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

摘要

目的：椎间盘（IVD）退行性变（IDD）是导致椎间盘突出、椎管狭窄、脊柱畸形等脊柱退行性疾病的主要原因之一。越来越多的证据强调了氧化应激在 IDD 发病机制中的作用，因此抗氧化治疗被认为是一种有前途的 IDD 治疗策略。本研究的目的是探讨自由基清除剂 N-叔丁基-α-苯基硝酮 (PBN) 是否可以通过减轻氧化应激来减轻 IDD 的病理变化。

材料和方法： 从兔腰椎间盘中分离出髓核（NP）细胞。采用 MTT 法、实时 PCR 和蛋白质印迹法评估 PBN 对 2,2′-偶氮二（2-脒基丙烷）二盐酸盐 (AAPH) 在 NP 细胞中诱导的氧化损伤的影响。

结果：AAPH 通过 ERK/MAPK 通路诱导 NP 细胞的氧化应激和随后的退行性变化。相反，由AAPH引起的氧化应激被PBN显着改善。此外，PBN 还减弱了 AAPH 诱导的基质降解蛋白酶和细胞凋亡的表达。PBN 抑制 AAPH 诱导的 ERK/MAPK 通路激活，这可能是 PBN 保护作用的潜在机制。

结论：我们的研究首次确定了 PBN 在保护 IVD 免受氧化应激、基质分解代谢和细胞凋亡方面的新作用和机制，这可能对其在对抗 IVD 退行性疾病中的进一步应用产生影响。

缩写： AAPH：2,2′-偶氮二（2-甲基丙脒）二盐酸盐；ADAMTS：具有血小板反应蛋白基序的去整合素和金属蛋白酶；AF：纤维环；CEP：软骨终板；DCF：2'7'-二氯荧光素；IDD：椎间盘退变；IVD：椎间盘；LPS：脂多糖；MMP：基质金属蛋白酶；MTT：甲基-噻唑基-四唑；NP：髓核；PBN：N-叔丁基-α-苯基硝酮；PGs：蛋白聚糖；ROS：活性氧；SDS：十二烷基硫酸钠