Effect of heme oxygenase 1 on the apoptosis of human degenerated nucleus pulposus cells induced by tumor necrosis factor α_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HOU Xiaozhong ¹ , XU Linfei ^2,3 , SHEN Jieliang ³ ,  HU Zhenming ³

1. Department of Orthopedics, Henan Provincial Hospital, Zhengzhou Henan, 450000, P.R.China;
2. Department of Orthopedics, Henan Provincial Chest Hospital, Zhengzhou Henan, 450003, P.R.China;
3. Department of Orthopedics, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, P.R.China;

Corresponding author：

HU Zhenming, Email: spinecenter_hu@126.com

Keywords：

Heme oxygenase 1; tumor necrosis factor α; nucleus pulposus cells; apoptosis

DOI：

10.7507/1002-1892.201709043

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Objective To investigate the effect of heme oxygenase 1 (HO-1) on the apoptosis of human degenerated nucleus pulposus (NP) cells induced by tumor necrosis factor α (TNF-α), and explore its possible molecular mechanism. Methods The intervertebral disc tissues were derived from patients with lumbar intervertebral disc herniation. Then, the NP cells were cultured in vitro and the third generation of NP cells were used for subsequent experiments. Cell counting kit 8 (CCK-8) method was used to observe the proliferative effect of TNF-α on the NP cells in vitro at the concentration of 10, 20, 50, 100, and 200 ng/mL. The most apropriate concentration was selected according to the result of CCK-8. The NP cells were cultured with basal medium (control group), TNF-α (TNF-α group), TNF-α and CoPP 10 μmol/L (CoPP group), and TNF-α and ZnPP 15 μmol/L (ZnPP group), respectively. After cultured, the cell poptosis was detected by Hoechst staining and flow cytometry; the expression of cleaved Caspase-3, epithelial membrane protein 1 (EMP-1), HO-1, and p-P65 proteins were detected by Western blot. In order to further explore the potential molecular mechanisms of HO-1 for cell apoptosis, the NP cells were cultured with TNF-α (TNF-α stimulated group), TNF-α and pyrrolidine dithiocarbamate (PDTC) 5 μmol/L (TNF-α+PDTC stimulated group), respectively. Then the cell apoptosis rate was measured by flow cytometry at 24 hours after cultured. Results The optimal concentration of TNF-α was 100 ng/mL. Hoechst staining showed that a few apoptotic cells could be observed in control group and CoPP group; the apoptosis-like nucleis were observed in TNF-α group and ZnPP group, which was the most significant in ZnPP group. Flow cytometry showed that the cell apoptosis rates of TNF-α group, CoPP group, and ZnPP group were significantly increased when compared with the control group (P<0.05). Compared with TNF-α group, the cell apoptosis rate in CoPP group decreased (P<0.05), while in ZnPP group it increased (P<0.05). Western blot showed that the expression of HO-1 protein in TNF-α group was decreased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were increased when compared with the control group (P<0.05). Compared with TNF-α group, the expression of HO-1 protein in CoPP group increased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were reduced (P<0.05); the expression of HO-1 protein in ZnPP group decreased (P<0.05), the expressions of cleaved Caspase-3 and EMP-1 proteins increased (P<0.05), and the expression of p-P65 protein was not significantly changed (P>0.05). Compared with TNF-α stimulated group, the cell apoptosis rate in TNF-α+PDTC stimulated group was significantly reduced (t=3.076, P=0.031). Conclusion HO-1 can inhibit the apoptosis of degerated NP cells induced by TNF-α, and its mechanism effect is by inhibiting the nuclear factor кB signaling pathway.

Citation： HOU Xiaozhong, XU Linfei, SHEN Jieliang, HU Zhenming. Effect of heme oxygenase 1 on the apoptosis of human degenerated nucleus pulposus cells induced by tumor necrosis factor α. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(1): 69-74. doi: 10.7507/1002-1892.201709043 Copy

1.	Maidhof R, Alipui DO, Rafiuddin A, et al. Emerging trends in biological therapy for intervertebral disc degeneration. Discov Med, 2012, 14(79): 401-411.
2.	Xu HG, Li ZR, Wang H, et al. Intermittent cyclic mechanical tension-induced down-regulation of ectonucleotide pyrophosphatase phosphodiesterase 1 gene expression is mainly dependent on TGF-β₁ in end-plate chondrocytes. Orthop Surg, 2013, 5(1): 40-45.
3.	Wang XH, Zhu L, Hong X, et al. Resveratrol attenuated TNF-α-induced MMP-3 expression in human nucleus pulposus cells by activating autophagy via AMPK/SIRT1 signaling pathway. Exp Biol Med (Maywood), 2016, 241(8): 848-853.
4.	Jiang W, Zhang X, Hao J, et al. SIRT1 protects against apoptosis by promoting autophagy in degenerative human disc nucleus pulposus cells. Sci Rep, 2014, 4: 7456.
5.	Miyazaki S, Kakutani K, Yurube T, et al. Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells. Arthritis Research & Therapy, 2015, 17: 253.
6.	Kim KW, Ha KY, Lee JS, et al. The apoptotic effects of oxidative stress and antiapoptotic effects of caspase inhibitors on rat notochordal cells. Spine (Phila Pa 1976), 2007, 32(22): 2443-2448.
7.	Poveda L, Hottiger M, Boos N, et al. Peroxynitrite induces gene expression in intervertebral disc cells. Spine (Phila Pa 1976), 2009, 34(11): 1127-1133.
8.	Planavila A, Rodríguez-Calvo R, Palomer X, et al. Atorvastatin inhibits GSK-3beta phosphorylation by cardiac hypertrophic stimuli. Biochim Biophys Acta, 2008, 1781(1-2): 26-35.
9.	Hu B, Shi C, Xu C, et al. Heme oxygenase-1 attenuates IL-1β induced alteration of anabolic and catabolic activities in intervertebral disc degeneration. Sci Rep, 2016, 6: 21190.
10.	Pfirrmann CW, Metzdorf A, Zanetti M, et al. Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine (Phila Pa 1976), 2001, 26(17): 1873-1878.
11.	Choi BM, Lim DW, Lee JA, et al. Luteolin suppresses cisplatin induced apoptosis in auditory cells: possible mediation through induction of heme oxygenase-1 expression. J Med Food, 2008, 11(2): 230-236.
12.	Wu BJ, Chen K, Barter PJ, et al. Niacin inhibits vascular inflammation via the induction of heme oxygenase-1. Circulation, 2012, 125(1): 150-158.
13.	谷颖, 王爱红, 屈福超, 等. HO-1 对 AngⅡ诱导的乳鼠心肌细胞凋亡的作用. 山东大学学报: 英文版, 2013, 51(6): 1-4.
14.	梁鹤, 董双海, 夏天, 等. NF-κB 信号通路的激活与椎间盘退变的的关系. 中国矫形外科杂志, 2015, 23(5): 458-462.
15.	Marvin KW, Fujimoto W, Jetten AM. Identification and characterization of a novel squamous cell-associated gene related to PMP22. J Biol Chem, 1995, 270(48): 28910-28916.
16.	胡明, 张传森, 陈道运, 等. 人退变椎间盘组织基因表达谱. 解剖学杂志, 2004, 27(4): 348-351.
17.	Hu M, Ma YZ, Feng HC, et al. Analysis of Gene Expression Pattern of Lumbar Intervertebral Disc Degeneration in Human. 中国康复理论与实践, 2006, 12(5): 420-422.
18.	孙中仪, 田纪伟. NF-κB 信号通路与椎间盘退变的研究进展. 中国矫形外科杂志, 2012, 20(23): 2162-2164.
19.	Lee H, Jeon J, Ryu YS, et al. Disruption of microtubules sensitizes the DNA damage-induced apoptosis through inhibiting nuclear factor κB (NF-κB) DNA-binding activity. J Korean Med Sci, 2010, 25(11): 1574-1581.
20.	孔敬波, 马信龙, 王涛, 等. Wnt/β-catenin 及 NF-кB 信号通路与椎间盘退变的研究进展. 中国修复重建外科杂志, 2013, 27(12): 1523-1528.
21.	Ko SH, Rho da J, Jeon JI, et al. Bacteroides fragilis Enterotoxin Upregulates Heme Oxygenase-1 in Intestinal Epithelial Cells via a Mitogen-Activated Protein Kinase- and NF-κB-Dependent Pathway, Leading to Modulation of Apoptosis. Infect Immun, 2016, 84(9): 2541-2554.

1. Maidhof R, Alipui DO, Rafiuddin A, et al. Emerging trends in biological therapy for intervertebral disc degeneration. Discov Med, 2012, 14(79): 401-411.
2. Xu HG, Li ZR, Wang H, et al. Intermittent cyclic mechanical tension-induced down-regulation of ectonucleotide pyrophosphatase phosphodiesterase 1 gene expression is mainly dependent on TGF-β₁ in end-plate chondrocytes. Orthop Surg, 2013, 5(1): 40-45.
3. Wang XH, Zhu L, Hong X, et al. Resveratrol attenuated TNF-α-induced MMP-3 expression in human nucleus pulposus cells by activating autophagy via AMPK/SIRT1 signaling pathway. Exp Biol Med (Maywood), 2016, 241(8): 848-853.
4. Jiang W, Zhang X, Hao J, et al. SIRT1 protects against apoptosis by promoting autophagy in degenerative human disc nucleus pulposus cells. Sci Rep, 2014, 4: 7456.
5. Miyazaki S, Kakutani K, Yurube T, et al. Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells. Arthritis Research & Therapy, 2015, 17: 253.
6. Kim KW, Ha KY, Lee JS, et al. The apoptotic effects of oxidative stress and antiapoptotic effects of caspase inhibitors on rat notochordal cells. Spine (Phila Pa 1976), 2007, 32(22): 2443-2448.
7. Poveda L, Hottiger M, Boos N, et al. Peroxynitrite induces gene expression in intervertebral disc cells. Spine (Phila Pa 1976), 2009, 34(11): 1127-1133.
8. Planavila A, Rodríguez-Calvo R, Palomer X, et al. Atorvastatin inhibits GSK-3beta phosphorylation by cardiac hypertrophic stimuli. Biochim Biophys Acta, 2008, 1781(1-2): 26-35.
9. Hu B, Shi C, Xu C, et al. Heme oxygenase-1 attenuates IL-1β induced alteration of anabolic and catabolic activities in intervertebral disc degeneration. Sci Rep, 2016, 6: 21190.
10. Pfirrmann CW, Metzdorf A, Zanetti M, et al. Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine (Phila Pa 1976), 2001, 26(17): 1873-1878.
11. Choi BM, Lim DW, Lee JA, et al. Luteolin suppresses cisplatin induced apoptosis in auditory cells: possible mediation through induction of heme oxygenase-1 expression. J Med Food, 2008, 11(2): 230-236.
12. Wu BJ, Chen K, Barter PJ, et al. Niacin inhibits vascular inflammation via the induction of heme oxygenase-1. Circulation, 2012, 125(1): 150-158.
13. 谷颖, 王爱红, 屈福超, 等. HO-1 对 AngⅡ诱导的乳鼠心肌细胞凋亡的作用. 山东大学学报: 英文版, 2013, 51(6): 1-4.
14. 梁鹤, 董双海, 夏天, 等. NF-κB 信号通路的激活与椎间盘退变的的关系. 中国矫形外科杂志, 2015, 23(5): 458-462.
15. Marvin KW, Fujimoto W, Jetten AM. Identification and characterization of a novel squamous cell-associated gene related to PMP22. J Biol Chem, 1995, 270(48): 28910-28916.
16. 胡明, 张传森, 陈道运, 等. 人退变椎间盘组织基因表达谱. 解剖学杂志, 2004, 27(4): 348-351.
17. Hu M, Ma YZ, Feng HC, et al. Analysis of Gene Expression Pattern of Lumbar Intervertebral Disc Degeneration in Human. 中国康复理论与实践, 2006, 12(5): 420-422.
18. 孙中仪, 田纪伟. NF-κB 信号通路与椎间盘退变的研究进展. 中国矫形外科杂志, 2012, 20(23): 2162-2164.
19. Lee H, Jeon J, Ryu YS, et al. Disruption of microtubules sensitizes the DNA damage-induced apoptosis through inhibiting nuclear factor κB (NF-κB) DNA-binding activity. J Korean Med Sci, 2010, 25(11): 1574-1581.
20. 孔敬波, 马信龙, 王涛, 等. Wnt/β-catenin 及 NF-кB 信号通路与椎间盘退变的研究进展. 中国修复重建外科杂志, 2013, 27(12): 1523-1528.
21. Ko SH, Rho da J, Jeon JI, et al. Bacteroides fragilis Enterotoxin Upregulates Heme Oxygenase-1 in Intestinal Epithelial Cells via a Mitogen-Activated Protein Kinase- and NF-κB-Dependent Pathway, Leading to Modulation of Apoptosis. Infect Immun, 2016, 84(9): 2541-2554.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of heme oxygenase 1 on the apoptosis of human degenerated nucleus pulposus cells induced by tumor necrosis factor α

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