Thursday, September 27, 2018 2:05 PM–3:05 PM Section on Biologics and Basic Research Abstract Presentations: 124. NF-κB dependent unfolded protein response in the survival and proliferation of nucleus pulposus cells under TNF-α stimulation

Abstract

BACKGROUND CONTEXT Intervertebral disc (IVD) degeneration is a degenerative disease closely related to inflammation of nucleus pulposus (NP) cells. Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine expressed by NP cells (NPCs) of degenerate IVD, which induces NPCs apoptosis and extracellular matrix (ECM) degradation, and accelerate IVD degeneration (IDD). The endoplasmic reticulum serves several important cell functions, which are essential for normal cell function and survival. Nuclear factor-kappaB (NF-κB) is important for genes involved in cell survival, adhesion, differentiation, and proliferation. However, the roles of ER stress and NF-κB in IDD remain to be elucidated. PURPOSE This study aims to clarify the roles of NF-κB and ER stress related unfolded protein response (UPR) in TNF-α-induced biological changes in rat NPCs and IVD degeneration. METHODS In vivo, different doses of exogenous TNF-α were injected into rat tail discs. Magnetic resonance imaging (MRI) yielding T2-weighted images were performed. Then, the discs were stained by Hematoxylin and Eosin, Masson's trichrome to visualize the histocytological changes, immunohistochemical staining was performed to evaluate the expression of ECM including aggrecan and collagen II. In vitro, we cultured rat NPCs with different concentrations of TNF-α, with or without UPR and NF-κB pathway small interfering RNA (siRNA). The mRNA expression and protein levels of UPR markers (XBP1s and p-eIF2α) and p-p65 were measured by immunofluorescence staining, qRT-PCR and western blot analysis respectively and were used to monitor UPR and NF-κB. Cell proliferation was evaluated by CCK-8 assay, cell-cycle analysis and cyclin D1 expression. Apoptosis was detected by flowcytometry and Western blot analyses. All the data were expressed as mean ± SD and from multiple independent experiments. The results were compared among different groups by using unpaired student-test. P-values RESULTS In vivo, after one month, a significant decrease in T2 value calculated from T2-mapping was observed in TNF-α injection groups along with clearly decrease in disc height and nucleus volumes in comparison to the control discs. Histological analysis showed degenerative changes with NPCs and ECM loss, while no degenerative changes were observed in control groups. In vitro, TNF-α induced the apoptosis of some NPCs in the early stage and then accelerated the proliferation of surviving cells. In addition, TNF-α stimulus up-regulated XBP1s, p-eIF2α and p-p65 at the protein level, which indicated that TNF-α activated UPR and NF-κB signals in rat NP cells. However, these effects could be reversed by UPR and NF-κB siRNA, and UPR interference decreased the expression of p-p65 notably. In parallel, both UPR and NF-κB interference reduced cell proliferation and enhanced apoptosis. CONCLUSIONS Our study demonstrated that TNF-α is an important early pathogenetic factor in disc degeneration. UPR reinforces the survival and proliferation of NPCs in TNF-α stimulus by activating NF-κB signaling, which could be an important therapeutic target in the future. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.