Fasudil alleviates LPS-induced lung injury by restoring aquaporin 5 expression and inhibiting in flammation in lungs

Jingjing Wang,Hui Kong,Jian Xu,Yanli Wang,Hong Wang,Weiping Xie

Departmentof Respiratory&Critical Care Medicine,the First Af fi liated Hospital of Nanjing Medical University,Nanjing,Jiangsu 210029,China.

AbstractFasudil,a selective rho kinase(ROCK)inhibitor,has been reported to play a bene fi cial role in systeMic in flammation in acute lung injury,but its mechanisMfor ameliorating pulmonary edema and in flammation remains unclear.Using hematoxylin-and-eosin(H&E)staining,immunohistocheMistry,enzyme-linked immunosorbentassay,quantitative real time PCR and Western blotting,we found that fasudil attenuated LPS-induced lung injury,decreased lung edema,and suppressed in flammatory responses including leukocyte in fi ltration and IL-6 production.Further,fasudil upregulated LPS-induced aquaporin 5 reduction and inhibited NF-κB activation in the lungs of mice.Our results suggest that fasudil could restore the expression of aquaporin 5 to eliminate LPS-induced lung edema and prevent LPS-induced pulmonary in flammation by blocking the in flammatory pathway.Collectively,blockadeof the ROCK pathway by fasudil may be a potential strategy for the treatment of acute lung injury.

Keywords:acute lung injury,aquaporin 5,fasudil,fliud transport,NF-κB

Introduction

Acute lung injury(ALI)or acute respiratory distress syndrome(ARDS)is a leading cause of acute respiratory failure in critically ill patients.It is characterized by the disruption of the alveolar-capillary barrier which results in accumulation of protein-rich fluid in thealveolar cavity and leukocyte in fi ltration into the lung parenchyma.These pathological alterations lead to gas exchange abnormalities and hypoxemia[1].Currently,there is no effective therapy for this critical illness,although a large number of patients are diagnosed with ARDS[2].Despite improvement in supportive care,it hasa high mortality among Intensive Care Unit patients[1,3].Thus,it is an urgent need to search for proMising therapeutic concepts that can reduce lung in flammation and permeability of the alveolar-capillary barrier in ALI.

Regulation of water movement in the lungs between the vascular,alveolar,and airway compartments is pivotal for normal lung function.Aquaporins(AQPs)are a family ofintegral membrane proteins and waterselective channels that mainly promotes osmotic water transport.To date,at least 4 types of AQPs(AQP1,AQP3,AQP4,and AQP5)are expressed in the airways and lungs[4].Among these AQPs,AQP5 is the predoMinant water channel expressed in type I alveolar epithelial cells[5-7].Previous investigators have shown that AQP5 de fi ciency in mice resulted in a 10-fold reduction of osmotic water permeability of thealveolarcapillary barrier in the lungs[8]and the expression of AQP5 decreased in the model of radiation,viral infection and severe acute pancreatitis-related lung injury[9-11].In addition,AQP5 deletion aggravates Pseudomonas aeruginosa-induced ALI[12].Together,these studies suggest that AQP5 plays a pivotal role in maintaining normal water movement and inhibiting pulmonary in flammation in ALI.

ROCK signaling,which is involved in important biological functions including cell adhesion,migration and gene expression,can be activated by in flammatory stimuli such as TNF-αor/and LPS[13].Several experimental researches suggest that fasudil,a ROCK selective inhibitor,is bene fi cial to ALI induced by lipopolysaccharide(LPS),ischeMia or reperfusioninduced ALI[14-15].However,the underlying mechanisMwas obscure.In this study,we explored the effects of fasudil on theexpression of AQP5 and in flammation in the lungs of LPS-treated mice.

Materials and methods

Animal models of ALI

All experimental protocols were approved by the Institutional Animal Care and Use ComMittee of Nanjing Medical University and were in accordance with the guidelines of the National Institutes of Health.Male C57BL/6 mice(20～22 g,Changzhou Canvas Laboratory,Animal Corporation Ltd.China)were orally intubated with a sterile plastic catheter followed by anesthesia,and then challenged with LPS(5 mg/kg,E.coli055:B5;Sigma)dissolved in 50μL normal saline or saline as control.Fasudil(10 mg/kg,CHASE SUN,China)wasgiven by intraperitoneal injection 1 h before LPS adMinistration.Another group of Mice without LPS instillation were also pretreated with fasudil to serve as controls for any side effects that Might be attributed to fasudil.At 24 hours after LPS adMinistration,mice were humanely killed by exsanguination under deep anesthesia to collect samples for analysis.

Histological exaMination

To evaluate lung pathological changes of Mice,the left upper lobes were fi xed in 4%paraformaldehyde after dehydration in gradient ethanol and embedded in paraf fi n,sectioned at 3μm,and stained with H&E.Images were taken under a light microscope(Leica,Germany)by two investigators blinded to group assignment.Ten randoMfi elds of each lung section were exaMined.

Lung W/D ratio analysis

To evaluate water content in lungs,the wet weight of the right upper lobes was measured.Then,dry weight was obtained after the fresh lung lobes were desiccated in an oven at 70°C for 72 h.The lung water content was de fi ned as the wet weight divided by the dry weight,namely lung W/D ratio.

ImmunohistocheMistry

Lung sections were deparaf fi nized followed by heatinduced antigen retrieval with 0.01 mol/L citrate buffer and quenching of the endogenous peroxidase activity with 3%hydrogen peroxide.A fter 1-hour blockagewith 3%BSA,the sections were incubated with primary antibodies of AQP5(1:200,Santa Cruz Biotechnology)and CD11b(1:100,Cell Signaling Technology,Danvers,MA)overnight at 4°C and goat anti-rabbit secondary antibody(1:2,000,Proteintech)for 1 h at rooMtemperature.The reactions were induced by a DAB substrate kit and hematoxylin as counterstain.Each slide was evaluated under a light microscope(Leica).

ELISA

Concentrations ofiL-6 in the bronchoalveolar lavage fluid(BALF),seruMand lung tissues were measured with murine cytokine-speci fi c ELISA kits(R&D Systems,Minneapolis,MN,USA)according to the manufacturer's instructions.Protein contents in the BALF were assayed by bicinchoninic acid(BCA)kit(Beyotime,China).IL-6 levels in the BALF and lung tissues were normalized by corresponding protein concentrations.

Myeloperoxidase(MPO)analysis

MPO activities in the BALF and lung tissues were measured with a commercial test kit(Jiancheng Bioengineering Institute,Nanjing,China)follow ing the manufacturer's directions.Protein concentrations were also measured with a BCA assay kit.MPO activities in the BALF and lungs were normalized by corresponding protein concentrations.

Quantitative RT-PCR

Total mRNA of lung tissueswasextracted with Trizol reagent(Gibco BRL,Grand Island,NY,USA).Reverse transcription was performed with 300 ng of total RNA with SYBR®PreMix Ex TaqTM(TaKaRa,Japan).Quantitative RT-PCR was performed with Eppendorf Mastercylcer EPRealplex Real-time PCRSystem.Twostep RT-PCRwasused to perforMrelativequanti fi cation of mRNA.The RT-PCR primer sequences are listed in Table1.The2-ΔΔCtmethod wasused to quantify mRNA expression relative toβ-actin.

Protein extraction and Western blotting analysis

Frozen lung tissues were homogenized in a Protein Extraction Reagent(Thermo Scienti fi c,Rockford),containing protease and phosphatase inhibitor cocktail.Twenty mg of protein was subjected to electrophoresis on 10%SDS-PAGE gels and transferred to PVDF membranes(Millipore).The membranes were blocked in TBST containing 5%skimmed Milk for 1 h at rooMtemperature and then incubated with primary antibodies against AQP5(1:500,Santa Cruz Technology),occludin(1:1,000,Proteintech,Rosemont,IL),NF-κB p-p65/p65,p-iκκαβ/iκκαβ and ICAM-1(1:1,000,Cell Signaling Technology)and goat anti-rabbit antibody(1:10,000,Proteintech),respectively.The immunoblots were developed with an ECL(Advansta).β-actin was used as loading control for each sample.Densitometric quanti fi cation was performed using Image Laboratory software to evaluate relative density of protein expressions againstβ-actin.

Statistical analysis

For all statistical analysis,SPSS 18.0 software(SPSS Inc.,Chicago,IL,USA)was used.All parametric data were compared by one-way ANOVA analysis followed by Turkey's-b test.Valueswereexpressed asmean±SE.A value of P＜0.05 was considered statistically signi fi cant.

Results

Fasudil attenuated LPS-induced lung injury

To characterize the effects of fasudil on LPS-induced lung injury,histological analysis was performed by H&E staining.Lung sections froMthe saline group showed normal alveolar septa without pulmonary interstitial edema,while those froMthe LPS-treated Micehad septal congestion in the lung tissues.However,fasudil signi fi cantly ameliorated lung injury induced by LPS(Fig.1A).Accordingly,the W/D ratio demonstrated that fasudil predoMinantly reduced the high water content induced by LPS in the lungs of mice(Fig.1B).

Fasudil ameliorated lung in flammation caused by LPS

Immunohistochemical analysis of CD11b,a marker of myeloid leukocytes,demonstrated that LPS induced a large number of leukocytes to in fi ltrate into the alveolar septa,which was signi fi cantly suppressed by fasudil(Fig.2A).Further,fasudil blocked the mRNA transcription of Rock2(Fig.2B)and decreased MPO activities in the BALFand lung tissues(Fig.2C and D)induced by LPS.Consistently,it inhibited theexudation of neutrophilsand protein in the BALF(Fig.2E and F)caused by LPS.Moreover,fasudil suppressed LPSinduced upregulation ofiL-6 in the BALF,lung tissues as well as in peripheral blood(Fig.2G-I).

Fasudil reversed reduction of AQP5 and occludin induced by LPS

AQP5 ismainly distributed attheapical membraneof type I alveolar epithelial cells in the lungs.Asshown in Fig.3A,LPSchallenge resulted in decreaseof AQP5 in the alveolar epithelia of mice which was corrected by fasudil.To further validate this result,both quantitative and seMiquantitative analysis of AQP5 expression by qRT-PCR and Western blotting were performed.Consistent with the results froMimmunohistochemistry,fasudil signi fi cantly increased or reversed LPS-induced downregulation of Aqp5 mRNA or protein in the lung tissues of mice(Fig.3B and C).

Intercellular tight junction(TJ)occludin plays an important role in maintaining the integrity of the alveolar-capillary barrier and was examined in the study.As shown in Fig.3D,LPS challenge led to a signi fi cant downregulation of occludin in the lungs,which,however,was restored by fasudil.

Fasudil suppressed LPS-induced NF-κB activation and ICAM-1 overexpression

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Fig.1 Fasudil attenuates LPS-induced pathological damageand lung W/D ratio in Mice.A:H&E staining shows that fasudil ameliorates severe lung in flammation caused by LPSinstillation(n=6～8).B:Lung W/D ratio demonstrates that LPS leadsto high water contents in the lungs of LPS-treated mice,which is abated by fasudil pretreatment(n=8～13).*P＜0.05,Scale bar:50μm.Con:control,Fas:fasudil.

Fig.2 Fasudil Mitigates LPS-induced lung in flammation.A:Immunohistochemical analysis shows that fasudil reduces leukocyte in fi ltration induced by LPS in the lungs of mice.B:Fasudil reverses LPS-induced high transcription of Rock2 mRNA.C and D:LPS challenge results in signi fi cant increases of MPO activities in the BALF and lung tissues which are downregulated by fasudil.E:Fasudil signi fi cantly inhibits LPS-induced neutrophil in fi ltration in the BALF.F:Fasudil downregulates LPS-induced high protein concentration in the BALF.G～I:LPS-induced upregulation ofiL-6 in the BALF and lung tissues as well as in seruMare decreased by fasudil.n=6～8,*P＜0.05,Scale bar:20 μm.Con:control,Fas:fasudil.

The NF-κB pathway is important in various in flammatory diseases.In this model of lung injury,LPSinduced NF-κB pathway activation as indicated by signi fi cant phosphorylation ofiκκαβ(Fig.4A)and NFκB p-p65(Ser535)(Fig.4B)was partly or completely inhibited by fasudil.ICAM-1 is oneof the most critical molecules regulated by NF-κB signaling and involved in leukocyte in fi ltration in ALI.Our resultsshowed that LPS challenge induced signi fi cant overexpression ofiCAM-1,but it was reversed by fasudil(Fig.4C).

Fig.3 Fausil reverses LPS-induced reduction of AQP5 and occludin in the lungs of Mice.A:Immunohistochemistry shows that fasudil signi fi cantly upregulates theexpression of AQP5 induced by LPSin the lung tissuesof mice.B and C:qRT-PCR and Western blotting show that LPS challenge causessigni fi cantdownregulationsof Aqp5 mRNA(n=6～8)and protein(n=3～4),which areupregulated or reversed by fasudil.D:Fasudil restores LPS-induced downregulation of TJs occludin in the lungs of LPS-treated mice.(n=4～5,*P＜0.05)Scalebar:20μm.Con:control,Fas:fasudil.

Fig.4 Fasudil inhibits theactivation of NF-κB signal induced by LPS.A and B:Western blotting analysisshows that fasudil partially or completely suppresses the phosphorylation ofiκκαβor NF-κB p65(Ser 535).C:The result shows that LPS challenge inducesoverexpression ofiCAM-1 in the lungs of mice which is reversed by fasudil.(n=4～5,*P＜0.05)

Discussion

ALIor ARDS is characterized by hyperpermeability of the alveolar-capillary barrier and recruitment ofin flammatory cells.In this study,we demonstrated that fasudil attenuated LPS-induced lung injury by inhibiting pulmonary edema formation and in flammation.Fasudil attenuated lung pathological damage,and suppressed the upregulation of ROCKα,neutrophil in fi ltration,protein concentration,MPO activities,IL-6 aswell as NF-κB activation in the lungsof LPS-treated mice.In addition,it restored LPS-induced reduction of AQP5 and occludin in the lungs of Mice.

Excessive fluid exudation and impaired fluid clearance in the alveolar spaces contribute to pulmonary edema in the development of lung injury.Previous studies suggest that dysfunction of alveolar fluid transport leads to poor prognosis in ARDS patients[16].Conversely,augmenting the clearance of edema fluid and enhancing transepithelial sodiuMtransportcould be more effective for prevention or treatment of certain pulmonary edema[17].Fluid transport in the lungs is mainly undertaken by the active pathway of Na,Kadenosine triphosphatase(Na,K-ATPase)[18]and the passive pathway facilitated by AQPs[5].AQP5 which is expressed at the apical membrane of type I alveolar epithelia and acinar epithelia in the submucosal glands osmotically drives water transport between alveolar airspaces and capillaries[19].Downregulation of AQP5 in lung injury/edema is caused by various insults such as thoracic irradiation[9],viral infection[11]and endotoxin[12],suggesting an important role of AQP5 under these conditions.Our data showed that LPS challenge induced the increase of W/D ratio and reduction of AQP5 in the lungsof Mice.However,thiseffect of LPS on water contentand theexpression of AQP5 wasoffset by fasudil.Inhibiting ROCK by fasudil has been reported to attenuate endotoxin-induced lung injury,butthemechanisMwasnotcon fi rmed.A lthough thereis a paucity ofinformation to date regarding the relationship between ROCK and AQP5,our resultsindicate that fasudil ameliorates LPS-induced pulmonary edema through upregulating the expression of AQP5.

The integrity of epithelial barrier is also crucial in lung fluid homeostasis.TJs between the epithelia maintain the low permeability of normal epithelial cells to paracellular transport.It is a belt-like interconnected strand structure between alveolar epithelial cells and separates the apical and basolateral epithelium[20].Two of the well-characterized proteins of TJs in the pulmonary epithelia are occludin,a transmembrane protein responsible for major barrier function and zonula occludens(ZO)-1,an intracellular protein linking occludin to cytoskeletal proteins[21-22].Low concentrationsof extracellular calciuMand intracellular ATPand other biological factors including infection[23],cytokines[24]and reactive oxygen species(ROS)[25]induce dissociation/disruption of TJs.In the present study,we showed that occludin was decreased by LPS instillation in the lungs of mice,but it was reversed by fasudil.ROCK inhibitor C3 transferase and Y-27632 partially reverse the reduction of occludin induced by human immunode fi ciency virus Tat in brain microvascular endothelial cells[26],indicating that ROCK is an upstreaMsignaling of occludin.Combining these fi ndings with our results,it is reasonable that fasudil attenuates lung edema by LPS via restoring the expression of occludin.Moreover,Kawedia et.al have demonstrated that AQP5 deletion leads to downregulation of TJoccludin,suggesting that AQP5 and occludin could coordinate to facilitatewater transport[27].Thus,it is suggested that fasudil ameliorates LPS-evoked pulmonary edema by increasing the expression of AQP5 and occludin.

It is widely accepted that lung in flammation is the major characteristic of lung injury.Apart froMthe classical proin flammatory cytokines IL-6,TNF-α,γ-IFN and TGF-β,in flammasome-regulated cytokines(such as caspase-1,IL-1βand IL-18)[28],IL-17A[29]and endocan[30]are also important mediators of ALI or ARDS.All of the resident lung cells including the alveolar epithelia,vascular endothelia,alveolar macrophages,and fi broblasts as well as leukocytesparticipate in the in flammatory process[31].Among them,the alveolar epithelia can release proin flammatory mediators,produce neutrophil chemotactic factors,and increase adhesion molecules induced by bacteria or endogenous factors IL-1βand TNF-α,while maintaining water balance[32].Previousstudies have shown that ROCK inhibition by fausdil prevents early lung in flammatory response induced by intratracheal instillation of LPS in mice through suppressing production ofiL-6 and TNF-α[33].SiMilarly,our data demonstrated that pretreatment with fasudil signi fi cantly inhibited leukocyte in fi ltration,MPO activities and cytokine production(IL-6)caused by LPS,indicating that fasudil could have a potent anti-in flammatory effect on LPSinduced lung injury.

NF-κB isawell-recognized signaling in LPS-induced lung in flammation which in turn promotes the transcription of cytokines,chemokines and adhesion molecules[34].Previous studies have shown that LPS challenge-induced in flammation in thebronchial epithelia is impaired by inhibiting NF-κB activation with a constructed IκBαmutant[35].Moreover,ROCK results in signi fi cant NF-κB activation in in flamed intestinal mucosa[36].Consistently,our resultsshowed that ROCK blockade by fasudil inhibited LPS-induced phosphorylation of NF-κB p65 and iκκαβ.Thus,it could be suggested that ROCK signal participates in LPSinduced NF-κB activation.

Ithasbeen reported that AQP5 knockout led to severe bacterial blood disseMination and lung injury in a mousemodel of Pseudomonasaeruginosa infection[12].Additionally,NF-κB inhibitors block the reduction of AQP5 in LPS-induced in flammation of salivary gland[37].In our study,fasudil inhibited NF-κB activation accompanied by reduction of AQP5 induced by LPS.Combining theabove fi ndingswith our results,fasudil could attenuate LPS-induced lung in flammation by inhibiting NF-κB activation and upregulating the expression of AQP5.

In conclusion,this is the fi rst report about the effects of fasudil on the expression of AQP5 in LPS-induced ALI.Our study suggests that fasudil attenuated LPSinduced pulmonary edema via upregulating the expression of AQP5 and TJ protein occludin.Moreover,fasudil ameliorated LPS challenged-lung in flammation by inactivating NF-κB and increasing AQP5.However,the exact mechanisMby which fasudil acts on the expression of AQP5 and theother effectsof thisprotein in ALIneeds to be further investigated.

Acknow ledgements

This study was supported by the National Natural Science Foundation of China(No.81273571),Jiangsu Clinical Research Center for Respiratory Diseases Project under grant No.BL2012012,and a Project Funded by the Priority AcadeMic PrograMDevelopment of Jiangsu Higher Education Institutions(PAPD)(No.JX10231802).