Effects Of Melatonin On The Expression Of Ps And Icam-1 In Acute Lung Injury Rats Caused By Oleic Acid

Objective: Acute lung injury (ALI) is a common disease induced by many factors including infection in clinic and 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 remains unclear. Studies had approved 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. Studies had approved that a great quantity of polymorphonuclear neutrophil (PMN) sequestrated in intrapulmonary were the important cells, which could result in inflammatory reaction of ALI. First, the activated neutrophils sequester within the pulmonary microvasculature. They then adhere to the endothelium and migrate out of the vasculature into the lung parenchyma, produce a series of inflammatory mediators and result in diffuse pulmonary parenchyma injury. P-selectin is regulated in part by its sequestration within theα-granules of platelets and Weibel-Palade bodies of endothelial cells. Within minutes after activation of cells, Ps is transported to the cell surface where it can bind leukocytes. It was discovered that intercellular adhesion molecule-1 (ICAM-1) was the foundation of molecular biology which mediated ALI by PMN; Another study showed that ICAM-1 can also be used as a marker of activity of pulmonary vascular endothelium. A large number of pro-inflammatory factors and inflammatory mediators produced by PMN and endothelial cells caused severe amplificatory reaction, which played a crutial role in the development of ALI. So it has important practical significance to the therapy of ALI through controlling inflammtory responses and keeping the balance of oxidation/antioxidation of pulmonary.The mitogen-activated protein kinase (MAPK) was an important signal transduction system in biosystem. The family, four isoforms of MAPK kinase, ERK, JNK, p38MAPK and ERK5 have been identified. Each has its own different MAPK upstream kinase and substrate specificity, representing cells of different physiological responses to stimulation of a common mechanism. From the extracellular stimulus to appear in the cell biology of the corresponding reaction, it includes through necessary MAPK, MAPKK and MAPKKK 3 kinase cascade in accordance with the Ras-Raf (MAPKKK)-MAPKK-MAPK activation downstream of the order kinase, and finally transferred to the nucleus, activating transcription factor Elk, ATF2 and AP-1 and so on, which are starting the related cytokines and inflammatory mediators of gene expression. This multi-kinase cascade regulation, not only signal "waterfall" amplification, but also through the interaction between the various kinase signal integration, thus ensuring the accuracy and specificity of the signal.Evidence has shown that p38MAPK pathway could protect 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 had 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. In 1959, melatonin was first extracted and purified from bovine pineal gland by Lerner. 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. Since melatonin had been demonstrated as a good antioxidant, it would further extend the application of melatonin against acute lung injury (ALI). Now, melatonin as a kind of antioxygen and anti-inflammatory, the mechanism and position of its function in signal transduction pathway 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 intravenous injection (iv) of oleic acid, 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 intravenous injection (iv) of oleic acid.Eighty-four male Sprague Dawley(SD)rats were divided into 7 groups randomly (n=12 in each group).(1) Control group was injected 0.2 ml/kg per rat, by intravenous route(iv)of normal saline (NS);(2) OA group was injected 0.2 ml/kg, iv of oleic acid;(3) MT+OA group was pre-treated with 20 mg/kg melatonin intraperitoneally for 30 min before oleic acid infusion;(4) OA+SB203580 (p38MAPK-specific inhibitor) group: a bolus dose SB203580 (5μmol/L, 0.2 ml/kg) was injected before oleic acid infusion;(5) MT+OA+SB203580 group: MT and SB203580 was injected before oleic acid infusion;(6) MT group: MT was injected intraperitoneally only;(7) SB203580 group: SB203580 was injected only.All rats of each group were sacrificed at different time points (1 h, 3 h and 6 h, respectively) after OA infusion, the blood was collected from left ventricular and lung tissues were harvested. Lung tissues were imbedded by paraffin to observe morphological changes and the expression of Ps and ICAM-1 in lung tissues by means of immunohistochemistry staining and image analytical system.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 Praxiology appearance: Rat become breathlessness, dispirited, shrinked into agglomerate, reared pelage and did not ingest after OA infusion.2 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 in OA group, there had many infiltrated inflammatory cells and collapsed alveoli of lung. Pathological changes alleviated in MT+OA and SB203580+OA group. MT and SB203580 group of rat lung tissue was normal.3 Lung coefficient: Compared with Control group, the lung coefficient increased significantly in OA group(P<0.05); Compared with OA group, the lung coefficient decreased obviously in MT+OA and SB203580+OA group(P<0.05), but it was still higher than that of control group(P<0.05). MT and SB203580 group of rat lung coefficient was normal(P>0.05).4 Observation of immunohistochemistry staining of lung tissues:①Ps: In control group, positive expression (buffy) of Ps resided in endothelial cell of airway mucosa, the endochylema was positive and positive signal was weaker, but positive expression hadn't been found in vascular endothelial cell; Compared with control group, positive expression was very obvious (P<0.05) in OA group, which mainly resided in vascular endothelial cell and endothelial cell of airway mucosa, all cells'endochylema were positive; The distribution of positive cells was similar to that of OA group in MT+OA group, OA+SB203580 group and MT+OA+SB203580 group, but positive expression decreased significantly (P<0.05), especially in vascular endothelial cell.②ICAM-1: In control group, positive expression (buffy) of ICAM-1 resided in endothelial cell of airway mucosa, the endochylema was positive and positive signal was weaker, but positive expression hadn't been found in vascular endothelial cell; Compared with control group, positive expression was very obvious (P<0.05) in OA group, which mainly resided in vascular endothelial cell and endothelial cell of airway mucosa, all cells'endochylema were positive; The distribution of positive cells was similar to that of OA group in MT+OA group, OA+SB203580 group and MT+OA+SB203580 group, but positive expression decreased significantly (P<0.05), especially in vascular endothelial cell.Conclusions:1 OA could lead to severe inflammatory reaction in lung tissues of rats, which indicated that animal model of ALI could be duplicated in success.2 Application of p38MAPK inhibitor SB203580 inhibited the expression of Ps and ICAM-1 in lung tissues, suggesting that p38MAPK pathway play an important role in ALI cause by OA.3 MT is an effective antioxidant, which relieves the inflammation in acute lung injury rats, possibly through the inhibition of p38MAPK over activation.