The Effection Of Resistin On NLRP3-NF-?B Signaling Pathway In RAW264.7 Cells

Resistin is an inflammatory cytokine that can participate in the immune regulation of the body,it is associated with the occurrence of various diseases such as type II diabetes and obesity.NLRs(NOD-like receptors),include NOD1(Nucleotide-binding oligomerization domains 1),NOD2,NLRP3,etc,are important receptors in the innate immune system,can accept the regulation of various molecules,and are associated with the activation of multiple immune-related signaling pathways.Recent studies have shown that resistin doesn't affect NOD1 expression in RAW264.7 cells,but it can promote NOD2 expression and activate NOD2-NF-?B signaling pathway,releasing inflammatory cytokines like IL-6,TNF-? and so on;while NOD2 is not a essential factor for the activation of NF-?B and the release of inflammatory cytokines induced by resistin.NLRP3 is the center of the NLRP3 inflammasome,it is regulated by NF-?B and involved in the inflammatory process.There is no report about the association between resisitin and NLRP3,Whether resistin can activate NLRP3 through the NF-?B signaling pathway and initiate NLRP3 inflammasome were still to be proved.Therefore,we used the RAW264.7(mouse macrophage)cells to study the effects of resistin on its NLRP3 gene and protein expression,and the activation of NF-?B signaling pathway to promote the release of inflammatory cytokines,define the mechanism of resistin and NLRP3.Methods and Results:(1)RAW264.7 were incubated with different resistin concentrations of 50 ng/mL,100 ng/mL,150 ng/mL,and 200 ng/mL for a predetermined time(0 h,3 h,6 h,12 h,24 h).Real-Time RCR and Western Blot techniques were used to detect the expression levels of NLRP3 and NF-?B in the cells.ELISA was used to detect the concentration of cytokines such as TNF-?,IL-6,IL-18 and IL-1? in cell supernatants.The results showed that resistin can upregulate the gene and protein expression levels of NLRP3 and NF-?B in RAW264.7,the concentrations of TNF-?,IL-6,IL-18 and IL-1? in supernatant are related to the final concentration of resistin(50-200 ng/mL)and culture time(0-24h)increased.(2)Three different siRNAs were designed to slience the NLRP3 gene.The transfection efficiency was detected by fluorescence microscopy and the silence effect was detected by Real Time-PCR.As a result,it was found that the silencing effect of the three siRNAs were poor under high transfection efficiency conditions.Subsequently,RAW264.7 cells were incubated with MCC950 at a final concentration of 10 nmol/L for0.5 h,then treated with 200 ng/mL resistin for 24 h.Detected the gene and protein expression changes of NLRP3 and NF-?B,and detected the concentration of IL-6,IL-18,IL-1? and TNF-? in supernatant.Results: After add MCC950,the gene and protein expression of NLRP3 in RAW264.7 cells was significantly down-regulated(P<0.001);TNF-?,IL-6,IL-1? and IL-18 in supernatant were significantly decreased(P<0.001).Which showed us that NLRP3 is a key regulator of the release of inflammatory cytokines(TNF-?,IL-6,IL-18 and IL-1?)induced by resistin in RAW264.7 cells,but it doesn't affect the expression of NF-?B.(3)RAW264.7 cells were pretreated with a final concentration of 25 ?mol/L parthenolide(specific inhibitor of NF-?B)for 1 h,then added 200 ng/mL resistin incubating for 24 h to detect the protein expression changes of NLRP3 and NF-?B and the concentrations of IL-6,IL-18,IL-1? and TNF-? in supernatant.Results: After adding parthenolide,the protein concentration of NLRP3 and NF-?B were significantly reduced(P<0.001),and the concentrations of TNF-?,IL-6,IL-18 and IL-1? in the supernatant were significantly decreased(P<0.001).It was suggested that NF-?B is an essential factor of resistin-promoted both NF-?B and NLRP3 protein expression and the release of cytokines in RAW264.7 cells(TNF-?,IL-6,IL-18 and IL-1?)induced by resistin.Conclusions:(1)Resistin promotes the NLRP3 gene expression and protein expression in RAW264.7 cells,activates NF-?B and promotes the release of pro-inflammatory cytokines such as IL-6,IL-18,TNF-? and IL-1?.(2)NLRP3 participates in the release of resistin-induced pro-inflammatory cytokines such as TNF-?,IL-6,IL-18 and IL-1? in RAW264.7 cells,but it is not a necessary factor for resistin-activated NF-?B.(3)NF-?B is one of the key regulators of NLRP3 expression and the release of pro-inflammatory cytokines induced by resistin in RAW264.7 cells.