| Background:Acute lung injury/acute respiratory distress syndrome(ALI/ ARDS)is the most common clinical acute critical illness with high morbidity and mortality.Even though some patients survived from the early acute inflammatory storm,pulmonary fibrosis occurred after discharge or accompanied with the inflammatory reaction in the early stage,which seriously affected patients' lung function and long-term survival rate.ALI is an early pathological change of ARDS.The specific mechanism of pulmonary fibrosis after ALI/ARDS is unclear.Still,the therapeutic effect of immunosuppressive drugs such as glucocorticoids is ineffective,suggesting that non-inflammatory mechanisms are involved.At present,oxidative stress is considered one of the main mechanisms underlying organ fibrosis development.There is a balance between the generation of oxygen free radicals with the organism's antioxidant capacity under physiological conditions.Under stress state,erythrocyte-derived nuclear factor E2-related factor 2(Nrf2)-antioxidant response element(ARE)signaling pathway(a principal sensor and regulator of the body's oxidative stress response)is activated,regulating the expression of hundreds of antioxidant and detoxification genes and improving the state of redox imbalance in vivo.But if the organism suffers from strong stimulation for a short time or chronic stimulation for a long time,which exceeded the metabolic ability,the antioxidant response capacity is insufficient,and tissue cells will suffer from oxidative stress damage.Lung tissue is more vulnerable to various oxidative stress due to the special innate structure.Exogenous oxidants,pollutants,inflammatory irritants(such as an endotoxin component of the bacterial cytoderm-lipopolysaccharide,LPS),could promote the production of oxygen free radicals,induce oxidative stress reaction,and finally damage lung epithelial cells.Repeated injury and repair failure of epithelial cells eventually lead to pulmonary fibrosis.Therefore,looking for antioxidants which may up-regulate Nrf2 and improve oxidative stress response is a possible target for the clinical treatment of pulmonary fibrosis.Melatonin is a natural antioxidant in organism,which has various biological functions,such as regulating inflammatory response,oxidative stress,and alleviating cell damage.It has been reported that melatonin has a protective effect on pulmonary fibrosis,the specific molecular mechanism is still unknown.On the other hand,the most cause of ALI is sepsis in actual clinical scenarios,although most of the current animal experimental studies were based on bleomycin-induced model to explore the mechanism and possible intervention targets of fibrosis after ALI.Currently,there has been no study focus on their related mechanisms.ObjectiveIn this study,we investigated the molecular mechanism of melatonin improving pulmonary fibrosis after ALI by analysing the correlation between the clinical data and related indicators of ARDS patients,demonstrating the effection of melatonin on animal model of pulmonary fibrosis after ALI induced by LPS,and its underlying mechanisms on cell experiments in vitro.Our study provides a theoretical basis for seeking a new therapeutic target and elucidating the underlying molecular mechanism of pulmonary fibrosis after ALI.Materials and MethodsThe study included three parts:(1)Clinical observation study: a total of 29 patients with a diagnosis of ARDS admitted to our hospital,and 24 mechanically ventilated control patients were recruited.BALF(Bronchoalveolar lavage fluid)and clinical data,such as the expression of inflammatory factors,oxidative stress indicators and fibroproliferative markers in each group were collected.By analyzing the correlation between patients' clinical data,oxidative stress response,and fibroproliferative markers,we demonstrated oxidative stress response is involved in lung fibroproliferative response after ARDS.(2)Animal experimental study: The mouse model(C57B6/L,6-8 weeks)of pulmonary fibrosis after ALI was established by using LPS three hits to observe the lung tissue pathology,oxidative stress indicators(MDA,SOD),Nrf2 signaling pathways(Nrf2,Heme oxygenase-1(HO-1)),the levers of fibrosis-related indicators(Hydroxyproline,Ecadherin,?-SMA),and their changes after melatonin intervention.We found that melatonin ameliorates pulmonary fibrosis after ALI via activating the Nrf2 signaling pathway and inhibiting oxidative stress response.(3)The cell experiment further has been investigated the underlying mechanisms: Human type II alveolar epithelial cell-derived A549 cells were incubated with LPS and melatonin alone or in combination for up to 24 h.The morphological changes of the treated cells were evaluated as well as indexes of oxidative stress.EMT-related proteins and the Nrf2 signaling pathway were detected by western blot analysis and immunofluorescence staining,respectively.To further investigate the underlying mechanisms,the effects of melatonin on cells transfected Nrf2 short hairpin RNA(sh RNA)and the PI3 K / GSK-3? signaling pathway were evaluated.Results(1)The patients with ARDS suffered double hits which come from inflammatory response and oxidative stress injury,and fibroproliferative response were present in the lung in the early stage.The level of NT-PCP-III in BALF was significantly increased within 24 h of admission and day 7,and higher in the non-surviving group than in the surviving group.The level of NT-PCP-III in BALF was correlated with the oxidative stress indicators(MDA and SOD)at either time point.Oxidative stress is involved in the early pulmonary fibroproliferative response in ARDS.(2)Successfully constructed an animal model of pulmonary fibrosis after LPS-induced ALI in mice.After LPS treatment for 1 week,the collagen deposition and the level of hydroxyproline in the lung tissue gradually were increased.Masson staining also showed that pulmonary fibrosis is mostly obvious at 4 weeks,accompanied by oxidative stress injury and EMT process in the lung tissue.While in the melatonin group,melatonin activated the Nrf2 signal pathway,up-regulated the expressions of Nrf2 and HO-1,decreased the level of oxidative stress in the lung,inhibited the EMT process,and finally alleviated the pulmonary fibrosis induced by LPS in mice.(3)Melatonin upregulated Nrf2 expression,inhibited LPS-induced cell morphological change,reversed the expressions of EMT-related proteins,and reduced ROS production in A549 cells,as well as the levels of MDA and anti-oxidative enzymes.Yet,the effects of melatonin were almost completely abolished in cells transfected Nrf2 sh RNA.Furthermore,the data demonstrated that melatonin could activate the PI3K/AKT signaling pathway,resulting in phosphorylation of GSK-3?(Ser9)and upregulation of the Nrf2 protein in A549 cells,which ultimately attenuated LPS-induced EMT.ConclusionsOxidative stress response is involved in pulmonary fibrosis after ARDS and closely related to the prognosis of patients.Melatonin ameliorates pulmonary fibrosis after inflammation by inhibiting the oxidative stress response and EMT occurrence in lung tissue cells through activating the Nrf2 signaling pathway.Melatonin may be one of the clinical interventions for pulmonary fibrosis after ARDS. |
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