丹皮酚对去卵巢骨质疏松大鼠叉头框蛋白O3a/Wnt信号通路及椎骨密度的影响

背景:丹皮酚可抑制炎症,减轻慢性骨关节炎临床症状,作为一种不良反应小、更为安全的天然药物成分,探讨其对骨质疏松症的治疗作用具有重要的临床意义。目的:探究丹皮酚对去卵巢骨质疏松大鼠叉头框蛋白O3a(forkhead box O3a,FoxO3a)/Wnt信号通路及椎骨密度的影响。方法:雌性SD大鼠随机分为6组,每组10只。假手术组仅切除卵巢旁部分脂肪组织;其余50只大鼠均采用双侧卵巢摘除法制备去卵巢骨质疏松大鼠模型,模型制备成功后分为模型组、雌激素组炔雌醇10μg/(kg·d)]、丹皮酚低、中、高剂量组丹皮酚125,250,500 mg/(kg·d)],每组10只,灌胃给药;假手术组、模型组灌胃等量生理盐水,持续干预12周。ELISA法测定血清雌激素、骨保护素、骨钙素水平;采用双能X射线动物骨密度测定仪检测大鼠股骨及椎骨骨密度;采用骨科生物力学测试仪检测大鼠股骨及椎骨生物力学指标:弹性模量、最大载荷、屈服载荷;实时荧光定量PCR检测椎骨组织FoxO3a、Wnt2 mRNA表达水平;免疫印迹法检测椎骨组织FoxO3a、Wnt2及核内β-catenin蛋白表达。结果与结论:①治疗后,与假手术组比较,模型组L4,5骨密度、弹性模量、最大载荷、屈服载荷、血清雌二醇、骨保护素水平、椎骨组织Wnt2 mRNA及蛋白水平、核内β-catenin蛋白水平均显著降低(P<0.05),骨钙素水平、椎骨组织FoxO3a mRNA及蛋白水平显著增加(P<0.05);②与模型组比较,丹皮酚低、中、高剂量组大鼠L4,5骨密度、弹性模量、最大载荷、屈服载荷、血清雌二醇、骨保护素水平、椎骨组织Wnt2 mRNA及蛋白水平、核内β-catenin蛋白水平依次增加(P<0.05),血清骨钙素水平、椎骨组织FoxO3a mRNA及蛋白水平依次降低(P<0.05),且丹皮酚高剂量组均优于雌激素组(P<0.05);③提示丹皮酚可增加去卵巢骨质疏松大鼠椎骨骨密度,改善其骨生物力学状况,减轻骨质疏松,该作用可能与抑制FoxO3a、促进Wnt2/β-catenin通路激活有关。

Effects of paeonol on forkhead box O3a/Wnt signal pathway and vertebral bone mineral density in ovariectomized osteoporosis rats

BACKGROUND: Paeonol can inhibit inflammation and relieve the clinical symptoms of chronic osteoarthritis. As a natural drug component with less side effectsand more safety, it is of great clinical significance to explore the treatment of osteoporosis.OBJECTIVE: To investigate the effects of paeonol on the signal pathway of forkhead box O3a/Wnt and the density of vertebrae in ovariectomized osteoporosis(OVX-OP) rats.METHODS: Female Sprague-Dawley rats were randomly divided six groups (n=10 per group). Rats in the sham operation only underwent removal of adiposetissue adjacent to the ovary, and the other 50 rats were used to make OVX-OP rat models by bilateral ovariectomy. The rat models were randomly divided intoOVX-OP group, estrogen group (ethinylestradiol 10 μg/kg/d), low- (paeonol 125 mg/kg/d), medium- (paeonol 250 mg/kg/d), and high-dose (paeonol 500 mg/kg/d)groups. The rats were administered by gavage. Sham operation group and OVX-OP group were given the same amount of normal saline. The intervention lastedfor 12 weeks. The levels of serum estrogen, osteoprotegerin and osteocalcin were measured by enzyme-linked immunosorbent assay. Bone mineral density ofthe femur and vertebrae was measured by dual energy X-ray animal bone mineral density meter. The biomechanical indexes of the femur and vertebrae, elasticmodulus, maximum load and yield load, were measured by orthopedic biomechanical tester. The expression levels of forkhead box O3a and Wnt2 mRNA weredetected by real-time fluorescence quantitative PCR. The expression levels of forkhead box O3a, Wnt2 and nucleus β-catenin proteins were detected by westernblot.RESULTS AND CONCLUSION: After the treatment, the bone mineral density, elastic modulus, maximum load, and yield load in the 4th and 5th lumbar vertebrae,serum estradiol and osteoprotegerin levels, Wnt2 mRNA and protein levels, and nucleus β-catenin protein level were significantly lower in the OVX-OP groupthan the sham operation group (P < 0.05), while serum osteocalcin and vertebral forkhead box O3a mRNA and protein levels were significantly higher in theOVX-OP group (P < 0.05). Compared with the OVX-OP group, low-, medium, and high-dose paeonol significantly increased the bone mineral density, elasticmodulus, maximum load, and yield load in the 4th and 5th lumbar vertebrae, serum estradiol and osteoprotegerin levels, Wnt2 mRNA and protein levels inthe vertebrae (P < 0.05), and decreased the levels of serum osteocalcin and vertebral FOXO3a mRNA and protein (P < 0.05). Moreover, the above-mentionedindexes were better in the high-dose paeonol group than the estrogen group (P < 0.05). To conclude, paeonol can increase bone mineral density of thevertebrae, improve the biomechanical condition, and relieve osteoporosis in OVX-OP rats, which may be related to the inhibition of forkhead box O3a and thepromotion of Wnt2/β-catenin pathway activation.