The Protective Of Glutamine On Smoke Inhalation Induced Acute Lung Injury And Its Underlying Mechanism In The Experiment Model

BackgroundAs a result of extensive burns, the immune function decreases when susceptibility to microorganism increases significantly, and the respiratory track becomes the important route of general infection, especially to patients suffering the burn combined with the inhalation injury. The inhalation injury is a respiratory and pulmonary parenchymal disease caused by heat and (or) smoke. It usually occurs to patients suffering the extensive burn and especially the facial burn. It is one of the major reasons for death caused by burn. As revealed by data in the past, the case fatality rate of patients suffering the inhalation injury is generally between50%-60%, and it can reach as high as90%for patients with the severe inhalation injury, which can lead to the serious acute lung injury (ALI) and further induce the acute respiratory distress syndrome (ARDS). Although new ventilation technologies and other treatment measures have been applied, the fatality rate of ARDS still reaches as high as40%-60%. Thus, great attention has been paid to how to prevent occurrence and development of lung injury to patients suffering the inhalation injury, which is especially key to reduce the fatality rate of burn sufferers. ALI caused by the inhalation injury is an inflammatory reaction out of control. It is characterized by extensive injury of pulmonary vascular endothelial cells and alveolar epithelial cells and accompanied by pulmonary hemorrhage, edema, excessive inflammatory cell infiltration and other features. According to the researches in recent years, heat shock proteins (HSP) has the protective effect on the injured or stressed body and certain protective effect on ALI, and can alleviate injury to lungs caused by oxidative stress, inflammation and ischemia reperfusion, so it may be a new strategy for clinical prevention and treatment of ALI to induce or enhance the body HSP.GLN is the richest free amino acid within the human body. In the stress state of sepsis and trauma, etc., the demand for GLN by intestinal mucosal cells and rapidly-proliferated cells (e.g. immune cell) increases greatly, and lungs will release a large amount of GLN within a short time. Afterwards, the low-concentration GLN may lead to pulmonary immune dysfunction and occurrence of ARDS. GLN supplement can promote the survival rate, enhance the immune function, reduce the bacterial infection, enhance the gut barrier function, and prevent atrophy of intestinal mucosa. As revealed by the recent researches, after GLN treatment is applied on the mouse sepsis model made through the means of caecum ligation, the expression of TNF-α and IL-6is reduced significantly. According to relevant researches, through intravenous injection of GLN, the lung tissue of a rat with sepsis can highly express HSP70to resist the endotoxin shock. In our present study,we produced the acute lung injury model by smoke inhalation and using the glutamine to investigate the protective of glutamine on smoke inhalation induced acute lung injury and its underlying mechanism.Our research was divided ino three parts. Part One Building a rat model of smoke inhalation injuryObjective:Smoke inhalation injury is the leading cause of acute respiratory failure in critical burn victims. Advances in the treatment of smoke inhalation injury have been limited in the past years. To further explore the pathogenesis, stable and practical animal models are necessary; develop a rat model of smoke inhalation injury.Methods:Part1,36rats were randomly divided into three groups:9,12, and15min based on the exposure time. The corresponding mortalities were recorded during the time exposure to smoke and the subsequent? days. Part2,54rats were randomly divided into two groups:normal group (ambient air exposure,6rats) and control group (9min time of smoke inhalation,48rats). After smoke inhalation, six rats were killed each time in the I group at2h,4h,6h,24h,48h,96h,7days and28days by overdosed pentobarbital sodium(100mg/kg body weight).The blood gas values, pro-inflammatory and protein concentration in bronchoalveolar lavage fluid and lung wet to dry weight ratio were assayed. Pathological evaluations of pulmonary were performed at24h,96h,7days and28days post-injury. Masson-Goldner trichrome staining was performed on day7and28post-injury.Results:In our present animal model, smoke inhalation caused a significant hypoxemia and CO poisoning. A surge of pro-inflammatory response and microvascular hyperpermeability with neutrophils accumulations were also found in our animal model. At24h post-smoke inhalation, the hematoxylin and eosin results exhibited that there were inflammatory exudates and diffuse hemorrhage in the lung tissue with significant edema. With the time going, the lung injuries appeared at alveolar collapse and alveolar septum thickening, which indicated that smoke inhalation further induced damage to lung parenchyma. Specially, the markedly collagen deposition appeared at28days post-injury indicated that pulmonary fibrosis happened.Conclusion:Rats model of smoke inhalation injury recommended in this article is a convenient, useful, stable and reduplicated animal model. Smoke generator could be used for acute and chronic lung injury experiments. Part Two The therapeutic efficacy of glutamine for smoke inhalation-induced lung injury in ratObjective:Smoke inhalation injury represents a major cause of mortality in burn patients and associated with a high incidence of pulmonary complications. Glutamine (GLN) is considered a conditionally essential amino acid during critical illness and injury. However, whether GLN could attenuate lung injury caused by smoke inhalation is still unknown. The purpose of this study is to investigate whether GLN has a beneficial effect on smoke inhalation induced lung injury.Methods:Fifty-four rats were randomized into3groups:normal group (Sham operation), control group (Smoke inhalation injury+physiological saline) and experiment group (Smoke inhalation injury+Glutamine).The rat in control and experiment group were induced ALI by Smoke inhalation injury. The rats in the experiment groups were given glutamine solution750mg/kg body weight and the rats in control group were given saline at the same volume. At24h post-injury,6rats in each group were killed and their lung tissues were isolated and assayed for malonaldehyde (MDA), super oxide dismutase activity(SOD), TNF-α, IL-1β, IL-8and histological examination. The remained12rats in each group were used for monitoring the survival rate of mice every12h for4daysResults:Our experiments exhibited that the survival rate of experiments group was higher than control group (P<0.05).After Smoke inhalation injury, MDA content in the lung tissues was increased in the control group,whereas it was decreased in the glutamine treatment group (P<0.01). After Smoke inhalation injury, SOD activity in the lung tissues was decreased in the control group,whereas it was increased in the glutamine treatment group (P<0.01). After Smoke inhalation injury, TNF-α, IL-1β and IL-8content in the lung tissues was increased in the control group, whereas they were decreased in the glutamine treatment group. Glutamine treatment further attenuated lung edema inflammatory cell infiltrations, widened alveolar septum, and diffuse hemorrhage.Conclusion:Our data demonstrated that Glutamine can relieve the acute lung injury induced by smoke inhalation, improve the symptoms of the lung injury induced by smoke inhalation and the oxygenation function of the lung, raise the survival rate of the rats, alleviate the pathologic change, prevent the development of pulmonary edema and alleviate the oxidative stress injury of the lungs as well as the acute inflammatory reaction of the lung. It proves that glutamine can exert protective effects on the lung injury induced by smoke inhalation. Part Three The protective of glutamine on smoke inhalation induced lung injury and its underlying mechanismObjective:The purpose of this study is to investigate the protective effects of glutamine treatment on smoke inhalation induced lung injury.Methods:In our present work,54rats were equally randomized into three groups: normal group (Sham operation, ambient air inhalation), Control group (Smoke inhalation plus physiological saline) and experiment group (Smoke inhalation injury plus GLN treatment). At sampling, bronchoalveolar lavage fluid was performed to determine total protein concentration and pro-inflammatory cytokine levels. Lung tissues were collected for wet/dry ratio, histopathology, hydroxyproline and western blotting measurement.Results:According to the arterial blood gas analysis, the PaO2of control group and experimental group is much lower than that of the normal group and the PaO2of the experimental group is much higher that of the control group (P<0.01). In terms of the changes of PaCO2and pH value, there is no obvious difference between the experimental group and control group. The pathological score of the experimental group is much lower than that of the control group (P<0.01). Comparing with the control group, the experimental group can reduce the W/D ratio and the protein concentration of BLAF significantly (P<0.01). The positive cell TUNEL of the lung tissue of the experimental group reduced obviously compared with the control group (P<0.01). The IL-8level in BALF of the experimental group is much lower than that of the control group (P<0.01). As the results of Western blot indicates, the P-HSF-1、HSP70、HO-1and IκB-β expression of the lung tissue of the experimental group increased evidently compared with that of the control group while the NF-κB expression of the lung tissue of the former reduced obviously compared with that of the latter(P<0.01). Masson stain and the hydroxyproline assaying show that the collagen deposition of lung tissue of the experimental group reduced evidently compared with that of the control group(P<0.01).Conclusion:Glutamine can relieve the lung injury induced by smoke inhalation, mitigated pulmonary edema, obviously curb the release of the inflammatory factor, improve the pulmonary function, alleviate the pathologic change and curb the pulmonary fibrosis in long term. Our data demonstrated that GLN protected rats against smoke inhalation-induced lung injury and its protective mechanism seems to involve in inhibition inflammatory response and enhancing HSPs expression.