Department of Spine Surgery, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, China.
Cell Biol Int. 2022 Apr;46(4):568-578. doi: 10.1002/cbin.11751. Epub 2022 Jan 12.
Acid-induced cellular senescence is a critical underlying mechanism of intervertebral disc (IVD) degeneration (IDD). Acid stimulation activates a variety of biological changes including autophagy, endoplasmic reticulum stress, and related unfolded protein response (UPR), which are important regulators of cellular senescence. However, the precise mechanism of acid-mediated UPR and autophagy in nucleus pulposus cell (NPC) senescence has not been fully elucidated. In this study, we used acid to mimic the acidic microenvironment of IVD, and rat NPCs were cultured with or without autophagy or UPR signaling small-interfering RNAs. The related proteins and genes were assessed by immunofluorescence staining assay, Western blot analyses, and quantitative real-time polymerase chain reaction to monitor the activation of these signals and classify the molecular mechanisms underlying the correlation between autophagy and UPR pathway. Cell cycle analyses, senescence-associated β-galactosidase staining, gene expression, and immunoblotting analyses were performed to observe NPC senescence. Results showed that acid stimulation not only induced NPC senescence, but also initiated UPR and autophagy. Silencing the binding immunoglobulin protein signaling of UPR or autophagy signaling promoted rat NPC senescence. Knock-down of the UPR also blocked NPC autophagy. Taken together, UPR inhibits NPC senescence under acidic condition by activating autophagy. Hence, UPR-dependent autophagy could be an effective biologic target for the treatment of IDD in the future.
酸诱导的细胞衰老（cellular senescence）是椎间盘（intervertebral disc，IVD）退变（degeneration）（IDD）的关键潜在机制。酸刺激激活了多种生物学变化，包括自噬（autophagy）、内质网应激（endoplasmic reticulum stress）和相关未折叠蛋白反应（unfolded protein response，UPR），这些都是细胞衰老的重要调节因子。然而，酸介导的 UPR 和核内体（nucleus pulposus，NP）细胞自噬在 NP 细胞衰老中的精确机制尚未完全阐明。在这项研究中，我们使用酸模拟 IVD 的酸性微环境，并用或不用自噬或 UPR 信号小干扰 RNA（small-interfering RNA，siRNA）培养大鼠 NP 细胞。通过免疫荧光染色、Western blot 分析和实时定量聚合酶链反应（quantitative real-time polymerase chain reaction，qRT-PCR）评估相关蛋白和基因，以监测这些信号的激活，并对自噬和 UPR 途径之间的相关性的分子机制进行分类。细胞周期分析、衰老相关β-半乳糖苷酶染色、基因表达和免疫印迹分析用于观察 NP 细胞衰老。结果表明，酸刺激不仅诱导 NP 细胞衰老，还引发 UPR 和自噬。抑制 UPR 或自噬信号的结合免疫球蛋白蛋白（binding immunoglobulin protein，BIP）信号可促进大鼠 NP 细胞衰老。敲低 UPR 也阻断了 NP 细胞的自噬。总之，在酸性条件下，UPR 通过激活自噬抑制 NP 细胞衰老。因此，UPR 依赖性自噬可能成为未来治疗 IDD 的有效生物学靶点。
