Influence Of Melatonin On P38 MAPK In Acute Lung Injury Rat Caused By LPS

Objective: Acute lung injury (ALI) is a common disease induced by many factors including infection in clinic, it can result in diffuse pulmonary parenchyma injury. ALI is also a typical appearance in lung for systemic inflammatory response syndrome (SIRS) or multiple organ dysfunction syndrome (MODS). The pathogenesis of ALI is very complicated, which has remained unclear. The majority of scholars thought that the unbalance of oxidation/antioxidation played an important role in body, especially in lung. When lung tissues were demolished by all kinds of etiological factors (trauma, shock, infection, et al), then severe oxidative stress was generated in lung and it also caused ALI accordingly. Gram-negative bacteria was one of important pathogens which could cause inflammation in airways and lung tissue. Lung alveolus macrophage,neutrophilic leukocyte and endothelial cell released a large of proinflammatory factor,excitatory transmitter and chemotatic factor caused by LPS. The process played an important role in Inflammatory reaction, but the signal transduction was vital in process of Inflammatory reaction .The mitogen-activated protein kinase (MAPK) was an important pathway by mediating nuclear reaction. The family, four isoforms of MAPK kinase, ERK, JNK, p38MAPK and ERK5 have been identified. p38MAPK had a close relationship with ALI which participated in Inflammatory reaction in cell. P38MAPK was activated by phosphorylation and promoted downstream substrate phosphorylated. The process could make signal transduction succeeded and started nuclear factors of NF-κB, AP-1, et all. This could promoted mediators of inflammation excreted and mediated Inflammatory reaction. But it was not clear about mechanism of activation of p38MAPK and downstream regulation.Evidence has shown that p38MAPK pathway could ptotect organism. But over avtivated p38MAPK may make TNF-1, IL-1 continue high level expression. This could induce phlegmasia process, especially LPS played a important role in cytokine synthesis process. Beaty has shown that p38MAPK pathway mediated signal transduction in neutrophilic leukocyte. p38MAPK was phosphorylated under the function of LPS. Acticate p38MAPK was shifted kytoplasm into nucleus, transcribed and produced a large of inflammatory factor. Some investigation has indicated that inhibition of p38MAPK pathway could efficiently suppress inflammatory reaction, obviously palliate inflammatory reaction of organism.Melatonin was synthesized and secreted primarily in pineal gland. In1959, melatonin was first extracted and purified from bovine pineal gland by Lerner. To date, research about the function of melatonin about physiology, pharmacolog has made great progress. Following research findings demonstrated that MT could regulate many physiological functions including endocrinium,nervous system,immune system and biologic rhythm, but until the 1990's, Tan reported that MT possessed antioxidative activity. MT was a potent endogenous free radical scavenger and has positive effect in anti-inflammatory. Now, melatonin as a kind of antioxygen and anti-inflammatory, the mechanism and position of its function in signal transduction pathway and physiological level has not been understood. Therefore, we solve these questions from molecular level in order to develop wide prospect for the application of melatonin. In the present study, we duplicated rat model of ALI by instilling lipopolysaccharide (LPS) intratracheally, and explored effect of MT on ALI and its possible mechanism. Through this study, theoretical and experimental evidence was provided for clinical application of MT.Methods: Rat model of ALI was established by instilling LPS intratracheally and MT was injected intraperitoneally. Effect of MT on lung tissues was researched by means of changes of malonaldehyde (MDA),superoxide dismutase (SOD),nitrogen monoxidum (NO) and pathological change of lung tissues at different time points.Senventy two male Sprague Dawley(SD)rats were divided into 3 groups randomly (n=24 in each group).①Control group: normal saline (NS) was instilled intratracheally (200μl / per rat) , and vehicle (1% alcohol normal saline, 1ml/kg) was injected intraperitoneally 30 min prior to and 30 min after NS instillation;②LPS group: LPS (200μg/200μl / per rat) was instilled intratracheally,and vehicle was injected intraperitoneally 30 min prior to and 30 min after LPS instillation;③LPS+MT group: LPS (200μg/200μl / per rat) was instilled intratracheally, and MT (10mg/kg) was injected intraperitoneally 30 min prior to and 30 min after LPS instillation. All rats of each group were sacrificed at different time points (3h,6h and 10h, respectively) after LPS instillation, lung tissues were harvested. Lung tissues were divided into three parts: one was homogenized to determine changes of MDA,NO content and SOD activity; The other was imbedded by paraffin to observe morphological changes and the expression of P38mapk in lung tissues by means of immunohistochemistry staining and image analytical system; The another was homogenized to determine content of p38MAPK by means of Western blotting and image analytical..Statistical analysis was carried out using SPSS 11.5. All data were expressed as means±SD (x±s). Group of data were compared with an analysis of variance (one way ANOVA) followed by Student-Newman-Kuels q tests. Values of P <0.05 were regarded as significant.Results: 1 Changes of MDA contents in lung tissues: Compared with control group, MDA contents in lung tissues increased significantly at 3h,6h and 10h after LPS instillation in LPS group (all P<0.01), and MDA contents reached the peak _ at10h, there were significant deviation between adjacent time points (all P<0.01); When compared with LPS group, MDA contents decreased obviously in LPS+MT group (all P <0.01), but it was still higher than that of control group (all P <0.01).2 Changes of SOD activity in lung tissues: Compared with control group, SOD activity in lung tissues decreased significantly at 3h,6h and 10h after LPS instillation in LPS group (all P<0.01), and SOD activity was the lowest at 10h, there were significant deviation between adjacent time points (all P <0.01); When compared with LPS group, SOD activity increased obviously in LPS+MT group (all P<0.01), but it was still lower than that of control group (all P <0.01).3 Changes of NO contents in lung tissues: Compared with control group, NO contents in lung tissues increased significantly at 3h,6h and 10h after LPS instillation in LPS group (all P<0.01), and NO contents reached the peak at 10h, there were significant deviation between adjacent time points (all P<0.01); When compared with LPS group, NO contents decreased obviously in LPS+MT group (all P<0.01), but it was still higher than that of control group (all P <0.01).4 Morphological changes in lung tissues: The light microscope results showed that the structure of lung alveolus was clear, alveolar wall was thin and there wasn't diffusate in alveolar space in control group; Alveolar septum thickened significantly at 3h after LPS instillation, there had many infiltrated inflammatory cells and collapsed alveoli of lung. The structure of lung alveolus could not be identified at 6h, lung tissues were infiltrated by many inflammatory cells, the changes of lung tissues at 10h were still prominent; the changes of lung tissues at 3h in LPS+MT group were similar to that of control group, the structure of lung alveolus was identified easily at 6h and 10h, alveolar septum had a little thicker, PMN infiltration was lessened significantly when compared with LPS group, there hadn't obvious effusion in alveolar space, but inflammation changes were outstanding in individual region of lung tissues.5 Observation of immunohistochemistry staining of lung tissues: There were rare p38 MAPK positive cells scattered in alveolar and airway epithelial cells in the control group (all P<0.01). The positive p38 MAPK cells in LPS injury groups increased obviously than those in the control group (P<0.05or P<0.01),and were mainly distributed in infiltrative inflammatory cells, airway epithelial cells, alveolar epithelial cells and pleurames epithelial cells. In LPS+MT group, the p38 MAPK positive cells in airway and lung tissue were much less than those in the acute lung injury group (all P<0.05).6 Western blotting analysis showed the expression of p38 MAPK in acute lung injury rats. Compared with control group, the expression of P38 in LPS group was much more ( P<0.05or P<0.01). Compared with LPS group, the expression of P38 in LPS+MT group was less (P<0.05or P<0.01), but it was more than control group (P<0.05or P<0.01 ). The Western blot results were consistent with those of immunohistochemical method.Conclusion: 1 Instilling LPS intratracheally could lead to severe inflammatory reaction in lung tissues of rats, which indicated that animal model of ALI could be duplicated in success.2 p38 MAPK expression increases in alveolar and airway epithelial cells in LPS induced acute lung injury rat models. The activation of p38MAPK is found in most lung tissues, suggesting that p38MAPK participates in the signal transduction in inflammatory and noninflammatory cells.3 MT is an effective antioxidant, which relieves the inflammation in acute lung injury rats, possibly through the inhibition of p38MAPK over activation.