泼尼松龙可抑制转化生长因子β激活激酶1诱导的成骨细胞凋亡
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摘要
背景:研究显示转化生长因子β激活激酶1(transforming growth factor beta activated kinase 1,TAK1)在骨、关节的发育以及骨形态信号转导中发挥着重要作用,与骨关节炎的发生也存在一定的相关性。目的:探究泼尼松龙通过抑制TAK1表达对诱导成骨细胞凋亡的影响。方法:将M3T3-E1成骨细胞经原代培养后传代培养。取第3代细胞分为3组,正常细胞组(对照组)、阴性转染+泼尼松龙组、TAK1 siRNA转染+泼尼松龙组。采用碱性磷酸酶染色和钙结节染色评估成骨细胞分化能力的变化;采用免疫印迹法(Western blot)检测细胞内磷酸化(p)-TAK1、TAK1、磷酸化c-jun氨基末端激酶(p-JNK)、JNK蛋白表达,MTT法检测M3T3-E1细胞增殖情况;流式细胞仪检测细胞周期以及细胞凋亡变化。结果与结论:①TAK1 siRNA转染+泼尼松龙组细胞的碱性磷酸酶红染程度较少,阴性转染+泼尼松龙组次之,正常细胞组碱性磷酸酶染色最多;钙结节染色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最少;②荧光显微镜显示,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数量明显增加;③Western Blot显示,3组间p-TAK1、p-JNK蛋白表达量逐渐降低(P <0.05);④MTT检测显示,3组间TAK1 siRNA转染+泼尼松龙组细胞增殖抑制率最高(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;⑤流式细胞仪检测结果显示,TAK1 siRNA转染+泼尼松龙组G_2期细胞比例高于其他2组,S期细胞比例显著低于其他2组(P <0.05);⑥TAK1 siRNA转染+泼尼松龙组细胞凋亡率明显高于正常细胞组、阴性转染+泼尼松龙组(P <0.05);⑦结果说明,沉默TAK1表达后能够增强泼尼松龙诱导成骨细胞凋亡的作用,可能与JNK信号通路相关。
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 G_2 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.
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 G_2 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.
引文
[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-βmediated 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.
[26]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]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-βmediated 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.
[26]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.