泼尼松龙通过抑制TAK1诱导成骨细胞凋亡的实验研究
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摘要
目的观察泼尼松龙通过抑制转化生长因子β激活激酶1(TAK1)表达对成骨细胞凋亡的影响。方法 2017年2月-2017年7月于长江航运总医院实验室进行实验,将M3T3-E1成骨细胞经原代培养后传代培养,将细胞分为3组,A组(正常组)、B组(阴性转染+泼尼松龙处理组)、C组(TAK1 siRNA转染+泼尼松龙处理组),采用碱性磷酸酶(ALP)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A组比较,B、C组细胞逐渐发生破裂,细胞数量逐渐减少,细胞内钙结节数量逐渐减少;B组细胞出现破碎、形态发生改变,C组破碎细胞数量明显增加。p-TAK1、p-JNK蛋白表达量A组>B组> C组(F/P=51.624/0. 000、21. 163/0. 000); A、B、C 3组细胞抑制率均随着时间的延长,细胞增值率呈现逐渐上升的趋势,其中48 h受抑制更显著。在12 h时,A组细胞抑制率明显高于B组和C组(q=5. 093、5. 821,P均<0. 05),在24 h、48 h、72 h时,细胞抑制率A组> B组> C组(F/P=74. 880/0. 000、117. 081/0. 000、116. 019/0. 000);3组处于G2期细胞比例C组> B组> A组(F/P=21.254/0. 000),处于S期细胞比例C组B组>A组(F/P=362.449/0.000)。结论沉默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。
Objective To observe the effect of prednisolone on osteoblast apoptosis by inhibiting the expression of transforming growth factor beta-activated kinase 1(TAK1). Methods The M3 T3 E1 osteoblasts were subcultured in the laboratory of Changjiang Shipping General Hospital from February 2017 to July 2017. The cells were divided into three groups:group A(normal group), group B(negative transfection + prednisolone treatment group), group C(TAK1 siRNA transfection + prednisolone treatment group). The differentiation of osteoblasts was evaluated by alkaline phosphatase(ALP) staining and calcium nodule staining. The expression of phosphorylated(p)-TAK1, TAK1, phosphorylated c jun amino terminal kinase(p-JNK) and JNK protein were detected by Western blot, the proliferation of M3 T3 E1 cells was detected by MTT, and the cell cycle and apoptosis were detected by flow cytometry. Results Compared with group A, cells in groups B and C gradually ruptured, the number of cells gradually decreased, and the number of intracellular calcium nodules gradually decreased.The cells in group B showed fragmentation and morphological changes, and the number of broken cells in group C increased significantly. p-TAK1, p-JNK protein expression level A group > B group > C group(F/P =51. 624/0. 000, F/P =21.163/0.000). The cell inhibition rates of group A, B and C all increased with time, and the cell proliferation rate showed a gradual increase trend, and 48 h was more significantly inhibited. At 12 h, the inhibition rate of group A was significantly higher than that of group B and group C(q =5.093,q=5. 821, P<0.05). At 24 h, 48 h, and 72 h, the cell inhibition rate was group A > group B > group C(F/P=74.880/0.000,F/P= 117.081/0.000,F/P = 116.019/0.000).The proportion of cells in group G2 was C group > B group > A group(F/P = 21.254/0. 000). The proportion of cells in the S phase was group C < group A < group B(F/P = 10.22/0.000). Apoptosis rate was group C > group B > group A(F/P = 362.449/0.000). Conclusions Silencing TAK1 expression can enhance the apoptosis of osteoblasts induced by prednisolone, which may be related to JNK signaling pathway.
Objective To observe the effect of prednisolone on osteoblast apoptosis by inhibiting the expression of transforming growth factor beta-activated kinase 1(TAK1). Methods The M3 T3 E1 osteoblasts were subcultured in the laboratory of Changjiang Shipping General Hospital from February 2017 to July 2017. The cells were divided into three groups:group A(normal group), group B(negative transfection + prednisolone treatment group), group C(TAK1 siRNA transfection + prednisolone treatment group). The differentiation of osteoblasts was evaluated by alkaline phosphatase(ALP) staining and calcium nodule staining. The expression of phosphorylated(p)-TAK1, TAK1, phosphorylated c jun amino terminal kinase(p-JNK) and JNK protein were detected by Western blot, the proliferation of M3 T3 E1 cells was detected by MTT, and the cell cycle and apoptosis were detected by flow cytometry. Results Compared with group A, cells in groups B and C gradually ruptured, the number of cells gradually decreased, and the number of intracellular calcium nodules gradually decreased.The cells in group B showed fragmentation and morphological changes, and the number of broken cells in group C increased significantly. p-TAK1, p-JNK protein expression level A group > B group > C group(F/P =51. 624/0. 000, F/P =21.163/0.000). The cell inhibition rates of group A, B and C all increased with time, and the cell proliferation rate showed a gradual increase trend, and 48 h was more significantly inhibited. At 12 h, the inhibition rate of group A was significantly higher than that of group B and group C(q =5.093,q=5. 821, P<0.05). At 24 h, 48 h, and 72 h, the cell inhibition rate was group A > group B > group C(F/P=74.880/0.000,F/P= 117.081/0.000,F/P = 116.019/0.000).The proportion of cells in group G2 was C group > B group > A group(F/P = 21.254/0. 000). The proportion of cells in the S phase was group C < group A < group B(F/P = 10.22/0.000). Apoptosis rate was group C > group B > group A(F/P = 362.449/0.000). Conclusions Silencing TAK1 expression can enhance the apoptosis of osteoblasts induced by prednisolone, which may be related to JNK signaling pathway.
引文
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[14]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. DOI:10. 3969/j. issn. 1672-5301.2012.12.015.
[15] Pathak S, Borodkin VS, Albarbarawi O, et al. O-GIcNAcylation of TABI modulates TAK1-mediated cytokine release[J]. Embo Journal, 2012, 31(6):1394-1404. DOI:10. 1038/emboj. 2012.8.
[16]王芳,江瑾,夏霖,等. PPARγ配体4i通过抑制NF-κB和MAPK信号通路抑制小鼠巨噬细胞炎性细胞因子的产生[J].细胞与分子免疫学杂志,2018, 34(6):23-28. DOI:10.13423/j.cnki. cjcmi. 008613.
[17]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-KB p65的影响[J].中国组织工程研究,2016, 20(46):6868-6877. DOI:10. 3969/j. issn. 2095-4344.2016.46.004.
[18]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. DOI:10. 3969.j. issn.1671-7856.2017.01.014.
[19] Zhang N, Wei G, Ye J, et al. Effect of curcumin on acute spinal cord injury in mice via inhibition of inflammation and TAK1 pathway[J]. Pharmacological Reports, 2017,69(5):1001-1006. DOI:10.1016/j. pharep.2017.02.012.
[20] 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[J]. Plos One, 2013, 8(4):e62087.DOI:10. 1371/journal. pone. 0062087.
[21] Zhang JX, Wei-Tan H, Hu CY, et al. Anticancer activity of 23,24-dihydrocucurbitacin B against the HeLa human cervical cell line is due to apoptosis and G2/M cell cycle arrest[J]. Experimental&Therapeutic Medicine, 2018, 15(3):2575-2582. DOI:10.3892/etm. 2018.5710.
[22]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. DOI:10. 3969/j. issn.1006-5725.2015.04.051.
[23]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. DOI:10. 13193/j. issn.1673-7717.2013.12.009.
[24] Hildebrandt S, Baschant U, Thiele S,et al. Glucocorticoids suppress Wnt16 expression in osteoblasts in vitro and in vivo[J]. Sci Rep,2018,8(1):8711. DOI:10. 1038/941598-018-26300-z.
[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.DOI:10.7661/CJIM.2015.08.1004.
[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. DOI:10. 19405/j. cnki. issn1000-1492. 2014. 03.016.
[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. DOI:10.3760/cma. j. issn. 1001-7097.2016.01.006.
[5]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(z1):114-115.DOI:10.3969/j.issn. 1002-1949.2016. z1. 089.
[6]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.DOI:10.3969/j.issn.1004-406X.2015.02.12.
[7] Chen Z, Xue J, Shen T, et al. Curcumin alleviates glucocorticoi d-induced osteoporosis by protecting osteoblasts from apoptosis in vivo and in vitro[J]. Clinical&Experimental Pharmacology&Physiology, 2016, 43(2):268-276. DOI:10. 1111/1440-1681.12513.
[8] Zanotti S, Yu J, Adhikari S,et al. Glucocorticoids inhibit notch target gene expression in osteoblasts[J]. J Cell Biochem, 2018,119(7):6016-6023. DOI:10.1002/jcb.26798.
[9]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.DOI:10.3969/j. issn. 1673-548X. 2012.03.035.
[10]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. DOI:10.3969/j. issn. 1006-7108.2014.09.024.
[11] 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[J]. Bone, 2011, 49(5):1080-1089.DOI:10.1016/j. bone. 2011.08.006.
[12] Suzuki A, Takayama T, Suzuki N, et al. Daily low-intensity pulsed ultrasound-mediated osteogenic differentiation in rat osteoblasts[J].Acta Biochim Biophys Sin(Shanghai), 2009,41(2):108-115.DOI:10.1002/jbm. b. 30583.
[13] Zhou C, Zhang X, Xu L, et al. Taurine promotes human mesenchymal stem cells to differentiate into osteoblast through the ERK pathway[J]. Amino Acids, 2014, 46(7):1673-1680. DOI:10. 1007/s00726-014-1729-8.
[14]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. DOI:10. 3969/j. issn. 1672-5301.2012.12.015.
[15] Pathak S, Borodkin VS, Albarbarawi O, et al. O-GIcNAcylation of TABI modulates TAK1-mediated cytokine release[J]. Embo Journal, 2012, 31(6):1394-1404. DOI:10. 1038/emboj. 2012.8.
[16]王芳,江瑾,夏霖,等. PPARγ配体4i通过抑制NF-κB和MAPK信号通路抑制小鼠巨噬细胞炎性细胞因子的产生[J].细胞与分子免疫学杂志,2018, 34(6):23-28. DOI:10.13423/j.cnki. cjcmi. 008613.
[17]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-KB p65的影响[J].中国组织工程研究,2016, 20(46):6868-6877. DOI:10. 3969/j. issn. 2095-4344.2016.46.004.
[18]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. DOI:10. 3969.j. issn.1671-7856.2017.01.014.
[19] Zhang N, Wei G, Ye J, et al. Effect of curcumin on acute spinal cord injury in mice via inhibition of inflammation and TAK1 pathway[J]. Pharmacological Reports, 2017,69(5):1001-1006. DOI:10.1016/j. pharep.2017.02.012.
[20] 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[J]. Plos One, 2013, 8(4):e62087.DOI:10. 1371/journal. pone. 0062087.
[21] Zhang JX, Wei-Tan H, Hu CY, et al. Anticancer activity of 23,24-dihydrocucurbitacin B against the HeLa human cervical cell line is due to apoptosis and G2/M cell cycle arrest[J]. Experimental&Therapeutic Medicine, 2018, 15(3):2575-2582. DOI:10.3892/etm. 2018.5710.
[22]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. DOI:10. 3969/j. issn.1006-5725.2015.04.051.
[23]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. DOI:10. 13193/j. issn.1673-7717.2013.12.009.
[24] Hildebrandt S, Baschant U, Thiele S,et al. Glucocorticoids suppress Wnt16 expression in osteoblasts in vitro and in vivo[J]. Sci Rep,2018,8(1):8711. DOI:10. 1038/941598-018-26300-z.