Effects Of Nebulized N-acetylcystein On The Expression Of HMGB1 And RAGE In Rats With Hyperoxia-induced Lung Injury

Research BackgroundOxygen therapy is one of the important clinical treatments for children with acute and critical illness.Hypoxemia induced by acute respiratory failure often needs to high concentration of oxygen to maintain arterial oxygen partial pressure to improve oxygen supply to the brain and other vital organs.However,prolonged breathing of very high FiO₂ uniformly causes the damage of the lungs’structure and the function injury,which result in acute lung injury,and we call it hyperoxia-induced lung injury（HALI）.High mobility group box 1（HMGB1）is a highly conserved nuclear transcriptional protein that can be involved in airway inflammation as an immunoregulatory factor and inflammatory factor.The receptor for advanced glycation end products（RAGE）is an important pattern recognition receptor,which participates in the body’s innate immunity and acquired immune response,and it plays an important role in the airway inflammatory response.Studies have shown that RAGE is one of the receptors for HMGB1 involved in cell signal transduction,and RAGE can combine HMGB1 to activate intracellular signal transduction pathways,which results in inducing pro-inflammatory cytokines,protease release and oxidative stress.N-acetylcysteine（NAC）is a precursor of glutathione（GSH）,which has been shown in many animal models that it can scavenge oxygen free radicals directly,and it also can regulate the production of some cytokines and the expression of adhesion molecules in endothelial cells and bronchial epithelial cells to improve lung tissue damage.At present,there are few studies on HMGB1/RAGE signaling pathway in hyperoxia-induced lung injury.The study of nebulized NAC on this signaling pathway has not been reported at home and abroad.In this study,we established a high-oxygen lung injury animal model to explore the expression changes of HMGB1and RAGE and the protective effect of NAC on lung injury,which provided a theoretical basis for the prevention and treatment of high-oxygen lung injury.Objective1.To investigate the role of high mobility group box 1（HMGB1）and advanced glycation end product receptor（RAGE）mRNA and protein in the lungs of SD rats with hyperoxia-induced lung injury.2.Explore the protective mechanism of N-acetylcysteine（NAC）on hyperoxia lung injury,and provide new research ideas for the prevention and treatment of hyperoxia lung injury.Methods45 male Sprague Dauley（SD）rats（aged 3 weeks and weight 54.97±5.32 g）were randomly divided into three groups（n=15）:control group,hyperoxia group and NAC intervention group.Rats in the hyperoxia group and the NAC intervention group were placed in a self-made plexiglass oxygen chamber,and pure oxygen（2.5 L/min）was continuously input.The oxygen and carbon dioxide concentrations were continuously measured to ensure that the oxygen concentration was≥90%and the carbon dioxide concentration was≤5%.We took 7th consecutive day to established this animal model of hyperoxia lung injury,and rats in the control group were housed in the same size homemade plexiglass box in the same room,and we continued to pass atmospheric air into the box.We opened the box once a day at 9:00 am（replacing soda lime,litter,water and feed）,30 min/time,and gave the intervention group rats nebulized NAC（300mg/3ml）,and the control group and hyperoxia group were given to inhale an equal volume of normal saline for a total of 7 days.The state and body weight changes of each group rats were observed and recorded every day.On the 7th day of the experiment,all animals were sacrificed and lung tissue samples were taken.The wet/dry（W/D）ratio of the lungs was determined to evaluate the edema of the lung tissues.Conventional hematoxylin-eosin（HE）staining was used to observe the pathological changes of lung tissues.Immunohistochemical staining was used to investigate the expression of HMGB1 and RAGE in the lung tissues.Quantitative real-time PCR and western blot were used to detect the changes in the mRNA and protein expression of HMGB1 and RAGE,respectively.Results1.During the experiment,the basic condition of the rats in the control group was normal;the rats in the hyperoxia group showed signs of mental wilting,slowing of reaction,decreased activity and diet,and the above-mentioned performance in the NAC intervention group was between the control group and the hyperoxia group.2.After exposed 7 days in the high oxygen environment,the weight gain of the rats in the hyperoxia group was significantly slower than that in the control group,and the weight gain of the intervention group was higher than that in the hyperoxia group（P<0.05）.The W/D ratio in hyperoxia group was significantly higher than that in control group,and the W/D ratio in the intervention group was lower than that in the hyperoxia group（P<0.05）.3.HE staining results showed:In the control group,the alveolar size was uniform,the structure was intact,the epithelial cells were arranged neatly,the alveolar space was narrow,and a small amount of inflammatory cells were infiltrated.In the hyperoxia group,the number of alveolar decreased,the alveolar size was not of uniform,the alveolar space became widened,congestion and edema were observed,the lung tissue structure was disordered,the alveolar wall was broken,and a large number of inflammatory cells infiltrated.The pathological changes of lung tissue in the NAC intervention group are between the two groups.4.The results of PCR and Western-blot showed the mRNA and protein expression of HMGB1 and RAGE in the hyperoxia group were significantly higher than control group,and the intervention group was significantly lower than hyperoxia group（P<0.05）.Conclusions1.HMGB1 and RAGE are involved in the pathogenesiss of hyperoxia-induced lung injury.2.Inhalation of N-acetylcysteine may alleviate hyperoxia-induced lung injury by regulating the expression of HMGB1 and RAGE.