目的 探讨仙茅苔黑酚类酚苷(orcinol glucoside in Curculigo orchioideson,COOG)对去卵巢骨质疏松大鼠骨代谢及破骨细胞形成分化的影响。方法 3月龄Sprague-Dawley大鼠被随机分为正常对照组和去卵巢组。再将去卵巢大鼠分为5组,连续12周分别灌胃给0.5%的羧甲基纤维素钠(sodium carboxymethyl cellulose,CMC-Na)溶液,1 mg·kg-1尼尔雌醇(nilestriol,Nyl-1),15、45和135 mg·kg-1 COOG。用双能X线吸收仪测定骨密度(bone mineral density,BMD),采用试剂盒测定血清和尿液的生化指标。RANKL诱导RAW264.7细胞分化为破骨细胞,抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色测定破骨细胞的数目;罗丹明-鬼笔环肽染色和激光共聚焦显微镜观察破骨细胞F-actin环的结构和形态;磷酸苯二钠法测定破骨细胞TRAP的活性;Elisa试剂盒测定破骨细胞组织蛋白酶K(cathepsin K,CK)和基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)的活性。结果 去卵巢大鼠骨密度显著降低,血清碱性磷酸酶(alkaline phosphatase,ALP)和抗酒石酸酸性磷酸酶活性升高,尿钙排出增加,血清抗氧化物水平降低。COOG在15、45和135 mg·kg-1剂量下,显著增加去卵巢大鼠的骨密度,显著降低血清抗酒石酸酸性磷酸酶活性,增加血清超氧化物歧化酶(superoxide dismutase,SOD)和还原型谷胱甘肽(fredused glutathione hormone,GSH)的水平,降低氧化产物丙二醛(malondialdehyde,MDA)的含量,减少尿钙排出。苔黑酚葡萄糖苷(sakakin,Sak)和苔黑酚龙胆二糖苷(orcinol gentiobioside,Og)显著抑制破骨细胞的形成分化、抑制破骨细胞抗酒石酸酸性磷酸酶的活性、CK的活性和MMP-9的活性,抑制破骨细胞F-Actin环的形成。结论 COOG能够减缓去卵巢大鼠的骨丢失,抑制破骨细胞的形成、分化和骨吸收作用。
Abstract
OBJECTIVE To investigate the efects of orcinol glycosides from Curculigo orchioides on (COOG) on bone metabolism in ovariectomized rats and formation and differentiation of osteoclast. METHODS 3-Month-old Sprague-Dawley rats were randomly divided into normal control group and ovariectomized group. Then ovariectomized rats were divided into five groups, and orally administered with 0.5% sodium carboxymethyl cellulose (CMC-Na) solution, 1 mg·kg-1 of nilestriol (Nyl-1), 15, 45, and 135 mg·kg-1 of COOG for 12 weeks. The bone mineral density of femur in rats was measured by Dual-Energy X-ray absorptiometry, and serum and urine biochemical biomarkers were assayed with regent kit. The osteoclasts were induced from RAW264.7 cells by RANKL, the number of osteoclasts was measured by tartrate-resistant acid phosphatase (TRAP) staining, and the structure and morphologic characteristic of F-Actin in osteoclast were stained with rhodamine-phalloidin and observed with laser confocal microscopy, and the activities of TRAP of osteoclasts were measured with phenyl disodium phosphate method, the activities of cathepsin K (CK) and matrix metalloproteinase-9 (MMP-9) were detected with an ELISA kit. RESULTS In ovariectomized rats, the bone mineral density was significantly reduced, and the activities of serum alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) were significantly increased, the excretion of urinary calcium was significantly increased, and the levels of serum antioxidant were decreased. COOG (15, 45 and 135 mg·kg-1) significantly increased bone mineral density, reduced the activities of tartrate-resistant acid phosphatase (TRAP), enhanced the serum levels of superoxide dismutase (SOD) and prototype glutathione hormone (GSH), and inhibited the production of malondialdehyde (MDA), reduced the excretion of urinary calcium. Sakakin (Sak) and orcinol gentiobioside (Og) significantly inhibited the formation and differentiation of osteoclasts, suppressed the activities of TRAP, CK and MMP-9, and inhibited the formation of F-Actin in osteoclast. CONCLUSION These results suggests that orcinol glycoside from Curculigo orchioides can attenuate bone loss in ovariectomized rats and inhibit the formation, differentiation and bone resorption of osteoclast.
关键词
仙茅苔黑酚类酚苷 /
卵巢切除术 /
骨质疏松 /
破骨细胞 /
骨吸收
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Key words
orcinol glycosides from Curculigo orchioides /
ovariectomy /
osteoporosis /
osteoclasts /
bone resorption
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脚注
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基金
国家自然科学基金项目资助(81973534)
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