辛伐他汀对肿瘤坏死因子α及放线菌素D诱导的MG63凋亡的保护作用及可能机制
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摘要
【目的】：探讨辛伐他汀对肿瘤坏死因子α(TNF-a)及放线菌素D(ActD)诱导的MG63凋亡的影响及可能的作用机制。
【方法】：人成骨样细胞(MG63)用DMEM+10%胎牛血清培养,分别用不同浓度的辛伐他汀、TNF-a、TNF-a+ActD作用于MG63,观察对细胞活性的影响,采用合适的浓度诱导细胞损伤,制作凋亡模型,将实验细胞分成5组,对照组,TNF-a+ActD组,另3组采用不同浓度的辛伐他汀(10-7—10-9mol／l)对MG63进行预处理,再加入TNF-a+ActD,用MTs比色法测定各种药物对细胞活性的影响,光镜下观察细胞形态的改变及典型的凋亡小体和细胞碎片,收集凋亡细胞进行蛋白裂解后行western blotting检测细胞凋亡途径。
[结果]
(1)TNF-a单独对MG63作用24小时细胞活性无明显减低。
(2)10-7—10-10mol／l浓度的辛伐他汀对MG63作用24小时后细胞活性无明显减低,但高浓度组(10-3—10-5mol／l)。细胞活性明显减低
(3)TNF-a对放线茵素D致敏的MG63细胞有明显的细胞毒作用,随着TNF-a浓度的增加,细胞活性明显减低。
(4)辛伐他汀可以抑制TNF-a／ActD诱导的人成骨样细胞(MG63)凋亡,经辛伐他汀(10-7—10-9mol／l)预处理后再与TNF-a／ActD作用,细胞活性较未加辛伐他汀有明显增加。在一定浓度范围内呈剂量依赖性,10-7mol／l组细胞活性增加较明显。光镜下观察凋亡小体和致密碎片核有明显减少。
(5)辛伐他汀通过激活AKT和抑制NF-KB途径,来抑制TNF-a／ActD诱导的人成骨样细胞(MG63)凋亡。
【结论】TNF-a能诱导ActD致敏的MG63细胞发生凋亡,而辛伐他汀则对这种细胞凋亡有明显的拮抗作用,以10-7mol／l作用最明显。辛伐他汀主要是通过激活AKT和抑制NF-KB途径来达到保护作用。
[Objective] The aim of the study is to probe the effects of simvastatin on the apoptosis of MG63 induced by TNF-a and Actinomycin D (ActD) and the possible mechanism.
[Methods] MG63 cells were cultured with DMEM essential medium supplemented with 10% fetal calf serum and antibiotics. Cell activity was measured by 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium,inner salt(MTs). The pyknotic fragmented nuclei and apoptosis bodies were carefully observed microscopically, and the apoptosis cells were collected to do proteolysis to make sure the accurate pathway by western blotting examination.
[Results] The cell activity was not decreased after putting TNF-a alone on MG63 for 24 hours, so was the 10-7-10-9mol/l concentration of simvastatin for 24 hours; in contrast, high concentration of 10-3-10-4mol/l could inhabit the cytogenesis.
TNF-a had an obvious cytotoxicity to MG63 cell that was sensitive to ActD, and with the rising in TNF-a concentration,the pyknotic fragmented nuclei and apoptosis bodies were increased.
After pre-reserving human osteoblast cell with 10-7-10-9mol/l of simvastatin, then interacted with TNF-a/ActD, and the reduction of the dense segments and apoptosis bodies were demonstrated. The protection related inversingly with the TNF-a concentration.
Simvastatin could inhabit the apoptosis of MG63 induced by TNF-a/ActD by means of activating AKT pathway and inhibiting NF-κB pathway。
[Conclusions] TNF-a could induce the apoptosis of MG63 sensitive to ActD, and simvastatin could be resistant to the phenomenon. The protection from simvastatin related inversingly with the TNF-a concentration, which primarily by means of activating AKT pathway and inhibiting NF-κB pathway。
【方法】：人成骨样细胞(MG63)用DMEM+10%胎牛血清培养,分别用不同浓度的辛伐他汀、TNF-a、TNF-a+ActD作用于MG63,观察对细胞活性的影响,采用合适的浓度诱导细胞损伤,制作凋亡模型,将实验细胞分成5组,对照组,TNF-a+ActD组,另3组采用不同浓度的辛伐他汀(10-7—10-9mol／l)对MG63进行预处理,再加入TNF-a+ActD,用MTs比色法测定各种药物对细胞活性的影响,光镜下观察细胞形态的改变及典型的凋亡小体和细胞碎片,收集凋亡细胞进行蛋白裂解后行western blotting检测细胞凋亡途径。
[结果]
(1)TNF-a单独对MG63作用24小时细胞活性无明显减低。
(2)10-7—10-10mol／l浓度的辛伐他汀对MG63作用24小时后细胞活性无明显减低,但高浓度组(10-3—10-5mol／l)。细胞活性明显减低
(3)TNF-a对放线茵素D致敏的MG63细胞有明显的细胞毒作用,随着TNF-a浓度的增加,细胞活性明显减低。
(4)辛伐他汀可以抑制TNF-a／ActD诱导的人成骨样细胞(MG63)凋亡,经辛伐他汀(10-7—10-9mol／l)预处理后再与TNF-a／ActD作用,细胞活性较未加辛伐他汀有明显增加。在一定浓度范围内呈剂量依赖性,10-7mol／l组细胞活性增加较明显。光镜下观察凋亡小体和致密碎片核有明显减少。
(5)辛伐他汀通过激活AKT和抑制NF-KB途径,来抑制TNF-a／ActD诱导的人成骨样细胞(MG63)凋亡。
【结论】TNF-a能诱导ActD致敏的MG63细胞发生凋亡,而辛伐他汀则对这种细胞凋亡有明显的拮抗作用,以10-7mol／l作用最明显。辛伐他汀主要是通过激活AKT和抑制NF-KB途径来达到保护作用。
[Objective] The aim of the study is to probe the effects of simvastatin on the apoptosis of MG63 induced by TNF-a and Actinomycin D (ActD) and the possible mechanism.
[Methods] MG63 cells were cultured with DMEM essential medium supplemented with 10% fetal calf serum and antibiotics. Cell activity was measured by 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium,inner salt(MTs). The pyknotic fragmented nuclei and apoptosis bodies were carefully observed microscopically, and the apoptosis cells were collected to do proteolysis to make sure the accurate pathway by western blotting examination.
[Results] The cell activity was not decreased after putting TNF-a alone on MG63 for 24 hours, so was the 10-7-10-9mol/l concentration of simvastatin for 24 hours; in contrast, high concentration of 10-3-10-4mol/l could inhabit the cytogenesis.
TNF-a had an obvious cytotoxicity to MG63 cell that was sensitive to ActD, and with the rising in TNF-a concentration,the pyknotic fragmented nuclei and apoptosis bodies were increased.
After pre-reserving human osteoblast cell with 10-7-10-9mol/l of simvastatin, then interacted with TNF-a/ActD, and the reduction of the dense segments and apoptosis bodies were demonstrated. The protection related inversingly with the TNF-a concentration.
Simvastatin could inhabit the apoptosis of MG63 induced by TNF-a/ActD by means of activating AKT pathway and inhibiting NF-κB pathway。
[Conclusions] TNF-a could induce the apoptosis of MG63 sensitive to ActD, and simvastatin could be resistant to the phenomenon. The protection from simvastatin related inversingly with the TNF-a concentration, which primarily by means of activating AKT pathway and inhibiting NF-κB pathway。
引文
[1].陆再英 钟南山 谢毅等内科学。人民卫生出版社。835-840
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[2]. Mundy G, Garrett R, Harris S, Chan J, Chen D, Rossini G, et al. Stimulation of bone formation in vitro and in rodents by statins. Science.1999;286:1946-9.
[3]. Silvia Ruiz-Gaspa, Xavier Nogues, Anna Enjuanes,et al.Simvastatin and Atorvastatin Enhance Gene Expression of Collagen Type 1 and Osteocalcin in Primary Human Osteoblasts and MG-63 Cultures. Journal of Cellular Biochemistry 101:1430-1438 (2007)
[4]. Rosenson RS, Tangney CC, Langman CB, et al. Shon term reduction in bone markers with high-dose simvastatin[J]. Osteoporos Int,2005; 16 (10): 1272-6
[5]. Sonobe M, Hattori K, Tomita N, et al. Stimulatory effects of statins on bone marrow2derived mesenchymal stem cells. Study of a new therapeutic agent for fracture. Biomed Mater Eng,2005,15:261-267
[6]. Hwang R, Lee EJ, Kim MH, et al. Calcyclin, a Ca2+ion2binding protein, contributes to the anabolic effects of simvastatin on bone. J Biol Chem,2004, 279 (20):21239-21247.
[7]. Wang X, Tokuda H, Hatakeyama D, et al. Mechanismof simvastatin oninduction of heat shock protein in osteoblasts. Arch BiochemBiophys,2003, 415 (1):6-13.
[8]. Maeda T, Kawane T, Horiuchi N. Statins augment vascular endothelial growth factor expression in osteoblastic cells via inhibition of protein prenylation. Endocrinology,2003,144:681-692.
[9]. Inoue I, Goto S, Mizotani K,et al. Lipophilic MHG2CoA reductase inhibitor has an anti2inflammatory effect:reduction of mRNA levels for IL21beta, IL26, COX22 and p22phox by regulation of peroxisome proliferator activated receptor alpha in primary endothelial cells. Life Sci,2000,67:863-876.
[10]. Garrett IR, Gutierrez G, Chen D, et al. Statins stimulate bone formation by enhancing eNOS expression. J Bone Miner Res,2001,16:S141.
[11]. Li X, Cui Q, Kao C, et al. Lovastatin inhibits adipogenic and stimulates osteogenic differentiation by suppressing PPARgamma2 and increasing CbfalPRunx2 expression in bone marrow mesenchymal cell cultures. Bone, 2003,33:652-659.
[12]. Glantsching,H.,Fisher,J.E.,Wesolowski.,et al.M-CSF,TNFalpha and RANK ligand promote osteoclast survival by signaling through mT0R/S6 kinase[J].Cell Death Differ,2003; 10,1165-1177
[13]. Yun-mei Yang, Wei-dong Huang, Qiang-min Xie, et al.Simvastatin attenuates TNF-a-induced growth inhibition and apoptosis in murine osteoblastic MC3T3-E1 cells. Inflammation Research 2009;59(2):151-157
[14]. Majima T, Komatsu Y, Fukao A, et al. Short-term effects of atorvastatin on bone turnover in male patients with hypercholesterolemia[J]. Endocr J,2007; 54(1):145-51.
[15]. Chua CC, Chua BH, Chen Z, Landy C, Hamdy RC. TGF-b1inhibits multiple caspases induced by TNF-a in murine osteo-blastic MC3T3-E1 cells. Biochim Biophys Acta.2002;1593(1):1-8.
[16]. R. Gerber, J.D. Ryan, D.S. Clark, Cell-based screen of HMG-CoA reductase inhibitors and expression regulators using LC-MS, Anal. Biochem.329 (2004)
[17]. Mullen MJ, Wright D, Donald AE, et al. Atorvastatin but not L-arginine improves endothelial function in type-I diabetes mellitus:a double-blind study[J]. Jam Coll Cardiol,2000,36:410-416.
[18]. Hu H, Sung A, Zhao G, Shi L, Qiu D, Nishimura T & Kao PN Simvastatin enhances bone morphogenetic protein receptor type II expression. Biochemical and Biophysical Research Communications 2006;339:59-64.
[19]. Axel DI, Riessen R, Runge H, et al. Effects of cerivastatin on human arterial
smooth muscle cell proliferation and migration in transfilter cocultures[J]. J Cardiovasc Pharmacol,2000,35:619-629.
[20]. Sang-Heng Kok, Kuo-Liang Hou, Chi-Yuan Hong, et al. Simvastatin Inhibits Cytokine-Stimulated Cyr61 Expression in Osteoblastic Cells:A Therapeutic Benefit for Arthritis. Arthritis & Rheumatism DOI 10.1002/art.27433
[21]. Sugiyama M, Kodama T, Konishi K, et al. Compactin and simvastatin,but not pravastatin, induce bone morphogenetic protein 2 in human osteosarcoma cells. Biochem Biophys Res Commun,2000,271:688-692.
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