| ABCA3(ATP-binding cassette class A3)is a transmembrane transporter that plays a positive role in chronic pulmonary inflammation by regulating lipid metabolism.However,it is not completely clear whether ABCA3 and its signaling factors are involved in chronic pulmonary inflammation induced by the combination of CSE(cigarette smoke extract)and LPS(lipopolysaccharide).In this study,we investigated the effects of combination of CSE and LPS on the expression of the efflux transporter ABCA3 associated with lipid metabolic transport and surfactant expression and revealed the role of the PPARγ and p38 MAPK signaling pathway in this mechanism,providing new targets for clinical treatment of chronic pulmonary inflammation.ObjectiveIn this study,we used the combination of CS and LPS to establish the pulmonary inflammatory model of SD rats in vivo,and the combination of CSE and LPS to establish the A549 cell inflammatory model In vitro,and explore the changes in the expression of ABCA3 in pulmonary inflammation state and its possible mechanism.Method(1)Establish the pulmonary inflammatory model of SD rats in vivo,analyze the pulmonary structure by pathology,and detect the expression changes of ABCA3 and related inflammatory factors.Divided into control group and CS+LPS model group.In the control group,we exposed rats to fresh air,and saline solution was injected directly into their tracheas.The model group was exposed to cigarette smoke and LPS stimulation.After establishing the model,observe the morphological changes of lung tissue,and detect the expression of ABCA3 and related inflammatory factors in lung tissue.(2)Establish the inflammatory model of A549 cells In vitro,and detect the expression changes of ABCA3 and related inflammatory factors.Divided into control group,CSE(0.5%)+LPS(10 μg/m L)group,CSE(1%)+LPS(10 μg/m L)group and CSE(2%)+LPS(10 μg/m L)group.The expression of ABCA3 and related inflammatory factors were detected after 24 hours of CSE with different concentrations and LPS stimulation.(3)Establish the inflammatory model of A549 cells In vitro,add p38 MAPK inhibitor SB203580,silence or overexpress MAPK 14,and detect the expression of ABCA3,p38,phosphorylated p38 and related inflammatory factors.In Experiment 1,control group,CSE-LPS model group and CSE-LPS model+SB203580group was divided to detect the expression of ABCA3,p38,phosphorylated p38 and related inflammatory factors.In Experiment 2,control group,CSE-LPS model group,CSE-LPS model+negative control group,CSE-LPS model+silent MAPK 14 group,or CSE-LPS model+overexpression MAPK14 group was divided to detect the expression of ABCA3,p38,phosphorylated p38 and related inflammatory factors.(4)Establish the inflammatory model of A549 cells In vitro,silence or overexpress PPARγ,and detect the expression of ABCA3,PPARγ,p38,phosphorylated p38 and related inflammatory factors.Control group,CSE-LPS model group,CSE-LPS model+negative control group,CSE-LPS model+silent PPARγ group or CSE-LPS model+overexpression PPARγ group was divided to detect the expression of ABCA3,PPARγ,p38,phosphorylated p38 and related inflammatory factors.(5)Establish the inflammatory model of A549 cells In vitro,overexpress PPARγ and inhibit p38 MAPK signaling pathway respectively,and detect the expression of ABCA3,PPARγ,p38,phosphorylated p38 and related inflammatory factors.Control group,CSE-LPS model group,CSE-LPS model+negative control group,CSE-LPS model+overexpression PPARγ group or CSE-LPS model+overexpression PPARγ+SB203580 group was divided to detect the expression of ABCA3,PPARγ,p38,phosphorylated p38 and related inflammatory factors.Results(1)In vivo experiments showed that rats in the CS-LPS model group had sparse hair,decreased activity,and respiratory symptoms compared to the control group.Histopathological analysis revealed more lung damage in the model group,with increased alveolar diameter and airway thickness,and higher levels of immune cells.Western blotting results showed lower levels of ABCA3 protein and higher levels of inflammatory factors in the CS-LPS group.(2)In vitro experiments showed that ABCA3 expression was lower and TNF-α and IL-6were higher in the CS+LPS model group,with expression levels dependent on CSE concentration.(3)In vitro experiments showed that ABCA3 expression was lower and p-p38 and related inflammatory factors were higher in the CSE+LPS group,while MAPK 14 si RNA increased ABCA3 expression and reduced p-p38 and related factors.The opposite was seen in the MAPK 14-OE group.(4)In vitro experiments showed that ABCA3 and PPARγ expression were lower and p-p38 and related inflammatory factors were higher in the CSE+LPS group,while PPARγ si RNA reduced ABCA3 expression and increased p-p38 and related factors.The PPARγ-OE group had higher ABCA3 expression and lower p-p38 and related factors than the model group.ConclusionThrough the investigation of In vitro and in vivo experimental results,it was found that the combined treatment of CSE and LPS can induce a rat lung tissue inflammation model and a lung epithelial cell A549 inflammation model.Moreover,in the chronic lung inflammation model,the expression of the lipid efflux transporter protein ABCA3 was downregulated.Additionally,the expression change of ABCA3 in chronic lung inflammation may be regulated by the PPARγ-mediated p38 MAPK signaling pathway.These findings may provide new insights into the study of therapeutic targets for lung-related diseases. |
