Senescent response in inner annulus fibrosus cells in response to TNFα, H2O2, and TNFα-induced nucleus pulposus senescent secretome.

Senescence, particularly in the nucleus pulposus (NP) cells, has been implicated in the pathogenesis of disc degeneration, however, the mechanism(s) of annulus fibrosus (AF) cell senescence is still not well understood. Both TNFα and H2O2, have been implicated as contributors to the senescence pathways, and their levels are increased in degenerated discs when compared to healthy discs. Thus, the objective of this study is to identify factor(s) that induces inner AF (iAF) cell senescence. Under TNFα exposure, at a concentration previously shown to induce senescence in NP cells, bovine iAF cells did not undergo senescence, indicated by their ability to continue to proliferate as demonstrated by Ki67 staining and growth curves and lack of expression of the senescent markers, p16 and p21. The lack of senescent response occurred even though iAF express higher levels of TNFR1 than NP cells. Interestingly, iAF cells showed no increase in intracellular ROS or secreted H2O2 in response to TNFα which contrasted to NP cells that did. Following TNFα treatment, only iAF cells had increased expression of the superoxide scavengers SOD1 and SOD2 whereas NP cells had increased NOX4 gene expression, an enzyme that can generate H2O2. Treating iAF cells with low dose H2O2 (50 μM) induced senescence, however unlike TNFα, H2O2 did not induce degenerative-like changes as there was no difference in COL2, ACAN, MMP13, or IL6 gene expression or number of COL2 and ACAN immunopositive cells compared to untreated controls. The latter result suggests that iAF cells may have distinct degenerative and senescent phenotypes. To evaluate paracrine signalling by senescent NP cells, iAF and TNFα-treated NP cells were co-cultured. In contact co-culture the NP cells induced iAF senescence. Thus, senescent NP cells may secrete soluble factors that induce degenerative and senescent changes within the iAF. This may contribute to a positive feedback loop of disc degeneration. It is possible these factors may include H2O2 and cytokines (such as TNFα). Further studies will investigate if human disc cells respond similarly.

衰老，特别是在髓核（NP）细胞中，与椎间盘退变的发病机制有关，然而，纤维环（AF）细胞衰老的机制仍未得到很好的理解。肿瘤坏死因子α（TNFα）和过氧化氢（H₂O₂）都被认为是衰老途径的促成因素，与健康椎间盘相比，它们在退变椎间盘中的水平升高。因此，本研究的目的是确定诱导内层纤维环（iAF）细胞衰老的因素。在TNFα暴露下，采用先前已证明可诱导NP细胞衰老的浓度，牛的iAF细胞未发生衰老，这通过Ki67染色和生长曲线所显示的其持续增殖能力以及衰老标记物p16和p21缺乏表达得以证明。尽管iAF表达的TNFR1水平高于NP细胞，但仍未出现衰老反应。有趣的是，iAF细胞在TNFα作用下细胞内活性氧（ROS）或分泌的H₂O₂没有增加，这与NP细胞形成对比。TNFα处理后，只有iAF细胞超氧化物清除剂超氧化物歧化酶1（SOD1）和超氧化物歧化酶2（SOD2）的表达增加，而NP细胞的NOX4基因表达增加，NOX4是一种可产生H₂O₂的酶。用低剂量H₂O₂（50μM）处理iAF细胞可诱导衰老，然而与TNFα不同的是，H₂O₂没有诱导类似退变的变化，因为与未处理的对照组相比，Ⅱ型胶原蛋白（COL2）、聚集蛋白聚糖（ACAN）、基质金属蛋白酶13（MMP13）或白细胞介素6（IL6）基因表达以及COL2和ACAN免疫阳性细胞数量没有差异。后一结果表明iAF细胞可能具有不同的退变和衰老表型。为了评估衰老的NP细胞的旁分泌信号传导，将iAF细胞和TNFα处理的NP细胞进行共培养。在接触共培养中，NP细胞诱导iAF细胞衰老。因此，衰老的NP细胞可能分泌可溶性因子，在iAF内诱导退变和衰老变化。这可能有助于形成椎间盘退变的正反馈回路。这些因子可能包括H₂O₂和细胞因子（如TNFα）。进一步的研究将调查人类椎间盘细胞是否有类似的反应。