Study On The Effect Of Trans-resveratrol In Regulating The Osteoblast-like Cell MC3T3-E1 And Its Molecular Mechanism

Partâ… The effect of trans-resveratrol on BMDs of OVX rats and proliferation,differentiation,mineralization and apoptosis of mice osteoblast-like cells MC3T3-E1Object:Trans-resveratrol a phytoestrogen found in grape skins and red wine,has been reported to have a wide range of biological and pharmacological properties.The objective of our study was to investigate the effect of trans-resveratrol on the bone mineral density (BMD)of Ovariectomized rats and the proliferation,differentiation, mineralization and apoptosis of mice osteoblast-like cells MC3T3-E1 in low-estrogen environment.Method:Forty-eight Sprague-Dawlery female rats were randomly divided into 6 groups and treated respectively as follows:group A,sham operated;group B,ovariectomized(OVX);group C,OVX supplemented with 0.03 mg·kgbw-1·d-1 diethylstilbestrol;and group D,E,F:OVX rats supplemented with 3 dosages of RES(5,15 and 45 mg·kgbw-1·d-1, respectively).The duration of exposure was 90 days and the BMDs of rats were measured.Phenol red-free DMEM was used to imitate estrogen withdrawal.Trans-resveratrol in different concentrations were administrated into culture medium to observe its effect on the mice osteoblast-like cells MC3T3-E1.WST-8 assay was used to measure the DNA synthesis of MC3T3-E1.The alkaline phosphatase(ALP)activities and nitric oxide(NO)concentration were determined to evaluate the cell differentiation.Bone gla protein(BGP)concentration and mineralization nodule counting(stained by Alizarin red)were used to identify the mineralization.Serum hungry was used to induce MC3T3-E1 apoptosis. RT-PCR was used to detect mRNA expression of bcl-2 and bax,two of the apoptosis related genes.Results:BMDs of ovariectomized rats were significantly lower than those of group sham(P＜0.05).Compared with group OVX,BMDs of the total body,lumbar vertebrae,femur and tibia of trans-resveratrol groups were increased significantly(P＜0.05),the most protective effect on bone mass was showed in group of 45 mg·kgbw^-1·d^-1trans-resveratrol. 10^-9～10^-6mol/L Trans-resveratrol increased DNA synthesis of MC3T3-E1 cells.In addition,10^-8～10^-5mol/L trans-resveratrol increased ALP activity, NO concentration,BGP level and mineralization nodule formation of MC3T3-E1 cells.Moreover,compared with serum hungry group,the expression of bcl-2 mRNA in trans-resveratrol groups were significant increased in 10^-8～10^-5mol/L trans-resveratrol groups(p＜0.05),while the expression of bax mRNA in these groups were significantly decreased (p＜0.05).On the other hand,10^-5mol/L trans-resveratrol inhibited proliferation of MC3T3-E1 cells.Conclusion:10^-8～10^-6mol/L trans-resveratrol can promote the proliferation,differentiation and mineralization of Mice obsteoblast-like cells MC3T3-E1,while protect from apoptosis induced by serum hungry. Partâ…¡The molecular mechanism of trans-resveratrol in regulating mice osteoblast-like cells MC3T3-E1Object:Osteoblast plays an important role in bone metabolism.This study is to investigate the regulation mechanism of trans-resveratrol, through estrogen receptor and OPG/RANKL pathways.Method:Phenol red-free DMEM was used to imitate estrogen withdrawal.Trans-resveratrol in different concentrations(10^-9mol/L, 10^-8mol/L,10^-7mol/L,10^-6mol/L及10^-5mol/L T-Res)were administrated into culture medium to observe its effect on expression of some gene and protein in mice osteoblast-like cells MC3T3-E1. antiestrogen ICI182780 was used to observe the estrogenic effect of resveratrol.RT-PCR was used to detect mRNA expression of ER-α, ER-β,transformation growth factorβ₁(TGF-β₁),collageâ… (Col-â… ), osteoprotegerin(OPG)and receptor activator of NF-κB(RANKL). Western blotting was used to detect protein expression of ER-β,OPG and RANKL.Result:MC3T3-E1 expressed ER-αand ER-β,while ICI182780 inhibited their expression.Compared with control group,10^-7～10^-5mol/L trans-resveratrol up-regulated the expression of ER-β,TGF-β₁,Col-â… mRNA,but no significant effect on ER-αmRNA was observed. 10^-7～10^-5mol/L trans-resveratrol down-regulated the expression of OPG and down-regulated the expression of RANKL,at both mRNA and protein level.Both up-regulation and down-regulation of trans-resveratrol on these factors were inhibited by ICI182780.Conclusion:The bone-protective effects of trans-resveratrol may is related to its regulating effect on the estrogen and OPG/RANKL pathway.