Nicotinamide Phosphoribosyltransferase Inhibitor APO866 Prevents IL-1β- Induced Human Nucleus Pulposus Cell Degeneration via Autophagy
Nicotinamide Phosphoribosyltransferase Inhibitor APO866 Prevents IL-1β- Induced Human Nucleus Pulposus Cell Degeneration via Autophagy
复制标题
烟酰胺磷酸核糖转移酶抑制剂 APO866 通过自噬防止 IL-1beta 诱导的人髓核细胞变性。
DOI：
10.1159/000493843
复制
发表时间：
2018-01-01
影响因子：
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通讯作者：
Yuan, Wen
中科院分区：
文献类型：
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作者：
Shi, Changgui;Wu, Huiqiao;Yuan, Wen
背景/目的：椎间盘由细胞外基质（ECM）和中心胶状髓核（NP）组成，其外层被称为纤维环。ECM代谢紊乱导致纤维环和NP之间的边界丧失，从而导致椎间盘退变（IDD）。促炎细胞因子，如白细胞介素-1β，介导IDD的进展。烟酰胺磷酸核糖基转移酶（Nampt）催化了烟酰胺腺嘌呤二核苷酸（NAD）生物合成的第一步，已知可由IL-1β诱导。APO866是一种NAD生物合成抑制剂，参与自噬。LC3（微管相关蛋白1轻链3）是自噬的关键调节因子，被用作自噬增加的指标。在此，我们研究APO866在调节NP细胞自噬和IL-1β介导的NP细胞变性和凋亡中的作用。方法：从IDD组织中提取NP细胞，在DMEM/F12培养基中培养。采用不同浓度的IL-1β（0、0.5、1、5、10 ng/mL）诱导Nampt作用24 h或10 ng/mL IL-1β作用NP细胞0、6、12、48 h，采用QRT-PCR和western blots检测IDD患者NP组织和NP细胞中Nampt和ecm相关蛋白的表达。共聚焦分析用于检测膜结合LC3， Aggrecan和Collagen II。结果：重度IDD （IV级和V级）患者与轻度IDD （II级和III级）患者相比，Nampt在NP组织中的表达水平更高。在NP细胞中，10 ng/mL IL-1β诱导Nampt表达48 h，降解相关蛋白ADAMTS4/5和MMP-3/13的表达增加，ecm相关蛋白Aggrecan和Collagen II的表达降低。然而，Nampt抑制剂APO866阻断了IL-1β的诱导，下调Nampt表达增加了被IL-1β抑制的ECM蛋白的表达。此外，自噬标志物LC3和Beclin-1提供的证据表明，APO866诱导NP细胞自噬。此外，虽然APO866抑制IL-1β下调ecm相关蛋白的表达，但这种功能被自噬抑制剂3-甲基腺嘌呤阻断。结论：APO866通过抑制il -1β诱导的NP细胞变性和凋亡来保护NP细胞并诱导自噬，可能具有治疗IDD的潜力。
Background/Aims: Intervertebral discs consist of an extracellular matrix (ECM) with a central gelatinous nucleus pulposus (NP) enclosed in an outer layer known as the annulus fibrosus. ECM metabolic disorders result in loss of boundary between the annulus fibrosus and NP, which can lead to intervertebral disc degeneration (IDD). Proinflammatory cytokines, such as interleukin (IL)-1β, mediate the progression of IDD. Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the first step in the biosynthesis of nicotinamide adenine dinucleotide (NAD) and is known to be induced by IL-1β. APO866 is an inhibitor of NAD biosynthesis and is involved in autophagy. LC3 (microtubule-associated protein 1 light chain 3) is a key regulator of autophagy and is used as an indicator of increased autophagy. Herein, we investigate the role of APO866 in regulating autophagy in NP cells and IL-1β mediated NP cell degeneration and apoptosis. Methods: NP cells were extracted from IDD tissues and cultured in DMEM/F12 medium. Nampt was induced by different concentrations of IL-1β (0, 0.5, 1, 5, 10 ng/mL) for 24 h or NP cells were treated with 10 ng/mL IL-1β for 0, 6, 12, 48 h. QRT-PCR and western blots were used to detect Nampt and ECM-related protein expression in NP tissue of patients with IDD and in NP cells. Confocal analysis was used to detect membrane-bound LC3, Aggrecan, and Collagen II. Results: Nampt is expressed in NP tissue at higher levels in severe grades of IDD (Grade IV and V) compared with low grades (Grade II and III). In NP cells, 10 ng/mL IL-1β induced Nampt expression for 48 h, increased expression of the degradative-associated proteins, ADAMTS4/5 and MMP-3/13, and decreased expression of ECM-related proteins, Aggrecan and Collagen II. However, the Nampt inhibitor APO866 blocked IL-1β induction, and the knockdown of Nampt expression increased the expression of ECM proteins that were inhibited by IL-1β. Moreover, evidence provided by the autophagic markers LC3 and Beclin-1 indicated that APO866 induced NP cell autophagy. Furthermore, although APO866 inhibited the downregulated expression of ECM-related proteins by IL-1β, this function was blocked by autophagy inhibitor, 3-methyladenine. Conclusion: APO866 protects NP cells and induces autophagy by inhibiting IL-1β-induced NP cell degeneration and apoptosis, which may have therapeutic potential in IDD.