Effect of melatonin on osteoblast differentiation under simulated microgravity

SUN Quan; XU Liqun; YU Tingfei; LIU Jingchun; HU Zebing; ZHANG Shu; SHI Fei

doi:10.13276/j.issn.2097-1656.2025.01.005

Abstract：Objective To investigate the effect of melatonin(MT)on osteoblast differentiation under simulated microgravity,and to further investigate the regulatory effect of MT on YTH N6-methyladenosine RNA binding protein F2(YTHDF2)and whether MT affects osteoblast differentiation through YTHDF2,in order to seek new intervention targets for the problem of microgravity-induced bone loss in spaceflight.Methods MC3T3-E1 cells were transfected with si-YTHDF2 or pEX-YTHDF2,and the expression changes of RUNX2 and COL1A1 in MC3T3-E1 cells were detected by qRT-PCR and Western blotting.MC3T3-E1 cells were cultured with 2D gyrator to investigate the changes of mRNA and protein contents of YTHDF2 and RUNX2 under simulated weightlessness.After transfection of pEX-YTHDF2 into MC3T3-E1 cells in 2D gyrator for 48 h,the expression levels of RUNX2 and COL1A1 were measured by qRT-PCR and Western blotting.The concentration gradients of 10 nmol/L,50 nmol/L,100 nmol/L,1 μmol/L and 100 μmol/L were set to study the concentration effect of MT on osteoblast differentiation.MC3T3-E1 cells were cultured in the medium containing MT(100 nmol/L)for 48 h,and the expression of YTHDF2 was detected.After transfection with si-YTHDF2,MT or negative control was given to detect the differentiation indexes RUNX2 and COL1A1.The changes of differentiation function were verified by alkaline phosphatase(ALP)staining and ALP activity detection.Results Transfection with si-YTHDF2 significantly inhibited MC3T3-E1 cell differentiation,and transfection with pEX-YTHDF2 promoted MC3T3-E1 cell differentiation(P＜0.05).The mRNA and protein expressions of YTHDF2 in MC3T3-E1 cells were significantly decreased under simulated microgravity(P＜0.01).Transfection with pEX-YTHDF2 partially alleviated the decreased differentiation function of MC3T3-E1 cells under simulated weightlessness(P＜0.05).MT treatment could alleviate the decreased mRNA and protein expression of YTHDF2 induced by simulated microgravity(P＜0.05).After transfection with si-YTHDF2 and MT treatment,the differentiation index was higher than that in si-YTHDF2+DMSO group,but lower than that in si-NC group(P＜0.05).Conclusion YTHDF2 promotes osteoblast differentiation.Simulated microgravity inhibited YTHDF2 expression and overexpressing YTHDF2 could partially rescue the inhibition of osteoblast differentiation caused by simulated microgravity.MT can alleviate the decreased expression of YTHDF2 under simulated microgravity.MT positively regulates osteoblast differentiation by partly promoting YTHDF2 expression.This study reveals for the first time that the MT/YTHDF2 axis plays a regulatory role in affecting osteoblast differentiation under simulated microgravity,which is expected to provide a potential strategy for the prevention of microgravity-induced bone loss.