Effects Of Resveratrol On Differentiation Of RAW264.7 Cells Into Osteoclasts By Autophagy

Objective: To investigate the effects of resveratrol and the role of autophagy on the differentiation of RAW264.7 cells into osteoclasts.Methods: The RAW264.7 cell viability was detected with different Resveratrol(RSV)concentrations at various checkpoints with the addition of RANKL by CCK8 kits,and the morphology of osteoclast differentiation was observed by TRAP staining.The osteoclast differentiation markers(TRAP,MMP-9,CTSK)mRNA and autophagy related mRNA(LC3,Beclin-1 and P62)were detected by RT-PCR,and the expression of autophagy related proteins LC3II/I,Beclin-1 and P62 were detected by Western Blot.Then adding to 3-methyladenin(3-MA)with selected RSV concentration,the mRNA expression of osteoclast and autophagy related markers were detected.Results: 1.There was no significant difference in the proliferation of RAW264.7 cells at lower RSV concentration and earlier intervention phase compared with the control.The proliferation of RAW264.7 cells decreased gradually with the higher RSV concentration and longer the intervention time compared with the untreated group.2.The cell proliferation firstly increased and then decreased gradually at concentration of 0.1-10?mol/L RSV with RANKL addition inducing OC differentiation,RSV(0.1,0.5?mol/L)increased TRAP positive osteoclast number and mRNA expression of CTSK,MMP9,TRAP,LC3,P62,Beclin-1 and autophagy related protein LC3II/I and beclin-1 expression were also elevated,and P62 expression was decreased.The higher increasing of RSV concentration,the less number of osteoclasts and the expression of related mRNA and protein LC3II/I and beclin-1,by contrast,the expression of protein P62 were increased.3.With the addition of 3-Methyladenin,the mRNA expression of osteoclast differentiation related markers CTSK,MMP9,TRAP and autophagy related markers LC3,P62,beclin-1 mRNA and autophagy related proteins LC3II/I and beclin-1 were decreased,while P62 protein expression were increased.Conclusion: In the process of differentiation of RAW264.7 cells into osteoclasts induced by RANKL,0.1-10?mol/L resveratrol promoted the differentiation of osteoclasts at low concentration and inhibited the differentiation of osteoclasts at high concentration.The mechanism of resveratrol's influence on osteoclast differentiation may be partly by regulating autophagy.