Effect Of NOD8on The Release Of NO、TNF-α And IL-1β Induced By LPS

Objective:To investigate the effect of NOD8on the release of nitric oxide (NO), interleukin-1beta (IL-1β)and tumor necrosis factor-alpha (TNF-α) induced by LPS in RAW264.7macrophages.Methods:The pEGFP-NOD8recombinant plasmids were transfected into mouse macrophag RAW264.7by JetPRIME and the optimal dose of the pEGFP-NOD8plasmid was chosen. pEGFP-C2emptyplasmids and pEGFP-NOD8plasmids were transfected into RAW264.7cells respectively, then theexpression of green fluorescent protein was observed under the inverted fluorescence microscopeand NOD8protein expression was detected by Western blotting.On this basis, the experiment wasdivided into four groups:(1) negative control group (NC): cells were cultured in fresh mediumwithout any treatment.(2) pEGFP-C2group: RAW264.7cells were transfected with pEGFP-C2empty vector for24h, then cultured with the LPS-free fresh medium;(3) pEGFP-C2+LPS group:RAW264.7cells were transfected with pEGFP-C2empty vector for24h, then cultured with freshmedium containing1mg/L LPS;(4) pEGFP-NOD8+LPS group: RAW264.7cells were transfectedwith pEGFP-NOD8plasmid for24h, then cultured with fresh medium containing1mg/L LPS, thenthese cells were continued to culture for0,6,12and24h. NO production was evaluated by Griessreagent assay, and the levels of IL-1β and TNF-α were measured by ELISA. The level of activatedcaspase-1was determined by fluorimetric method and the expression of NF-kappa B p65subunit incytoplasma was detected by Western blotting.Result:(1) The3μg/mL pEGFP-NOD8plasmid was chosen as the optimal dose of transfection byobserving the effect of pEGFP-NOD8on RAW264.7cell viability and the effect of differentconcentrations of pEGFP-NOD8plasmids on the release of NO in LPS-stimulated RAW264.7cells.Meanwhile, pEGFP-C2and pEGFP-NOD8plasmids were transfected into RAW264.7cells, greenfluorescence could be observed under the inverted fluorescence microscope, confirming successful transfection. Furthermore, the expression of NOD8was detected by Western blotting and the resultshowed thatNOD8protein level in pEGFP-NOD8group was significantly increased comparedwith pEGFP-C2group.(2) The releases of NO and IL-1β in pEGFP-C2+LPS group were increasedin a time-dependent manner after RAW264.7cells were treated with LPS for6,12and24hcompared with the negative control and pEGFP-C2groups, while the secretions of NO andIL-1βwere significantly reduced in pEGFP-NOD8+LPS group at12,24h or6,12and24hrespectively. On the other hand, no significant difference of NO production was observed betweenpEGFP-C2+LPS and pEGFP-NOD8+LPS groups at6h (P>0.05). In addition, the TNF-α contentin cell supernatant of pEGFP-C2+LPS group was significantly increased at6h,12h and24h; butno significant difference of TNF-α release was found between pEGFP-C2+LPS andpEGFP-NOD8+LPS groups at different time points (P>0.05).(3) RAW264.7cells were stimulatedby LPS for6,12and24h, the activation of caspase-1in pEGFP-C2+LPS group was significantlyincreased. By contrast, the activation of caspase-1in pEGFP-NOD8+LPS group was significantlydecreased at different time points compared with the pEGFP-C2+LPS group.(4) the expression ofNF-κB p65subunit in cytoplasma of pEGFP-C2+LPS group was gradually reduced after RAW264.7cells were treated with LPS for6,12and24h, while the expression of NF-κB p65subunit incytoplasma of pEGFP-NOD8+LPS group was significantly increased at different time points.Conclusion:(1) pEGFP-NOD8plasmids were successfully transferred into the RAW264.7macrophages.and theNOD8protein was significantly increased compared to pEGFP-C2empty plasmid group.(2) RAW264.7macrophages were stimulated for6,12and24h, overexpression of NOD8inhibitsthe release of NO at12and24h,and reduced the secretion of IL-1β at6,12and24h, but there wasno effect on the release of TNF-α at different time points.(3) Overexpression of NOD8can inhibit LPS-induced activation of caspase-1and NF-kappa B p65subunit nuclear translocation.In summary, NOD8could suppress the release of LPS-induced NO and IL-1β in macrophages, themechanism may be associated with inhibiting the activation of caspase-1and NF-κB.