Protective Effect Of Ouabain On LPS Induced Acute Lung Injury And Underlying Mechanisms In Mice

BackgroundAcute lung injury(ALI)is the damage of alveolar epithelial cells and capillary endothelial cells,which is often caused by sepsis,burns,pneumonia,or severe trauma.The pathophysiological characteristics of ALI include alveolar capillary endothelial barrier destruction,inflammatory cell infiltration,uncontrolled inflammatory responses,and oxidative stress injury.Although researchers have conducted in-depth studies on the strategies for the early diagnosis and treatment of ALI,the morbidity and mortality of ALI remain high,which has a significant impact on public health.Ouabain is a kind of sodium-potassium ATPase inhibitor,which is used for the treatment of congestive heart failure.Recent years,many studies found that ouabain plays an important role in organ damage protection,immune responses,inflammatory reaction,and anti-tumor treatment.However,whether ouabain has a protective effect on acute lung injury has not been reported yet.The purpose of this study is to investigate whether ouabain inhibits lipopolysaccharide-induced acute lung injury and to investigate its underlying mechanisms.MethodsMale C57BL/6 mice(20g-25g),8-12 weeks,were randomly divided into three groups.Control group;LPS group;Ouabain group.Mice in the control and LPS group were injected intraperitoneally with sterile PBS solution for 3 consecutive days.Ouabain group mice were intraperitoneal injection of ouabain for 3 consecutive days.One hour after the third day of ouabain/PBS injection,mice in LPS group and ouabain group were anesthetized with sevoflurane and inhaled LPS,mice in control group were inhaled the same volume of PBS.6 h and 24 h after the inhalation of PBS or LPS,mice were killed using carbon dioxide inhalation.Lung tissues and bronchoalveolar lavage fluid were collected to detect lung injury and other related indicators.Results1.Histopathological examination of the lungs at 6 and 24 h after LPS treatment showed that pulmonary interstitial edema,hemorrhage,increased pulmonary interstitial thickeness,and inflammatory cells infiltration in LPS group were significantly.However,H&E staining of lung tissues in ouabain group showed lessened lung tissue lesion.Pathological score of lung injury in LPS group was significantly higher than the pathological score in control group and ouabain group,which means that ouabain could reduce lung tissue pathological scores caused by LPS.2.LPS significantly increased the expression of TNF-?,IL-1?,and IL-6 in BALF.However,ouabain could decrease the levels of these cytokines in BALF.qRT-PCR was used to detect the mRNA expression of these cytokines in lung tissues.These cytokines in LPS group were the highest at transcriptional level.Ouabain inhibited the transcription of TNF-?,IL-1?,and IL-6.3.After LPS inhalation,the lung wet/dry ratio increased significantly,while ouabain significantly inhibited the wet/dry ratio of ALI mice.LPS inhalation also increased the concentration of protein in BALF,whereas the protein concentration in ouabain group was decreased.Evans blue in lung tissues was detected one hour after the tail vein injection of Evans blue and the content of Evans blue in lung tissues was the highest in LPS group.4.Compared with control group,the number of total cells,neutrophils,and macrophages in BALF increased significantly in LPS group.However,ouabain could reduce these cells infiltration.Ouabain also reduced MPO activity in mouse lung tissues.5.The phosphorylation of NF-?B p65 in LPS group was higher than that in control group,but ouabain could reduce the phosphorylation of NF-?B p65.Besides,the phosphorylation of ERK,JNK and p38 in ouabain group was higher than that in control group,but lower than that in LPS group.ConclusionOuabain protects mice against LPS-induced acute lung injury,and its mechanism may be associated with the inhibition of ERK,JNK,p38 and NF-?B p65 phosphorylation.Thereby it may regulate the secretion of inflammatory cytokines and the infiltration of inflammatory cells.