| Objective:To explore the regulatory mechanism and role of NF-κB signal pathway of NLRP3 inflammatory bodies and macrophage pyroptosis in bleomycin induced pulmonary fibrosis model in mice,To explore whether inhibition of NF-KB signal pathway can inhibit cell scorch and improve the degree of fibrosis in bleomycin induced pulmonary fibrosis mouse model.Methods:The wild-type C57 mice were divided into 4 groups(1)blank control group(Control group): normal wild-type mice,without treatment.(2)Negative control group(QNZ group): QNZ(1mg/kg)was intraperitoneally injected into wild type mice for 21 days.(3)Model group(BLM group): wild-type mice were injected with BLM(5mg/kg).(4)reatment group(BLM+QNZ group): wild type mice were intraperitoneally injected with QNZ(1mg/kg)for 21 days after intratracheal injection of BLM(5mg/kg).(all male,6-8 weeks old,6 mice/group)to establish BLM-induced pulmonary fibrosis model.Lung tissue samples were taken for freezing,paraffin,electron microscope embedding and sectioning.HE staining,Masson staining,immunofluorescence staining and tissue protein detection were used to evaluate the pathological changes and inflammatory activation of lung tissue.Collect the bronchoalveolar lavage fluid of mice in the inf Tlammatory phase for flow cytometry,and determine the percentage of neutrophils and macrophages in the total cells.Determination of IL-1β in bronchoalveolar lavage fluid of mice by ELISA and IL-6 expression and exudation.The NR8383 macrophage line from human and A549 cells from mice were divided into four groups.(1)Blank control group(control group): normal cells are not treated.(2)Negative control group(QNZ group): cells were incubated with QNZ(11n M)for 8 hours.(3)Model group(LPS group): The cells were stimulated with lipopolysaccharide(LPS,1 ug/ml)for 5 hours,and then incubated with ATP(4 m M)for 2 hours to trigger scorching.(4)Treatment group(LPS+QNZ group): cells were pre-incubated with QNZ(11n M)for 1 hour,then added with lipopolysaccharide(LPS,1ug/ml)for 5 hours,and then incubated with ATP(4m M)for 2 hours to trigger scorching.LPS was used to induce scorch death model.The expression of related target proteins secreted by macrophages and type II alveolar epithelial cells was determined by immunoblotting analysisResults:The relationship between the degree of pulmonary interstitial fibrosis in four groups of mice on the 21 st day after modeling was compared,The HE section and Masson staining results showed that the normal morphology and structure of the alveoli in BLM group had been damaged compared with that in the blank control group,and the alveolar wall was also significantly thickened.Inflammatory cells could be seen in the alveolar cavity,and a large number of collagen fibers were accumulated in the lung interstitium,and the fibrosis was very obvious;Compared with BLM group,the degree of fibrosis in BLM+QNZ group was significantly improved.Western blot and immunofluorescence detection The expression of two fibrosis-associated proteins,α-SMA and Col Ⅰ,showed a significant increase in BLM group.The expression in BLM+QNZ group was significantly lower than that in BLM group.In the mouse model induced by BLM for 3 days,the HE staining of lung tissue and the wet/dry ratio(W/D)of lung tissue showed that the degree of lung injury and the level of pulmonary edema in BLM+QNZ were significantly lower than those in BLM group.The flow cytometry results of bronchoalveolar lavage fluid in mice showed that the percentage of neutrophils in lung tissue in BLM mice was significantly increased to 61.7% compared with the control group,the percentage of macrophages in BLM+QNZ group was significantly decreased to 11.8% compared with the BLM group,and the percentage of macrophages was significantly decreased by 16.6% compared with the control group,while the percentage of neutrophils in BLM+QNZ group rose to 45.9%.Western blot and ELISA showed that NLRP3,GSDMD,caspase-1,pro-capase-1 and in lung tissue of BLM mice NF-κB,IL-6,IL-1β Compared with the control group,the expression of the above protein in the treatment group and the model group was significantly decreased at the same time.Western blot analysis of macrophages and type II alveolar epithelial cells in vitro showed that the treatment group had NLRP3,caspase-1,pro-caspase-1,GSDMD,The expression of NF-κB was significantly decreased.Conclusion:(1)NF-κB inhibitor QNZ can reduce inflammation of lung tissue,reduce infiltration of macrophages and neutrophils,activation of inflammatory bodies,cell scorching,and ultimately reduce BLM-induced lung fibrosis.(2)The inhibition of NF-κB pathwaywas further verified in macrophages in vitroreduces pulmonary fibrosis mainly due to inhibition of immune cell scorch,activation of inflammatory bodies and release of inflammatory factors.(3)It was confirmed in vitro that type II alveolar epithelial cells were also activated by inflammatory corpuscles and charred cells,which affected the development of pulmonary fibrosis. |
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