| Objective:To investigate the relationship between autophagy and the proinflammatory effect of interleukin-33(IL-33)in acute respiratory distress syndrome(ARDS).Method:1.Collect plasma samples from normal healthy controls and clini cal ARDS patients,detect the expression of IL-33 by ELISA,and de tect TNF-α,IL-1β,IL-2,IL-6,IL-10 expression levels by cytokine m agnetic bead panel kit.1.20 male C57BL/6 wild type mice were randomly divided into four groups,blank control group(N group),lipopolysaccharide treatment group(LPS group),lipopolysaccharide + rapamycin pretreatment group(RAPA group),lipopolysaccharide + 3-methyl adenine pretreatment group(3-MA group),n=5/group.A lung injury model was prepared by intratrachealinstillation of PBS containing LPS in LPS group.Thirty minutes before operation,the RAPA group was intraperitoneally injected with rapamycin 4mg/kg according to the body weight ratio,and the 3-MA group was intraperitoneally injected with 3-methyladenine 15 mg/kg according to the body weight ratio.The mice were observed at 24 hours after surgery.The blood,lung tissue,and alveolar lavage fluid samples were obtained.HE staining was performed to evaluated the degree of injury in mouse lung tissue and the morphological changes were recorded under microscope;Immunohistochemistry and qRT-PCR were performed to detect the expression of IL-33 protein and mRNA in lung tissue of mice;Immunofluorescence and Western blot were performed to detect the expression of NF-κB in lung tissue of mice.The expression of LC3-Ⅱ and P62 in lung tissue was detected by Western blot.The level of IL-33,CXCL-1,TNF-α,IL-10,IL-13,IL-17 A,IL-4,IL-6,CXCL-5 in Serum and alveolar lavage were detected by ELISA.Result:1.Changes in IL-33 levels in human plasma and their correlation with other cytokinesThe level of IL-33 in plasma with ARDS patients was higher than that in healthy controls(P<0.0001).Correlation analysis showed that IL-33 in plasma of ARDS was positively correlated with TNF-α,IL-1β,IL-2 and IL-6,and negatively correlated with IL-10.2.Pathological changes in mouse lung tissueThe lung tissues stained with HE were observed under light microscope.The lung tissues of LPS group,RAPA group and 3-MA group showed obvious edema,extensive alveolar wall thickening,alveolar collapse,alveolar hemorrhage and massive inflammatory cell infiltration.The pathological changes of lung tissue in the 3-MA group were significantly less than those in the LPS group.The pathological changes in the lung tissue of the RAPA group were aggravated compared with the LPS group.No obvious lung injury and inflammation were observed in the N group.3.The changes of IL-33 level in mouse serum and alveolar lavage fluidCompared with the N group,the levels of IL-33 in serum and alveolar lavage fluid of LPS group,RAPA group and 3-MA group changed.Compared with the LPS group,the level of IL-33 in the serum and alveolar lavage fluid of mice increased after RAPA pretreatment(P<0.0001,P<0.0001).After 3-MA pretreatment,the level of IL-33 in serum and alveolar lavage fluid increased slightly,but was lower than that in LPS group(P<0.05,P<0.05).4.Expression of IL-33 protein in mouse lung tissueCompared with the N group,IL-33 in the lung tissue of LPS group,RAPA group and 3-MA group had a certain increase.Compared with theLPS group,the level of IL-33 protein in the lung tissue of the RAPA group after rapamycin pretreatment increased,and decreased in 3-MA group after3-methyladenine pretreatment.5.Expression of IL-33 mRNA in mouse lung tissueqRT-PCR data showed that IL-33 messenger RNA(mRNA)in the lungs of LPS group began to elevate,the expression of mRNA in the3-MA group was lower than that in the LPS group(P<0.001).IL-33 mRNA in the lung tissue of the RAPA group was higher than the LPS group(P<0.05).6.Changes in the levels of cytokines in serum and alveolar lavage fluid of miceThe expression levels of CXCL-1,TNF-α,IL-17 A,IL-6 and CXCL-5in serum and alveolar lavage fluid of RAPA group were increased,which were higher than those of LPS group(P<0.05).The expression levels of CXCL-1,TNF-α,IL-17 A,IL-6 and CXCL-5 in serum and alveolar lavage fluid of 3-MA group were decreased compared with LPS group(P<0.05).The expression of IL-10 in serum and alveolar lavage fluid of RAPA group was lower than that of LPS group(P<0.05).The expression of IL-10 in serum and alveolar lavage fluid of 3-MA group was higher than that of LPS group.(P<0.01).There was no significant difference in the expression of IL-4 and IL-13 in serum and alveolar lavage fluid between LPS group,RAPA group and 3-MA group.7.Expression levels of LC3-Ⅱ and P62 in mouse lung tissueCompared with the blank control group,the expression of LC3-Ⅱ in the lung tissue of the LPS group increased,ratio of LC3B-II/LC3B-I increased.The expression of LC3-Ⅱ in the lung tissue of the RAPA group was higher than that in the LPS group,ratio of LC3B-II/LC3B-I increased.The expression of LC3-Ⅱ in the lung tissue of the 3-MA group was lower than that in the LPS group,ratio of LC3B-II/LC3B-I decreased.The expression of P62 in the lung tissue of the RAPA group was lower than that in the LPS group,ratio of p62/actin decreased.The expression of P62 in the lung tissue of the 3-MA group was higher than that in the LPS group,ratio of p62/actin increased.8.Changes of nuclear translocation of NF-κB in mouse lung tissueThe nuclear translocation of NF-κB in the lung tissue of mice increased after airway instillation of LPS,the nuclear translocation of NF-κB in the LPS group was higher than that in N group.Compared with the LPS group,the nuclear translocation level of NF-κB in the RAPA group increased.The nuclear translocation of NF-κB in the 3-MA group increased was lower compared to the LPS group.Conclusion:Autophagy can affect the progression of uncontrolled inflammation in acute lung injury in mice.Excessively enhanced autophagy can aggravate lung inflammation in mice,inhibit autophagy could reduce lunginflammation in mice;The effect that autophagy cause uncontrolled inflammation of acute lung injury in mice may be achieved by regulating the expression of IL-33 by the NF-κB pathway. |
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