CCK-8 to determine the exposure dose of BaP.Cells were treated with BaP at a concentration of 10μM.Cell proliferation and cell area changes were detected by cell counting and phalloidin staining.mRNA expression levels of cardiac hypertrophy marker genes were detected by qPCR.To determine the levels of AhR target genes and the location of AhR in the cytoplasm and nucleus,qPCR,Western blot,and immunofluorescence were used.Mitochondrial morphology was observed by transmission electron microscopy and mitotracker staining,and the overall levels of ROS,mtROS and ATP were detected.At the same time,the transcript of genes involved in mitochondrial fission/fusion and REDOX was examined using qPCR.DNA was extracted to detect the changes of mtDNA copy number.The expression changes of mitochondrial genome and nuclear genome after exposure to BaP were detected by transcriptome sequencing and qPCR.Mitochondrial proteins were isolated and examined by Western blot and immunofluorescence to detect co-localization of AhR with mitochondria.The global 5mC content was detected by immunofluorescence and dot blot,and the mRNA expression of DNA methylation-related enzymes was detected by qPCR.After the treatment of folic acid,the indexes were tested again as above,and the binding site of FA to AhR was predicted by molecular structure simulation.Results:1.FA reversed BaP-induced cardiac hypertrophy.BaP exposure at a concentration of 10μM had no significant effect on cell viability and proliferation,and 10μM was chosen as the effect concentration in the next experiments.BaP exposure for 48h resulted in an increase in cell area(150.56%±8.94%,P<0.001),and the mRNA expression levels of the cardiac hypertrophy markers Bnp and Acta2 were significantly upregulated,and FA could alleviate the increase in cell area caused by BaP.2.BaP affected mitochondrial quality control.Transmission electron microscopy showed that BaP treatment resulted in swelling of mitochondria and disappearance of cristae,accompanied by morphological changes from filamentous to punctate.BaP upregulated the expression of mitochondrial fission/fusion-related genes such as Drpl and Opal and stimulated an increase in mtDNA copy number(158%±7.24%,P<0.001).BaP treatment resulted in a decrease in cellular ATP production level(68.67%±6.91%,P<0.05),increased levels of ROS and mtROS,and abnormal mRNA expression of REDOX-related genes such as Sodl,Sod3,Gpx1,Cat,and Nrf2.3.BaP altered the transcriptional pattern of mitochondrial and nuclear genomes.BaP increased the expression of mitochondrial encoding genes mt-Ndl and mt-Co1.For nuclear coding genes,120 genes were downregulated and 281 genes were upregulated(Fold change>2)after BaP treatment.The down-regulated genes were primarily abundant in the cell cycle and DNA replication signaling pathways,according to KEGG pathway enrichment analysis.The up-regulated genes were mainly enriched in arrhythmogenic right ventricular cardiomyopathy,dilated cardiomyopathy and metabolism of xenobiotics by cytochrome P450.4.FA inhibited the activation of AhR by BaP.BaP treatment activated AhR,FA treatment inhibited AhR transfer from cytoplasm to nucleus,and down-regulated mRNA expression levels of AhR target genes Cyp1a1 and Cyp1b1.Molecular structure simulation revealed that FA has a binding site in the PAS-B structural domain of AhR with a binding energy of-12.14 kj/mol,presumably affecting AhR activation by possibly competitively binding ligands.5.FA had a protective effect on mitochondria.FA treatment could significantly alleviate the effects of BaP-induced decrease of ATP level,increase of ROS and mtROS level,etc.Meanwhile,FA treatment restored REDOX related genes Nrf2,Cat and mitochondrial fission/fusion related genes Drp1 to normal levels.AhR was partially localized in mitochondria,BaP reduced the degree of co-localization between AhR and mitochondria,and FA could reverse the effect to some extent.6.BaP exposure altered DNA methylation status.BaP reduced the global 5mC levels.At the mRNA level,BaP exposure down-regulated the expression of Dnmtl and up-regulated the expression of Tet2,and failed to alleviate the abnormal expression of these genes after FA supplementation;at the protein level,BaP exposure up-regulated the expression of TET2,and FA treatment restored the abnormal expression of TET2.Conclusion:Abnormal activation of AhR signaling pathway is involved in cardiomyocyte toxicity induced by BaP.FA plays a cardioprotective role by inhibiting AhR entry into the nucleus and regulating AhR localization in mitochondria.In addition,FA also has a certain effect on DNA methylation fluctuations caused by exposure to BaP.