SERGAS, Santiago University Clinical Hospital Research Laboratory 9 (NEIRID LAB: Neuroendocrine Interactions in Rheumatology and Inflammatory Diseases), Institute of Medical Research (IDIS), Santiago de Compostela, 15706, Spain.
J Orthop Res. 2013 Jul;31(7):1046-52. doi: 10.1002/jor.22331. Epub 2013 Mar 11.
Lipocalin 2 (LCN2) has recently emerged as a novel adipokine involved in different processes including arthritis and chondrocyte inflammatory response. However, little is known about its activity on chondrocyte homeostasis and its regulation by nitric oxide (NO) Hence, we performed a set of experiments aimed to achieve a better understanding of this relationship. Cell vitality was tested in the ATDC5 cell line by the MTT colorimetric assay. Protein expression and gene expression was evaluated by Western blot and real time RT-PCR, respectively. NO production (determined as nitrite accumulation) was assayed by the Griess reaction. First, we demonstrated that LCN2 decreased murine chondrocytes vitality. Next, LCN2 co-stimulation with LPS enhanced NOS2 protein expression by murine chondrocytes. In addition, inhibition of LPS-induced nitric oxide production by aminoguanidine, a selective NOS2 inhibitor, significantly reduced LPS-mediated LCN2 expression. In contrast, treatment of murine chondrocytes with sodium nitroprussiate (SNP), a classic NO donor, scarcely induced LCN2 expression. Intriguingly, SNP addition to LPS-challenged chondrocytes, treated with aminoguanidine, provoked a strong induction of LCN2 expression. Finally, murine ATDC5 cells, co-cultured with LPS pre-challenged macrophages, had higher LCN2 expression in comparison with murine chondrocytes co-cultured with non pre-challenged macrophages. In this work we have described for the first time that NO is able to exert a control on LCN2 expression, suggesting the existence of a feedback loop regulating its expression.
脂联素 2（LCN2）最近作为一种新的脂肪因子出现，参与包括关节炎和软骨细胞炎症反应在内的不同过程。然而，其对软骨细胞稳态的活性及其对一氧化氮（NO）的调节作用知之甚少。因此，我们进行了一系列实验，旨在更好地了解这种关系。通过 MTT 比色法测试 ATDC5 细胞系中的细胞活力。通过 Western blot 和实时 RT-PCR 分别评估蛋白质表达和基因表达。通过格里斯反应测定 NO 产生（确定为亚硝酸盐积累）。首先，我们证明 LCN2 降低了鼠软骨细胞活力。接下来，LCN2 与 LPS 共同刺激增强了鼠软骨细胞的 NOS2 蛋白表达。此外，氨基胍，一种选择性 NOS2 抑制剂，抑制 LPS 诱导的一氧化氮产生，显著降低了 LPS 介导的 LCN2 表达。相比之下，用经典的 NO 供体硝普钠（SNP）处理鼠软骨细胞几乎不会诱导 LCN2 表达。有趣的是，SNP 加入到用氨基胍处理的 LPS 刺激的软骨细胞中，会强烈诱导 LCN2 表达。最后，与与未预先刺激的巨噬细胞共培养的鼠 ATDC5 细胞相比，与预先用 LPS 刺激的巨噬细胞共培养的鼠 ATDC5 细胞具有更高的 LCN2 表达。在这项工作中，我们首次描述了 NO 能够对 LCN2 表达进行控制，表明存在调节其表达的反馈回路。
