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

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 ( >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 ( <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 ( <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 ( <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 ( <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.