Simvastatin-induced Attenuation Of The Lung Ischemia Reperfusion Injury And The Underlying Mechanism

lung ischemia-reperfusion injury （LIRI） is one of the major clinicalcomplication problem in thoracic and cardiovascular surgery, usually lead tosignificant morbidity and mortality. With lung transplantation, sleeve resection,cardiopulmonary bypass and other technology widely available, the postoperativepulmonary dysfunction of LIRI has caused widespread concern of Thoracic Surgeons.Accumulating evidence has indicated that ATII cells are the progenitors of ATI cellsin the alveoli, ATII cells are believed to play a pivotal role in maintaining tissuehomeostasis via epithelium restoration.The lung alveolar epithelium covers99%of the internal surface area of the lungand is composed of two morphologically and functionally distinct types of cells:alveolar type I (ATI) and type II (ATII). ATI cells are broad flat cells which cover95%of the alveolar surface and comprise40%of the alveolar epithelium and8%ofthe peripheral lung cells. However, ATII cells are small, cuboidal cells which cover5%of the alveolar surface and comprise60%of the alveolar epithelium and15%ofthe peripheral lung cells. They are characterized by the unique ability to secretesurfactant protein-C (SP-C) and by a distinct cell organ, the lamellar bodies (LBs).They can regulate alveolar fluid levels, host defense and immune response. Moreimportantly, increasing studies indicated that ATII cells are progenitor cells for ATIcells in the alveoli, which plays a pivotal role in maintaining tissue homeostasis viaepithelium restoration. Once ATI cells were damaged after lung injury, ATII cells canproliferate and transdifferentiate into ATI cells and thereby repair lung epithelium. Statins are a well-established class of drugs that effective decrease serumcholesterol levels by inhibiting the action of3-hydroxy-3-methylglutaryl coenzyme Areductase. In addition to its cholesterol-lowering activity, simvastatin which is a kindof statins also has pleiotropic properties, such as, separated from cholesterol lowering,including anti-inflammatory, angiogenic effects and its capacity to improve vascularendothelial cell functions.Recently, it has been shown that simvastatin protectsagainst ischemia-reperfusion injury, such as heart, brain, and etc. However, the effectsof simvastatin on lung ischemia-reperfusion injury are unknown.In terms of lung injury, Naidu et al first demonstrated that simvastatin amelioratedlung ischemia-reperfusion injury by modulating the endothelial nitric oxide synthase.Müller and coworkers reported that high dose simvastatin can attenuateventilator-induced injury by reducing pulmonary inflammation and hyperpermeabilityin mice. However, the protective effects of statins on the lung injury are stillcontroversial. Some clinical trials found that statins had no influence on the progressof lung injury and others reported that statins did improve organ dysfunction in acutelung injury and decrease the lipopolysaccharide-induced pulmonary inflammation inhealthy volunteers. Therefore, it is still worth to further elucidated whether statinsreally attenuates acute lung injury and in which what kind of mechanisms areinvolved. In the present study, we provided new evidences that the function of ATIIcells is restored by simvastatin both in vitro and in vivo, investigate the protectiveeffects of simvastatin on ATII and the underlying mechanisms of the simvastatin.Part I: Simvastatin Attenuates Lung Ischemia-Reperfusion Injury and theUnderlying MechanismsBackground: Alveolar type (AT) II cells, characterized by the presence of lamellarbodies (LBs) and surfactant protein-C (SP-C) secretion, can transdifferentiate intoATI cells and as such represent a promising source for regenerating lung epitheliumfollowing lung injury. Lung ischemia-reperfusion injury (LIRI) causes a distinct impairment of ATII cell function, subsequently hindering lung repair by loss of ATItransdifferentiation. In this study, we provide new evidence that the HMG-CoAreductase inhibitor simvastatin can restore function of impaired ATII cells in vitro andin vivo.Methods: ATII cell lines, A549(human) and MLE-12(mouse), were subjected tohypoxia reoxygenation (H/R) injury. Blank, control, variant doses simvastatin-treatedgroups (5,10,20,30,50,100μmol/L), L-mevalonate group, simvastatin+L-mevalonate group, simvastatin+Wortmannin group and Wortmannin group weredesigned in the present study. Different doses of simvastatin (5-100μmol/L) weregiven before H/R injury. A hypoxia condition was created by incubating cells in ahypoxia chamber with an atmosphere of95%N2/5%CO2at37℃for2h in each group.Different items were dectected at0h,2h,4h,6h,12h and24h after reoxygenation.The proliferation of ATII cells was evaluated by Cell Counting Kit-8(CCK-8) assay.The percentage of apoptotic cells was assessed by flow cytometry AV/PIdouble-staining. The protein levels of SP-C, PCNA, Akt, P70, mTOR, Caspase-3inATII cells were measured by Western blot. Rats were subjected to LIRI by left hilarclamping for60mins and were randomized into sham, IR and two simvastatin-treatedgroups (orally treated with0.5and5mg/kg/d, respectively).The lung tissue werecollected at baseline1h after ischemia,4h,1day,3days and7days after reperfusionrespectively. Lung injury was evaluated by hematoxylin and eosin (HE) staining andmyeloperoxidase (MPO) activity assay. The status of ATII cells’ proliferationdetermined by SP-C/PCNA immunofluorensence doubles staining. Ultrastructure andstereological analysis was used to quantify the alterations of lamellar bodies (LBs)and profiles of ATII cells, it contained Vv-lb、Sv-lb、Rsv-lb and Nv-lb. Western blotwas used to measure the protein levels of phosphorylated-Akt, phosphorylated-P70,caspase-3, and surfactant protein-C at4h and three days after reperfusion in the lungtissue and the surfactant protein C (SP-C) levels of bronchoalveolar lavage (BAL)fluid.Results: Simvastatin pretreatment at low (5-20μmol/L), but not high (50-100μmol/L) dose markedly reduced apoptosis and increased proliferation and expression of SP-C、PCNA、p-Akt、p-P70and mTOR protein levels. Administration of L-meva(20μmol/L) significantly blocked the SP-C-increasing effect of simvastatin byrescuing simvastatin-induced mevalonate depletion in MLE-12and A549treated cells,as compared to the simvastatin only treated groups at24h after reoxygenation.Treatment with Wort (100nmol/L) in combination with simvastatin reversed thesimvastatin-induced improvement of the percentage of SP-C-positive cells and theSP-C protein levels at24h after reoxygenation in both cell lines. SP-C/PCNAdouble-positive cell numbers in the LSIM and HSIM groups were found to besignificantly increased at4h, and even more increased (by>2-fold) at three days afterreperfusion, as compared with the I/R group. The nuclei and LBs of ATII cells werefound to be intact in the sham group at4h and three days after reperfusion. ApoptoticATII cells were also present at4h after reperfusion. The results of quantitativecomparison by stereological analysis are shown that at4h and3days after reperfusion,as compared with the I/R group, both the Vv-LB and Nv-LB were significantly higherin LSIM and HSIM groups. The Rsv-LB levels were remarkably lower in LSIM andHSIM groups. At4h after reperfusion, p-Akt levels in the LSIM and HSIM groupswere significantly higher than in the I/R group. Subsequently, the downstream p-P70level was also found to be remarkably increased. At three days after reperfusion,p-Akt levels further increased in the LSIM and HSIM groups, as did the downstreamp-P70levels. There appeared to be no difference in the caspase-3level between eachgroup; however, the SP-C levels in the LSIM and HSIM groups increased remarkably.In both LSIM and HSIM groups, SP-C protein levels in BAL fluid significantlyincreased at4h and3days after reperfusion as compared with the I/R groups.Conclusions: These data demonstrated that an appropriate dose of simvastatin has aprotective effect on LIRI in vitro and in vivo, due at least partially to restored ATIIcell function via the HMG-CoA reductase pathway-dependent activation of PI3K/Aktsignaling in a mevalonate pathway dependent manner.Part II: Simvastatin promotes the Transdifferentiation of Type II AlveolarEpithelial Cells and the Underlying Mechanisms Background: Accumulating evidence reveals that statins possess pleiotropicproperties beyond cholesterol reduction, which may contribute to the attenuation oflung ischemia-reperfusion injury (LIRI). And it may play an importmant role in thetransdifferentiation of Type II Alveolar Epithelial Cells. This study was designed toprove the hypothesis that in the ATII cells hypoxia and reoxygenation injury model,simvastatin can promotes the ATII cells transdifferentiation into ATI cells in vitro andin vivo.Methods: MLE-12(mouse ATII cell line), were subjected to hypoxia reoxygenation(H/R) injury. An appropriate dose of simvastatin (20μmol/L) was given before H/Rinjury. The percentage of Caveolin-1/Pro-SP-C expression in the different points wasassessed by flow cytometry double-staining. The Pro-SP-C and Caveolin-1expression of ATII cells was evaluated by immunofluorescence double staining. Theprotein levels of pro-surfactant protein-C (Pro-SP-C)、Caveolin-1、TGF-β1andSmad-4in ATII cells was determined by Western blot. Rats were subjected to LIRI byleft hilar clamping for60mins and were randomized into sham, IR andsimvastatin-treated group (orally treated with0.5mg/kg/d). Ultrastructure analysiswas used to quantify the alterations of nuclei and lamellar bodies (LBs) of ATII cellsand the ATII transdifferentiatied into ATI.Results: As compared with the sham group, the percentage of Caveolin-1/Pro-SP-Cexpression in Sim20group significantly decreased at the early phase（0d and1d） ofLIRI and gradually increased in three days and7days after reperfusion.Pro-SP-C/Caveolin-1double positive cell number significantly increased at1d,peaked at3d and decreased to normal until7days after reperfusion. The expressionof Pro-SP-C increased at0d, peaked at1d and gradually decreased in3d and7d. Inthe contrast, the expression of Caveolin-1increased at0d, peaked at7d and graduallydecreased in1d and3d. Compared with the Sim20group, after added inSB431542(an inhibitor of TGF-β/Smad signaling pathway), the expression ofPro-SP-C and Caveolin-1in the Sim20+SB group have no difference. As comparedwith Sim20group, the expression of TGF-β1and Smad-4in the SB and Sim20+SBgroup was significantly decreased. Compared with the Sim20group, after added in L-mevalonate (an inhibitor of HMG-COA), the expression of Pro-SP-C andCaveolin-1in the Sim20+L-meva group have no difference. Ultrastructure analysis ofATII cells showed that at3d, the LBs were remarkably decreased, nucleus wasstretched, microvilli was disappeared, pinocytotic vesicles was increased. When at7days after reperfusion, the LBs showing emptying phenomenon, microvilli wasdisappeared, organelles significantly reduced, and began to appear contains a lot ofpinocytotic vesicles thin squamous ATI cells.Conclusion: Our results demonstrated that simvastatin may promotes thetransdifferentiation of type II alveolar epithelial cells in vitro and in vivo, possiblythrough the activation of TGF-β/Smad signaling pathways, and the mechanisms is notin a L-mevalonate pathway-dependent manner.