Influence of endoplasmic reticulum stress on smoking-induced nucleus pulposus cells apoptosis and inflammatory response_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HOU Xiaozhong ¹ , XU Linfei ^2,3 , YI Weiwei ³ , CHEN Yanyang ³ ,  SHEN Jieliang 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, 400042, P.R.China;

Corresponding author：

SHEN Jieliang, Email: franklinspine@163.com

Keywords：

Smoking; intervertebral disc degeneration; cell apoptosis; inflammatory response; endoplasmic reticulum stress

DOI：

10.7507/1002-1892.201901094

Video：

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Objective To investigate the influence of endoplasmic reticulum stress (ERS) on smoking-induced nucleus pulposus cells apoptosis and inflammatory response.Methods Between October 2016 and October 2018, 25 patients with cervical disc herniation receiving discectomy were collected and divided into smoking group (14 cases) and non-smoking group (11 cases). The baseline data of age, gender, herniated segment, and Pfirrmann grading showed no significant difference between the two groups (P>0.05). The obtained nucelus pulposus tissues were harvested to observe the cell apoptosis via detecting the apoptosis-related proteins (Caspase-3 and PRAP) by TUNEL staining and Western blot test. The nucleus pulposus cells were isolated and cultured with enzyme digestion, of which the third generation cells were used in follow-up experiments. Then, the expressions of inflammatory factors [interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α)] were detected by ELISA; the nuclear translocation of P65 was monitored by cell immunofluorescence staining. Furthermore, ERS-related proteins (GRP78 and CHOP) were detected by Western blot; and endoplasmic reticulum ultrastructure was observed under transmission electron microscope. To verify the regulatory effect of ERS, cells were pretreated by ERS specific inhibitor (4-PBA), then cell apoptosis and inflammatory response were tested.Results The nucleus pulposus tissue observation showed that the cell apoptotic rate and the expressions of apoptosis-related proteins (Caspase-3 and PARP) were obviously higher in smoking group than in non-smoking group (P<0.05). The nucleus pulposus cells observation indicated that the expressions of the inflammatory factors (IL-1β and TNF-α) and the ERS-related proteins (GRP78 and CHOP) were also higher in smoking group than in non-smoking group (P<0.05). The results of cell immunofluorescence staining further confirmed that smoking stimulated nuclear translocation of P65 in nucleus pulposus cells. The ERS injury was much more serious in smoking group than in non-smoking group. Furthermore, after 4-PBA inhibiting ERS, the expressions of GRP78, CHOP, IL-1β, TNF-α, and P65 were significantly decreased (P<0.05), and flow cytometry results showed that cell apoptotic rate in smoking group was decreased, showing significant difference compared with the non-smoking group (P<0.05).Conclusion Somking can stimulate cell apoptosis and inflammatory response in nucleus pulposus cells via ESR pathway. Suppressing ESR may be a novel target to suspend smoking-induced intervertebral disc degeneration.

Citation： HOU Xiaozhong, XU Linfei, YI Weiwei, CHEN Yanyang, SHEN Jieliang. Influence of endoplasmic reticulum stress on smoking-induced nucleus pulposus cells apoptosis and inflammatory response. Chinese Journal of Reparative and Reconstructive Surgery, 2019, 33(6): 736-742. doi: 10.7507/1002-1892.201901094 Copy

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15.	Withrow J, Myrphy C, Liu Y, et al. Extracellular vesicles in the pathogenesis of rheumatoid arthritis and osteoarthritis. Arthritis Res Ther, 2016, 18(1): 286.
16.	蓝海洋, 杨智杰, 夏辉强, 等. 锌指蛋白 A20 对内毒素刺激时人椎间盘髓核细胞炎症及退变的影响. 第三军医大学学报, 2019, 41(6): 543-548.
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18.	Iurlaro R, Muñoz-Pinedo C. Cell death induced by endoplasmic reticulum stress. FEBS J, 2016, 283(14): 2640-2652.
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20.	蒋虹, 李隽, 李斌, 等. 髓系细胞触发受体-1 在脂多糖急性肺损伤大鼠肺组织中的表达及其与内质网应激和炎性反应的关系. 现代生物医学进展, 2018, 18(14): 2657-2660.

1. Hurwitz EL, Randhawa K, Yu H, et al. The Global Spine Care Initiative: a summary of the global burden of low back and neck pain studies. Eur Spine J, 2018, 27(Suppl 6): 796-801.
2. Sun Y, Leung VY, Cheung KM. Clinical trials of intervertebral disc regeneration: current status and future developments. Int Orthop, 2019, 43(4): 1003-1010.
3. 肖琳, 杨杰, 万霞, 等. 中国人群的吸烟率究竟有多高—三项全国烟草流行调查结果比较. 中华流行病学杂志, 2009, 30(1): 30-33.
4. Smith MA, Jackson A. Tobacco use, tobacco cessation, and musculoskeletal health. Orthop Nurs, 2018, 37(5): 280-284.
5. 潘福敏, 王善金, 麻彬, 等. 吸烟与腰椎间盘退变的相关性. 中国脊柱脊髓杂志, 2015, 25(8): 746-749.
6. Chen Z, Li X, Pan F, et al. A retrospective study: Does cigarette smoking induce cervical disc degeneration? Int J Surg, 2018, 53: 269-273.
7. Sher I, Daly C, Oehme D, et al. Novel application of the Pfirrmann disc degeneration grading system to 9.4T MRI: Higher reliability compared to 3T MRI. Spine (Phila Pa 1976), 2018. [Epub ahead of print].
8. 沈皆亮, 胡侦明, 钟小明, 等. 白藜芦醇恢复去分化正常髓核细胞表型的实验研究. 中国修复重建外科杂志, 2013, 27(5): 547-553.
9. 张昕, 王宸, 芮云峰. 吸烟与骨骼肌肉系统疾病. 东南大学学报(医学版), 2015, 34(6): 1023-1028.
10. Teraguchi M, Yoshimura N, Hashizume H, et al. Progression, incidence, and risk factors for intervertebral disc degeneration in a longitudinal population-based cohort: the Wakayama Spine Study. Osteoarthritis Cartilage, 2017, 25(7): 1122-1131.
11. Ma K, Chen S, Li Z, et al. Mechanisms of endogenous repair failure during intervertebral disc degeneration. Osteoarthritis Cartilage, 2019, 27(1): 41-48.
12. 陈琪, 石芳芳, 杨曦, 等. 不同代次髓核细胞生物学特性的比较研究. 中国修复重建外科杂志, 2018, 32(6): 660-667.
13. Johnson ZI, Schoepflin ZR, Choi H, et al. Disc in flames: Roles of TNF-α and IL-1β in intervertebral disc degeneration. Eur Cell Mater, 2015, 30: 104-117.
14. Shen JL, Xu SX, Zhou H, et al. IL-1β induces apoptosis and autophagy via mitochondria pathway in human degenerative nucleus pulposus cells. Sci Rep, 2017, 7: 41067.
15. Withrow J, Myrphy C, Liu Y, et al. Extracellular vesicles in the pathogenesis of rheumatoid arthritis and osteoarthritis. Arthritis Res Ther, 2016, 18(1): 286.
16. 蓝海洋, 杨智杰, 夏辉强, 等. 锌指蛋白 A20 对内毒素刺激时人椎间盘髓核细胞炎症及退变的影响. 第三军医大学学报, 2019, 41(6): 543-548.
17. Navone SE, Marfia G, Giannoni A, et al. Inflammatory mediators and signalling pathways controlling intervertebral disc degeneration. Histol Histopathol, 2017, 32(6): 523-542.
18. Iurlaro R, Muñoz-Pinedo C. Cell death induced by endoplasmic reticulum stress. FEBS J, 2016, 283(14): 2640-2652.
19. Krupkova O, Sadowska A, Kameda T, et al. p38 MAPK facilitates crosstalk between endoplasmic reticulum stress and IL-6 release in the intervertebral disc. Front Immunol, 2018, 9: 1706.
20. 蒋虹, 李隽, 李斌, 等. 髓系细胞触发受体-1 在脂多糖急性肺损伤大鼠肺组织中的表达及其与内质网应激和炎性反应的关系. 现代生物医学进展, 2018, 18(14): 2657-2660.

Chinese Journal of Reparative and Reconstructive Surgery

Influence of endoplasmic reticulum stress on smoking-induced nucleus pulposus cells apoptosis and inflammatory response

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