Protective Effect Of Hif-1α/Claudin-1 Axis On The Epithelial Barrier In Lung Injury Induced By Seawater Drowning In Mice

BackgroundDrowning is a very important but often overlooked public safety issue.The Global Burden of Diseases（GBD）study states that drowning is one of the most common causes of accidental death,with about 500,000 people worldwide dying from drowning every year.Due to the lack of effective treatment,nearly 1/3 patients develop acute respiratory distress syndrome（ARDS）.Drowning induced lung injury is the result of a variety of factors,including destruction of alveolar surfactant,dysfunction of alveolar epithelium and increased permeability of pulmonary capillaries.Claudin-1 is a cell-cell adhesion molecule located at tight junction（TJ）between epithelial cells,responsible for the para-cellular barrier function of TJ.Previous studies have shown that hypoxia inducible factor-1α（HIF-1α）can selectively regulate claudin-1,thus coordinating the integrity of the epithelial barrier.Given the important role of HIF-1α/claudin-1 signaling pathway in maintaining epithelial barrier function,its role in lung injury caused by seawater drowning deserves further exploration.The purpose of this study was to clarify the role of HIF-1α/claudin-1 signaling pathway in the treatment of seawater drowning lung injury,in order to provide new ideas for the treatment of seawater drowning lung injury.MethodsA.The model of lung injury induced by seawater drowning was established in mice to explore the effect of seawater inhalation on tight junctions between alveolar epithelial cells1.Artificial seawaterSeawater was prepared according to the major compositions of the East China Sea provided by the Chinese Ocean Bureau（osmolality 1300 mmol/L,p H 8.2,specific weight 1.05,Na Cl 26.518 g/L,KCl 0.725 g/L,Ca Cl₂1.141 g/L,Mg Cl₂2.447 g/L,Mg SO₄3.305 g/L,and Na HCO₃0.202 g/L）.2.The model of lung injury induced by seawater drowning was established in mice（1）C57 BL/6 mice,male,8 to 12 weeks old,20 g to 25 g,were randomly divided into control group and sea water drowning group.Before the experiment,the mice were fasted for 12 h without restriction of water intake.（2）During modeling,mice were put into artificial seawater with a depth of 6 cm and a water temperature of 25℃.After 28 s,mice were quickly removed from the water.The vital signs of mice were closely observed,and the mice were divided into dead group and alive group according to their survival conditions.The survival group was divided into the following subgroups according to the observation time point:30 min after drowning,1 h after drowning,3 h after drowning and 6 h after drowning.3.Lab test indexes（1）The survival rate of mice was analyzed.（2）Arterial blood gas analysis,lung wet/dry ratio and lung histopathology were performed.（3）The mRNA levels of claudin-1,ZO-1 and occludin,important components of TJ in the lung of mice were detected by polymerase chain reaction.（4）The changes of claudin-1 protein in the lung of mice were detected by western blotting.（5）The specific molecule CD324 of epithelial cells was double stained with claudin-1 by immunofluorescence double labeling assay to detect the change of claudin-1protein content in the alveolar epithelial cells of mice after seawater drowning.B.To explore whether HIF-1αhas a regulatory effect on claudin-1in mice with lung injury induced by seawater drowning1.Randomly grouping design（1）C57 BL/6 male mice,8 to 12 weeks of age,20 g to 25 g,were randomly divided into control group,2ME2（2-methoxyestradiol,HIF-1αinhibitor,20 mg/kg）group,seawater drowning group and seawater drowning group after 2ME2 administration.（2）The model of lung injury induced by seawater drowning was established according to the method described above.Then,the survived mice in seawater drowning group were divided into three subgroups according to the observation time point:1 h after drowning group,3 h after drowning group and 6 h after drowning group.The survived mice in seawater drowning group after 2ME2 administration were divided into the same group.2.Lab test indexes（1）The changes of HIF-1αprotein in the lungs of mice at 1h,3h and 6h after seawater drowning were detected by western blotting and immunohistochemistry.（2）The specific molecule CD324 of epithelial cells was double-stained with HIF-1αto detect the change of HIF-1αprotein content in alveolar epithelial cells after seawater drowning.（3）The transcriptional activity of HIF-1αwas inhibited by 2ME2,and the expression level of claudin-1 protein in the lung of mice was detected by PCR and western blotting.（4）Claudin-1 protein content in mouse alveolar epithelial cells was detected by double-staining CD324,a specific molecule of epithelial cells with claudin-1,in a double-labeling immunofluorescence assay.C.To explore the effect of HIF-1α/claudin-1 signaling pathway in mice with lung injury induced by seawater drowning1.Randomly grouping designC57 BL/6 male mice,8 to 12 weeks old,20 g to 25 g,were randomly divided into control group,2ME2（2-methoxyestradiol,HIF-1αinhibitor,20 mg/kg）group,seawater drowning group and seawater drowning group after 2ME2 administration.Mice model of lung injury induced by seawater drowning was established according to the method mentioned above,and the surviving mice were used for follow-up experiments.2.Lab test indexes（1）The survival rate of mice was analyzed.（2）The moisture/dry ratio of lung tissue,protein concentration of bronchoalveolar lavage fluid and Evans blue staining analysis of lung tissue were investigated to explore the changes of lung tissue permeability in seawater drowned group after 2ME2pretreatment.（3）Lung histopathology was used to investigate the change of lung injury severity in seawater drowning group after 2ME2 pretreatment.ResultsA.Seawater inhalation disrupts the tight junctions between alveolar epithelial cells1.The experimental results showed that the death of mice mostly occurred within 3h after drowning.The results of arterial blood gas analysis showed that severe hypoxemia,acidosis and hypercapnia occurred after seawater drowning.Compared with the control group,the pathological changes of lung tissue were also obvious,the normal structure of alveoli was destroyed,and the edema of alveoli and pulmonary interstitial was obvious.Most of the alveolar lumen saw uniform red staining,with pulmonary hemorrhage performance;There were more inflammatory cell infiltration in lung interstitium and alveolar cavity.These pathological features were found at 1 h,3 h and 6 h after drowning,and the most serious were found at 1 h.2.The inhalation of cold,hypertonic,bacterialized seawater into the lungs creates a strong,damaging irritation that damages the integrity of the alveolar epithelium.TJ is an important connection mode between alveolar epithelial cells and plays a key role in maintaining the barrier function of alveolar epithelium.The results of this part of the experiment indicated that the mRNA level of claudin-1,the key tight junction protein,decreased significantly after seawater flooding,followed by ZO-1,and occludin did not change significantly.The protein level of claudin-1 in the lung of mice began to decrease at 1 h after drowning,and was more obvious at 3 h,but showed a trend of recovery at 6 h.The results of double immunofluorescence assay showed that the change of claudin-1protein level in the alveolar epithelial cells was consistent with that in the lung tissue.Although claudin-1 is not the only protein molecule that makes up TJ,it is suggestive of decreased adhesion between alveolar epithelial cells and epithelial barrier dysfunction.B.HIF-1αhas a regulatory effect on claudin-1 in mice with lung injury induced by seawater drowning1.The hypertonic stimulation of seawater and the synergistic effect of pulmonary hypoxia resulted in the increase of HIF-1αprotein level in the lung of mice,which showed an increasing trend at 1 h after drowning,was more significant at 3 h,and remained at a high level at 6h.At the same time,HIF-1αprotein in alveolar epithelial cells showed the same trend of change,suggesting that HIF-1αis involved in the pathological process of seawater drowning lung injury.2.Compared with the control group,the mRNA and protein levels of claudin-1 in the lung of mice pretreated with 2ME2 were significantly decreased,suggesting that claudin-1 expression was HIF-1α-dependent.Compared with the seawater drowning group,the level of claudin-1 protein in the lung of mice pretreated with 2ME2 in the seawater drowning group decreased more significantly.Pretreatment with 2ME2 also reduced claudin-1 protein levels in alveolar epithelial cells.After comprehensive analysis of the above results,it can be concluded that HIF-1αmay have a regulatory effect on claudin-1 in mice with seawater drowning lung injury.C.HIF-1α/claudin-1 signaling pathway plays a protective role in lung injury induced by seawater drowning in miceCompared with the seawater drowning group,the 2ME2 pretreatment showed more significant increase in lung permeability,more fluid and inflammatory cell exudation,more severe pulmonary hemorrhage and pulmonary edema,and increased mortality within 6h after drowning.These results suggest that HIF-1αcan selectively regulate claudin-1 to maintain the barrier function of alveolar epithelium in mice with seawater drowning lung injury,that is,HIF-1α/claudin-1 signaling pathway plays a protective role.ConclusionEpithelial barrier dysfunction is one of the important mechanisms of seawater drowning lung injury.In this study,it was found that hypoxia-inducing factor HIF-1αwas up-regulated in the early stage of seawater drowning lung injury.At the same time,HIF-1αcould affect the tightness of the connection between alveolar epithelial cells by regulating claudin-1,an important protein molecule of TJ,thus affecting the barrier function of the alveolar epithelial cells.Therefore,HIF-1αplays an important role in the maintenance of alveolar epithelial barrier function in the mouse model of seawater drowning lung injury.The HIF-1α/claudin-1 signaling pathway is one of the regulatory axons related to the function of the alveolar epithelial barrier,and may be a potential target for the treatment of seawater drowning lung injury.