Protective Effects And Mechanism Of Fasudil On Acute Lung Injury In Mice

Acute lung injury(ALI)/acute respiratory distress syndrome(ARDS)is characterized by overproduction and malreabsorption of alveolar edema fluid,infiltration of massive leukocytes into lung interstitium or parenchyma,and release of pro-inflammatory mediators.These pathological alterations,which cause deficiency of gas and blood exchange and decreased oxygenation,result in respiratory failure.It caused by multiple insults including severe pneumonia,aspiration of gastric contents,sepsis,burns and trauma.In the USA,ARDS leads to 40%mortality annually in approximately 200,000 Intensive Care Units(ICU)patients with a high incidence and serious clinical presentations,posing a threat to public health.During the past three decades,although substantial basic researches and clinical trials have proved that certain drugs or stem cell therapy could attenuate lung injury in animal models or populations,to date Food and Drug Administration(FDA)has not yet approved any effective treatments for ARDS except formechanical ventilation and supportive care.Thus,it is an urgent need to explore the molecular targets associated with the development of ALI or ARDS,and find out the potential prevention and treatment for ALI/ARDS.Rho kinase(ROCK)is an effector of small GTP-binding protein Rho.Studies have shown that Rho/ROCK signaling plays an important role in various types of cells,such as smooth muscle cell contraction,actin cytoskeleton rearrangement,cell adhesion and motility,inflammatory cells migration,cytokinesis,and gene expression.ROCK involves in the pathogenesis of stroke,vasospasm,arteriosclerosis,hypertension,heart failure,pulmonary hypertension,and ischemia reperfusion injury.Studies on respiratory diseases have shown that ROCK inhibitors was effective for pulmonary hypertension,bronchial asthma and pulmonary fibrosis.Moreover,several experimental researches have shown that ROCK inhibition alleviated airway inflammation and lung injury.But the mechanism of ROCK inhibitor for preventing lung injury is unclear.Since pulmonary microvascular endothelial cell is one of the most important components of the alveolar-capillary barrier,it occupies a critical position in lung edema and inflammation.ROCK,highly expressed in PMECs is related to inflammatory response.Therefore,by using the well-recognized mice model of ALI,we observed whether the selective ROCK inhibitor fasudil could attenuate lung histological damage,pulmonary inflammation,lung hyperpermeability,and endothelia apotosis induced by LPS.Moreover,by establishing neutrophil-endothelial co-culture system and inducing human pulmonary microvascular endothelial cells(HPMECs)inflammatory responses,endothelial barrier disruption,and endothelial apoptosis with LPS in vitro,we explored the cellular and molecular mechanisms for beneficial effects of fasudil on ALI.Part I the Protective Effects of Fasudil on Acute Lung Injury in MiceObjective:To observe the effects of fasudil on ROCK activities,pathological damage,neutrophils infiltration,cytokines release,pulmonary permeability,lung water transport,and pulmonary endothelia injury/apoptosis induced by LPS in lung tissues of mice.Methods:Mice were challenged with intra-tracheal instillation of LPS(5mg/kg)with or without intra-peritoneal injection of fasudil(10 mg/kg)1 hour before LPS administration.Twenty four hours after LPS challenge,mice were killed for sample collection including blood,lung tissues and bronchoalveolar lavage fluid(BALF).Hematoxylin-and-eosin(HE)staining was used to observe the pathological damage in lung of mice;The levels of IL-6,TNF-cain serum or in lungs were measured by enzyme-linked immunosorbent assay(ELISA);Immunohistochemistry(IHC)was used to observe the neutrophils infiltration;Myeloperoxidase(MPO)activities and contents of evans blue dye(EB)were measured by a microplate reader.Protein concentrations in BALF were determined by BCA assay;Western blotting was used to measure ROCK activities and the expression of aquaporin 5(AQP5)in lung tissues;Pulmonary endothelial cells apoptosis/injury was determined by immunofluorescence double staining.Results:Firsly,LPS challenge induced ROCK activation was inhibited by fasudil;Fasudil pretreatment significantly alleviated LPS-induced lung injury,inhibited neutrophil infiltration,and down-regulated MPO activities as well as neutrophils in BALF.LPS-induced overproductions of IL-6 and TNF-a in serum and lung tissues were suppressed by fasudil;Secondly,fasdil inhibited LPS-induced protein and EB exudation in lung parenchymal andattenuated lung endothelial cells apoptosis/damage;Finally,fasudilreversed LPS-induced down-regulation of AQP5.Conclusion:In the invivostudy,fasudil prevented LPS-induced lung injury by inhibiting the infiltration of inflammatory cells,suppressing the release of pro-inflammatory mediators,decreasing pulmonary permeability and reducing lung endothelia apoptosis as well aspromoting lung water transport.Part ? the:Mechanisms for Beneficial Effects of Fasudil on LPS-induced ALIObjective:In vitro,by establishing neutrophil-endothelial co-culture system and inducing PMECs inflammation,barrier dysfunctionand endothelial apoptosis with LPS,we explore the potential cellular and molecular mechanisms for protective effects of fasudil on ALI.Methods:Primary HPMECs between passage 4 to 6 were treated as following:control group,1%ECM;fasudil group:1%ECM(containing 1 ?g/mlfasudil);LPS group:1%ECM(containing 1 ?g/mlLPS);LPS+fasudil group(containing 1?g/mlLPSand 1?g/ml[or 0.04 ?g/ml,0.2 ?g/ml,1 ?g/ml]fasudil).HPMECs transfected with lentiviral(LV)-BMPRIIand LV-BMPRII-siRNA were also treated with LPS and fasudil.All cells were pretreated with fasudil 0.5 h prior to LPS exposure and further cultured for indicated time intervals based on specific methods and aims,then the cell samples and the suernatants were collected for measurements.Neutrophil chemotaxis and adhesion to HPMECs were examined by a co-culture system;The transcriptions of CXCL1,CXCL2,CXCL8 was detected byquantitative real time PCR(qRT-PCR)and production of CXCL8 in supernatant was measured by ELISA.Western blottinganalysisexaminedthe expression of intercellular adhesion molecule(ICAM)-1and bone morphogenic protein receptor(BMPRII),the activation of ROCK,NF-?Band MAPK cell signalings,and the expression of vascular endothelial(VE)-cadherin and zonula occludens(ZO)-1 as well as the activation of proapoptosisproteins;HPMECs permeability to dextran was determined by a fluorescence microplate;Immunofluorescence was used to observethe distribution of VE-cadherin,ZO-land nuclear translocation of p65.Hoechest33342 staining was used to detect cell apoptosis.Results:Firstly,Fasudil dose-dependentlyinhibitedthe transcriptions of CXCL1,CXCL2,CXCL8,reduced CXCL8 production induced by LPS,and suppressed neutrophil chemotaxis mediated by activated PMECs.Secondly,fasudil inhibited neutrophil-endothelial adhesion by decreasing the expression of ICAM-1 through up-regulating BMPRII.Fasudil also blocked the activation of ROCK,NF-?Bp65 and p38 MAPK cell signalings.Thirdly,faudil restored the down-regulation of VE-cadherin and ZO-1,and reduced the permeability of HPMECs.Additionally,fasudil suppressed the cleavage of caspase3 and reversed HPMECs apoptosis.Conclusion:Fasudil attenuates LPS-induced ALI of mice through preventing HPMECs dysfunction.Fasudil protects LPS-challenged HPMECs against LPSvia the following machanisms:a)inhibiting neutrophil chemotaxis by decreasing the production of CXCL1,CXCL2 and CXCL8;b)suppressing neutrophil-endothelial adhesion by down-regulating the expression of ICAM-1;c)decreasing HPMECs monolayer permeability by restoring the tight junctions and adherens junctions between cells;d)reducing HPMECs apoptosis via suppressing the cleavage of pro-caspase3.