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. 2019 Jun 15;33(6):736-742.
doi: 10.7507/1002-1892.201901094.

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

[Article in Chinese]
Xiaozhong Hou  1 , Linfei Xu  2 , Weiwei Yi  3 , Yanyang Chen  3 , Jieliang Shen  4
Affiliations

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

[Article in Chinese]
Xiaozhong Hou et al. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. .

Abstract
in English, Chinese

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.

目的: 探讨内质网应激对吸烟诱导的髓核细胞凋亡与炎性反应的影响。.

方法: 以 2016 年 10 月—2018 年 10 月 25 例接受椎间盘摘除术的颈椎间盘突出症患者为研究对象，分为吸烟组（14 例）和非吸烟组（11 例）。两组患者年龄、性别、突出节段、Pfirrmann 分级比较，差异均无统计学意义（ P>0.05）。将术中获取的髓核组织，行 TUNEL 染色和 Western blot 检测，观察细胞凋亡情况。然后，采用酶序贯消化法分离培养髓核细胞并传代，取第 3 代细胞行 ELISA 检测炎性因子 IL-1β 和 TNF-α 含量，免疫荧光染色观察细胞中 P65 核转移情况，Western blot 检测内质网应激相关蛋白 GRP78 和 CHOP 表达，透射电镜观察细胞内质网超微结构。最后，为验证内质网调控作用，采用特异性抑制剂 4-PBA 处理吸烟组髓核细胞后，Western blot 检测 GRP78、CHOP、IL-1β、TNF-α 及 P65 蛋白相对表达量，流式细胞术检测细胞凋亡率；并以吸烟组未作处理的髓核细胞作对照。.

结果: 髓核组织观测显示，吸烟组细胞凋亡率及凋亡相关蛋白 Caspase-3 和 PARP 相对表达量均明显高于非吸烟组（ P<0.05）。髓核细胞观测显示，吸烟组髓核细胞分泌 IL-1β、TNF-α 水平以及 GRP78、CHOP 蛋白相对表达量均明显高于非吸烟组（ P<0.05）；免疫荧光染色示吸烟组细胞 P65 主要表达在细胞核，而非吸烟组在细胞质；透射电镜观察显示，与非吸烟组相比，吸烟组内质网处于应激损伤状态。吸烟组髓核细胞经 4-PBA 处理后，GRP78、CHOP、IL-1β、TNF-α 及 P65 蛋白相对表达量以及细胞凋亡率均较处理前明显降低（ P<0.05）。.

结论: 吸烟可通过内质网应激导致髓核细胞凋亡和炎性反应加剧，抑制内质网应激有望成为延缓吸烟导致椎间盘退变的新靶点。.

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

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Figures

图 1
图 1
The observation of nucleus pulposus in 2 groups 两组髓核组织观测
图 2
图 2
The expressions of ERS-related proteins in 2 groups detected by Western blot Western blot检测两组细胞内质网应激相关蛋白表达水平
图 3
图 3
Location expression of P65 under immunofluorescence staining in 2 groups (Fluorescence microscope×200) 两组P65免疫荧光染色观察（荧光显微镜×200）
图 4
图 4
Endoplasmic reticulum ultrastructure of 2 groups observed by transmission electron microscope (×15 k) 透射电镜观察两组细胞内质网超微结构（×15 k）
图 5
图 5
The expressions of the inflammatory- and ERS-related proteins detected by Western blot before and after 4-PBA treatments Western blot检测4-PBA处理前后髓核细胞相关蛋白表达
图 6
图 6
The relative expressions of the inflammatory- and ERS-related proteins before and after 4-PBA treatments 4-PBA处理前后髓核细胞相关蛋白相对表达量
图 7
图 7
Cell apoptosis detected by flow cytometry before and after 4-PBA treatments 流式细胞仪观察4-PBA处理前后髓核细胞凋亡
See this image and copyright information in PMC

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