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Zheng Yuanyuan
THE EFFECT OF NOD8 ON PRODUCTION OF NO, TNF-a AND IL-1P IN LPS-TREATED MACROPHAGES12
Цзинанъский университет, город Гуанъчжоу
[ABSTRACT] AIM: To investigate the effect of NOD8 on lipopolysaccharide (LPS) -induced releases of nitricoxide (NO), tumor necrosis factor a (TNF-a) and interleukin-1p (IL-1p) in RAW264. 7 cells. METHODS: The plas-mids of pEGFP-C2 and pEGFP-NOD8 were transfected into RAW264. 7 cells respectively. The transfected and non-trans-fected cells were stimulated by LPS for 0, 6, 12 and 24 h. NO production was evaluated by Griess reagent assay, and the levels of IL-1 p and TNF-a were measured by ELISA. The protein expression of NOD8 and the nuclear translocation of nu-clear factor kB (NF-kB) p65 subunit were detected by Western blotting. The level of activated caspase-1 was determined by fluorimetric method. RESULTS: Compared with pEGFP-C2 group, the protein expression of NOD8 was
12 NOD8 ^LPS |!®IlfflW&NO, TNF-afPIL-1p
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significantly elevated in pEGFP-NOD8 + LPS group. The releases of NO, IL-1 p and TNF-a were obviously increased after RAW264. 7 cells were treated with LPS for 6 h, 12 h and 24 h, and while the secretion of NO was significantly reduced in the cells transfected with pEGFP-NOD8 and induced by LPS for 12 h and 24 h, and the release of IL-ip was also significantly reduced at 6 h, 12 h and 24 h. However, no significant difference of TNF-a release was observed between pEGFP-C2 + LPS group and pEGFP-NOD8 + LPS group. The activation of caspase-1 in RAW264. 7 cells stimulated with LPS for 6 h, 12 h and 24 h was markedly increased, and the expression of NF-kB p65 subunit in the cytoplasm was significantly decreased, indicating that p65 nuclear translocation was increased. In addition, the activation of caspase-1 and the nuclear translocation of p65 were significantly inhibited in pEGFP-NOD8 + LPS group. CONCLUSION: NOD8 suppresses the releases of LPS-induced NO and IL-1p in RAW264. 7 cells by inhibiting the activation of caspase-1 and NF-kB.
Key words: RAW264.7 cells; Lipopolysaccharides; NOD8 protein; Nuclear factor-kappa B; Nitric oxide; Interleukin-1p.
