The Alteration Of Na~+-K~+-ATPase Activity And The Effect Of Different Dexamethasone Dosage On Different Acute Lung Injure Models Of Rats

Objective: Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized of hypoxemia respiratory failure as a result of the loss of barrier function of the alveolar epithelial and pulmonary capillary endothelial cells induced by different predisposing factors. ALI / ARDS are caused by a lot primary diseases, and the pathogenesis is complex. AECC classified ALI / ARDS into direct causes and indirect causes. Severe sepsis and aspiration are the most common causes of ARDS. The Na⁺-K⁺-ATPase plays an essential role in active alveolar epithelial fluid resorption. The purpose of the study was to investigate the role of the Na⁺-K⁺-ATPase in the resolution of pulmonary edema in lung epithelial cells. It was shown that different -subunits of the Na⁺-K⁺-ATPase have different effects on ALI.Due to its powerful anti-inflammatory property, GCs have been used in ARDS for many years. Clinical physicians have found that different response of GCs in ALI patients induced by various risk factors, the mechanism need to be further studied. What's the more effective dose of GCs for different ALI has not yet been determined even more and more doctors like to use mild-moderate dose GCs in ARDS.Therefore, our experiment established three ALI animal models with Lipopolysaccharide (LPS) one hit ,Hydrochloric acid (HCl) one hit and LPS-HCl two hit respectively. By comparing differences of the Na⁺-K⁺-ATPase hydrolytic activity and expression of Na⁺-K⁺-ATPaseαisoforms and the efficacy of GCs ,we hope to explore the pathomechanism and GCs curative efficacy of ALI induced by different causes and investigate its potential mechanism.Methods: 72 male SD rats were divided randomly into twelve groups: (1) NS group: 0.9% saline(0.6ml) was injected intravenously via a tail vein, (2) LPS group: LPS(8mg/kg) was injected intravenously via a tail vein, (3) HCl group: pH 1.8 HCl(2ml/kg) was instilled into trachea, (4) LPS-HCl group: pH 1.8 HCl(0.5ml/kg) was instilled into trachea at 4 hours after intravenous injection of LPS(4mg/kg), (5) NS+Dex1 group, (6) NS+Dex8 group, (7) LPS+Dex1 group, (8) LPS+Dex8 group, (9) HCl+Dex1 group, (10) HCl+Dex8 group, (11) LPS-HCl+Dex1 group, (12) LPS-HCl+Dex8 group. (5),(7),(9),(11)four groups were intraperitoneally injected with Dexamethasone (1mg/kg) immediately after replicating model,and(6),(8),(10),(12)four groups were injected with Dex (8mg/kg).SD rats were anaesthetized by intraperitoneal injection 4% chloral hydrate(4ml/kg)at 4 hours after replicating model. The anterior lobe of the right lung was used for the hydrolytic activity of the Na⁺-K⁺-ATPase. Na⁺-K⁺-ATPase activity was measured through spectrophotometry. About 100mg tissue from posterior lobe of right lung was used for extracting total mRNA. The mRNA of Na⁺-K⁺-ATPaseα-isoform was detected with RT-PCR. The accessory lobe of the right lung was placed in 10% formalin fluid, embedded in paraffin, stained with hematoxylin-eosin and examined under a light microscope. The wet/dry was measured with the accessory lobe and accessory lobe of the right lung(80℃, 48h). A trachea catheter was used for bronchoalveloar lavage in left lung. BALF was collected and centrifuged (1200rpm, 4℃, 10min). Cellular sediment was suspended in 0.9% saline 200ul and counted the total leukocytes.Results: (1) In the group of LPS, HCl and LPS+HCl, histological grade were remarkably higher than that in NS group(P<0.05). However, there was no difference among the histological grade of the three ALI model groups (P >0.05). The grade in the groups of LPS+Dex1(Dex8) and LPS-HCl+Dex1 (Dex8) were decreased than that in the corresponding groups(P <0.05). When compared to LPS+Dex1 group, the histological grade of LPS-HCl+Dex1 group was significantly increased (P <0.01).There was no significant difference between Dex1 groups and Dex8 groups. (2) In the three groups of LPS , HCl and LPS-HCl, W/D, the total leukocytes, protein concentration in BALF were higher than those of NS group(P<0.05). When compared among the three ALI model groups, there was no obvious difference in the above parameters except the protein concentration in BALF(P>0.05). These parameters in LPS+Dex1(Dex8) group were lower than those in LPS groups(P <0.05). Protein concentration in HCl+Dex1 group were decreased when compared with HCl group(P<0.05). Protein concentration and W/D in LPS-HCl+Dex1 group were decreased when compared with LPS-HCl group(P<0.05). There was no significant difference between Dex1 groups and Dex8 groups. (3) The Na⁺-K⁺-ATPase hydrolytic activity were significantly decreased in the three model groups compared with NS group (P<0.01). Compared to LPS-HCl group, the Na⁺-K⁺-ATPase hydrolytic activity of LPS group and HCl group were significantly increased(P<0.05). The parameters in the groups of LPS+Dex1(Dex8), HCl+Dex1 and LPS-HCl+Dex1 were increased than that in the corresponding groups(P <0.05). There was no significant difference between Dex1 groups and Dex8 groups for all above the parameters. (4) The expressions ofα₁ andα₂ isoforms of Na⁺-K⁺-ATPase in LPS group , HCl group and LPS-HCl group were significantly lower than that in NS group(P<0.01). The expression of Na⁺-K⁺-ATPaseαisoforms in the groups of LPS+Dex1(Dex8), HCl+Dex1(Dex8) and LPS-HCl+Dex1 (Dex8) were increased than that in the corresponding groups(P<0.05 or 0.01),without significant difference between Dex1 groups and Dex8 groups.Conclusion:â‘ In the three model groups, the Na⁺-K⁺-ATPase hydrolytic activity and expressions of Na⁺-K⁺-ATPaseαisoforms are significantly decreased than the NS group. It shows that the ALI is associated with Na⁺-K⁺-ATPase hydrolytic activity , and both theα₁- andα₂-subunits of the Na⁺-K⁺-ATPase should have important effect on alveolar epithelial fluid clearance in ALI. But in our experiment, it not is shown that different dexamethasone dosage make difference of expression between 1- and 2-subunits.â‘¡Dexamethasone can increase the Na⁺-K⁺-ATPase hydrolytic activity and expressions of Na⁺-K⁺-ATPaseαisoforms in the three model groups, and we can learn that the decrease of Na⁺-K⁺-ATPase hydrolytic activity and expressions of Na⁺-K⁺-ATPaseαisoforms is one of the important reasons that lead to the formation of the pulmonary edema. But it is not the only reason. The underlying mechanism was not known yet.â‘¢GCs can alleviate the pulmonary injury in three model groups, but the efficacy of GCs therapy on LPS group and LPS-HCl group is better than HCl group. That may be related to the differences in pathogenesis of three model groups. Compared to low dose GCs, high dose GCs does not show better therapeutic efficacy.