The Relationship Between All-trans Retinoic Acid And COX-2 Regulating BMP9-induced Osteogenic Differentiation Of Mesenchymal Stem Cells Via Wnt/?-catenin Signaling

Non-steroidal anti-inflammatory drugs(NSAIDs)have antipyretic?analgesic?anti-inflammatory and anti-rheumatic effects,and they widely used in clinical applications.Studies have shown that the use of NSAIDs can affect bone metabolism,such as delayed bone healing,decreased bone strength,especially selective cyclooxygenase-2(COX-2)inhibitors.Therefore,how to overcome the side effects of COX-2 inhibitors on bone metabolism is an urgent problem to be solved in improving the therapeutic effect of orthopedic related diseases.BMP9 has the ability to induce stem cell differentiation efficiently,and it has a good clinical application prospect.In this study,BMP9 induced stem cell bone differentiation into a basic model,and explored possible prevention and treatment measures to overcome the adverse effects of COX-2 inhibition on bone metabolism.Previous studies showed that the research team further analyzed the reversal effect of all-trans retinoic acid(ATRA)on the inhibition of BMP9-induced stem cell bone differentiation by COX-2 inhibitor(NS398)and the possible related molecular mechanisms.In this study,RT-PCR,Western blot,alkaline phosphatase(ALP)histochemical staining,alizarin red S staining,IP and ChIP were used to conduct related research.First,this study confirmed the effect of COX-2 on BMP9-induced osteoblast differentiation.Then,the effect of COX-2 inhibitor on BMP9-induced osteogenic differentiation and the reversal effect of ATRA on NS398-induced BMP9-induced stem cell differentiation were analyzed.To analyze the relationship between ATRA and the reversal effect of NS398 on BMP9-induced stem cell bone differentiation and Wnt/?-catenin signaling.The results showed that COX-2 could significantly promote BMP9-induced ALP activity,OPN expression and cell mineralization in C3H10T1/2 cells.Inhibition of COX-2 or silencing COX-2 significantly attenuated BMP9 osteogenic differentiation induction;ATRA was concentration dependent Promote BMP9-induced ALP activity,OPN expression and cell mineralization in C3H10T1/2 cells,and significantly reverse the inhibitory effect of NS398.BMP9 can significantly increase the level of ?-catenin protein.NS398 can attenuate the effect of BMP9 on ?-catenin levels.ATRA can not only increase the protein level of ?-catenin,but also significantly reverse the inhibitory effect of NS398 on the increase of ?-catenin in BMP9.In addition,NS398 had no significant effect on BMP9-induced CREB protein levels,but decreased p-CREB protein levels;conversely,ATRA up-regulated BMP9-induced p-CREB protein levels and reversed NS398 down-regulation of p-CREB.The results suggest that COX-2 inhibitors can attenuate the osteogenic differentiation of BMP9,but ATRA can reverse this inhibition;this effect of ATRAmay be mediated by the interaction between RXRa and p-CREB to promote the expression of ?-catenin and enhance Wnt/?-catenin signal activity.