地塞米松通过线粒体途径诱导成骨细胞凋亡的研究
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摘要
目的观察地塞米松对离体成骨细胞凋亡的影响,探讨地塞米松对成骨细胞凋亡的分子作用机制。方法采用不同浓度地塞米松(10~(-8)、10~(-6)、10~(-4)mol/L)干预SD大鼠离体成骨细胞,DAPI染色观察细胞核形态,透射电镜观察细胞核及线粒体形态,流式细胞术检测细胞凋亡率,JC-1荧光探针检测线粒体跨膜电位。结果 10~(-8)、10~(-6)、10~(-4)mol/L地塞米松干预24 h后,DAPI染色发现,10~(-8)mol/L地塞米松组的成骨细胞很少发生凋亡,10~(-6)mol/L和10~(-4)mol/L地塞米松组的成骨细胞凋亡明显,地塞米松的浓度越高,核固缩、核裂解等凋亡现象越明显;透射电镜观察发现,随着地塞米松浓度增加,细胞核固缩明显,线粒体肿胀、空泡样变化现象增多;成骨细胞的凋亡率随着地塞米松浓度的增加而逐渐增高,与空白对照组相比,10~(-8)mol/L地塞米松组细胞凋亡率无明显升高(P>0.05),10~(-6) mol/L和10~(-4)mol/L地塞米松组的成骨细胞凋亡率分别较空白对照组增加7.240%和31.173%(P<0.05);随着地塞米松浓度的增加,线粒体膜电位逐渐减低,与空白对照组相比较,10~(-8)mol/L地塞米松组细胞膜电位下降不明显(P>0.05),10~(-6) mol/L和10~(-4)mol/L地塞米松组膜电位下降分别较空白对照组降低6.814%和17.846%(P<0.05)。结论地塞米松通过激活线粒体途径诱导成骨细胞凋亡,存在浓度依赖性。
Objective To investigate the effect of dexamethasone(Dex) induced apoptosis of osteoblasts in vitro and to explore the molecular mechanism of dexamethasone in the apoptosis of osteoblasts. Methods Different concentrations of dexamethasone(10~(-8) mol/L, 10~(-6) mol/L, 10~(-4)mol/L) were used in the isolated osteoblasts of SD rats. The nucleus morphology was observed by DAPI staining. The morphology of nuclei and mitochondria was observed by transmission electron microscopy. The apoptosis rate of osteoblasts was detected by flow cytometry, and the mitochondrial transmembrane potential was detected by JC-1 fluorescence probe. Results After intervention for 24 hours using 10~(-8) mol/L, 10~(-6) mol/L, and 10~(-4)mol/L dexamethasone, DAPI staining showed that the osteoblasts of 10~(-8)mol/L dexamethasone group were rarely apoptotic, and the apoptosis of osteoblasts in 10~(-6)mol/L and 10~(-4)mol/L dexamethasone groups was obvious, the higher the concentration of dexamethasone, the more obvious apoptotic phenomena, such as nuclear condensation and nuclear cracking. The transmission electron microscopy showed that with the increase of dexamethasone concentration, the nuclear condensation was obvious, the swelling and vacuolar changes of mitochondria increased. And the apoptosis rate of osteoblasts gradually increased with the increase of dexamethasone concentration. Compared with the blank control group, the apoptosis rate of 10~(-8)mol/L dexamethasone group was not significantly changed(P>0.05). The apoptosis rate of osteoblasts in 10~(-6 )mol/L and 10~(-4)mol/L dexamethasone groups was increased by 7.240% and 31.173% respectively(P<0.05). With the increase of dexamethasone concentration, the mitochondrial membrane potential decreased gradually. Compared with the blank control group, the decrease of the cell membrane potential of 10~(-8)mol/L dexamethasone group was not obvious(P>0.05), The membrane potential of 10~(-6 )mol/L and 10~(-4)mol/L dexamethasone groups decreased by 6.814% and 17.846% respectively(P<0.05). Conclusion Dexamethasone induces apoptosis of osteoblasts through activation of mitochondrial pathway in a concentration dependent manner.
Objective To investigate the effect of dexamethasone(Dex) induced apoptosis of osteoblasts in vitro and to explore the molecular mechanism of dexamethasone in the apoptosis of osteoblasts. Methods Different concentrations of dexamethasone(10~(-8) mol/L, 10~(-6) mol/L, 10~(-4)mol/L) were used in the isolated osteoblasts of SD rats. The nucleus morphology was observed by DAPI staining. The morphology of nuclei and mitochondria was observed by transmission electron microscopy. The apoptosis rate of osteoblasts was detected by flow cytometry, and the mitochondrial transmembrane potential was detected by JC-1 fluorescence probe. Results After intervention for 24 hours using 10~(-8) mol/L, 10~(-6) mol/L, and 10~(-4)mol/L dexamethasone, DAPI staining showed that the osteoblasts of 10~(-8)mol/L dexamethasone group were rarely apoptotic, and the apoptosis of osteoblasts in 10~(-6)mol/L and 10~(-4)mol/L dexamethasone groups was obvious, the higher the concentration of dexamethasone, the more obvious apoptotic phenomena, such as nuclear condensation and nuclear cracking. The transmission electron microscopy showed that with the increase of dexamethasone concentration, the nuclear condensation was obvious, the swelling and vacuolar changes of mitochondria increased. And the apoptosis rate of osteoblasts gradually increased with the increase of dexamethasone concentration. Compared with the blank control group, the apoptosis rate of 10~(-8)mol/L dexamethasone group was not significantly changed(P>0.05). The apoptosis rate of osteoblasts in 10~(-6 )mol/L and 10~(-4)mol/L dexamethasone groups was increased by 7.240% and 31.173% respectively(P<0.05). With the increase of dexamethasone concentration, the mitochondrial membrane potential decreased gradually. Compared with the blank control group, the decrease of the cell membrane potential of 10~(-8)mol/L dexamethasone group was not obvious(P>0.05), The membrane potential of 10~(-6 )mol/L and 10~(-4)mol/L dexamethasone groups decreased by 6.814% and 17.846% respectively(P<0.05). Conclusion Dexamethasone induces apoptosis of osteoblasts through activation of mitochondrial pathway in a concentration dependent manner.
引文
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[14] Liu W, Zhao Z, Na Y, et al. Dexamethasone-induced production of reactive oxygen species promotes apoptosis via endoplasmic reticulum stress and autophagy in MC3T3-E1 cells[J]. Int J Molecul Med,2018,41(4):2028-2036.
[15] Ding DW, Zhang YH, Huang XE, et al. Bufalin induces mitochondrial pathway-mediated apoptosis in lung adenocarcinoma cells[J]. APJCP,2014,15(23):10495-10500.
[16] Mane SD, Thoh M, Sharma D, et al. Ascorbyl Stearate Promotes Apoptosis Through Intrinsic Mitochondrial Pathway in HeLa Cancer Cell[J]. Anticancer Res,2016,36(12):6409-6417.
[17] Qiu Y, Yu T, Wang W, et al. Curcumin-induced melanoma cell death is associated with mitochondrial permeability transition pore (MPTP) opening[J]. Biochem Biophys Res Communicat,2014,448(1):15-21.
[18] 赵硕,李成.糖皮质激素通过线粒体途径诱导成骨细胞凋亡[J].中国生化药物杂志, 2015, 35(7): 35-38.
[2] Moriishi T, Komori T. Glucocorticoid and Bone The inhibition of osteoblast differentiation and induction of osteocyte apoptosis through the regulation of Bcl-2 by glucocorticoids [J]. Clin Calcium,2014,24(9):1329-1336.
[3] Zhang JY, Yi T, Liu J, et al. Quercetin induces apoptosis via the mitochondrial pathway in KB and KBv200 cells[J].J Agricult Food Chem,2013,61(9):2188-2195.
[4] 夏正坤.糖皮质激素临床应用再认识[J].医学研究生学报,2018,31(2):113-117.
[5] Overman RA, Yeh JY. Prevalence of oral glucocorticoid usage in the United States: a general population perspective[J].Arthritis Care Res,2013,65(2):294-298.
[6] Yang J, Wu Q, Lv J. 4-Phenyl butyric acid prevents glucocorticoid-induced osteoblast apoptosis by attenuating endoplasmic reticulum stress[J]. J Bone Mineral Metabol,2017,35(4):366-374.
[7] Lin H, Gao X, Chen G, et al. Indole-3-carbinol as inhibitors of glucocorticoid-induced apoptosis in osteoblastic cells through blocking ROS-mediated Nrf2 pathway[J]. Biochem Biophys Res Communicat,2015,460(2):422-427.
[8] 吴丽婷,蔡劲薇,潘吉铭,等.不同剂量和作用时间地塞米松对大鼠骨密度和成骨细胞作用的研究[J].中国骨质疏松杂志,2018,24(6):762-768.
[9] Yu W, Zhu C, Xu W, et al. Neuropeptide Y1 receptor regulates glucocorticoid-induced inhibition of osteoblast differentiation in murine MC3T3-E1 cells via ERK signaling[J]. Int J Molecul Sci,2016,17(12):1-13.
[10] Ding S, Peng H, Fang HS, et al. Pulsed electromagnetic fields stimulation prevents steroid-induced osteonecrosis in rats[J]. BMC Musculoskel Disorders,2011 (12):215.
[11] Zhou C, Yu C, Guo L, et al. Study of the Effects of ER on Apoptosis and Proliferation of Hormone-Independent Prostate Cancer Cell Lines PC-3M[J]. Bio Med Res Int,2018,6(19):1-10.
[12] Kerr JF, Wyllie AH. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics[J]. Br J Cancer,1972,26(4):239-257.
[13] Won SJ, Ki YS, Chung KS, et al.3α,23-isopropylidenedioxyolean-12-en-27-oic acid, a triterpene isolated from Aceriphyllum rossii, induces apoptosis in human cervical cancer HeLa cells through mitochondrial dysfunction and endoplasmic reticulum stress[J]. Bio Pharmaceut Bulletin,2010,33(9):1620-1626.
[14] Liu W, Zhao Z, Na Y, et al. Dexamethasone-induced production of reactive oxygen species promotes apoptosis via endoplasmic reticulum stress and autophagy in MC3T3-E1 cells[J]. Int J Molecul Med,2018,41(4):2028-2036.
[15] Ding DW, Zhang YH, Huang XE, et al. Bufalin induces mitochondrial pathway-mediated apoptosis in lung adenocarcinoma cells[J]. APJCP,2014,15(23):10495-10500.
[16] Mane SD, Thoh M, Sharma D, et al. Ascorbyl Stearate Promotes Apoptosis Through Intrinsic Mitochondrial Pathway in HeLa Cancer Cell[J]. Anticancer Res,2016,36(12):6409-6417.
[17] Qiu Y, Yu T, Wang W, et al. Curcumin-induced melanoma cell death is associated with mitochondrial permeability transition pore (MPTP) opening[J]. Biochem Biophys Res Communicat,2014,448(1):15-21.
[18] 赵硕,李成.糖皮质激素通过线粒体途径诱导成骨细胞凋亡[J].中国生化药物杂志, 2015, 35(7): 35-38.