| Objective To investigate the protective role of folic acid on osteoporosis in ovariectomized rats and its mechanism and to explore new ways of thinking for the prevention and treatment of osteoporosis.Methods Forty-eight healthy three-month-old female Sprague-Dawley rats were either sham-operated(Sham)or ovariectomized(Ovx)and randomly divided into six groups consisting of eight rats in each group:sham-operated group(SHAM), ovariectomized group(OVX),Ovx + diethylstilbestrol(ERT group),Ovx + resveratrol(RES group)and Ovx + folic acid(FA1 or FA2 group).One week post-surgery,rats were treated daily by oral gavage with distilled water or drugs.The dose of diethylstilbestrol,resveratrol and folio acid was 0.03mg,20mg,5mg and 20mg kg-1body weight day-1.After 10 weeks of treatment,rats were sacrificed. Plasma samples were collected before sacrifice to determine plasma total homocysteine(tHcy),malondialdehyde(MDA),glutathione peroxidase(GSH-Px), total antioxidant capacity(TAC)and nitric oxide(NO)levels.The bone homogenates were made and used for the estimation of MDA,GSH-Px,TAC,NO,nitric oxide synthase(NOS),alkaline phosphatase(ALP)and tartrate-resistant acid phosphatase (TRACP)activity.The L5 vertebra and right femur were removed for the determination of bone mineral density(BMD)and bone biomechanics.HE slice of pathology were made of the L6 vertebra and left femur to observe the bone histological changes.Results 1.Plasma tHcy concentrations were increased significantly by 94.6%by Ovx(P＜0.01).The increase in tHcy concentrations were suppressed by diethylstilbestrol,resveratrol and folic acid,and the mean values of ERT and RES group were not different from the SHAM group,while the mean values of FA1 and FA2 group were lower than the SHAM group(p＜0.05).2.Ovx decreased the activity of TAC and GSH-Px and increased the levels of MDA in plasma and homogenate, respectively compared to Sham(p＜0.01).Diethylstilbestrol,resveratrol and high-dose folic acid treatment improved the activity of TAC and GSH-Px and decreased the levels of MDA,and the mean values of ERT,RES and FA2 group were not different from the SHAM group.Homogenate GSH-Px activity was increased significantly by low-dose folic acid treament,and the mean value of FA1 group was lower than the ERT and SHAM group(p＜0.01).The MDA levels of plasma and homogenate were decreased by low-dose folic acid treatment as the mean values were lower than the OVX group but higher than the ERT and SHAM group(p＜0.01).3. Ovx caused a significant reduction in NO concentrations of plasma and homogenate (p＜0.01).This reduction in NO concentrations was prevented by diethylstilbestrol, resveratrol and high-dose folic acid treatment,as the mean values of ERT and FA2 group were not statistically different from the SHAM group and it was lower in the RES group than in the SHAM group(p＜0.05).Ovx significantly decreased homogenate NOS activity(p＜0.01).Diethylstilbestrol and high-dose folic acid treatment improved the activity of NOS,and the mean value was not statistically different from the SHAM group.Low-dose folic acid did not have any effect on NO and NOS of Ovx rats.4.Homogenate ALP and TRACP activity were increased significantly by Ovx,the increase in ALP activity was further augmented by diethylstilbestrol,resveratrol and high-dose folio acid treatment.However,the increase in TRACP activity were suppressed by diethylstilbestrol,resveratroi high-dose and low-dose folic acid,and the mean values of ERT group were not statistically different from the SHAM group,but the mean values of RES,FA1 and FA2 group were higher than the SHAM group.Low-dose folic acid did not have any effect on ALP activity of Ovx rats.5.Ovx caused a significant reduction in BMD of lumbar vertebra and femur.This reductions in lumbar vertebra and femur BMD were prevented by diethylstilbestrol,resveratrol and high-dose folic acid treatment,and the mean values of ERT group were not statistically different from the SHAM group,and BMD of RES and FA2 group were lower than the SHAM group.Low-dose folic acid did not have any effect on BMD of Ovx rats.Plasma tHcy concentrations were negatively related to lumbar vertebra BMD(r=-0.342,p=0.033).6.Ovx decreased bone modulus elasticity,maximum stress,yield strength stress,maximum load and yield strength load of lumbar vertebra,respectively compared to sham(p＜0.01).The lumbar vertebra bone biomechanics parameter values which were mentioned above were increased by diethylstilbestrol,resveratrol and high-dose folic acid treatment. Femur bone modulus elasticity,yield strength stress and yield strength load were decreased significantly by Ovx(P＜0.01).The femur bone biomechanics parameter values which were mentioned above were increased by diethylstilbestrol,resveratrol and high-dose folic acid treatment.Conclusions 1.Hyperhomocysteinema is involved in osteoporosis pathogenesis in ovariectomized rats.2.Folic acid is effective in protectiving against osteoporosis perhaps by lowering plasma tHcy levels and improving oxidative stress state.
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