Effects And Mechanisms Of Simvastatin Pretreatment On Lung Injury Induced By Ischemia-reperfusion Of Hind Limbs In Rats

IntroductionIschemia of the extremities is an important and common clinical event,such as aortic aneurysm surgery,embolism or injury of extremity large blood vessels,extremity mutilation of superior position,serious extremity crush injury,replantation of extremity, long-time application of tourniquet in extremity operation and etc.Restoration of blood flow is the key point for survival of ischemic extremity.Ischemia-reperfusion injury of the extremities is not only occurrence in ischemic tissue,but also results in remote organ injury,even multiple organ dysfunction syndromes.Serious pulmonary dysfunction after ischemia-reperfusion injury of the extremities is still a major cause of morbidity and mortality,characterized by acute respiratory distress syndrome.The mechanisms of lung injury following extremity ischemia-reperfusion are unknown at present.Statins are a well-established class of drugs that effectively decrease serum cholesterol levels by inhibiting the action of 3-hydroxy-3-methylglutaryl coenzyme A reductase.In addition to its cholesterol-lowering activity,simvastatin which is a kind of statins also has pleiotropic properties.Recently,it has been shown that simvastatin protects against ischemia-reperfusion injury,such as heart,brain and etc.However,the effects of simvastatin on lung injury induced by ischemia-reperfusion of extremity are unknown.In view of the previous reasons,the purpose of this experimental study was to examine the effectiveness of pretreatment with simvastatin in preventing lung injury induced by ischemia-reperfusion of the hind limbs in a rat model and,in the light of the results,to discuss its possible mechanisms of action.This experiment was divided into three parts.Part 1:effect of simvastatin pretreatment on lung injury induced by ischemia-reperfusion of rat hind limbs;Part 2:effect of simvastatin pretreatment on oxygen free radicals and metabolism of nitric oxide in lung injury induced by ischemia-reperfusion of rat hind limbs;Part 3:effect of simvastatin pretreatment on heme oxygenase-1 in lung injury induced by ischemia-reperfusion of rat hind limbs.Materials and methods1.Materials(1)Experimental animalsOne hundred and forty-four male Sprague-Dawley rats each weighing 250-300g were purchased from the Experimental Animal Centre of China Medical University.(2)Experimental instrumentsPCR amplification device(Biometra,Germany),DYY-6B electrophoresis apparatus(Beijing,China),AE-240 electronic balance(Mettler,Japan),722 spectrophotometer(Shanghai,China),UV-300 ultraviolet spectrophotometer(Unicam, USA),3K15 high speed centrifuger(Sigma,Germany),OT-701 infusion pump(JMS, Germany),Gem Premier-3000 blood gas analyzer(IL,USA),ES-1000 SPM ultrasound blood flow detector(Hayashi Denki,Japan),gel imaging analysis system(Kodak, USA),BX-41 microphotograph system(Olympus,Japan),Scion Image analysis software(Apple,USA).(3)Experimental agents and drugsSimvastatin(Merck Sharp & Dohme,UK);NO kit,NOS kit,MDA kit,SOD kit, MPO kit and Coomassie brilliant blue kit(Nanjing Jiancheng Bioengineering Institute, China);Trizol kit(Invitrogen,USA);DNA Marker,RT-PCR kit and Primer synthesis (TaKaRa,Japan);Goat polyclonal anti-heme oxygenase-1(HO-1)antibody,Rabbit polyclonal anti-inducible nitric oxide synthase(iNOS)antibody and Rabbit polyclonal anti-endothelial nitric oxide synthase(eNOS)antibody(Santa Cruz Biotechnology, USA);Alkaline phosphatase conjugated rabbit anti-goat IgG and goat anti-rabbit IgG antibody(Chemicon,USA).2.Methods(1)Experiment 1Forty-eight male Sprague-Dawley rats were randomly divided into 6 groups(n=8): (1)Sham operation group(group C):bilateral femoral artery were exposed but not occluded;(2)Ischemia-reperfusion group(group IR):bilateral femoral artery were occluded for 2h with atraumatic arterial clamps and the released for 3h reperfusion;(3) Simvastatin groups(group S₁,group S₅ and group S₁₀):rats were pretreated with 1mg or 5mg or 10 mg·kg^-1·day^-1simvastatin in 1ml of distilled water for 3 days via an orogastric tube,and the last dose was given 2 h before the experiment,then other underwent the same procedure as IR group;(4)Simvastatin control group(group S): rats were pretreated with 10 mg·kg^-1·day^-1simvastatin in 1ml of distilled water for 3 days via an orogastric tube,and the last dose was given 2 h before the experiment,then other underwent the same procedure as group C.In group C and group IR,the rats received same amount of distilled water instead of simvastatin.At the end of reperfusion period,rats were killed by exsanguinations after 1ml of blood through carotid artery was taked for blood gas analysis.Then,lung samples were collected immediately for pathologic examination and measurements of Wet weigh/dry weigh (W/D),myeloperoxidase(MPO)activity and polymorphonuclear neutrophils(PMN) counting between pulmonary alveoli and blood capillary.(2)Experiment 2Forty-eight male Sprague-Dawley rats were randomly allocated,and methods of the grouping and model building were the same as Experiment 1.At the end of reperfusion period,lung samples were collected immediately after death by exsanguinations for measurements of superoxide dismutase(SOD)activity,nitric oxide synthase(NOS)activity,malondialdehyde(MDA)content and nitric oxide(NO) content.Meanwhile,the lungs were also assessed for expression of eNOS mRNA and protein or iNOS mRNA and protein using RT-PCR and Western blot analysis respectively.(3)Experiment 3Forty-eight male Sprague-Dawley rats were randomly allocated,and methods of the grouping and model building were the same as Experiment 1.At the end of reperfusion period,lung samples were collected immediately after death by exsanguinations for measurements of expression of HO-1 mRNA and HO-1 protein using RT-PCR and Western blot analysis respectively.Results1.Experiment 1The light microscopic findings in group C and group S revealed normal morphology of the lung tissues.In contrast,the lung tissues in group IR showed marked destruction with severe alveolar edema,thickening of the alveolar wall, capillary dilation,localized atelectasis and a large amount of PMN infiltration.These changes were significantly less prominent in the rats which received simvastatin,and the morphology in group S₁₀was almost close to the normal lung tissue.PO₂ and PCO₂ were significantly attenuated by group IR as compared with group C(P＜0.01).PO₂ and PCO₂ were significantly higher in the three simvastatin groups than in group IR(P＜0.01).PH changes between all the groups did not have statistical differences.PMN counting,W/D and MPO activity in the lung were significantly increased by group IR as compared with group C(P＜0.01).PMN counting,W/D and MPO activity in the lung were significantly attenuated by simvastatin pretreatment in a dose-dependent manner as compared with group IR(P＜0.01).2.Experiment 2MDA content in the lung was significantly increased by group IR as compared with group C(P＜0.01).SOD activity in the lung was significantly decreased by IR group as compared with group C(P＜0.01).The I/R-induced increase in MDA content were significantly attenuated by simvastatin pretreatment(P＜0.01).NOS activity and NO content were significantly higher in the three simvastatin groups than in group C or group IR(P＜0.01).The I/R-induced decrease in eNOS mRNA expression and increase in iNOS mRNA expression were significantly attenuated by simvastatin pretreatment in a dose-dependent manner(P＜0.05 or 0.01).The I/R-induced decrease in eNOS protein expression and increase in iNOS protein expression were significantly attenuated by simvastatin pretreatment in a dose-dependent manner(P＜0.01).3.Experiment 3HO-1 mRNA expression and HO-1 protein expression were significantly higher in the three simvastatin groups than in group C or group IR in a dose-dependent manner (P＜0.01).Conclusion1.Simvastatin pretreatment may ameliorate the lung injury associated with ischemia-reperfusion of the hind limbs in rats in a dose-dependent manner.2.Simvastatin pretreatment provides protective effects against lung injury by reduction of inflammatory reaction and oxidative damage,metabolism regulation of NO in the lung.3.Simvastatin pretreatment provides protective effects against lung injury by increase of eNOS mRNA and eNOS protein expression in the lung.4.Simvastatin pretreatment provides protective effects against lung injury by decrease of iNOS mRNA and iNOS protein expression in the lung.5.Simvastatin pretreatment provides protective effects against lung injury by increase of HO-1 mRNA and HO-1 protein expression in the lung.