ABSTRACT

Objectives: Inflammation is an important predisposing and progressive factor in chronic kidney disease (CKD). Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is associated with many fundamental cellular processes, but in chronic inflammatory pathologies remains unclear.

Methods: An in vitro peripheral inflammation model was established using lipopolysaccharide (LPS)-stimulated mouse RAW264.7 macrophages, followed by inflammasome activation by ATP treatment. Knockdown of hnRNPK by sihnRNPK and FLICE-like inhibitory protein (FLIP) by siFLIP transfection were achieved in Raw264.7 macrophages. ELISA was used to determine the expression of IL-1β, IL-18 and TNF-α. Real time PCR was applied to detect the mRNA levels of hnRNPK, NOD-like receptors family pyrin domain-containing 3 (NLRP3), FLIP, Caspase-1, IL-1β and IL-18. Western blot and immunofluorescence were performed to detect relevant protein expressions. Co-immunoprecipitation (Co-IP) was used to assess the interaction of hnRNPK with FLIP.

Results: Results showed that LPS plus ATP activated NLRP3 inflammasome, which evidenced by the up-regulation of TNF-α, IL-1β and IL-18. Notably, hnRNPK and FLIP were significantly up-regulated in activated NLRP3 inflammasome of macrophages. HnRNPK or FLIP knockdown significantly suppressed the activation of NLRP3 inflammasome, as reflected by down-regulation of Caspase-1, IL-1β and IL-18. Importantly, hnRNPK could directly bind to FLIP in activated NLRP3 inflammasome.

Discussion: Our findings suggest that hnRNPK could promote the activation of NLRP3 inflammasome by directly binding FLIP, which might provide potential new therapeutic targets for CKD.

摘要

目的：炎症是慢性肾脏病（CKD）的重要诱发和进展因素。异质核核糖核蛋白 K (hnRNPK) 与许多基本细胞过程有关，但在慢性炎症病理中仍不清楚。

方法：一种体外周炎症模型是使用脂多糖成立（LPS）刺激的小鼠巨噬细胞的RAW264.7，接着通过ATP治疗炎性活化。在 Raw264.7 巨噬细胞中实现了 sihnRNPK 对 hnRNPK 的击倒和通过 siFLIP 转染的 FLICE 样抑制蛋白 (FLIP)。采用ELISA法测定IL-1β、IL-18和TNF-α的表达。应用实时 PCR 检测 hnRNPK、NOD 样受体家族含 pyrin 结构域 3 (NLRP3)、FLIP、Caspase-1、IL-1β 和 IL-18 的 mRNA 水平。进行蛋白质印迹和免疫荧光检测相关蛋白表达。免疫共沉淀 (Co-IP) 用于评估 hnRNPK 与 FLIP 的相互作用。

结果：结果表明，LPS 加 ATP 激活 NLRP3 炎症小体，这可以通过 TNF-α、IL-1β 和 IL-18 的上调来证明。值得注意的是，hnRNPK 和 FLIP 在活化的巨噬细胞 NLRP3 炎性体中显着上调。HnRNPK 或 FLIP 敲低显着抑制了 NLRP3 炎性体的激活，如 Caspase-1、IL-1β 和 IL-18 的下调所反映的。重要的是，hnRNPK 可以直接与激活的 NLRP3 炎症小体中的 FLIP 结合。

讨论：我们的研究结果表明，hnRNPK 可以通过直接结合 FLIP 来促进 NLRP3 炎症小体的激活，这可能为 CKD 提供潜在的新治疗靶点。