杯苋甾酮抑制破骨分化和促进成骨分化的双向作用治疗骨质疏松症
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摘要
目的研究杯苋甾酮对破骨和成骨分化的影响,探讨其在改善骨质疏松中的作用.方法以CCK8检测杯苋甾酮对小鼠源性单核巨噬细胞(BMMs)及前成骨细胞MC3T3E1的毒性作用;流式检测药物对细胞凋亡的影响.以TRAP染色观察破骨分化;以ALP染色评估成骨分化,以Real-Time PCR检测TRAP及ALP的表达.15只小鼠分为假手术组、OVX组、治疗组.治疗组给予杯苋甾酮干预,余2组以生理盐水处理.4周后取胫骨,Micro-CT检测骨质及微观结构变化.结果杯苋甾酮≤10 mg/L时对细胞无毒性(P>0.05),不影响凋亡(P>0.05).其可显著抑制破骨分化(P<0.05),促进成骨分化.杯苋甾酮可抑制TRAP表达(P<0.05),促进ALP表达(P<0.05).杯苋甾酮可改善雌激素缺乏导致的骨质疏松.结论杯苋甾酮通过抑制破骨、促进成骨的双向作用达到改善骨质疏松的作用.
Objective To explore the influence of cyasterone on the osteoclast and osteoblast differentiation and then to investigate its effect on the bone quality in the osteoporosis mice. Me thods CCK8 assay was firstly used to detect the toxic effect of cyasterone on the mouse bone marrow derived mononuclear macrophages(BMMs) and anterior osteoblast lines MC3 T3 E1. Cell apoptosis was measured by flow cytometry. Then TRAP staining and ALP staining were employed to detect osteoclast differentiation and osteoblast differentiation, respectively. Realtime PCR was carried out to test the expression of osteoclast special gene TRAP and osteogenesis crucial gene ALP. In vivo, 15 mice were divided into three groups: sham-operated group, OVX group and OVX + cyasterone treatment group. In treatment group, cyasterone was used as 5 mg/kg every day. Sham-operated group and OVX group were treat with saline solution. After 4 weeks, the tibia was collected for Micro-CT detection to observe the bone quality and microstructure changes. Re s ults Cyasterone with the concentration of less than 10 mg/L had no significant cytotoxicity nor influence on the apoptosis(P >0.05). Cyasterone could significantly inhibit the osteoclast differentiation of BMMs(P<0.05), simultaneously, it also had the effect to promote the osteoblast differetiation of MC3 T3 E1. Real-time PCR indicated that cyasterone could block the expression of TRAP and increase the expression of ALP(P<0.05). In vivo, cyasterone was able to obviously improve the osteoporosis status caused by estrogen deficiency without general toxicity. Conclus ion cyasterone could provide a good treatment for osteoporosis through the bidirectional effect of inhibiting osteoclast differetiation and promoting osteoblast differentiation.
Objective To explore the influence of cyasterone on the osteoclast and osteoblast differentiation and then to investigate its effect on the bone quality in the osteoporosis mice. Me thods CCK8 assay was firstly used to detect the toxic effect of cyasterone on the mouse bone marrow derived mononuclear macrophages(BMMs) and anterior osteoblast lines MC3 T3 E1. Cell apoptosis was measured by flow cytometry. Then TRAP staining and ALP staining were employed to detect osteoclast differentiation and osteoblast differentiation, respectively. Realtime PCR was carried out to test the expression of osteoclast special gene TRAP and osteogenesis crucial gene ALP. In vivo, 15 mice were divided into three groups: sham-operated group, OVX group and OVX + cyasterone treatment group. In treatment group, cyasterone was used as 5 mg/kg every day. Sham-operated group and OVX group were treat with saline solution. After 4 weeks, the tibia was collected for Micro-CT detection to observe the bone quality and microstructure changes. Re s ults Cyasterone with the concentration of less than 10 mg/L had no significant cytotoxicity nor influence on the apoptosis(P >0.05). Cyasterone could significantly inhibit the osteoclast differentiation of BMMs(P<0.05), simultaneously, it also had the effect to promote the osteoblast differetiation of MC3 T3 E1. Real-time PCR indicated that cyasterone could block the expression of TRAP and increase the expression of ALP(P<0.05). In vivo, cyasterone was able to obviously improve the osteoporosis status caused by estrogen deficiency without general toxicity. Conclus ion cyasterone could provide a good treatment for osteoporosis through the bidirectional effect of inhibiting osteoclast differetiation and promoting osteoblast differentiation.
引文
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[12]YUAN X,BI Y,YAN Z,et al.Psoralen and Isopsoralen ameliorate sex hormone deficiency-induced osteoporosis in female and male mice[J].Biomed Res Int,2016,2016(4):6869452.
[13]HUANG Q,GAO B,JIE Q,et al.Ginsenoside-Rb2 displays anti-osteoporosis effects through reducing oxidative damage and bone-resorbing cytokines during osteogenesis[J].Bone,2014,66(9):306-314.
[14]国家药典委员会.中华人民共和国药典(第一部)[M].北京:化学工业出版社,2000:28.
[15]黄太康主编.常用中药成分与药理手册[M].北京:中国医用科技出版社,1994:410-413.
[2]彭永德.骨质疏松症研究年度报告(2012-8至2013-8)[J].中国内分泌代谢杂志,2014,30(8):639-642.
[3]LIM S Y,BOLSTER M B.Current approaches to osteoporosis treatment[J].Curr Opin Rheumatol,2015,27(3):216-224.
[4]DE LUCA A,LAMURA L,GALLO M,et al.Pharmacokinetic evaluation of zoledronic acid[J].Expert Opin Drug Metab Toxicol,2011,7(7):911-918.
[5]SILVERBERG S J,LINDSAY R.Postmenopausal osteoporosis[J].Med Clin North Am,1987,71(1):41-57.
[6]GRAY T K,FLYNN T C,GRAY K M,et al.17 beta-estradiol acts directly on the clonal osteoblastic cell line UMR106[J].Proc Natl Acad Sci U S A,1987,84(17):6267-6271.
[7]KOMM B S,TERPENING C M,BENZ D J,et al.Estrogen binding,receptor m RNA,and biologic response in osteoblast-like osteosarcoma cells[J].Science,1988,241(4861):81-84.
[8]OURSLER MJ,OSDOBY P,PYFFEROEN J,et al.Avian osteoclasts as estrogen target cells[J].Proc Natl Acad Sci U S A,1991,88(15):6613-6617.
[9]MANO H,YUASA T,KAMEDA T,et al.Mammalian mature osteoclasts as estrogen target cells[J].Biochem Biophys Res Commun,1996,223(3):637-642.
[10]ALDEN J C.Osteoporosis a review[J].Clin Ther,1989,11(1):3-14.
[11]邵雄杰,叶志强,罗春晓等.大豆异黄酮对绝经后骨质疏松的疗效观察[J].中山大学学报(医学科学版),2007,28(S1):241-242.
[12]YUAN X,BI Y,YAN Z,et al.Psoralen and Isopsoralen ameliorate sex hormone deficiency-induced osteoporosis in female and male mice[J].Biomed Res Int,2016,2016(4):6869452.
[13]HUANG Q,GAO B,JIE Q,et al.Ginsenoside-Rb2 displays anti-osteoporosis effects through reducing oxidative damage and bone-resorbing cytokines during osteogenesis[J].Bone,2014,66(9):306-314.
[14]国家药典委员会.中华人民共和国药典(第一部)[M].北京:化学工业出版社,2000:28.
[15]黄太康主编.常用中药成分与药理手册[M].北京:中国医用科技出版社,1994:410-413.