叶酸对去卵巢大鼠骨质疏松的保护作用
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摘要
目的探讨叶酸对去卵巢大鼠骨质疏松的保护作用及机制,为防治骨质疏松提供新思路。
方法48只健康3月龄雌性SD大鼠随机分为6组,每组8只:假手术组(SHAM)、去卵巢组(OVX)、乙烯雌酚组(ERT,0.03mg.kg~(-1).d~(-1))、白藜芦醇组(RES,20 mg.k8~(-1).d~(-1))、低剂量叶酸组(FA1,5 mg.kg~(-1).d~(-1))和高剂量叶酸组(FA2,20 mg.kg~(-1).d~(-1))。大鼠去卵巢术后1周开始灌胃给药,用药10周后处死全部大鼠。测定血浆总同型半胱氨酸(tHcy)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)和总抗氧化能力(TAC)和一氧化氮(NO)的水平;同时测定腰椎骨匀浆中MDA、GSH-Px、TAC、NO、一氧化氮合酶(NOS)、碱性磷酸酶(ALP)和抗酒石酸酸性磷酸酶(TRACP)的水平;取L5椎体和右股骨测定骨密度(BMD)和骨生物力学指标;取L6椎体和左股骨制备HE病理切片,观察骨组织形态学变化。
结果1.OVX组大鼠血浆tHcy浓度较SHAM组明显升高达94.6%(p＜0.01);FA1和FA2组血浆tHcy浓度较OVX组均显著降低(p＜0.01),并低于SHAM组水平(p＜0.05);ERT和RES组较OVX组明显降低(p＜0.01),接近SHAM组水平;2.与SHAM组相比,OVX和FA1组大鼠血浆和骨匀浆TAC和GSH-Px活性明显降低(p＜0.01),MDA浓度均显著升高(p＜0.01);ERT、RES和FA2组大鼠血浆和骨匀浆TAC和GSH-Px活性较OVX组明显提高,与SHAM组相近(p＞0.05),MDA浓度较OVX组明显降低(p＜0.01),血浆MDA浓度仍高于SHAM组(p＜0.01),骨匀浆MDA浓度接近SHAM组(p＞0.05);FA1组大鼠骨匀浆GSH-Px活性较OVX组增加(p＜0.05),低于ERT和SHAM组(p＜0.01,p＜0.05),血浆和骨匀浆MDA浓度较OVX组降低(p＜0.05),仍高于ERT和SHAM组(p＜0.01,p＜0.05);3.OVX和FA1组大鼠血浆和骨匀浆NO浓度较SHAM组均明显降低(p＜0.01);ERT、RES和FA2组大鼠血浆和骨匀浆NO浓度较OVX组均显著增加,ERT和FA2组接近SHAM组,RES组血浆NO浓度低于ERT和SHAM组(p＜0.05);OVX和FA1组大鼠骨匀浆NOS活性较SHAM组明显降低(p＜0.01),ERT和FA2组骨匀浆NOS活性较OVX组显著提高(p＜0.01),与SHAM组相近(p＞0.05);4.OVX和FA1组骨匀浆ALP和TRACP活性较SHAM组均显著增加(p＜0.01);ERT、RES和FA2组较OVX组骨匀浆ALP活性均明显增高(p＜0.01),TRACP活性均显著降低(p＜0.01),ERT组TRACP活性与SHAM组相近(p＞0.05),RES和FA2组TRACP活性仍高于SHAM组(p＜0.01);FA1组ALP活性与OVX组相当(p＞0.05),TRACP活性低于OVX组(p＜0.05);5.与SHAM组相比,OVX和FA1组大鼠腰椎和股骨BMD均明显降低(p＜0.01);ERT、RES和FA2组大鼠腰椎和股骨BMD较OVX组均有增高,ERT组接近SHAM组,RES和FA2组仍低于SHAM组(p＜0.01)。大鼠血浆tHcy浓度与腰椎BMD呈负相关(r=-0.342,p=0.033);6.与SHAM组相比,OVX组大鼠腰椎骨弹性模量、最大值应力、屈服强度应力、最大值载荷和屈服强度载荷均明显降低(p＜0.01);ERT、RES和FA2组与OVX组比较,大鼠上述腰椎骨生物力学参数值均明显增高(p＜0.01).OVX组大鼠股骨弹性模量、屈服强度应力和屈服强度载荷较SHAM组均明显降低(p＜0.01);与OVX组比较,ERT、RES和FA2组大鼠上述股骨生物力学参数值均明显增高(p＜0.01)。
结论1.高同型半胱氨酸(hHcy)参与了去卵巢大鼠骨质疏松的发生;2.叶酸对去卵巢大鼠骨质疏松具有保护作用,其机制可能与改善hHcy代谢和抗氧化作用有关。
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~(-1)body 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.
方法48只健康3月龄雌性SD大鼠随机分为6组,每组8只:假手术组(SHAM)、去卵巢组(OVX)、乙烯雌酚组(ERT,0.03mg.kg~(-1).d~(-1))、白藜芦醇组(RES,20 mg.k8~(-1).d~(-1))、低剂量叶酸组(FA1,5 mg.kg~(-1).d~(-1))和高剂量叶酸组(FA2,20 mg.kg~(-1).d~(-1))。大鼠去卵巢术后1周开始灌胃给药,用药10周后处死全部大鼠。测定血浆总同型半胱氨酸(tHcy)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)和总抗氧化能力(TAC)和一氧化氮(NO)的水平;同时测定腰椎骨匀浆中MDA、GSH-Px、TAC、NO、一氧化氮合酶(NOS)、碱性磷酸酶(ALP)和抗酒石酸酸性磷酸酶(TRACP)的水平;取L5椎体和右股骨测定骨密度(BMD)和骨生物力学指标;取L6椎体和左股骨制备HE病理切片,观察骨组织形态学变化。
结果1.OVX组大鼠血浆tHcy浓度较SHAM组明显升高达94.6%(p＜0.01);FA1和FA2组血浆tHcy浓度较OVX组均显著降低(p＜0.01),并低于SHAM组水平(p＜0.05);ERT和RES组较OVX组明显降低(p＜0.01),接近SHAM组水平;2.与SHAM组相比,OVX和FA1组大鼠血浆和骨匀浆TAC和GSH-Px活性明显降低(p＜0.01),MDA浓度均显著升高(p＜0.01);ERT、RES和FA2组大鼠血浆和骨匀浆TAC和GSH-Px活性较OVX组明显提高,与SHAM组相近(p＞0.05),MDA浓度较OVX组明显降低(p＜0.01),血浆MDA浓度仍高于SHAM组(p＜0.01),骨匀浆MDA浓度接近SHAM组(p＞0.05);FA1组大鼠骨匀浆GSH-Px活性较OVX组增加(p＜0.05),低于ERT和SHAM组(p＜0.01,p＜0.05),血浆和骨匀浆MDA浓度较OVX组降低(p＜0.05),仍高于ERT和SHAM组(p＜0.01,p＜0.05);3.OVX和FA1组大鼠血浆和骨匀浆NO浓度较SHAM组均明显降低(p＜0.01);ERT、RES和FA2组大鼠血浆和骨匀浆NO浓度较OVX组均显著增加,ERT和FA2组接近SHAM组,RES组血浆NO浓度低于ERT和SHAM组(p＜0.05);OVX和FA1组大鼠骨匀浆NOS活性较SHAM组明显降低(p＜0.01),ERT和FA2组骨匀浆NOS活性较OVX组显著提高(p＜0.01),与SHAM组相近(p＞0.05);4.OVX和FA1组骨匀浆ALP和TRACP活性较SHAM组均显著增加(p＜0.01);ERT、RES和FA2组较OVX组骨匀浆ALP活性均明显增高(p＜0.01),TRACP活性均显著降低(p＜0.01),ERT组TRACP活性与SHAM组相近(p＞0.05),RES和FA2组TRACP活性仍高于SHAM组(p＜0.01);FA1组ALP活性与OVX组相当(p＞0.05),TRACP活性低于OVX组(p＜0.05);5.与SHAM组相比,OVX和FA1组大鼠腰椎和股骨BMD均明显降低(p＜0.01);ERT、RES和FA2组大鼠腰椎和股骨BMD较OVX组均有增高,ERT组接近SHAM组,RES和FA2组仍低于SHAM组(p＜0.01)。大鼠血浆tHcy浓度与腰椎BMD呈负相关(r=-0.342,p=0.033);6.与SHAM组相比,OVX组大鼠腰椎骨弹性模量、最大值应力、屈服强度应力、最大值载荷和屈服强度载荷均明显降低(p＜0.01);ERT、RES和FA2组与OVX组比较,大鼠上述腰椎骨生物力学参数值均明显增高(p＜0.01).OVX组大鼠股骨弹性模量、屈服强度应力和屈服强度载荷较SHAM组均明显降低(p＜0.01);与OVX组比较,ERT、RES和FA2组大鼠上述股骨生物力学参数值均明显增高(p＜0.01)。
结论1.高同型半胱氨酸(hHcy)参与了去卵巢大鼠骨质疏松的发生;2.叶酸对去卵巢大鼠骨质疏松具有保护作用,其机制可能与改善hHcy代谢和抗氧化作用有关。
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~(-1)body 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.
引文
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[41]许惠英,黎旸.治疗骨质疏松症药物的应用进展.广东药学.2004,14:35-39.
[42]陈容清,谭礼蓉.骨质疏松症的药物治疗进展.中国药业.2003,12:79-80.
[43]蒋利华.骨质疏松症药物治疗进展.中国药业.2006,15:3-4.
[44]邱贵兴,荣国威.高级医师案头丛书.骨科学.北京:中国协和医科大学出版社.2002,504.
[45]朱汉民.骨质疏松非性激素类药物新进展.骨质疏松和骨矿盐疾病临床基础研究,2003,2:130.
[46]Doshi SN,McDowell IF,Moat SJ,et al.Folate improves endothelial function in coronary artery disease:an effect mediated by reduction of intracellular superoxide? Arterioscler Thromb Vase Biol.2001,21:1196-1202.
[47]Joshi R,Adhikari S,Patro BS,et al.Free radical scavenging behavior of folic acid:evidence for possible antioxidant activity.Free Radic Biol Med.2001,30:1390-1399.
[48]张华,王峰,陆伟,等.叶酸与四氢生物喋呤对高脂血症兔内皮功能的影响.实用医药杂志.2005,11:997-999.
[49]Moat SJ,Madhavan A,Taylor SY,et al.High- but not low-dose folic acid improves endothelial function in coronary artery disease.Eur J Clin Invest.2006,36:850-859.
[50]Bednarek-Tupikowska G,Tupikowski K,Bidzinska B,et al.The effect of estrogen deficiency,estrogen and estro-progestagene therapy on total plasma homocysteine and serum lipid peroxide levels in postmenopausal women.Ginekol Pol.2005,76:687-692.
[51]el-Swefy SE,Ali SI,Asker ME,et al.Hyperhomocysteinaemia and cardiovascular risk in female ovariectomized rats:role of folic acid and hormone replacement therapy.J Pharm Pharmacol.2002,54:391-397.
[52]陈健,黎明,舒研文,等.叶酸对高同型半胱氨酸血症大鼠单核细胞趋化蛋白-1和白细胞介素-8释放的影响.临床心血管病杂志.2005,21:338-340.
[53]Lean JM,Jagger CJ,Kirstein B,et al.Hydrogen peroxide is essential for estrogen-deficiency bone loss and osteoclast formation.Endocrinology.2005,146:728-735.
[54]Upchurch GR Jr,Welch GN,Fabian AJ,et al.Homocyst(e)ine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase.J Biol Chem.1997,272:17012-17017.
[55]Huang RF,Yaong HC,Chen SC,et al.In vitro folate supplementation alleviates oxidativestress,mitochondria-associated death signalling and apoptosis induced by 7-ketocholesterol.BJ Nutr.2004,92:887-894.
[56]VanTH,Ralston SH.Nitric oxide and bone.Immunology.2001,103:255-261.
[57]宋宁,富玲,厚银栋,等.去势成年雌性大鼠骨质疏松模型血清NO和NOS 的水平及其意义.中国骨质疏松杂志.2004,10:135-137.
[58]Faraci FM,Lentz SR.Hyperhomocysteinemia.oxidative stress and cerebral vascular dysfunction.Stroke.2004,35:345-347.
[59]Stroes ES,van Faassen EE,Yo M,et al.Folio acid reverts dysfunction of endothelial nitric oxide synthase.Circ Res.2000,86:1129-1134.
[60]Herrmann M,Schmidt J,Umanskaya N,et al.Stimulation of osteoclast activity by low B-vitamin concentrations.Bone.2007,41:584-591.