Research Of The Resistin-induced Effect On NODs-NF-κB Signaling Pathway In RAW264.7 Cells

Resistin is a new adipokine found in vivo in recent years, and its can contribute to the development of insulin resistance and mediate inflammatory reaction via different signal pathways. However, because of the specific receptor of resistin has not been identified in the body so far, the molecular mechanism of resistin is still unclear. NODs (NOD1 and NOD2) are the important receptors in innate immunity, which can be regulated by endogenous signaling molecules to induce aseptic inflammation. However, whether NODs play a role as resistin receptor has not been reported.To explore whether the proinflammatory effects of resistin is via NODs-NF-κB signaling pathway, the relationship between resistin and NODs receptor was studied in this experiment.Results:(1)To detect the expression of NODs at mRNA and protein level,the RAW264.7 cells were incubated with different concentrations of resistin (50 ng/mL、100 ng/mL、150 ng/mL、200 ng/mL), then the cells were collected to extract RNA and protein to detect the mRNA level and protein level of R1P2, IKKβ, NF-κB and the supernatant was collected to detect the content of cytokines when the predetermined culture time (0 h、 3 h、6h、12 h、24 h) is over. The results showed that the resistin treatment significantly increased the expression of NOD2, RIP2 and IKKβ at mRNA level in RAW264.7 cells. However, resistin has no effect on the expression of NOD1. The results aslo showed that resistin treatment significantly up-regulated the protein expression level of NOD2 and RIP2 in RAW264.7 cells. Furthermore, the nuclear transfer of NF-κB was also significantly increased. And resistin treatment significantly promoted the expression of TNF-a, IL-6 and IL-1β in RAW264.7 cells.(2) To screen the transfection conditions, the RAW264.7 cells were transfected with diffenrent concentration of NOD2-siRNA (20 nM,50 nM,80 nM) and the mRNA and protein levels of NOD2 were been detected. The results showed that the mRNA level of NOD2 was decreased by 75% and the protein level was decreased by 60% with 80 nM NOD2 transfected into RAW264.7 cells.(3) The RAW264.7 cells were transfected with NOD2-siRNA (80 nM) and then added to resistin (200 ng/ml). the level of NOD2 and its downstream signal molecules were been detected when the predetermined culture time is over. The results showed that the mRNA and protein level of NOD2 and RIP2 of group of NOD2-siRNA+ resistin remarkablely decreased compared to the group of resistin, and the mRNA level of IKKβ and the nuclear translocation of NF-κB was not significantly affected. The content of TNF-a. IL-6 and IL-1β was not significantly affected as well.(4) The RAW264.7 cells were incubated with parthenolide (25 μmol/L) for 1 hour, and then add resistin (200 ng/ml) in cultured for 24 hour. The cell supernatants were collected to detect the content of TNF-a, IL-6 and IL-1β. The results showed that the content of TNF-a, IL-6 and IL-1β were significantly decreased, indicating that the release of inflammatory cytokines such as TNF-a, IL-6 and IL-1β were inhibited by parthenolide which inhibite the nuclear translocation of NF-κB.Conclusion:(1) Resistin treatment can promote the expression of NOD2 at both mRNA and protein level, but has no effect on the expression of NOD1 in RAW264.7 cells. (2) Resistin exert proinflammatory its effects through promoting the expression of NOD2 and its downstream signal molecules, activating the NOD2 signaling pathway in RAW264.7 cells, but NOD2 isn’t the receptors of resistin. (3) NF-κB is the key transcription factor for resistin-induced proinflammatory effect in RAW264.7 cells.