Role Of MMP-9 In Phosgene Induced Lung Injury

BackgroundPhosgene is a kind of colorless,rotten hay smelled and asphyxiating gas,widely used in rubber,plastics,dyes,pesticides,medicine,and the production of other chemical raw materials.This gas was originally manufactured as an agent for chemical warfare during World War I.Therefore,the United Nations Disarmament Commission determined phosgene as a"dual-use agent".Because of its wide range of uses, accessibility,there is often an accident of improper protection or accidental leakage. When phosgene poisoning happened,it would cause acute lung injury(acute lung injury,ALI),and even developed into acute respiratory distress syndrome(acute respiratory distress syndrome,ARDS),even life-threatening.However,the pathogenesis of lung injury induced by phosgene inhalation is unclear now,with no effective therapy,high mortality,which make the study of pathogenesis of phosgene poisoning more significant.Phosgene toxicity is mainly damage to the respiratory system,with delayed pulmonary edema or even acute respiratory distress syndrome(ARDS).Phosgene toxicity depends on its inhaled concentration and time.Maximum allowable concentration of phosgene in air is about 0.5mg/m~3.At lower concentration(20mg/m,1 min),there is no significant signs of local stimulation,after a period of symptomatic relief,symptoms of alveolar-capillary membrane damage emerged,leading to pulmonary edema.Phosgene poisoning can cause early protective inflammatory response occur,but excessive or out-of-control systemic inflammatory response may be caused by"inflammatory cascade waterfall",which happened with acute lung injury(ALI), developed into systemic inflammatory response syndrome(SIRS)and ended in multiple organ dysfunction syndrome(MODS).To date,the mechanism of phosgene toxicity is still unclear,but there is a lot of hypothesis,including acylation,phosgene direct role,hydrochloric acid and neural reflex of pulmonary hemodynamic changes and so on.But any kind of hypothesis can not satisfactorily explain the occurrence of pulmonary edema and its progressive process.Matrix metalloproteinase-9(MMP-9),also known as gelatinase B,is one kind of the family of matrix metalloproteinases,produced by a variety of inflammatory cells, including neutrophils,alveolar macrophages,as well as connective tissue cells.MMP -9 was directly involved in pulmonary inflammation and in the process of extracellular matrix breakdown and remodeling,which played great role in acute lung injury, but its role in phosgene-induced lung injury has not been reported.At present,the important role of matrix metalloproteinases-9 in lung injury has been proved by a number of experimental studies.Fligiel showed that MMP-9,MM P-8,MMP-2 were increased in bronchoalveolar lavage fluid of 28 cases of ALI and ARDS patients.Zhang Xiang-Feng set up the experiment animal model with acute lung injury in hyperxia environment,and proved increase of MMP-9mRNA in hyperoxia -induced lung injury by the method of RT-PCR.KIM,found expression of MMP-9 and MMP-2 were increased in lung tissue and BALF of rats with bleomycin-induced lung injury,with MMP-9 increasing more obviously,and the main effect of MMP-9 at the early stage of lung injury was damagement,while MMP-9 and MMP-2 also increased in the late stage of lung injury,with the main role of repairment,leading to pulmonary fibrosis.Phosgene induced lung injury caused toxic pulmonary edema majorly,and we presumed that there existed a common pathway mediated by MMP-9 between ALI induced by phosgene and other factors such as LPS,hyperoxia and so on.In this experiment,we will set up animal model of rats with phosgene-induced lung injury,and evaluate the expression and significance of MMP-9 in phosgene-induced lung injury by use of molecular biological methods.Objective To establish rat phosgene inhalation lung injury animal model and observ -e the expression and role of matrix metalloproteinase-9(MMP-9)in rats with phosgene induced lung injury and dynamicly monitor the expression of MMP-9 along the time course.Methods 50 male SPF level Sprague Dawley(SD)rats(180-220g)were randomly divided into 2 big groups,five subgroups.Rats were randomly divided into the air control and phosgene exposure groups.We set up phosgene-induced lung injury animal models according to reference and improved this method.Rats in phosgene exposu- re group were home in dynamic exposure cabinet,accessible to a concentration of 8.33mg/L phosgene(phosgene purity is 100%,production of triphosgene dissolved in cyclohexane generated by N,N-dimethylformamide,according to the references) and were exposed to phosgene for 5 min,while the control group animals were home in the exposure cabinet,with the exception that exposure was the same air flow.And we sacrificed the rats respectively 2,4,6,8 h after exposure,dynamicly determined expression of MMP-9 in lung tissue by method of immunohistochemistry and realtime quantitative reverse transcription polymerase chain reaction(RT-PCR),meanwhile we monitored the lung wet / dry weight ratio,protein concentration in bronchoalveolar lavage fluid,neutrophils count and histopathological changes in lung tissue dynamicly.Results Compared to the control group,pulmonary edema,hemorrhage and inflammatory cells infiltration appeared in the phosgene exposure groups.Wet-to-dry lung weight ratio,protein content in BALF,PMNs in BALF in phosgene exposure groups were higher compared to control group(P＜0.05).Comparing Wet-to-dry lung weight ratio,protein content in BALF,PMNs in BALF between the four phosgene exposure groups by one-way ANOVA,differences were statistically significant(P＜0.05),which was correlated with lasting of time.Expression of MMP-9 in phosgene exposure groups were higher than control group observed under light scope by method of immunohistochemistry (P＜0.05).Acute lung injury caused by phosgene exposure was accompanied with increased expression of MMP-9mRNA in lung tissues(P＜0.01),which was correlated with lasting of time and aggravation of lung injury.Conclusion MMPs activation and the excessive expression of inflammatory factors play an important role in the development of phosgene-induced acute lung injury in rats.