| Background:Phosgene is also known as carbonyl chloride,which can be generated by the reaction of carbon monoxide and chlorine under strong light irradiation.It is a colorless gas with the smell of rotten hay at room temperature.As an important chemical raw material,phosgene is widely used in pharmaceuticals,plastics,chemical fibers,leather,and agricultural production.Meanwhile,phosgene is a typical chocking chemical warfare agent,which was widely used in World War I and the Japanese invasion of China,causing hundreds of thousands of people deaths.After phosgene inhaling,the respiratory tract is the major damage target because of its poor water solubility.Evidences show that phosgene exposure easily leads to acute lung injury(ALI),which can develop into acute respiratory distress syndrome(ARDS)in severe cases,resulting in death from poisoning.At present,the annual output of phosgene in China is 6 million tons,which is prone to leakage during production,storage,transportation,and usage.There are many people engaged in related production.In addition,as an important chemical warfare agent,the risk of using phosgene in future warfare still exists.Therefore,the research on the treatment of phosgene-induced ALI is not only useful for ensuring the chemical production safety,but also has potential value for national defense.To date,the mechanism of phosgene-induced ALI and pulmonary edema is still unclear internationally,and clinical treatment mainly relies on respiratory support measures such as oxygen inhalation and mechanical ventilation,which has shown limited effect.Therefore,to investigate the mechanism of phosgene-induced ALI and the specific treatment drugs is necessary.Many literatures have shown that the renin-angiotensin system(RAS)is a humoral regulation system composed of a series of peptide hormones and related enzymes,which plays an important role in regulating the homeostasis of blood vessels and the occurrence of pulmonary edema.Among them,angiotensin Ⅱ-angiotensin-converting enzyme 2-angiotensin-(1-7),which is short for Ang Ⅱ-ACE2-Ang-(1-7)axis,is an important part of the RAS system.Meanwhile,overexpression or activation of ACE2 is proved to be effective for antagonizing ALI caused by biochemical factors such as hydrochloric acid or lipopolysaccharide.Decreased Ang Ⅱ and increased protective Ang-(1-7)levels,ultimately attenuate lung injury.Howe ver,the changes and mechanisms of the Ang Ⅱ-ACE2-Ang-(1-7)axis during phosgene-induced ALI are still unclear.Relevant research is expected to provide novel strategies for the treatment of patients with phosgene poisoning.Our paper aims to systematically study whether the ACE2 agonist diminazene aceturate(DIZE)has a protective effect on phosgene-induced ALI,and further explore the potential role of Ang Ⅱ-ACE2-Ang-(1-7)axis.This work may provide novel strategies for the treatment of phosgene inhali ng patients.Objective:1.The SD rat ALI model was established by dynamic exposure to phosgene,and the expression profile of Ang Ⅱ-ACE2-Ang-(1-7)axis was clarified.2.To determine the role of ACE2 agonist DIZE in treatment phosgene-induced ALI,and to explore the molecular mechanism of the Ang Ⅱ-ACE2-Ang-(1-7)axis in this procession.Methods:First,SD rats ALI model was constructed by dynamic inhalation phosgene to study the expression profile of Ang Ⅱ-ACE2-Ang-(1-7)axis at different time points after phosgene exposure.Fifty SD rats were divided into 5 groups(10 rats per group),and the animals were sacrificed 3,6,12,and 24 h after exposure to phosgene at41mg/m~3·30min(total 1230 mg·min/m~3)to collect blood,Bronchoalveolar lavage fluid Fluid(BALF)and lung tissue,and set the control group.Tested the pathological damage in lung tissue(hematoxylin-eosin,HE),pulmonary edema(lung wet weight,lung-to-body ratio,total protein concentration in BALF,total cell number in BALF),respiratory function(enhanced pause).Then,ACE2 expression and contents in lung tissues were detected at different time points,AngⅡ,Ang-(1-7)changes.Second,60 mg/kg DIZE was administered 8,16 and 24 hours before phosgene exposure at 41 mg/m~3,respectively.Forty rats were randomly divided into 4 groups with 10 rats in each group,namely contro l group,DIZE group,Phosgene group,and DIZE+Phosgene group.The lung tissue pathological damage,pulmonary edema,respiratory function were analyzed between different groups.The inflammatory indicators(TNF-α,IL-6)in lung and serum with ELISA assay.MDA,4-HNE and 8-OH-d G level in lung tissues were tested to indicate oxidative damage.Meanwhile,the expression of ACE2,Ang Ⅱ,Ang-(1-7)levels were also tested by Immunofluorescence and Immunohistochemistry assays.Results1.After 3-24 hours of dynamic inhalation of 41 mg/m~3·30min(1230 mg·min/m~3)phosgene in rats,the lung tissue showed hemorrhage and edema,and the wet weight of the lung tissue was significantly increased.BALF showed increased protein and cell content,and the enhanced expiratory pauses was significantly increased.Pathological results showed interstitial thickening,inflammatory cell s infiltration and increased exudate in lung tissue after phosgene exposure.It is suggested that the lung tissue is acutely damaged after phosgene exposure,and gradually develops into pulmonary edema with the passage of time,and the lung function is damaged.In addition,the contents of myeloperoxidase(MPO)and malondialdehyde(MDA)in lung tissue also was significantly increased after phosgene exposure,indicating that inflammatory reaction and oxidative stress damage occurred in the lung tissue of animals.Western-blot and immunofluorescence results showed that the protein expression of ACE2 in lung tissue and BALF of animals wa s significantly increased after phosgene exposure.The expression or content of Ang-(1-7)downstream of ACE2 decreased gradually with phosgene exposure,while the expression or content of Ang Ⅱ in lung tissue and BALF gradually increased with phosgene exposure.Therefore,ACE2 and the downstream Ang Ⅱ-ACE2-Ang-(1-7)axis were significantly inhibited after phosgene exposure,suggesting that activation of ACE2 may have a protective effect on ALI induced by phosgene exposure.2.DIZE is an agonist of ACE2.After exposure to phosgene,60 mg/kg of DIZE was injected intraperitoneally.The results showed that DIZE(60 mg/kg)administration could significantly inhibit phosgene-induced hemorrhage and edema in animal lung tissue,reduce lung tissue pathological damage and inflammatory cells infiltration,block the increase in protein and cell content in BALF,and reverse phosgene induced lung wet weight and lung-to-body ratio increases.DIZE also significantly inhibited the phosgene-induced enhanced pause,end expiratory pause,oxidative damage and inflammatory responses in animals.The above results indicated that DIZE treatment could inhibit phosgene exposure-induced lung tissue damage,improve pulmonary edema,and improve lung respiratory function.In addition,we also found that the expression and activity of ACE2 in lung tissue were significantly enhanced after DIZE treatment,the expression or content of Ang Ⅱ in lung tissue and BALF was decreased,while the expression or content of Ang-(1-7)in lung tissue and BALF was increased.Therefore,DIZE has a protective function against phosgene-induced acute lung injury,in which the Ang Ⅱ-ACE2-Ang-(1-7)axis plays a key role.It is suggested that DIZE may be a potential salvage drug for the treatment of phosgene-induced ALI.Conclusion:1.Phosgene dynamic inhalation exposure can induce ALI and pulmonary edema in rats,and the mechanism may be related to oxidative stress and inflammatory response.Phosgene exposure can time-dependently inhibit the Ang Ⅱ-ACE2-Ang-(1-7)axis,thereby affecting the function of the RAS system.Our work suggested that the Ang Ⅱ-ACE2-Ang-(1-7)axis may be involved in the occurrence of phosgene-induced ALI.2.The ACE2 agonist DIZE can significantly inhibit phosgene-induced oxidative stress and inflammatory response in lung tissue,and improve ALI and edema.The lung protective function of DIZE may be achieved by regulating the Ang Ⅱ-ACE2-Ang-(1-7)axis.This work suggested that DIZE may be a potential salvage drug for the treatment of phosgene-induced ALI. |
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