Benzo [a] Pyrene Impairs Self-renewal And Differentiation Of Mesenchymal Stem Cells And Influence Fracture Healing

BackgroudMesenchymal stem cells(MSCs)are implicated in the bone-forming process during fracture repair.Benzo[a]pyrene(Ba P)—a cigarette smoke component and powerful motivator of the aryl hydrocarbon receptor(Ahr)—unfavorably influences bone condition and osteoblast differentiation.ObjectiveWe explored collected clinical samples in vitro and in vivo and investigated the effect of BaP on MSCs and the possible mechanism involved in influencing fracture healing.MethodsThe experiment was divided into three parts1.We collected human bone marrow mesenchymal stem cells(hBMSCs)in smokers,the amount of blue/purple precipitates designates alkaline phosphatase(ALP)staining,ALP activity and red stain of calcium accumulation were detected.Ahr signaling and the Ahr-aimed at gene cytochrome p4501B1(CYP1B1)which were triggered by Ba P detected by Western Blot.2.We employed C3H10T1/2 and Bone marrow mesenchymal stem cells(BM-MSCs)withBaP,MTT and CFU to show self-renewal of MSCs.Besides MSCs were separately cultured by adipogenic differentiation media and osteogenic differentiation media,then cells marked with ALP,Alizarin Red S or Oil Red O were detected,which showed the multiple differentiation potential of Mesenchymal Stem Cells.To verify that whether Ba P was motivated by Ahr-dependent signaling,we prevented receptor signaling using knocked-down siRNAs.The interference efficiency of the siRNAs targeting Ahr was verified by Western blotting.The suppressive effect of siAhr to ALP activity and matrix mineralization was observed counteracting withBaP.The expression of Runt-related transcription factor 2(RUNX2),SMAD-dependent signaling pathways TGF-β1/SMAD4 and SMAD-independent TGF-β1/ERK/AKT signaling pathways were decreased.To verify TGF-β1 signaling pathways whether play a major role during BaP inhibition of osteogenic differentiation of MSCs,we rescued the Ahr-dependent effects of BaP by inhibiting osteogenic differentiation with siAhr and then employed specific inhibitors of TGF-β1(LY2157299).Resveratrol is an aggressive obstructor of Ahr ligands,the osteogenic differentiation potential of Mesenchymal Stem Cells withBaP and resveratrol were detected.The effect of related signal pathways was detected by Western Blot.In vivo studies,rats were fed BaP daily by gavage and then given tibial fractures.We detected CYP1B1 in Ba P-exposed group,radiographs from 2 and 6 weeks after fractures.Histological features from 6 weeks were detected.At last the influence on tibial fractures by resveratrol at every specified time was detected.3.Bone Morphogenetic Protein,(BMPs)were detected in differentiation process of mesenchymal-type cells into osteoblast cells.ResultsWe noticed decreased self-renewal and differentiation of hBM-MSCs in smokers and activated Ahr signaling in hBM-MSCs by upregulating the Ahr-aimed at CYP1B1.In vitro studies,we employed C3H10T1/2 and BM-MSCs withBa P and discovered that BaP impaired innate properties of MSCs.Further investigation into MSCs showed that exposure to BaP activated Ahr signaling and inhibited TGF-β1/SMAD4 and TGF-β1/ERK/AKT signaling pathways.This effect of BaP was abrogated by resveratrol,a natural Ahr antagonist,in vivo tibial fracture calluses produced by Ba P-administered rats appeared delayed healing.Resveratrol inhibited MSCs from adverse effects caused by BaP.Bone Morphogenetic Proteins(BMPs)induced osteogenic differentiation of MSCs by reducing the adverse effects caused by Ba P.Conclusion1.Ahr may play a key role in BaP-impaired innate properties by inhibiting SMAD-dependent signaling pathways TGF-β1/SMAD4 and SMAD-independent TGF-β1/ERK/AKT signaling pathways.Furthermore,resveratrol inhibits the adverse effect on MSCs caused by BaP.2.Ba P altered healing in rat tibial fractures and restored by resveratrol.