Protective Effects Of Imipramine On Alveolar Epithelial Barrier Function In Murine Acute Lung Injury

Objective Acute lung injury(ALI)/ Acute respiratory distress syndrome(ARDS)is a common respiratory critical disease,with high mortality.At present,there is lack of effective clinical treatment.The most prominent pathological changes in the development of ALI are diffuse alveolar epithelial cell injury,therefore,enhancing the barrier function of alveolar epithelial cells can be a target for treatment of ALI.Imipramine is a common tricyclic antidepressant and acid sphingomyelinase(acid sphingomyelinase,ASMase)inhibitor with anti-inflammatory and anti-apoptotic effects.In this study,we investigated whether imipramine has protective effects on pulmonary alveolar epithelial barrier function in lipopolysaccharide(LPS)-induced in murine ALI and to explore its possible mechanism.Methods SPF male Balb/c mice were randomly divided into four groups: control group,Imipramine group,LPS group,LPS + Imipramine group.Mice were treated with imipramine at 25 mg/kg 30 min prior to LPS administration and injected via the tail vein with FITC-FD4 10 min before being sacrificed at 12 hours after LPS administration.Bronchoalveolar lavage fluid(BALF)and lung tissue were obtained for further analysis.HE staining was chosen to observe histopathological changes;lung tissue wet-to-dry weight ratio and BALF/serum FD4 ratio were used to assess alveolar epithelial permeability;Real-time PCR,western blot and immunochemistry were chosen to detect the m RNA and protein levels of Occludin,Claudin-4 and ZO-1.Immunofluorescence was chosen to detect the activity of ASMase in each group.One way analysis of variance(ANOVA)was used to compare multiple sets of variables and the intergroup comparisons were analyzed by the least-significant-difference(LSD)tests with SPSS 16.0 software.Results Intraperitoneal injection of LPS is simple,stable and well accepted experimental model of ALI.The histopathological changes of ALI were consistent with pathological changes of ALI in mice.There was no significant change in lung tissue between control group and Imipramine group;the lung tissue pathological score of LPS + Imipramine group was less than LPS group(P<0.001);the wet-to-dry weight ratio of LPS + Imipramine group was less than LPS group,the difference was statistically significant(P=0.001);the ratio of BALF/serum FD4 in LPS+Imipramine was less than LPS group,the difference was statistically significant(P=0.002);relatived to the control group,the m RNA and protein levels of Occludin,Claudin-4 and ZO-1 in LPS group were significantly decreased(P<0.05).Moreover,to LPS group,the tight junction proteins of LPS + Imipramine group were significantly increased(P<0.05).Immunochemistry showed that Occludin,Claudin-4 were mainly in alveolar epithelial cell membrane,Z0-1 were mainly in cytoplasm of alveolar epithelial cell.In the control group and Imipramine group,tight junction proteins were obviously expression;LPS group decreased(P<0.05);to LPS group,LPS+Imipramine group tight junction proteins had been restored(P<0.05).The results of immunofluorescence assay showed that ASMase activity in LPS + Imipramine group was lower than that in LPS group(P <0.05),which indicated that imipramine could inhibit the activity of ASMase in stress state and decrease the generation of ceramide.Conclusion The establishment of murine ALI by intraperitoneal injection of LPS is recommended in the ALI model;Imipramine can improve the pathological changes of ALI and reduce the degree of pulmonary edema;the protective effects of imipramine on alveolar epithelial barrier function by up-regulating tight junction proteins expression though inhibiting the activity of ASMase in stress response and reducing the production of ceramide in murine LPS-induced ALI.