| Galectin-1(Gal-1)is expressed in both inside(nucleus,cytoplasm,and inner surface of plasma membrane)and outside(outer surface of cell membrane and extracellular matrix)of cell.It plays a profound role in regulating inflammation by affecting processes such as immune cell adhesion,migration,activation,signaling,proliferation,differentiation,and apoptosis.In this study,we aim at investigating the effect and mechanism of Gal-1on inflammatory responses in vitro and in vivo.1.The effect of Gal-1 on NF-?B inflammation in S.aureus-infected macrophagesBackgroundGal-1 plays an important role in inflammatory response.Recent studies have found that Gal-1 has an anti-inflammatory effect on Trypanosoma cruzi-infected macropahsges.However,the anti-inflammatory effects of Gal-1 on NF-?B inflammation have not confirmed in S.aureus-infected macrophages.Objective1.To study the activated Gal-1 and NF-?B in S.aureus-infected macrophages.2.To explore the roles of Gal-1 in S.aureus-mediated NF-?B inflammation in macrophages.Methods1.Measured activation of Gal-1 and NF-?B in S.aureus-infected macrophages.We infected the mouse macrophage cell line RAW264.7 with S.aureus for 20 min,40 min,60 min,and 120 min.The expression levels of correlative inflammatory signaling proteins were detected by Western Blotting(WB),and GFP-p-P65 were observed by laser scanning confocal microscope(LSCM).2.Measured NF-?B inflammatory during administration of r Gal-1 in S.aureusinfected macrophagesRAW264.7 macrophages pretreated by r Gal-1 were infected with S.aureus,the expression levels of correlative inflammatory signaling proteins were detected by WB,GFP-p-P65 were observed by LSCM,and the secretion levels of IL-1? and TNF-? in cell supernate were measured by ELISA.3.Measured NF-?B inflammatory during administration of Gal-1 si RNA in S.aureus-infected macrophagesRAW264.7 macrophages were pretreated by Gal-1 si RNA,and then were infected with S.aureus,the expression levels of related inflammatory signaling proteins were detected by WB,GFP-p-P65 were observed by LSCM,and the cellular secretion levels of IL-1? and TNF-? in supernate were examined by ELISA.4.Measured NF-?B inflammatory during administration of r Gal-1 and U0126 in S.aureus-infected macrophagesRAW264.7 macrophages pretreated by r Gal-1 and U0126 were infected with S.aureus,the expression levels of correlative inflammatory signaling proteins were detected by WB and the secretion levels of IL-1? and TNF-? in cell supernate were measured by ELISA.Results1.The expression levels of p-ERK,p-P65 and Gal-1 are significantly increased in S.aureus-infected RAW264.7 cellsInfected RAW264.7 with S.aureus,the expression levels of p-ERK,p-P65 and Gal-1 were significantly increased by WB,and the expression levels reached a peak at 60 min after infection,and then the expression levels gradually dcreased.Similarly,the expression levels of GFP-p-P65 were significantly increased by LSCM.The expression levels peaked at 60 min point,and then the expression levels gradually decreased.2.r Gal-1 reduces S.aureus-induced NF-?B inflammation in RAW264.7 macrophagesRAW264.7 macrophages pretreated by r Gal-1 were infected with S.aureus for 60 min.Compared to the untreated group,the protein expression levels of p-ERK and p-P65 were significantly reduced in the r Gal-1 treated group.Similarly,significantly decreased expression levels of GFP-p-P65 were observed by LSCM in the r Gal-1 treated group.Furthermore,the production levels of IL-1? and TNF-? in cell supernate were significantly decreased in the r Gal-1 treated group.3.Gal-1 si RNA enhances S.aureus-induced NF-?B inflammation in RAW264.7 macrophages.RAW264.7 macrophages pretreated by Gal-1 si RNA were infected with S.aureus for 60 min.Compared to the control si RNA group,the protein expression levels of p-ERK were significantly dereased,however,the expression levels of p-P65 were significantly increased in the Gal-1 si RNA group.Similarly,significantly elevated expression levels of GFP-p-P65 were observed by LSCM in the Gal-1 si RNA group.Furthermore,the production levels of IL-1? and TNF-? in cell supernate were significantly increased in the r Gal-1 treated group.4.r Gal-1 decrease S.aureus-induced NF-?B inflammation through ERK signal pathway in RAW264.7 macrophagesCompared to the r Gal-1 pre-treated cells,the protein expression levels of p-ERK and pP65 were significantly increased in the r Gal-1 and U0126 pre-treated RAW264.7 macrophages.Furthermore,the production levels of IL-1? and TNF-? in cell supernate were significantly elevated in the r Gal-1 and U0126 pre-treated RAW264.7 macrophages.Conclusion1.S.aureus activates NF-?B inflammation and Gal-1 expression in RAW264.7 macrophages.2.Gal-1 reduces S.aureus-induced NF-?B inflammation through ERK signal pathway in RAW264.7 macrophages.2.The effect and mechanism of Gal-1 on allergic airway inflammation in murine.BackgroundAsthma is an airway inflammatory disease involving a variety of cells and cellular components.Statistically,there are approximately 30 million patients with asthma in China.Recent studies have shown that Gal-1 can be reckened as an anti-inflammatory molecular in vivo and in vitro.Gal-1 has immunomodulatory activities in various allergic inflammatory disorders,but its potential anti-inflammatory properties on allergic airway diseases have not been confirmed.Obejective1.To study the expression levels of Gal-1 in a mouse model of allergic airway inflammation.2.To explore the role and mechanism of Gal-1 in a mouse model of allergic airway inflammation.Methods1.Establish an allergic airway inflammation model,and measure the expression levels of Gal-1 in the lung tissues of mice.C57BL/6 mice were sensitized with OVA by intraperitoneal injection and challenged with OVA by nebulizer to establish an allergic airway inflammation model.We measured the expression levels of Gal-1 m RNA in the lung tissues of mice by RT-PCR,and detected the expression levels of Gal-1 protein in the lung tissues by immunohistochemistry and WB respectively,and measured the production levels of Gal-1 in BALF by ELISA.2.Measure the related inflammatory parameters in murine model of allergic airway inflammation after administration of r Gal-1.Mice were pretreated with r Gal-1 before challenged.The airway inflammatory scores were measured by H&E staining,and airway goblet cell hyperplasia and mucus secretion were detected by PAS staining,and the classification and number of inflammatory cells in BALF were measured by Wright's staining,and the production levels of IL-4 and IL-5 in BALF were measured by ELISA.3.Detect the infiltration of eosinophils and the expression levels of epithelial cellderived cytokines in the lung tissues after administration of r Gal-1.Mice were pretreated with r Gal-1 before challenged.We observed the infiltration of eosinophils in the lung by high resolution microscope in PAS staining pictures.The m RNA expression levels of eotaxin,EPX,IL-25,IL-33 and TSLP in lung tissues were detected by RT-PCR,and the protein expression levels of eotaxin,EPX and IL-25 were detected by WB.4.Measure the expression levels of MAPKs and p-P65 in the lung tissues after administration of r Gal-1.Mice were pretreated with r Gal-1 before challenged.We measured the protein expression levels of p-MAPKs including p-ERK,p-JNK,p-P38 and p-P65 in the lung tissues by WB.5.Detect the production levels of systemic inflammation in allergic mice after administration of r Gal-1.Mice were pretreated with r Gal-1 before challenged.We detected the plasma levels of IL-17 and anti-OVA Ig E in allergic mice.Results1.The expression levels of Gal-1 are induced in OVA-challenged mice.The m RNA and protein expression levels of Gal-1 in the lungs of OVA-challenged mice were elevated compared to that in control mice.Similarly,the production levels of Gal-1 in BALF of OVA-challenged mice were also increased compared to that of control mice.2.r Gal-1 attenuates OVA-induced allergic airway inflammation in mice.Administration of r Gal-1 showed a significant decrease in goblet cell proliferation,mucus secretion,BALF inflammatory cell inflitration,and cytokine production compared to that in untreated mice.3.Treatment of r Gal-1 decreases the infiltration of eosinophils and the expression levels of IL-25 in allergic lung.Treatment of r Gal-1 decreases the infiltration of eosinophils and the m RNA and protein expression levels of the endogenous adhesion molecules eotaxin and EPX.The m RNA expression levels of IL-25,IL-33 and TSLP were significantly increased in the lungs of OVA-challenged mice.However,only the m RNA and protein expression levels of IL-25,neither IL-33 nor TSLP,were significantly decreased by treatment of r Gal-1 relative to those of the untreated mice.4.r Gal-1 induces the expression of the p-ERK and decreases the expression of p-P65 in allergic lungs.Only the expression levels of p-ERK,neither p-JNK nor p-P38,were significantly increased by treatment with r Gal-1 relative to the untreated OVA-challenged mice.Moreover,the expression levels of p-P65 were marked decreased in r Gal-1 treated mice.5.r Gal-1 reduces the plasma levels of IL-17 and anti-OVA Ig E in allergic mice.Treatment of r Gal-1 markedly decreased the plasma levels of IL-17 and OVA-induced Ig E relative to the untreated OVA-challenged mice.Conclusions1.The expression levels of Gal-1 is induced in the lungs of OVA-challenged allergic mice.2.r Gal-1 treatment was effective in the reduction of allergic airway inflammation through activation of the ERK signaling pathway in acute asthma mice. |
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