Prednisolone inhibites osteoblast apoptosis induced by transforming growth factor beta activated kinase 1

doi:10.3969/j.issn.2095-4344.1306

Abstract

Abstract:

BACKGROUND: Transforming growth factor beta activated kinase 1 (TAK1) has been shown to play important roles in development of bone and joint and bone morphology signal transduction, and is related to osteoarthritis.
OBJECTIVE: To explore the influence of prednisolone on osteoblast apoptosis induced by inhibiting TAK1 expression.
METHODS: M3T3-E1 osteoblasts were cultured and passaged. The third generation of cells were divided into three groups: control group (normal group), negative transfection + prednisolone group, and TAK1 siRNA transfection + prednisolone group. The changes of osteoblast differentiation ability were evaluated by alkaline phosphatase staining and calcium nodule staining. Intracellular phosphorylation (p)-TAK1, and TAK1, phosphorylated c-jun amino, terminal kinase (p-JNK), and JNK protein expression levels were detected by western blot assay. MTT assay was used for M3T3-E1 cell proliferation. Flow cytometry was used to detect the cell cycle and apoptosis.
RESULTS AND CONCLUSION: (1) Alkaline phosphatase staining showed that the number of stained cells in the TAK1 siRNA transfection + prednisolone group was least, followed by negative transfection + prednisolone group, and most in the control group. Calcium nodule staining showed that the number of intracellular calcium nodules in the negative transfection + prednisolone and TAK1 siRNA transfection + prednisolone groups decreased compared with the control group, especially the TAK1 siRNA transfection + prednisolone group. (2) Fluorescence microscope showed that the cells in the negative transfection + prednisolone group were broken and the morphology changed. The number of broken cells in the TAK1 siRNA transfection + prednisolone group increased significantly. (3) Western blot assay showed that the expression levels of p-TAK1 and p-JNK protein decreased gradually (P < 0.05). (4) MTT assay showed that TAK1 siRNA transfection + prednisolone group had highest cell inhibition rate (P < 0.05). Within 12 to 48 hours, the cell inhibition rate was on a rise, and then decreased at 72 hours. (5) The results of flow cytometry showed that the proportion of G2 phase cells in the TAK1 siRNA transfection + prednisolone group was higher than that in the other two groups, and the proportion of S phase cells was lower than that in the other two groups (P < 0.05). (6) The apoptosis rate in the TAK1 siRNA transfection + prednisolone group was higher than that in the other two groups (P < 0.05). (7) These results suggest that silencing TAK1 expression can increase the apoptosis of osteoblasts induced by prednisolone, which may be related to JNK signaling pathway.

Key words: transforming growth factor beta activated kinase 1, prednisolone, osteoblast, apoptosis, JNK signaling pathway, TAK1, gene silencing

CLC Number:

R446

Zhang Wen, Zhang Lei, Ren Shouzhong, Liu Risheng . Prednisolone inhibites osteoblast apoptosis induced by transforming growth factor beta activated kinase 1[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(23): 3630-3635.

Figures/Tables 1

2.1 M3T3-E1细胞碱性磷酸酶染色、钙结节染色结果通过大体拍摄采集图片和显微镜下观察，发现不同培养方式处理的细胞其组间碱性磷酸酶染色程度有很大差异，主要表现为：稳定沉默TAK1表达的TAK1 siRNA转染+泼尼松龙组细胞的碱性磷酸酶红染程度较少，仅有约10%的部分细胞可呈现出阳性表达；阴性转染+泼尼松龙组细胞的红染现象明显，阳性率可高达20%；而正常细胞组碱性磷酸酶染色最多，可达40%。将钙化结节所占面积比例量化显示，与正常细胞组相比，阴性转染+泼尼松龙组钙结节数量明显减少，TAK1 siRNA转染+泼尼松龙组结节数量少于阴性转染+泼尼松龙组。免疫荧光检测，阴性转染+泼尼松龙组细胞出现破碎，形态发生改变，TAK1 siRNA转染+泼尼松龙组破碎细胞明显增加。见图1。 2.2 M3T3-E1细胞中p-TAK1、TAK1、p-JNK、JNK 蛋白的表达 Western Blot显示，正常细胞组、阴性转染+泼尼松龙组、TAK1 siRNA转染+泼尼松龙组3组间TAK1、JNK蛋白表达量并无明显变化，差异无显著性意义(P > 0.05)。p-TAK1、p-JNK 蛋白表达量3组间逐渐降低，与正常细胞组相比，阴性转染+泼尼松龙组p-TAK1、p-JNK 蛋白表达量降低，TAK1 siRNA转染+泼尼松龙组蛋白表达量低于阴性转染+泼尼松龙组(P < 0.05)。见图2。 2.3 沉默TAK1后泼尼松龙对M3T3-E1细胞增殖的影响 MTT检测显示，与正常细胞组相比，阴性转染+泼尼松龙组和TAK1 siRNA转染+泼尼松龙组细胞增殖抑制率均有所升高，且TAK1 siRNA转染+泼尼松龙组升高更为明显(P < 0.05)；在12-48 h随着时间的延长，细胞增殖抑制率呈逐渐上升趋势，在72 h时开始下降。见图3。 2.4 沉默TAK1后泼尼松龙对M3T3-E1细胞周期的影响流式细胞仪检测结果显示，TAK1 siRNA转染+泼尼松龙组处于G2期细胞比例明显高于正常细胞组、阴性转染+泼尼松龙组[(15.34±4.66)%，(8.23±2.64)%，(4.23±1.64)%]，处于S期细胞比例明显低于正常细胞组、阴性转染+泼尼松龙组[(2.18±0.45)%，(3.56±1.03)%，(4.73±0.87)%，P < 0.05]。见图4。 2.5 沉默TAK1后泼尼松龙对M3T3-E1细胞凋亡的影响流式细胞仪检测显示左下象限为正常细胞，右下象限代表晚期凋亡细胞，右上象限代表早期凋亡细胞，正常细胞组中细胞凋亡不明显，阴性转染+泼尼松龙组细胞少量出现细胞凋亡，TAK1 siRNA转染+泼尼松龙组凋亡细胞数量明显增多。TAK1 siRNA转染+泼尼松龙组细胞凋亡率[(33.64±6.37)%]显著高于正常细胞组、阴性转染+泼尼松龙组[(13.16± 0.87)%，(3.83±0.76)%，P < 0.05]。见图5。"

References

[1]韩亚军,帖小佳,伊力哈木•托合提.中国中老年人骨质疏松症患病率的Meta分析[J].中国组织工程研究, 2014,18(7):1129-1134.

[2]许争光,杨峻,吕志平,等. 芫花根醇提颗粒联合甲基泼尼松龙对脊髓损伤大鼠BDNF、NMDA表达及行为学的影响[J].中国中西医结合杂志, 2015,35(8):1004-1010.

[3]张琪琪,胡勇,周定,等.ADAMTS-4与TAK1在骨关节炎软骨组织中表达的相关性研究[J].安徽医科大学学报, 2014,49(3): 343-346.

[4]冯世尧,徐兴欣,邵云侠,等. TAK1信号通路在高糖诱导骨髓来源巨噬细胞激活中的作用[J].中华肾脏病杂志, 2016,32(1):37-42.

[5]Ismail HM,Didangelos A,Vincent TL,et al.Articular cartilage injury rapidly activates TAK1 in chondrocytes by inducing its phosphorylation and K63-polyubiquitination. Arthritis Rheumatol. 2017;69(3):565-575.

[6]程锦,胡晓青,段小宁,等.转化生长因子β活化激酶1诱导软骨细胞发生骨关节炎相关病变机制[J].中国生物化学与分子生物学报, 2016, 32(12):1326-1333.

[7]宋志刚. 不同剂量甲基泼尼松龙治疗急性脊髓炎的效果对比[J].中国急救医学, 2016, 36(1):114-115.

[8]崔健超,杨志东, 江晓兵,等.泼尼松龙与地塞米松介导腰椎骨量降低的差异及其对成骨成脂基因表达的影响[J].中国脊柱脊髓杂志, 2015, 25(2):168-173.

[9]Chen Z, Xue J, Shen T, et al. Curcumin alleviates glucocorticoid-induced osteoporosis by protecting osteoblasts from apoptosis in vivo and in vitro. Clin Exp Pharmacol Physiol.2016;43(2):268-276.

[10]袁赤亭,朱敏,范利荣,等.糖皮质激素受体在3种人成骨肉瘤细胞株MG63、U2-OS及HOS中表达及差异[J].医学研究杂志, 2014, 43(11):144-146.

[11]粱军波,徐春丽,潘伟波,等.高血糖对成骨细胞增殖分化影响的实验研究[J].医学研究杂志,2012, 41(3):117-120.

[12]任辉,魏秋实,江晓兵,等.糖皮质激素性骨质疏松的研究新进展[J].中国骨质疏松杂志, 2014,20(9):1138-1142.

[13]Wang FS,Wu RW,Ko JY,et al.Heat shock protein 60 protects skeletal tissue against glucocorticoid-induced bone mass loss by regulating osteoblast survival. Bone. 2011;49(5): 1080-1089.

[14]Suzuki A, Takayama T, Suzuki N, et al.Daily low-intensity pulsed ultrasound-mediated osteogenic differentiation in rat osteoblasts. Acta Biochim Biophys Sin (Shanghai). 2009; 41(2):108-115.

[15]Zhou C,Zhang X,Xu L,et al.Taurine promotes human mesenchymal stem cells to differentiate into osteoblast through the ERK pathway. Amino Acids, 2014, 46(7): 1673-1680.

[16]刘化刚,王春,谷天祥,等. 转化生长因子-β1/TGFp激活激酶信号表达变化在心房颤动心肌纤维化中的作用[J].中国心血管病研究, 2012, 10(12):932-936.

[17]Pathak S,Borodkin VS,Albarbarawi O,et al.O -GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release. EMBO J. 2012;31(6):1394-1404.

[18]潘建科,曹学伟,刘军,等.龙鳖胶囊对骨关节炎软骨细胞p38MAPK信号通路及NF-κB p65的影响[J].中国组织工程研究, 2016, 20(46):6868-6877.

[19]欧阳春,张里克,卢远航,等. TAK1抑制剂对糖尿病大鼠MAPK与NF-κB信号通路的影响及其对肾脏保护机制[J]. 中国比较医学杂志, 2017, 27(1):67-72.

[20]Kerzic PJ, Pyatt DW, Zheng JH, et al.Inhibition of NF-kappaB by hydroquinone sensitizes human bone marrow progenitor cells to TNF-alpha-induced apoptosis.Toxicology. 2003;187(3): 127-137.

[21]Duan J,Yu Y,Yang L,et al.Toxic Effect of Silica Nanoparticles on Endothelial Cells through DNA Damage Response via Chk1-Dependent G2/M Checkpoint. Plos One.2013; 8(4): 62087-62093.

[22]Ji Y,Dou Y,Zhao Q,et al.Paeoniflorin suppresses TGF-β mediated epithelial-mesenchymal transition in pulmonary fibrosis through a Smad-dependent pathway.Acta Pharmacologica Sinica.2016; 37(6):794-804.

[23]Liao PC, Lieu CH. Cell cycle specific induction of apoptosis and necrosis by paclitaxel in the leukemic U937 cells. Life Sciences.2005; 76(14):1623-1639.

[24]袁芳,何晓瑾,邱亦江,等. 骨关节炎的软骨细胞凋亡机制[J]. 实用医学杂志, 2015, 31(4):666-668.

[25]徐晓东,邓洋洋,郑洪新. 糖皮质激素诱导肾虚骨质疏松症大鼠骨组织中OPG/RANKLmRAN及蛋白表达的影响[J].中华中医药学刊, 2013,31(12):2623-2627.

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.