Inhibitory Effect And Mechanism Of Galectin-1 On The Activation Of NLRP3 In LTA-induced Macrophages

Background Bronchial asthma is a chronic airway inflammation involving a variety of inflammatory cells and structural cells and cell components characterized by airway hyperresponsiveness and reversible airflow limitation.Its symptom is cough,wheeze,night early morning more.According to current surveys,asthma attacks affect 1-18% of the population in different countries.Currently,there are 45 million adults with asthma in China.In the lungs,the NLRP3 inflammarosomes recruit and activate the pro-inflammatory protease Caspase-1 by identifying pathogen-associated molecular patterns(PAMP)or host source molecular patterns of red flags(DAMP);Activated caspase-1 cleaved the precursors of IL-1? and IL-18 to produce the corresponding mature cytokines IL-1? and IL-18,which induced inflammation.Studies have shown a significant increase in NLRP3 inflammasome in patients with acute asthma,suggesting that NLRP3 inflammasome plays an important role in the pathogenesis of asthma.Studies have shown that galactosin-1 has a wide range of anti-inflammatory and immunomodulatory effects.Although Gal-1 has been proved to have a wide range of anti-inflammatory effects in macrophage inflammation,the specific molecular mechanism is still unknown.Therefore,macrophages were used in this study to construct an inflammatory response model,and exogenous gal-1 interfered with the NF-?B-NLRP3 signaling pathway.Objective Effect of galactosagglutinin-1 on inhibition of NLRP3 in LTA-induced macrophages of staphylococcus aureus and its mechanism.Methods Raw264.7 macrophages were cultured to a stable state with 96-well plate,and the effects of recombinant mouse galactosagglutinin-1(rm Gal-1)at different concentrations on cell viability were detected by tetramethylazolizate microenzyme reaction colorimetry(MTT),and the state of cells at different concentrations was observed under an optical microscope.In cultured macrophages and 12-well plates,LTA stimulated the expression of Gal-1,NLRP3 and p65 proteins for 1h,3h and 6h,respectively.Macrophages were cultured and 12-well plates were divided into four groups,namely the blank Control group(Control group),the inflammation model group(LTA group),the drug Control group(rm Gal-1 group),and the intervention group(LTA+ rm Gal-1 group).The other four groups were:Blank Control group(Control group),inflammatory model group(LTA group),drug Control group(JSH-23 group),and intervention group(LTA+ JSH-23 group).Raw264.7 cells were pretreated with rm Gal-1 and NF-?B inhibitor JSH-23,respectively,in the model group and the intervention group.The expressions of P-p65 and NLRP3 in cells were observed by laser confocal microscopy.The protein expression levels of p65,P-p65,NLRP3,ASC,Caspase-1 and Pro-Caspase-1 were further determined by Western blot.Results Under the light microscope,the state of the cells remained basically unchanged after the intervention with recombinant mouse galactosagglutinin-1(0.04,0.4)?g/ml.When the concentration of the drug increased to 4 ?g/ml,the cells were significantly inhibited and overstimulated to produce antennae.MTT further demonstrated that,compared with the control group(0 ?g/ml),the concentration of rm Gal-1 at 0.04?g/ml and 0.4 ?g/ml had no significant effect on cell viability,while the concentration at 4?g/ml had a significant effect on cell viability(P<0.01).LTA stimulated Raw246.7 macrophages to activate NF-?B-NLRP3 pathway in a time-dependent manner,and the expression levels of P-p65,NLRP3 and Gal-1 peaked 3 h after stimulation,and the expression levels of endogenous Gal-1 increased significantly with the extension of time(P<0.05).Immunofluorescence results showed that after LTA stimulation of Raw264.7cells for 3 h,the expression levels of NLRP3 and P-p65 in cytoplasm were significantly increased compared with the control group.Meanwhile,Western blot results also showed that the expression levels of NLRP3,P-p65,ASC and Caspase-1 in LTA group were significantly increased(P <0.01).The expression levels of NLRP3,P-p65,ASC and Caspase-1 in intracellular NLRP3 were significantly decreased(P <0.01).Conclusions Gal-1 May inhibit LTA-induced macrophage inflammation through the NF-?B-NLRP3 signaling pathway.