hnRNPK knockdown alleviates NLRP3 inflammasome priming by repressing FLIP expression in Raw264.7 macrophages.
hnRNPK knockdown alleviates NLRP3 inflammasome priming by repressing FLIP expression in Raw264.7 macrophages.
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hnRNPK 敲低通过抑制 Raw264.7 巨噬细胞中的 FLIP 表达来减轻 NLRP3 炎性体启动。
DOI：
10.1080/13510002.2020.1857157
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发表时间：
2020-12
期刊：
影响因子：
--
通讯作者：
Zhang Y
中科院分区：
文献类型：
--
作者：
Feng J;Li H;Li J;Meng P;Wang L;Liu C;Zhao S;Sun W;Zhang Y
目的：炎症是慢性肾脏病（CKD）的重要易感因素和进展因素。异质性核核糖核蛋白K（hnRNPK）与许多基本的细胞过程，但在慢性炎症病理仍不清楚。研究方法：使用脂多糖（LPS）刺激的小鼠RAW264.7巨噬细胞建立体外外周炎症模型，随后通过ATP处理进行炎性小体活化。在Raw 264.7巨噬细胞中实现了sihnRNPK对hnRNPK的敲低和siFLIP转染FLICE样抑制蛋白（FLIP）。ELISA法检测IL-1β、IL-18和TNF-α的表达。采用真实的实时荧光定量PCR检测hnRNPK、NOD样受体家族pyrin domain-containing 3（NLRP 3）、FLIP、Caspase-1、IL-1β和IL-18的mRNA水平。Western blot和免疫荧光法检测相关蛋白的表达。使用免疫共沉淀（Co-IP）来评估hnRNPK与FLIP的相互作用。结果：LPS + ATP可激活NLRP 3炎性体，并可上调TNF-α、IL-1β和IL-18的表达。值得注意的是，hnRNPK和FLIP在活化的巨噬细胞NLRP 3炎性小体中显著上调。HnRNPK或FLIP敲低显著抑制NLRP 3炎性体的活化，如通过Caspase-1、IL-1β和IL-18的下调所反映的。重要的是，hnRNPK可以直接与活化的NLRP 3炎性体中的FLIP结合。讨论内容：提示hnRNPK可通过与FLIP直接结合，促进NLRP 3炎性小体的活化，这可能为CKD的治疗提供新的靶点。
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.
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