Mechanisms Of Resolvin D1 Attenuates Mechanical Stretch-induced Pulmonary Fibrosis

Part One Resolvin D1 attenuates mechanical-stretch induced epithelial mesenchymal transition(EMT)through inhibiting Smad2/3 phosphorylationObjective: Mechanical ventilation induced pulmonary fibrosis plays an important role in high mortality rate of acute respiratory distress syndrome(ARDS).Resolvin D1(Rv D1)displays potent pro-resolving activity.This study aimed to investigate the potential mechanisms whether Rv D1 could attenuate mechanical-stretch induced EMT in vitro.Methods: In vitro,EMT was induced by mechanical stretch in human lung epithelial cells(BEAS-2B).BEAS-2B cells were exposed to HCl(40 m M)at a p H of 4.0 in serum-free DMEM/F12.Control cells were incubated with the same amount of PBS in serum-free DMEM/F12.After 10 min of HCl treatment,cells were then subjected to cyclic stretch for 48 hours at a frequency of 20 cycles/min and 20% elongation using the FX-5000 loading system.Four groups were divided:(1)control group(2)HCl group(3)PBS +stretch group(4)HCl+stretch group.The morphologic changes of BEAS-2B cells was observed by phase contrast microscopy.The changes of EMT proteins were gauged by western blot.Different concentrations of Rv D1 and BOC-2 were treated with BEAS-2B,and the expression of EMT-related markers were evaluated by western blot and immunofluorescence.Then,BEAS-2B cells were treated with ALK-5 inhibitor A8301.The activation of Smad2/3 and Slug were observed by western blot and TGF-?1 was detected by ELISA.Results: 1.BEAS-2B cells treated with mechanical stretch after HCl priming changed in morphology from oval,polar and closely connected to long spindular with increased intercellular space.Exposure to mechanical stretch and HCl resulted in lower protein expression of E-Cadherinand higher expression of vimentin and ?-SMA.2.Low concentration of Rv D1 had no effect on BEAS-2B cells.The medium concentration of Rv D1 significantly inhibited EMT.The high concentration of Rv D1 did not inhibit EMT significantly.3.BOC-2 reversed the effect of Rv D1 on mechanical stretch-induced EMT.4.Smad2/3 phosphorylation and Slug activation were significantly suppressed,and the levels of TGF-?1 were decreased after treatment with ALK-5 inhibitor A8301.Conclusion: Resolvin D1 attenuates mechanical-stretch induced EMT through inhibiting Smad2/3 phosphorylation.Part two Resolvin D1 attenuates mechanical ventilation induced pulmonary fibrosis in mice via EMTObjective: EMT plays an important role in the process of pulmonary fibrosis.This part aims to uncover the underlying mechanisms whether resolvin D1 could inhibit mechanical ventilation-induced pulmonary fibrosis in vivo.Methods: 24 male C57 mice,6-8 weeks old,23-23 g body weight,were used in this experiment.Mice were randomly allocated to 4 groups(n=6).The animals were subjected to intratracheal instillation of HCl(p H 1.2,2 ml/kg),Equal volumes PBS served as control.After 24 hours,the mice were anesthetized,orotracheally intubated,and ventilated for 2 hours with peak inspiratory pressure(PIP)of 22 cm H2 O,PEEP of 2 cm H2 O,respiratory rate(RR)of 120 breaths per minute.The animals were then extubated observed for 14 days.Mice were randomly divided into four groups:(1)control group(2)HCl group(3)ventilation group(MV)(4)HCl + ventilation(MV)group.Hematoxylin-eosin and Masson's trichrome staining were performed to observe the changes of lung tissues and evaluate the severity of lung fibrosis.Fibrosis was also quantified by the determination of collagen-1,hydroxyproline in lung tissues and TGF-?1 levels in BALF.E-Cadherin,vimentin and ?-SMA expression was confirmed by western blot.Different doses of Rv D1 and BOC-2 were injected into mice,and the expression of EMT-related markers were evaluated by western blot and immunofluorescence.The activation of Smad2/3 and Slug were observed by western blot after ALK-5 inhibitor A8301 treated with mice.Results: 1.Mice were subjected to mechanical ventilation for 2 hours following intratracheal instillation of HCl.Lung tissues were analyzed by HE staining and Masson's trichrome staining.The classical histological presentation of lung fibrosis was more evident in the HCl+MV group with larger areas of collagen deposition in the alveolar epithelium than in the control group.Lung fibrosis scores,collagen-1 levels,hydroxyproline levels in the lungs and TGF-?1 in BALF significantly increased in the HCl+MV group.In addition,the epithelial marker E-Cadherinwas downregulated,and vimentin was upregulated in the MV+HCl group.However,?-SMA was not changed in the MV+HCl group.2.Different doses of Rv D1 can reduce the degree of pulmonary fibrosis.Tissue content of hydroxyproline was not significantly decreased,and protein level of E-Cadherinwas not significantly increased in both Rv D1-0.01?g group and Rv D1-1?g group. 3.BOC-2 reversed the effect of Rv D1 in mechanical ventilation-induced lung fibrosis.4.Pulmonary fibrosis was attenuated after treatment with ALK-5 inhibitor A8301,and the phosphorylation levels of Smad2/3 and Slug were down-regulated.Conclusion: Rv D1 attenuates mechanical ventilation induced pulmonary fibrosis in mice via EMT.