CHNQD-00603 Promotes Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells By MiR-452-3p Mediated Autophagy Pathway

Objective: periodontitis is a chronic and progressive disease accompanied by bone resorption and tooth loss.The restoration of bone structure in periodontitis remains a challenge.Bone marrow mesenchymal stem cells(BMSCs)have the ability of osteogenic,chondrogenic and adipogenic differentiation,among which osteogenic differentiation plays an important role in bone repair and regeneration.Marine natural products have been proved to have a variety of biological activities,including anti-tumor,anti-inflammatory.However,the exploration of Marine natural products and their derivatives in bone metabolism is far from sufficient.Materials and methods: We obtained a series of derivatives through structural optimization from 4-phenyl-3,4-dihydroquinolin-2(1H)-one alkaloid isolated from Scopulariopsis sp.Some preliminary cytological experiments showed that CHNQD-00603 might promote the osteogenic differentiation of BMSCs.To further investigate the effects of CHNQD-00603 on BMSCs,we performed cytotoxicity assay and quantificational real-time polymerase chain reaction(q RT-PCR),alkaline phosphatase staining(ALP),and alizarin red S staining to assess the cytotoxicity and the ability of osteogenic differentiation of CHNQD-00603.The autophagy level was assessed and validated by western bloting(WB),q RT-PCR,and transmission electron microscopy.Then,3-methyladenine(3-MA)was added to further examine the role of autophagy.Based on the expression of autophagy-related genes,we predicted and examined the potential miRNAs by bioinformatics.Further,the correlation between miR-452-3p and autophagy and the role of miR-452-3p in the osteogenic differentiation of BMSCs were detected by transfection of miR-452-3p mimics.Results: CCK-8 assay showed that CHNQD-00603 at 1 μg/m L did not influence BMSCs activity.However,the proliferation rate decreased at day 7.q RT-PCR,ALP staining,ALP activity assay,and Alizarin red S staining showed that CHNQD-00603 at 0.5μg/m L and 1μg/m L promotes osteogenic differentiation of BMSCs,but the best concentration of CHNQD-00603 to promote osteogenic differentiation was 1 μg/m L.Further investigations indicated that CHNQD-00603 activated autophagy,and the inhibition of autophagy by 3-MA attenuated CHNQD-00603-enhanced osteogenic differentiation.Subsequently,the findings from bioinformatics and q RT-PCR indicated that miR-452-3p might be a regulator of autophagy and osteogenesis.Furthermore,we transfected BMSCs with miR-452-3p NC and mimics separately to further determine the function of miR-452-3p.The data showed that the overexpression of miR-452-3p moderated the level of autophagy and osteogenic differentiation of CHNQD-00603-treated BMSCs.Conclusion: Our data showed that CHNQD-00603 promoted the osteogenic differentiation of BMSCs by enhancing autophagy.Meanwhile,miR-452-3p played a regulatory role in this process.