High mechanical stretch stress promotes degeneration of the human nucleus pulposus cells through NF-κb signaling pathway

Abstract

Objective: To investigate the effect of high mechanical stretch stress(HMS)on human nucleus pulposus cells and its regulatory mechanism. Methods: The non-degenerated nucleus pulposus tissue (Pfirrmann<grade Ⅲ) removed from the patient's surgery was harvested and the human nucleus pulposus cells were isolated and cultured. In the presence or absence of pretreatment with the NF-κB specific blocker Bay11-7082, the cultured human nucleus pulposus cells were loaded cyclic mechanical stretch stress(CMS) with different parameters using the Flexercell system.The cell culture medium was collected and the secretion of inflammatory cytokines was detected by Elisa. The nucleus pulposus cells loaded with cyclic mechanical stretch stress(CMS) was collected, the changes of NF-κB/P65 signal pathway were detected, The mRNA and protein levels' expression changes were detected by RT-PCR and WB; after human nucleus pulposus cells were exposed to IL-1β, with or without Bay11-7082, the changes of P65 were detected by immunofluorescence. Results: Those mechanical stretch stress of high amplitude (9%, 19%), low frequency (0.01 Hz) and long duration (72 h) led to degeneration of human nucleus pulposus cells, while the mechanical stretch stress of low amplitude (3%), low frequency and long duration could not promote the degeneration process; the mechanical stretch stress of high amplitude(19%), low-frequency(0.01 Hz) could promote the release of inflammatory cytokines of human nucleus pulposus cells after 24 h duration; high-amplitude, low-frequency mechanical stretch stress could activate the NF-κB signaling pathway in human nucleus pulposus cells, Bay11-7082 could block the process; immunofluorescence showed that IL-1β could promote the phosphorylation of P65 in the cytoplasm of human nucleus pulposus cells and promote the entry of P65 into the cell nucleus process, Bay11-7082 could block those processes; Bay11-7082, the specific blocking agent of NF-κB signaling pathway, could block the degeneration process of human nucleus pulposus cells induced by high cyclic mechanical stretch stress(CMS) in a dose-dependent. Conclusions: High cyclic mechanical stretch stress promotes human nucleus pulposus cells degeneration through NF-κB signaling pathway.