The Role Of HO-1/CO In Melatonin Attenuating Endotoxic Shock-induced Acute Lung Injury In Rats

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 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.There was a rapid increase in anti-oxidative substances such as heme oxyenase-1.HO catalyzes the heme to bilirubin , CO and iron.which is the first and rate-limiting step in the process,and it has a better protective effect of tissues and organs. And it has important biological significance. Especially in recent years research has shown that HO-1/CO is an important component endogenous protection system in body, and it involved in many disease and pathophysiological processes through the intracellular signal transduction system. 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 MAPKKK3 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.Studies have shown that in the course of acute lung injury, HO-1 protein expression in lung tissue increased, and it is related to MAPK activity, therefore, MAPK activation involved in HO-1 expression and regulation. At HO-1 gene sequence of the 5 'end of transcription starting upstream promoter contains nuclear transcription regulatory factor-activated protein -1 (activatorprotein-1, AP-1) binding site .AP-1 is composed of the Fos/Jun heterodimer or Jun/Jun homologous dimer constitute, and c-Jun/c-Jun composed of AP-1 for the most common form.AP-1 is controlled by MAPK signaling pathways, and the JNK is the only effectively phosphorylated c-Jun protein kinase in the MAPK family.Melatonin has been found that they are strong anti-oxidative stress in the role of hormones, but whether it enhanced endogenous HO-1/CO activity related protection system is through the JNK/c-jun pathway and, at present, both at home and abroad was no correlation reported in the literature.Melatonin was synthesized and secreted primarily in pineal gland. the function of melatonin about physiology, pharmacolog has made great progress. 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. Therefore, this issue through the cecal ligation and puncture (CLP)rat ALI model, in vivo experiments designed to observe the ALI and the JNK/c-jun signaling pathway and the relationship between the MT anti-ALI mechanism for the clinical application of MT to provide a theoretical and experimental evidence.Method: By using a wistar rat model of CLP,56 rats were randomly divided into seven groups:①the Sham group:only traversed a string at cecal root,did not ligate and puncture.②CLP group③CLP +SP600125(JNK-specific inhibitor)group: a bolus dose(15mg/kg,ip)of SP600125 was injected before 2h of CLP.④MT+CLP group,MT (10mg/kg) was injected intraperitoneally 30 min prior to and 30 min after CLP.⑤MT+CLP+SP600125group,MT and SP600125was injected before CLP administration,⑥MT group,MT was injected intraperitoneally only.⑦MT+Sp600125 group,Sp600125 was injected before MT. Parameters were observed respectively 6h after sham operation or CLP in each group. The right carotid vein and left common carotid artery of were exposed to collect blood that represented in-flowing pulmonary blood (IPB) and out-going pulmonary blood (OPB) respectiively Carpbxyhemoglbin(COHb) levels in-flowing pulmonary blood (IPB) and out-going pulmonary blood (OPB)were detected. The level of COHb represented CO tontent.Carotid artery bled to death animals, collecting blood for the detection of malondialdehyde (MDA) content, superoxide dismutase (SOD) activity .Lung tissues were harvested. Lung tissues were divided into two parts: one was imbedded by paraffin to observe morphological changes and the expression of HO-1 in lung tissues by means of immunohistochemistry staining and image analytical system; The another was homogenized to determine content of HO-1 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 The changes of COHb level in IPB and OPB:COHb level in OPB was higher than that in IPB in each group(P <0.05).Compared with control group, the level of COHb in OPB and IPB increased in CLP group and MT group.the difference of COHb level between OPB and IPB also increased (P <0.05). After the application of SP600125 , CLP + SP600125 group, MT+CLP+SP600125 group and MT+ SP600125 group of IPB and OPB and the difference of COHb level between OPB and IPB when compared with the corresponding control group were reduced (P < 0.05).2 The MDA content of plasma: Compared with control group, MDA contents in plasma increased significantly after CLP(P < 0.01). The contents of MDA decreased obviously in MT+CLP group compared with CLP group(P <0.01). Only using MT.the MDA contents decreased compared with control group. After the application of SP600125 , CLP + SP600125 group, MT + CLP + SP600125 group and MT + SP600125 group of MDA contents were increased when compared with the corresponding control group (P < 0.01).3 Changes of SOD activity in plasma: Compared with control group, SOD activity in plasma decreased significantly after CLP(P <0.01). SOD activity in plasma increased obviously in MT+CLP group compared with CLP group(P <0.01). Only using MT.the SOD activity increased compared with control group. After the application of SP600125, CLP+SP600125 group, MT+CLP+SP600125 group and MT+ SP600125 group of MDA contents were decreased when compared with the corresponding control group (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 6h after CLP, there had many infiltrated inflammatory cells and collapsed alveoli of lung. Pathological changes alleviated in MT+CLP group . In CLP+SP600125 group ,the structure of lung alveolus could not be identified , lung tissues were infiltrated by many inflammatory cells. MT and MT+SP600125 group of rat lung tissue was normal, no abnormal changes.5 Observation of immunohistochemistry staining of lung tissues: The positive cells only exist in endothelial cell of small vessels in lung tissue in the control group.Compared with control group,the HO-1expression of CLP group significantly incresaed ( p<0.01 ) ,and the positive cells were mainly distributed in alveolar epithelial cells and alveolar capillary endothelial cells. There were more HO-1 positive cells in MT + CLP group than CLP group(p<0.01).Only using MT can also increase the expression of HO-1(p<0.01). After the application of SP600125, CLP + SP600125 group, MT + CLP + SP600125 group and MT + SP600125 group of HO-1expression were decreased when compared with the corresponding control group .6 Western blotting analysis showed the expression of HO-1 in acute lung injury rats. Compared with control group, Compared with control group,the HO-1expression of CLP group significantly incresaed(p<0.01). There were more HO-1 positive cells in MT+ CLP group than CLP group(p<0.01).Only use MT can also increase the expression of HO-1(p<0.01). After the application of SP600125, CLP + SP600125 group, MT + CLP + SP600125 group and MT + SP600125 group of HO-1expression were decreased when compared with the corresponding control group .Conclusion:1 CLP could induce ALI, HO-1 protein expression in the lung tissue increased,and caused increase of CO level in blood. MT can improve lung injury, HO-1 protein expression in the lung tissue and the CO level in blood increased significantly. MT play a role in cell protection. .2 Application of JNK inhibitor SP600125 inhibited MT and (or) CLP-induced HO-1 expression, suggesting that JNK/ c-jun pathway in CLP-induced increase in MT in the lung tissue. HO-1 expression play an important role in the process.3 MT has apparent protective effect of the lungs of ALI, the protection mechanisms may be related to MT through the activation of JNK/c-jun signal transduction pathway and the increase in lung HO-1 protein expression, activation of endogenous HO-1/CO protection system.