PBN protects NP cells from AAPH-induced degenerative changes by inhibiting the ERK1/2 pathway

Abstract

ABSTRACT Aim: Intervertebral disc (IVD) degeneration (IDD) is one of the main causes for spinal degenerative diseases, such as disk herniation, spinal canal stenosis, and spinal deformities. Growing evidence has highlighted the contribution of oxidative stress in pathogenesis of IDD, and antioxidant treatment is thus considered to be a promising therapeutic strategy for IDD. The aim of this study was to investigate whether N-tert-butyl-α-phenylnitrone (PBN), a free radical scavenger, could attenuate the pathological changes of IDD by alleviating oxidative stress. Materials and Methods: Nucleus pulposus (NP) cells were isolated from rabbit lumbar disks. MTT assay, real-time PCR and western blotting were employed to evaluate the effects of PBN on oxidative damages induced by 2,2ʹ-azobis (2-amidinopropane) dihydrochloride (AAPH) in NP cells. Results: AAPH induced oxidative stress and the subsequent degenerative changes in NP cells via the ERK/MAPK pathway. On the contrary, the oxidative stress induced by AAPH was significantly ameliorated by PBN. Moreover, PBN also attenuated AAPH-induced expression of matrix degradation proteases and apoptosis. PBN suppresses AAPH-induced activation of ERK/MAPK pathway, which may be the underlying mechanism for the protective effects of PBN. Conclusions: Our study for the first time identified a novel role and mechanism for PBN in protecting the IVD against oxidative stress, matrix catabolism and apoptosis, which may have implications for its further application in combating IVD degenerative diseases. Abbreviations: AAPH: 2,2ʹ-azobis(2-methylpropanimidamidine) dihydrochloride; ADAMTS: a disintegrin and metalloproteinase with thrombospondin motifs; AF: annulus fibrosus; CEP: cartilage endplate; DCF: 2ʹ7’-dichlorofluorescein; IDD: intervertebral disc degeneration; IVD: intervertebral disc; LPS: lipopolysaccharide; MMP: matrix metalloproteinase; MTT: methyl-thiazolyl-tetrazolium; NP: nucleus pulposus; PBN: N-tert-butyl-alfa-phenylnitrone; PGs: proteoglycans; ROS: reactive oxygen species; SDS: sodium dodecyl sulfate