The Effect And Mechanism Of Benzo[a]pyrene On BMP2-induced Osteogenic Differentiation Of Mesenchymal Stemcells

Objective: The aim of this study was to investigate the effect of Benzo[a]pyrene on bone morphogenetic proteins 2(BMP2)-induced osteogenic differentiation of mesenchymal stem cells(MSCs)and the regulatory mechanism involved.Methods: MSCs were infected with Ad-BMP2,and the expression of BMP2 and aryl hydrocarbon receptor(AhR)were detected by RT-PCRafter 48 hours.Then the effect of BaP on BMP2-induced osteogenic differentiation in MSCs was examined after being treatedwith different concentrations of BaP.And then under the same treatment,the effect of BaP on the early stage of differentiation was detected by ALP staining and ALP activity,and the effect of it on the later stage of differentiation was detected by Alizarin Red S(ARS)staining.The protein expression levels of Runx2,OPN,OCN and p-Smad1/5/8 were determined by Western Blot.Then the markers of osteogenic differentiation and the pathway of BMP/Smad were measured after being pretreated witharyl hydrocarbon receptor antagonists(CH223191).To further clarify its mechanism,we found that BMPRII and Hey1 are target genes of BaP by bioinformatics analysis.The expression of BMPR? and Hey1 were detected by RT-qPCR.The protein expression levels of BMPR?was detected by Western blot and Immunofluorescence.Then the expression of BMPR? and Hey1 were measured after being pretreated with CH223191.Results: After being infected with Ad-BMP2,the mRNA level of BMP2 significantlyincreased while the mRNA level of AhR did not alter obviously.After being infectedwith BMP2 and treated with different concentrations of BaP in MSCs,we found that BaP could suppress BMP2-induced ALP activity and calcium deposition in a dose-dependent manner.The results of Western Blot showed BaP could inhibit BMP2-induced the protein expression levels of Runx2,OPN,OCN and p-Smad1/5/8 in a dose-dependent manner.However,when treated MSCs with CH223191,we found that CH223191 could partly rescue the toxicologic effects of BaP on oesteogenic differentiation.At the mean while,it partly rescued the inhibition effects of BaP on BMP2/Smad signal pathway.BaP inhibited the protein expression of BMPR?,and promoted the mRNA expression of Hey1;The CH223191 rescued the inhibitory effect of BaP on expression of BMPR?,and inhibited the activity of Hey1.Conclusion: BaP could suppress BMP2-Induced osteogenic differentiation of MSCs through AhR;The mechanism may be that AhR inhibited the expression of BMPRII,while activating Hey1 to enhance its negative feedback regulation on BMP2,thereby affecting the activation of BMP2/Smad signaling pathway.