| Part 1:Establishment of LPS-induced ALI rat model and study on the levels of inflammatory cytokines from the BALFBackground:Acute lung injury(ALI)/Acute respiratory distress syndrome(ARDS)is a serious respiratory disorder caused by intense and uncontrolled systemic inflammation directly or indirectly,in which alveolar macrophages plays a critical role.The acute injury of lung epithelium and endothelial barriers as well as inflammatory response in alveolus could be reproduced by animal model with inhaled or systemic administration of LPS,which is an ideal model for study on the pathological mechanisms of ALI/ARDS.Objective:To investigate the levels of inflammatory cytokines in LPS-induced ALI ratsMethods:1.The ALI model was established by intraperitoneal injection of LPS.2.The degree of acute lung injury was evaluated by measuring wet/dry ratio of lung tissues,BALF protein concentration,and inflammatory cells in BALF.3.Hematoxylin-eosin(HE)staning was applied for histological examination of lung tissues.4.ELISA was used to determine the levels of inflammatory cytokines in BALF and culture medium.5.Flow cytometry was used to purify macrophages from BALF.6.qPCR was applied to quantify the mRNA of IL-33 in the macrophages purified from BALF.7.Western blot was performed to measure the protein levels of ST-2 and IL-1RAP.Results:1.The wet/dry ratio of lung tissues,BALF protein concentration,and total cells/macrophages in the BALF from LPS-induced ALI rats were significantly increased.2.There was widespread alveolar wall thickness caused by edema,alveolar collapse and prominent inflammatory cell infiltration in the lung tissues from LPS-induced ALI rats.3.The levels of IL-33,TNF-a,MMP-2,MMP-9,and TIMP1 were upregulated in the BALF from LPS-induced ALI rats.4.In the macrophages purified from the BALF of LPS-induced ALI rats,the mRNA level of IL-33 was increased,and as the receptor of IL-33,the protein level of ST-2 was also upregulated but IL-1RAP showed no change.5.The levels of IL-33,TNF-?,MMP-2,and MMP-9 were evidently increased in the culture medium of primary alveolar macrophages treated with LPS.6.The protein level of ST-2 but not IL-1RAP was upregulated in the primary alveolar macrophages treated with LPS.7.The secretions of IL-33,TNF-?,MMP-2,MMP-9,and TIMP1 by NR8383 cells were enhanced after LPS stimulation,in which,the elevated concentrations of TNF-?,MMP2,MMP9,and TIMP1 were detected in a time-dependent manner after LPS treatment,while IL-33 was elevated to peak at 12 h after LPS treatment.Conclusion:Secretions of several inflammatory cytokines by alveolar macrophages were induced in LPS-induced ALI rats.Part 2:Mechanistic study on the role of IL-33/STAT3 signaling in the LPS-induced secretion of MMP-2 and MMP-9 by alveolar macrophagesBackground:As a pro-inflammatory cytokines,IL-33 displays a critical role in innate immunity,allergic inflammation,and tissue injury.The levels of IL-33 in patients with asthma,chronic obstructive pulmonary disease,idiopathic pulmonary fibrosis,or rheumatic arthritis were observed increased.Our previous work also showed that the serum IL-33 levels in ALI patients were significantly higher than in the normal,suggesting an association between ALI/ARDS and IL-33.As the intracellular signal transducer,STAT3 could be activated by extracellular stimulation,contributing to the signal transduction in inflammatory response.MMP-2 and MMP-9,belonging to metalloproteinase family,were widely known as a driver of ALI/ARDS.Objective:To investigate the role of IL-33 in LPS-induced secretions of MMP-2 and MMP-9 in alveolar macrophages.Methods:1.ELISA was used to determine the levels of inflammatory cytokines in the culture medium of NR8383 cells.2.Western blot was performed to measure the protein levels of indicated genes in NR83 83 cells.3.qPCR was applied to quantify the mRNA of indicated genes in NR8383 cells.Results:1.IL-33 induced the secretions of MMP-2 and MMP-9 in NR8383 cells.2.Blocking STAT3 signaling by p-STAT3 inhibitors or STAT3 siRNA reduced the IL-33-induced expressions and secretions of MMP-2 and MMP-9.3.Neutralizing IL-33 in culture medium with specific antibody inhibited LPS-induced secretions of MMP-2 and MMP-9.Conclusion:IL-33/STAT3 signal pathway was required for the LPS-induced secretions of MMP-2 and MMP-9 in alveolar macrophages.Part 3:Study on the protective role of IL-33 antibody against LPS-induced ALIBackground:The expression of IL-33 has been discussed in preclinical ALI animal models.In the ventilator-induced lung injury model,IL-33 was upregulated in the lung tissues of rats.IL-33 could promote peripheral monouclear cells,pulmonary endothelial cells,and epithelial cells to secret IL-8 and IL-6,which consequently recruited neutrophil into alveolus.Except for regulating cytokines,IL-33 could increase the permeability of alveolar endothelial barrier and induce the apoptosis of alveolar epithelial cells.Therefore,IL-33 accelerates the development of lung injury.Objective:To verify the protective role of IL-33 neutralizing antibody against LPS-induced ALI.Methods:1.The degree of acute lung injury was evaluated by measuring wet/dry ratio of lung tissues,BALF protein concentration,and inflammatory cells in BALF.2.ELISA was used to determine the levels of MMP-2 and MMP-9 in BALF.3.Hematoxylin-eosin(HE)staning was applied for histological examination of lung tissues.4.Immunohistochemistry(IHC)was used to determine the levels of p-STAT3,MMP-2,and MMP-9 in lung tissues.Results:1.IL-33 neutralizing antibody suppressed the LPS-induced upregulation of wet/dry ratio,BALF protein concentration,and total cells/macrophages in the BALF.2.IL-33 neutralizing antibody decreased the secretions of MMP-2 and MMP-9 in LPS-induced ALI rats.3.LPS induced the pathological changes of lung tissues,such as alveolar wall thickness,alveolar collapse,and prominent inflammatory cell infiltration,and the expressions of p-STAT3,MMP-2,and MMP-9,which were attenuated by IL-33 antibody.Conclusion:IL-33 neutralizing antibody attenuated LPS-induced ALI with inhibiting the secretions of MMP-2 and MMP-9. |
PDF Full Text Request