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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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.	陳琪, 石芳芳, 楊曦, 等. 不同代次髓核細胞生物學特性的比較研究. 中國修復重建外科雜志, 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.	藍海洋, 楊智杰, 夏輝強, 等. 鋅指蛋白 A20 對內毒素刺激時人椎間盤髓核細胞炎癥及退變的影響. 第三軍醫大學學報, 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, Mu?oz-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.	蔣虹, 李雋, 李斌, 等. 髓系細胞觸發受體-1 在脂多糖急性肺損傷大鼠肺組織中的表達及其與內質網應激和炎性反應的關系. 現代生物醫學進展, 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, 楊杰, 萬霞, 等. 中國人群的吸煙率究竟有多高—三項全國煙草流行調查結果比較. 中華流行病學雜志, 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. 吸煙與腰椎間盤退變的相關性. 中國脊柱脊髓雜志, 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, 胡偵明, 鐘小明, 等. 白藜蘆醇恢復去分化正常髓核細胞表型的實驗研究. 中國修復重建外科雜志, 2013, 27(5): 547-553.
9. 張昕, 王宸, 芮云峰. 吸煙與骨骼肌肉系統疾病. 東南大學學報(醫學版), 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. 陳琪, 石芳芳, 楊曦, 等. 不同代次髓核細胞生物學特性的比較研究. 中國修復重建外科雜志, 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. 藍海洋, 楊智杰, 夏輝強, 等. 鋅指蛋白 A20 對內毒素刺激時人椎間盤髓核細胞炎癥及退變的影響. 第三軍醫大學學報, 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, Mu?oz-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. 蔣虹, 李雋, 李斌, 等. 髓系細胞觸發受體-1 在脂多糖急性肺損傷大鼠肺組織中的表達及其與內質網應激和炎性反應的關系. 現代生物醫學進展, 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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