Induced senescence of healthy nucleus pulposus cells is mediated by paracrine signaling from TNF-α-activated cells

Intervertebral disc degeneration is an irreversible process associated with accumulation of senescent nucleus pulposus (NP) cells. This study investigates the hypothesis that Tumor necrosis factor-alpha (TNF-alpha)-treated senescent NP cells propagate senescence of neighboring healthy cells via a paracrine effect that involves p-Stat3 signaling and the cytokine interleukin-6 (IL-6). NP cells isolated from bovine caudal intervertebral disc (IVD) were treated with TNF-alpha to induce senescence which was confirmed by demonstrating upregulation of senescence-associated beta-galactosidase and p16. This was correlated with downregulation of NP-associated markers, Aggrecan, Col2A1, and Sox9. Direct contact and non-contact co-culture of healthy and senescent cells showed that TNF-alpha-treated cells increased the senescence in healthy cells via a paracrine effect. The senescent cells have a secretory phenotype as indicated by increased gene and protein levels of IL-6. Phosphorylated Signal Transducer and Activator of Transcription 3 (pStat3) levels were also high in treated cells and appeared to upregulate IL-6 as inhibition of Stat3 phosphorylation by StatticV downregulated IL-6 mRNA expression in cells and protein levels in the culture media. All trans retinoic acid, an IL-6 inhibitor, also decreased the secretion of IL-6 and reduced the paracrine effect of senescent cells on healthy cells. Decreased pStat3 levels and inhibition of IL-6 secretion did not fully restore NP gene expression of Col2A1 but importantly, appeared to cause senescent cells to undergo apoptosis and cell death. This study demonstrated the paracrine effect of senescent NP cells which involves Stat3 and IL-6 and may explain why senescent NP cells accumulate in IVD with age. The role of pSTAT3 and IL-6 in mediating NP senescence requires further study as it may be a novel strategy for modulating the senescent-inducing effects of TNF-alpha.

椎间盘退变是一个与衰老的髓核（NP）细胞积累相关的不可逆过程。本研究验证了肿瘤坏死因子 -α（TNF -α）处理的衰老NP细胞通过旁分泌效应传播邻近健康细胞衰老这一假说，该旁分泌效应涉及p - Stat3信号传导和细胞因子白细胞介素 - 6（IL - 6）。从牛尾椎间盘（IVD）分离的NP细胞用TNF -α处理以诱导衰老，这通过衰老相关β - 半乳糖苷酶和p16的上调得以证实。这与NP相关标志物聚集蛋白聚糖、Col2A1和Sox9的下调相关。健康细胞和衰老细胞的直接接触和非接触共培养表明，TNF -α处理的细胞通过旁分泌效应增加了健康细胞的衰老。衰老细胞具有分泌表型，表现为IL - 6的基因和蛋白质水平升高。磷酸化信号转导和转录激活因子3（pStat3）水平在处理后的细胞中也较高，并且似乎上调IL - 6，因为用StatticV抑制Stat3磷酸化可下调细胞中IL - 6 mRNA的表达以及培养基中的蛋白质水平。全反式维甲酸，一种IL - 6抑制剂，也减少了IL - 6的分泌，并降低了衰老细胞对健康细胞的旁分泌效应。pStat3水平降低和IL - 6分泌抑制并未完全恢复Col2A1的NP基因表达，但重要的是，似乎导致衰老细胞发生凋亡和细胞死亡。本研究证明了衰老NP细胞的旁分泌效应涉及Stat3和IL - 6，并可能解释了为什么随着年龄增长衰老NP细胞在椎间盘内积累。pSTAT3和IL - 6在介导NP衰老中的作用需要进一步研究，因为它可能是调节TNF -α诱导衰老效应的一种新策略。