银杏叶提取物对破骨细胞分化和骨吸收的作用及其机制

doi:10.13481/j.1671-587x.20170612

摘要/Abstract

摘要： 目的：探讨不同浓度银杏叶提取物（GBE）对破骨胞分化和骨吸收的作用，并阐明其作用机制。方法：体外培养RAW264.7细胞，采用核因子κB受体活化因子配体（RANKL）和不同浓度GBE处理细胞，分为空白对照组（0μg·L^-1 RANKL）、RANKL组（100 μg·L^-1RANKL）和RANKL+75mg·L^-1 GBE和RANKL+150 mg·L^-1 GBE组。抗酒石酸酸性染色（TRAP）法观察各组破骨细胞的形态及数量，骨吸收陷窝面积评估GBE对破骨细胞骨吸收能力的影响，流式细胞术检测细胞凋亡率及细胞周期，RT-PCR法检测RAW264.7细胞中破骨细胞相关基因活化T细胞核因子c1（NFATc1）、树突状细胞特异性跨膜蛋白（DC-STAMP）、组织蛋白酶K （Cathepsin K）、基质金属蛋白酶9（MMP-9）、B淋巴细胞瘤2（Bcl-2）、Bcl-2相关X蛋白（Bax）、P27和细胞周期蛋白D1（Cyclin-D1）的表达水平。结果：与空白对照组比较，RANKL组破骨细胞的数量明显增加（P < 0.05）；与RANKL组比较，RANKL+75 mg·L^-1 GBE和RANKL+150 mg·L^-1 GBE组破骨细胞数量明显降低（P < 0.05）。与空白对照组比较，RANKL组骨片中骨吸收陷窝面积明显升高（P < 0.05）；与RANKL组比较，RANKL+75 mg·L^-1 GBE和RANKL+150 mg·L^-1 GBE组骨片中骨吸收陷窝面积明显降低（P < 0.05）。与空白对照组比较，75和150 mg·L^-1 GBE组RAW264.7细胞凋亡率升高（P < 0.05），RAW264.7细胞中Bcl-2基因表达水平明显降低（P < 0.05），Bax基因表达水平明显升高（P < 0.05）。与RANKL组比较，RANKL+75 mg·L^-1 GBE和RANKL+150 mg·L^-1 GBE组RAW264.7细胞G₀-G₁期阻滞明显缩短（P < 0.05）；RAW264.7细胞中P27基因表达水平明显降低（P < 0.05），Cyclin-D1基因表达水平明显升高（P < 0.05）。与空白对照组比较，RANKL组RAW264.7细胞中破骨细胞相关基因NFATc1、DC-STAMP、Cathepsin K和MMP-9表达水平明显升高（P < 0.05）；与RANKL组比较，RANKL+75 mg·L^-1 GBE和RANKL+150 mg·L^-1 GBE组RAW264.7细胞中破骨细胞相关基因NFATc1、DC-STAMP、Cathepsin K和MMP-9表达水平明显降低（P < 0.05）。结论：GBE可抑制破骨细胞分化和骨吸收能力，其机制可能与促进RAW264.7细胞凋亡、缩短RANKL诱导的RAW264.7细胞的G₀-G₁期有关。

关键词: 银杏叶提取物, 破骨细胞, 细胞周期, 细胞凋亡

Abstract: Objective:To study the effects of Ginkgo Biloba extract(GBE) on the differentiation and bone resorptionof the osteoclasts, and to clarify their mechanisms. Methods:The RAW264.7 cells were cultured in vitro, then were treated with receptor activator for nuclear factor-κB ligand (RANKL) and different concentrations of GBE. The cells were divided into blank control group(0 μg·L^-1 RANKL), RANKL group(100 μg·L^-1 RANKL), RANKL+75 mg·L^-1 GBE group,and RANKL+150 mg·L^-1 GBE group.The morphology and number of osteoclasts were assessed with TRAP staining assay, and bone resorption of GBE was examined with bone resorption pits assay. Flow cytometry was applied to analyze the the apoptotic rate of RAW264.7 cells and the cell cycle;the expression levels of nuclear factor of activated T cells 1 (NFATc1), DC-STAMP, Casthepin K,matrix metalloprotein 9(MMP-9), Bcl-2, Bax,P27 and Cyclin-D1 in the RAW264.7 cells were analyzed by RT-PCR method. Results:Compared with blank control group, the number of osteoclasts in RANKL group were significantly increased(P<0.01); compared with RANKL group,the number of osteoclasts in RANKL+75 mg·L^-1 GBE and RANKL+150 mg·L^-1 GBE groups were significantly decreased (P<0.05). Compared with blank control group,the area of bone resorption pit of bone slice in RANKL group was significantly increased(P<0.05);compared with RANKL group, the areas of bone resorption pit of bone slice in RANKL+75 mg·L^-1 GBE and RANK+150 mg·L^-1 GBE groups were significantly decreased (P<0.05). Compared with blank control group, the apoptotic rates of the RAW264.7 cells in 75 and 150 mg·L^-1 GBE groups were increased, and the expression levels of Bcl-2 in the RAW264.7 cells were significantly decreased and the expression levels of Bax were significantly increased (P<0.05). Compared with RANKL group, the G₀-G₁ phase arrest of the RAW264.7 cells in RANKL+75 mg·L^-1 GBE and RANKL 150 mg·L^-1 GBE groups were shortened;the expression levels of P27 in the RAW264.7 cells were significantly decreased and the expression levels of Cyclin-D1 were significantly increased (P<0.05). Compared with blank control group, the expression levels of NFATc1, DC-STAMP, Casthepin K and MMP-9 in the RAW264.7 cells in RANKL group were significantly increased(P<0.05); compared with RANKL group, the expression levels of NFATc1, DC-STAMP, Casthepin K and MMP-9 in the RAW264.7 cells in RANKL+75 mg·L^-1 GBE and RANKL+150 mg·L^-1 GBE groups were significantly decreased (P<0.05). Conclusion:GBE could inhibit the differentiation and bone resorption of the osteoclasts, and their mechanisms may be related to promoting the apoptosis of RAW264.7 and shortening the G₀-G₁ phase of the RAW264.7 cells.

Key words: Ginkgo Biloba extract, osteoclast, cell cycle, apoptosis

中图分类号:

R329.28

王俊妹, 孙千月, 吴哲, 李彤, 李婧. 银杏叶提取物对破骨细胞分化和骨吸收的作用及其机制[J]. 吉林大学学报(医学版), 2017, 43(06): 1130-1136.

WANG Junmei, SUN Qinyue, WU Zhe, LI Tong, LI Jing. Effects of Ginkgo Bitoba extract on differentiation and bone resorption ofosteoclasts and their mechanisms[J]. Journal of Jilin University Medicine Edition, 2017, 43(06): 1130-1136.

参考文献

[1] Chrcanovic BR, Albrektsson T, Wennerberg A. Bone quality and quantity and dental implant failure:a systematic review and meta-analysis[J].Int J Prosthodont,2017,30(3):219-237.
[2] Feihl S, Renz N, Schutz M, et al. Antibiotic strategies in trauma surgery:Treatment of implant-associated infections[J].Unfallchirurg,2017,120(6):486-493.
[3] Mungenast F, Aust S, Verqote L, et al. Clinical significance of the estrogen-modifying enzymes steroid sulfatase and estrogen sulfotransferase in epithelial ovarian cancer[J].Oncol Lett,2017,13(6):4047-4054.
[4] Gross C, Weber M, Creutzburg K, et al. Osteoclast profile of medication-related osteonecrosis of the jaw secondary to bisphosphonate therapy:a comparison with osteoradionecrosis and osteomyelitis[J].J Transl Med,2017,15(1):128-130.
[5] Zhou X, Qi Y, Chen T. Long-term pre-treatment of antioxidant Ginkgo biloba extract EGb-761 attenuates cerebral-ischemia-induced neuronal damage in aged mice[J].Biomed Pharmacother,2017,85(1):256-263.
[6] Guzel N,Sayit E,Aynaci O, et al. Ginkgo Biloba improves bone formation during fracture healing:an experimental study in rats[J]. Acta Ortop Bras, 2017,25(3):95-98.
[7] 张甲弟,时舒曼,张海燕,等.银杏叶提取物对骨髓间充质干细胞增殖及成骨分化的影响[J].口腔医学研究,2015,31(6):575-579.
[8] 时舒曼,袁浩天,张晓晓,等.银杏叶提取物诱导骨髓间充质干细胞成骨分化过程中P38信号通路的研究[J].口腔医学研究,2016,32(9):912-915.
[9] Matsuo K, Irie N. Osteoclast-osteoblast communication[J].Arch Biochem Biophys,2008,473(2):201-209.
[10] Zeng XZ, He LG, Wang S, et al. Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-κB and NFATc1 activation and DC-STAMP expression[J].APS, 2016, 37(2):255-263.
[11] Walsh MC, Kim N, Kadono Y, et al. Osteoimmunology:interplay between the immune system and bone metabolism[J]. Annu Rev Immunol,2006,24:33-63.
[12] Takayanagi H. Osteoimmunology:shared mechanisms and crosstalk between the immune and bone systems[J].Nat Rev Immunol,2007,7(4):292-304.
[13] Negishi-Koga T, Takayanagi H. Ca²⁺-NFATc1 singnaling is an essential axis of osteoclast differentiation[J]. Immunol Rev,2009,231(1):241-256.
[14] Wisitrasameewong W, Kajiya M, Movila A, et al. DC-STAMP is an osteoclast fusogen engaged in periodontal bone resorption[J].J Dent Res,2017,96(6):685-693.
[15] Nagase Y, Iwasawa M, AkiyamaT, et al. Anti-apoptotic molecule Bcl-2 regulates the differentiation, activation, and survival of both osteoblasts and osteoclasts[J].J Biol Chem,2009,284(52):366659-366669.
[16] Kwon M,Kim JM,LecK, et al. Synchronized cell cycle arrest promotes osteoclast differentiation[J].MolSci,2016,17(8):1292-1300.
[17] Li A, Wang J, Wu M, et al. The inhibition of activated hepatic stellate cells proliferation by arctigenin through G₀/G₁ phase cell cycle arrest:persistent p27(Kip1) induction by interfering with PI3K/Akt/FOXO3a signaling pathway[J].Eur J Pharmacol,2015,747:71-87.
[18] 章振林,夏维波,金小岚,等.骨质疏松诊治进展及分级诊疗制度之讨论[J].中国实用内科杂志,2016,36(11):928-934.
[19] 赵兰岚,姚军,高莹,等.促甲状腺激素抑制治疗及其与分化型甲状腺癌骨代谢相关性研究进展[J].中国实用内科杂志,2017,37(2):162-165.