Background

NOD-like receptor family CARD domain containing 3 (NLRC3) plays an important role in both innate and adaptive immunity. This study was to explore the function and related mechanisms of NLRC3 in a hypoxia/reoxygenation (H/R)-induced inflammatory response in RAW264.7 cells.

Methods

Liver ischemia-reperfusion (I/R) model in mice and H/R model in RAW264.7 cells were constructed. Western blotting was used to determine the protein expression level of NLRC3 in liver tissue and NLRC3, TRAF6, p–p65, p65, IκB–α, and the K63-linked ubiquitination level of TRAF6 in cells. The immunofluorescence assay was performed to evaluate the nuclear level of the NF–κB (p65). ELISA was conducted to measure the content of IL–1β in serum and cell supernatant. The interaction between NLRC3 and TRAF6 in cells was analyzed by the Co-IP assay.

Results

The NLRC3 protein level in liver tissue was decreased with the prolongation of reperfusion time (P < 0.05). The expression of NLRC3 and IκB–α protein in RAW264.7 was decreased gradually, while the expression of p–p65 and TRAF6 proteins and K63-linked ubiquitination of TRAF6 were increased gradually with the prolongation of reoxgenation time (P < 0.05). The Co-IP assay revealed that NLRC3 and TRAF6 can bind to each other directly. However, NLRC3 had no effect on the expression of TRAF6 protein. The ubiquitination test results showed that the K63-linked ubiquitination level of TRAF6 in H/R + Lv–NLRC3 group was significantly lower than that in the H/R + negative control (NC) group (P < 0.05). Moreover, the activation of NF–κB in H/R + Lv–NLRC3 group was inhibited compared with that in the H/R + NC group, and the level of the inflammatory factor IL–1β in the cell culture supernatant was also decreased accordingly (P < 0.05).

Conclusions

NLRC3 might alleviate H/R-induced inflammation in RAW264.7 cells by inhibiting K63-linked ubiquitination of TRAF6.

中文翻译：

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NLRC3 通过抑制 K63 相关的 TRAF6 泛素化来减轻 RAW264.7 细胞中缺氧/复氧诱导的炎症。

背景

NOD 样受体家族 CARD 域包含 3 (NLRC3) 在先天免疫和适应性免疫中都起着重要作用。本研究旨在探讨 NLRC3 在缺氧/复氧 (H/R) 诱导的 RAW264.7 细胞炎症反应中的功能和相关机制。

方法

构建小鼠肝缺血再灌注（I/R）模型和RAW264.7细胞H/R模型。Western印迹被用于测定肝组织和NLRC3，TRAF6，对P65，P65，我在NLRC3的蛋白质表达水平κ B- α，并且在细胞TRAF6的K63连接的遍在蛋白化的水平。进行了免疫荧光实验，以评估NF-的核液位κ B（P65）。ELISA法进行测定IL-1的含量β在血清和细胞上清液。通过 Co-IP 测定分析细胞中 NLRC3 和 TRAF6 之间的相互作用。

结果

肝组织NLRC3蛋白水平随着再灌注时间的延长而降低（P  < 0.05）。NLRC3的表达和我κ B- α在RAW264.7蛋白质逐渐降低，而p p65和TRAF6蛋白和TRAF6的K63连接的遍在蛋白化的表达用的复氧时间的延长逐渐升高（P  <0.05） . Co-IP 分析表明 NLRC3 和 TRAF6 可以直接相互结合。然而，NLRC3对TRAF6蛋白的表达没有影响。泛素化试验结果显示，H/R+Lv-NLRC3组TRAF6的K63连锁泛素化水平显着低于H/R+阴性对照（NC）组（P < 0.05)。此外，NF-的激活κ在H / R +吕NLRC3 B组被抑制与所述H / R + NC组相比，和炎性因子的水平的IL-1 β的细胞培养物上清液也相应降低（P  < 0.05）。

结论

NLRC3 可能通过抑制 K63 相关的 TRAF6 泛素化来减轻 RAW264.7 细胞中 H/R 诱导的炎症。