| Porcine contagious pleuropneumonia caused by the Actinobacillus pleuropneumoniae(A.pleuropneumoniae)is recognized as one of the major diseases that endanger the modern pig industry in China.It is generally held that the uncontrolled release of pro-inflammatory mediators/ cytokines in porcine alveolar macrophages is an important pathological reason for A.pleuropneumoniae-induced lung injury,but the specific mechanism is not clear.Previous studies have investigated that large amount of pro-inflammatory cytokines,like IL-1,IL-6 and TNF-α were expressed in diseased lungs and hilar lymph nodes of A.pleuropneumoniae-infected pigs.A noteworthy observation was that the resistin gene expression was significantly up-regulated in A.pleuropneumoniae-infected lungs.Some studies have demonstrated that resistin could up-regulate the expression levels of pro-inflammatory cytokines in peripheral blood mononuclear cells(PBMC)via TLR4/NF-κB mediated signaling pathway.Besides,the TLR4/NF-κB pathway is the main pathway,by which Gram-negative bacterial LPS could activate inflammatory cells.Porcine alveoli macrophages(PAMs)are predominant macrophages in lungs that can eliminate the invasive pathogens and play important roles both in pro-and anti-inflammatory reactions of pulmonary infection.Thus this study was aimed to investigate the mechanisms of A.pleuropneumoniae LPS/resistin on PAMs production of IL-1β,IL-6 and TNF-α via TLR4/NF-κB mediated pathway.1.Activation of porcine alveolar macrophages by Actinobacillus pleuropneumoniae LPS via the Toll-like receptor 4/ NF-κB mediated pathwayPAMs were exposed to 0 ng/mL,10 ng/mL,100 ng/mL,and 1000 ng/mL A.pleuropneumoniae LPS for 6 h,respectively.PAMs were also incubated with 100 ng/mL A.pleuropneumoniae LPS for 0 h,1.5 h,3 h,6 h,and 12 h,respectively.The levels of tested cytokines(IL-1β,IL-6,TNF-α,and Resistin)in the supernatant were measured by ELISA,and the mRNA expression levels of cytokines were tested by qRT-PCR.After 70% cells were attached to flask,PAMs were pretreated with 150 nmol/L TLR4-siRNA,MyD88-siRNA,and TRAM-siRNA for 48 h,respectively,and then exposed to 100 ng/mL A.pleuropneumoniae LPS for 6 h.The levels of tested cytokines in the supernatant were measured by ELISA,and the mRNA expression levels of genes(TLR4,MyD88,TRAM,NF-κB,IL-1β,IL-6,TNF-α,and Resistin)were tested by qRT-PCR.After 100% cells were attached to flask,PAMs were pretreated with 5 μmol/L Bay11-7082 for 2 h,and then exposed to 100 ng/mL A.pleuropneumoniae LPS for 6 h.The levels of cytokines were tested by ELISA and qRT-PCR.Results in this study showed that the expression levels of cytokines were increased when PAMs were exposed to different concentrations of A.pleuropneumoniae LPS for 6 h,and the expression levels of cytokines were increased when PAMs were incubated with 100 ng/mL A.pleuropneumoniae LPS for the indicated times.Due to the cytopathic effect of PAMs caused by A.pleuropneumoniae LPS,the expression levels of cytokines were decreased dramatically when PAMs were exposed to 1000 ng/mL A.pleuropneumoniae LPS for 6 h.After PAMs were pretreated with siRNA or Bay11-7082,the cytokines induced by A.pleuropneumoniae LPS were decreased significantly(P< 0.05).These results showed that A.pleuropneumoniae LPS might promote the expression levels of IL-1β,IL-6,TNF-α,and Resistin,and the mechanism of which was closely correlated to the genes expression of TLR4,MyD88,TRAM,and NF-κB in PAMs.2.Activation of the porcine alveolar macrophages via Toll-like receptor 4/NF-κB mediated pathway provides a mechanism of resistin leading to inflammationPAMs were exposed to 0 ng/mL,1 ng/mL,10 ng/mL,and 100 ng/mL resistin for 6 h,respectively.PAMs were also incubated with 10 ng/mL resistin for 0 h,1.5 h,3 h,6 h,12 h,and 24 h,respectively.The levels of tested cytokines(IL-1β,IL-6,and TNF-α)in the supernatant were measured by ELISA,and the mRNA expression levels of cytokines were tested by qRT-PCR.After 70% cells were attached to flask,PAMs were pretreated with 150 nmol/L TLR4-siRNA,MyD88-siRNA,and TRAM-siRNA for 48 h,respectively,and then exposed to 10 ng/mL resistin for 6 h.The levels of tested cytokines in the supernatant were measured by ELISA,and the mRNA expression levels of genes(TLR4,MyD88,TRAM,NF-κB,IL-1β,IL-6,and TNF-α)were tested by qRT-PCR.After 100% cells were attached to flask,PAMs were pretreated with 5 μmol/L Bay11-7082 for 2 h,and then exposed to 10 ng/mL resistin for 6 h.The levels of cytokines were tested by ELISA and qRT-PCR.Results in this study showed that the expression levels of cytokines were increased when PAMs were exposed to different concentrations of resistin for 6 h,and the expression levels of cytokines were increased when PAMs were incubated with 10 ng/mL resistin for indicated times.After PAMs were pretreated with siRNA or Bay11-7082,the cytokines induced by resistin were decreased significantly(P< 0.05).These results showed that resistin might promote the expression levels of IL-1β,IL-6,and TNF-α,and the mechanism of which was closely correlated to the genes expression of TLR4/NF-κB mediated pathway in PAMs.3.Resistin competes with A.pleuropneumoniae LPS in porcine alveolar macrophages via Toll-like receptor 4/NF-κB-mediated pathwayPAMs were pretreated with three different concentrations of resistin(1,10 and 100 ng/mL)for 2 h,and then incubated with A.pleuropneumoniae LPS(100 ng/mL)for 12 h.The levels of tested cytokines(IL-1β,IL-6,and TNF-α)in the supernatant were measured by ELISA,and the mRNA expression levels of cytokines were tested by qRT-PCR.After 70% cells were attached to flask,PAMs were pretreated with 150 nmol/L TLR4-siRNA,MyD88-siRNA,and TRAM-siRNA for 48 h,respectively,then exposed to 10 ng/mL resistin for 2 h,and finally treated with 100 ng/mL A.pleuropneumoniae LPS for 12 h.The levels of tested cytokines in the supernatant were measured by ELISA,and the mRNA expression levels of genes(TLR4,MyD88,TRAM,NF-κB,IL-1β,IL-6,and TNF-α)were tested by qRT-PCR.After 100% cells were attached to flask,PAMs were pretreated with 5 μmol/L Bay11-7082 for 2 h,then exposed to 10 ng/mL resistin for 2 h,and finally treated with 100 ng/mL A.pleuropneumoniae LPS for 12 h.The levels of cytokines were tested by ELISA and qRT-PCR.Results in this study showed that the expression levels of cytokines induced by A.pleuropneumoniae LPS were decreased when PAMs were exposed to different concentrations of resistin.After PAMs were pretreated with siRNA or Bay11-7082,the cytokines induced by resistin and A.pleuropneumoniae LPS were decreased significantly(P< 0.05).These results showed that the competitive effect of resistin on A.pleuropneumoniae LPS-induced IL-1β,IL-6,and TNF-α expression in PAMs was mediated through interference of TLR4 and its downstream molecules such as MyD88,TRAM and NF-κB.In conclusion1.The mechanism that A.pleuropneumoniae LPS promoted the release of pro-inflammatory cytokines IL-1β,IL-6,TNF-α and Resistin was positively correlated with the up-regulation of TLR4,MyD88,TRAM,and NF-κB in PAMs.2.The mechanism that resistin promoted the release of pro-inflammatory cytokines IL-1β,IL-6,and TNF-α was positively related to the up-regulation of TLR4/NF-κB mediated pathway in PAMs.3.The mechanism that resistin exerted inhibition role on the releasing of pro-inflammatory cytokines IL-1β,IL-6,and TNF-α was correlated with the inhibition of TLR4,MyD88,TRAM,and NF-κB in PAMs. |
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