Regulation Mechanism Of Aquaporin 5 Through Mitogen-Activated Protein Kinases Signaling Pathways In Hyperoxia-Induced Lung Injury

Mechanical ventilation with high concentration of oxygen is often used in the treatment of severe respiratory failure, especially acute respiratory distress syndrome (ARDS). However, prolonged exposure to high concentration of oxygen may cause lung epithelial cell injury, cell death and induce acute lung injury (ALI), respiratory failure. The rate of bronchopulmonary dysplasia (BPD) has ever been increasing in recent years. Patients with BPD often have lung development impairement and lung dysfunction. There were no effective methods to prevent this disease currently, although massive clinical data have related to its etiology with hyperoxia-induced lung injury.It has been found that the early changes of hyperoxic lung injury is characterized by alveolar inflammatory edema, followed by increased deposition of extracellular matrix and lung tissue fibrosis, which may eventually lead to complete loss of respiratory function. Early interventions to enhance the lung water reabsorption may ameliorate hypoxia, shorten the application of high concentration oxygen, improve hyperoxic lung injury, block the process of lung interstitial tissue hyperplasia and fibrosis.Aquaporins (AQPs) is a family of specific cell membrane water channel proteins correlated to water permeability. At least four types of AQPs have been found in lung tissues and respiratory airways. AQP5 is one of the markers of alveolar epithelial cells type I. The effect and mechanism of AQP5 in alveolar water balance have become a hot topic in recent years. A few studies demonstrated abnormal AQP5 gene expression in lung inflammation and lung edema, which imply the possible important role of AQP5 in regulating water equilibrium of alveolar cells. Therefore, we hypothesizes that the oxygen-derived free radicals attack leads to imbalance of the AQP5 gene expression in hyperoxia-induced lung injury, which decreases alveolar fluid clearance (AFC) during pulmonary edema.Mitogen-activated protein kinases (MAPK) is a superfamily including at least three members, the extracellular signal-regulated kinase (ERK), the c-Jun N-terminal kinase /stress-activated protein kinase(JNK/SAPK) and the p38-MAPK. The MAPKs is important modulator in reactive oxygen species (ROS) regulating gene expressions through proteins phosphorylation. Evidences have shown that AQPs expression can be regulated by growth factors, cytokines and osmotic stresses. Studies have demonstrated the AQPs gene expression associated with MAPK signaling pathway in response to different stimuli. However, the relationship between AQPs and MAPK under oxidative stress is not clear. It is unknown whether MAPKs regulate AQPs gene expression in hyperoxia-induced lung injury.ROS, the important signal transduction molecules, may regulate AQP5 expression through MAPK pathways in hyperoxia-induced lung injury. It may become a possible choice to prevent and to treat hyperoxia-induced lung injury and BPD through regulating ROS levels or MAPK pathways to enhance water reabsorption in the future. We studied the AQP5 expressions of hyperoxia-injuried lung tissues in vivo and one alveolar epithelial cell line in vitro respectively in order to understand the specific roles of AQP5 in hyperoxia-induced lung injury and to explore the regulation mechanism of MAPK pathways on AQP5.Part 1 Regulation of MAPKs pathways on AQP5 expression in MLE-12 cells under H2O2-stressBackgroundThe mouse lung epithelial cell line (MLE-12) has good specificity for studying the expression and regulation of alveolar epithelial cell membrane proteins. Studies have shown that sustained activation of transcription factor AP-1, p38 and JNK may be triggered by 95% oxygen and H2O2 and mediate the hyperoxia induced oncotic cells death and cell apoptosis. The cell survival rate is improved prominently by specific MAPK inhibitors. AQP5 plays key roles in maintaining lung liquid equilibrium and lung edema development. Lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α) have been reported to affect the AQP5 mRNA and protein expression in MLE-12 cells. However, it has little known about the expressions of AQP5 under hyperoxia stress. This study proposed to simulate the condition of oxidative attacking cell injury and to investigate the alteration of oxidative AQP5 gene expression in vitro and to observe the effect of MAPK on AQP5 expression under H2O2-stress through MAPK specific inhibitors to block the activation of the protein kinase in vitro in order to find a new options for the ALI repairment.Objective1. To build cell injury model of oxidative stress.2. To observe the effects of MAPK (ERK, JNK and p38) in MLE-12 cells under different concentration and duration of H2O2-stress.3. To observe the effects of AQP5 expression under different concentration and duration of H2O2-stress.4. To study the possible role of MAPK in regulating AQP5 expression by specific inhibitors of the MAPK pathway.Methods 1. Using H2O2 as the exogenous ROS, adherent MLE-12 cells were exposed to different concentration (125μM, 250μM, 500μM or 1000μM) of H2O2 for 1h or 250μM for different duration (0, 15, 30, 60, 180, or 300min).2. The levels of MAPKs (ERK, JNK and p38) protein were detected by Western blot. MAPK signaling pathway inhibitors (PD98059, SB203580, and SP600125) were used to inhibit MAPK protein expression.3. The expression of AQP5 protein and mRNA of cells under H2O2-stress or/and MAPK inhibitors pretreatment were detected by Western blot and FQ-PCR.Results1. The phophorylated MAPK protein levels of MLE-12 cells in response to H2O2-stress were dose-dependent, increasing with higher H2O2 concentrations. Compared with the control, the protein levels of ERK, p38 and JNK were significantly increased (P<0.05) at 125μM H2O2 and peaked at the highest tested concentration (1000μM).2. The phophorylated MAPK protein levels increased with prolonged H2O2 incubation. The levels of ERK and p38 increased to highest at 1h. The ERK level decreased to normal at 3h, while the p38 level elevated consistantly for up to 5h. The JNK expression reached maximal values at 3h and returned to normal at 5h.3. The ERK inhibitor PD98059 (20μM), the p38 inhibitor SB203580 (20μM) and the JNK inhibitor SP600125 (25μM) decrease the phosphorylation protein levels of ERK, p38 and JNK respectively.4. The expression of AQP5 protein decreased after 1h with 250μM of H2O2 compared with the control group.The decrease was more with higher concentration of H2O2. Under 250μM of H2O2, the level of AQP5 protein expression had a gradual decline generally when the time of H2O2 treatment was prolonged, compared with the control. Only the p38 inhibitor (SB203580) increased the AQP5 protein level compared with the corresponding H2O2 stimulated group.5. According to the results of FQ-PCR, the mRNA level of AQP5 decreased generally with higher concentration of H2O2. It was the lowest at 500μM H2O2 concentration. Under 250μM H2O2, The mRNA level of AQP5 was lower with the increased time of H2O2 incubation, compared with the control group. Only the p38 inhibitor SB203580 affected the AQP5 mRNA level.ConclusionsBy using H2O2 to attack MLE-12 cells under different concentration and duration as the model of oxidant stress in vitro, H2O2 can trigger the activation of three members of MAPK (ERK, p38 and JNK). The inhibitors, PD98059 and SB203580 and SP600125, decreased the corresponding protein level of MAPK. H2O2 decreased the mRNA and protein levels of AQP5 in MLE-12 cells. Inhibition of p38 prevented the decrease of AQP5 gene expression under H2O2 stimulation.Part 2 Regulation of MAPKs pathways on AQP5 expression in lung tissue under hyperoxia-induced lung injuryBackgroundAcute lung injury(ALI) and acute respiratory distress syndrome (ARDS), which are clinically serious diseases caused by many high risk factors. The role of inflammatory response in ALI and ARDS etiology has been paid more and more attention. Oxygen free radicals are important inflammatory factors. Polymorphonuclear neutrophils (PMN), mononuclear cells, macrophages and eosinophils all can generate oxygen free radicals and may play important roles in hyperoxia-induced lung injury. With the extension of reactive oxygen concept, oxygen molecules can be considered as reactive oxygen. It has long been recognized that high concentration oxygen inhalation can lead to hyperoxia-induced lung injury. Although massive animal studies have shown that AQP5 plays important roles in lung injury and edema, limited studies of AQP5 in hyperoxia-induced lung injury exist, and the correlation of MAPK signaling pathway and hyperoxia-induced lung injury is rarely reported. The aim of this study is to establish a hyperoxia-induced ALI model and to explore the alteration of AQP5 expression. Using MAPK inhibitors, the possible roles of MAPK in regulating AQP5 gene expression were studied. It will provide experimental evidence for effective control AQP5 expression disturbance in ALI.Objectives1. To establish hyperoxia-induced ALI model in the juvenile rat.2. To study the effects of hyperoxia on MAPK expression, and to establish the effective concentration of the MAPK inhibitors in vivo.3. To study the effects of hyperoxia on AQP5 gene expression, and to investigate the molecular mechanism of regulating AQP5 by use of specific MAPK inhibitors.Methods1. Animal and grouping: 3-week-old Wistar rats were used in the study. The animals were randomized into following groups (n=6): group A (room air); group O3 (exposed to high concentration of oxygen for 3 days); group O7 (exposed to high concentration of oxygen for 7 days); group O14 (exposed to high concentration of oxygen for 14 days); group O7 + PD (venous injection of the ERK inhibitor PD98059 followed by exposing to high concentration of oxygen for 7 days); group O7+SB (venous injection of the p38 inhibitor SB203580 followed by exposing to high concentration of oxygen for 7 days); group O7+SP, (intraperitoneal injection of the JNK inhibitor SP600125 followed by exposing to high concentration of oxygen for 7 days); and the corresponding control groups of using inhibitors(A+PD, A+SB, A+SP).2. Phosphorylating MAPK protein (ERK, P38, JNK) levels in lung tissue were detected by Western blot and the effective concentrations of the MAPK inhibitors were explored. MAPK distribution in lung tissue was detected by IHC.3. AQP5 protein expression in lung tissue of hyperoxia-induced lung injury was detected by Western blot and by IHC. AQP5 mRNA expression was determined by FQ-PCR method. MAPK inhibitors were administered to investigate the possible role of MAPK in regulating AQP5 gene expression in hyperoxia-induce lung injury.Results1. Hyperoxia-induced ALI model: The rats were kept in cabinet with high concentration of oxygen (≥95%) monitored continuously. Lung injury developed in hyperoxia exposure groups, as evidenced by severe alveolar edema, inflammatory cell infiltration, RBC leakage and alveolar structure destruction.2. Protein levels of ERK, p38 and JNK markedly elevated in rats exposed to high concentration of oxygen compared with those in room air controls. The levels of p-ERK and p-SAPK/JNK were at highest in group O7 (P<0.05), while the level of p-p38 was at highest in group O14 (P<0.05). Depressed ERK protein expression was detected after 0.3mg/kg of PD98059 intravenously. Effective inhibited protein expressions of p38 and JNK were detected after 0.2mg/kg of SB203580 intravenously and 15mg/kg of SP600125 intraperitoneally administered, respectively. The inhibition effect increased with increasing inhibitor concentrations.3. The positive staining of MAPK protein was found in alveolar and airway epithelial cells only in the room air group, while the number of MAPK-positive cells in hyperoxia exposure groups was significantly more than those in room air-exposure controls and widely distributed in various types cells in lung tissue. The p38-positive staining was more in inflammatory cells particularly.4. AQP5 was expressed mainly in AEC I and airway secretory epithelium plasma membrane. The number of AQP5-positive cells in hyperoxia exposure groups was significantly less than those of the controls. The protein level of AQP5 detected by Western blot decreased with longer exposure to hyperoxia, especially in group O7. The mRNA level of AQP5 changed similarly to the protein level.5. The histological characteristics of the lung tissues in hyeroixa exposure 7 days were significantly improved under inhibitions of p38 and JNK. Furthermore, the protein and mRNA levels of AQP5 were increased markedly compared with group O7. There was no significant change in the AQP5 gene expression for PD98059, the inhibitor of ERK.ConclusionsThis study simulated successfully hyperoxia-induced ALI model. Exposure to high concentration of oxygen activated the MAPK signaling pathways. Appropriate concentrations of inhibitors (PD98059 / SB203580/SP600125) could block the activity of ERK/p38/JNK, three members of MAPK family. The protein and mRNA expressions of AQP5 were decreased in hyperoxia-induced ALI rats. SB203580 and SP600125 improved the AQP5 gene expression, which demonstrated that p38 and JNK pathways regulated the decrease of AQP5 expression in hyperoxia-induced ALI animals.