Protective Effects And Possible Mechanisms Of Simvastatin Pretreatment On Acute Pulmonary Thromboembolism In Rats

BackgroundAcute right-sided heart failure and circulatory shock due to increased pulmonary vascular resistance are the prime cause of death in pulmonary thromboembolism embolism (PTE).Pharmacologic blockade of the pulmonary vasoconstriction can attenuate the hemodynamic changes during acute pulmonary embolism(APE) and has been suggested as adjuvant therapy.Statins have exhibited many other therapeutic effects beyond their cholesterol lowering effect, such as protecting against the risk of developing venous thromboembolism,reducing ischemic brain injury and perfusion deficits in an embolic model of stroke,and attenuating pulmonary hypertension induced by either monocrotaline treatment or chronic hypoxia.To investigate the effect of simvastatin pretreatment on APE and its possible mechanisms,the following study was carried out.Objective1. To investigate the effects of simvastatin pretreatment on PTE induced by jugular vein injection of autologous blood clots in a rat model.2. To further explore the possible mechanisms of the protective effects of simvastatin pretreatment on APE-induced pulmonary hypertension.Methods1.72 male Sprague-Dawley rats were randomly divided into three groups:Sham group,PTE group and Statins group.Each group including three subgroups:2h subgroup,6h subgroup and 24h subgroup. Sham group and PTE group received with normal saline by daily oral gavage for 2 weeks,Statins group received with simvastatin(10mg/kg) by daily oral gavage for 2 weeks.On the fourteenth day, experimental pulmonary thromboembolism was induced in 48 rats of PTE group and Statins group by injection of auto-blood clots into jugular vein,an additional 24 rats in the Sham group underwent sham operations.2. Right ventricular systolic pressure(RVSP) and mean pulmonary arterial pressure (mPAP)were measured by right heart catheter at different time points following the induction of PTE.All rats were killed by exanguination after hemodynamic measurement and their lungs were harvested.3. Immunohistochemical analysis was used to study the changes of eNOS protein at pulmonary vascellum.4. ELISA kits were used to evaluate the proinflammatory cytokines (IL-6, TNF-a) in plasma at protein level.5. The protein expression of MMP-2 and MMP-9 at pulmonary vascellum were examined by immunohistochemistry. The enzymic activity of MMP-2 and MMP-9 of plasma or lung tissue were detected through gelatin zymography.6. The protein expression of ROCK1 and ROCK2 in lung tissue were determined by Western blot.The level of phophorylated MYPT1 of lung tissue was determined by Western blot.Results1. Effects of simvastatin pretreatment on hemodynamic parameter:Compared with Sham group,the mPAP, RVSP at every time points in the PTE group increased significantly(P<0.01); Compared with PTE Group, the mPAP, RVSP at every time points in the Statins group decreased significantly(P<0.05),but were still higher than those of Sham Group(P<0.05).2. Effects of simvastatin pretreatment on artery blood gas:Arterial oxygen tension (PaO2) was significantly lower and alveolar-arterial difference in oxygen partial pressure (A-aDO2) was significantly higher in the PTE 6h subgroup than in the Sham 6h subgroup(P<0.01).Compared with PTE 6h subgroup, simavastatin pretreatment prevented the change in worsening of PaO2 and A-aDO2 in the Statins 6h subgroup(P<0.05).3. Effects of simvastatin on expression of eNOS protein at pulmonary vascellum:eNOS protein expression at small pulmonary arteries in PTE 6h subgroup decreased significantly when compared with Sham 6h subgroup(P<0.01).After simavastatin pretreatment, the expression of eNOS protein at small pulmonary arteries in Statins 6h subgroup increased significantly when compared with PTE 6h subgroup(P<0.05).4. Influence of simvastatin pretreatment on plasma levels of IL-6 and TNF-α:Compared with Sham group,the plasma levels of IL-6 and TNF-αat every time point in the PTE group increased slightly(P>0.05).After simavastatin pretreatment, the plasma levels of IL-6 and TNF-αat every time point in the Statins group decreased, but no significant difference was found among three groups.5. Influence of simvastatin pretreatment on plasma MMP-2 and MMP-9 activity:Compared with Sham Group, the plasma MMP-9 activity at every time point in PTE Group increased significantly(P<0.05).Compared with PTE Group,the plasma MMP-9 activity at every time point in Statins Group decreased significantly(P< 0.05),but was still higher than that of Sham Group(P<0.05).No significant difference in MMP-2 activity at every time point was observed among three groups(P>0.05).6. Influence of simvastatin pretreatment on MMP-2 and MMP-9 expression and activity in the lung tissue:MMP-2 and MMP-9 expression of small pulmonary arteries in PTE Group increased significantly when compared with Sham Group; After simavastatin intervension, the expression of MMP-2 and MMP-9 of small pulmonary arteries in Statins Group decreased. Compared with Sham Group,the MMP-2 and MMP-9 activity of lung tissue at every time point in PTE Group increased significantly(P< 0.01).Compared with PTE Group,the MMP-2 and MMP-9 activity of lung tissue at every time point in Statins Group decreased significantly(P<0.05),but were still higher than those of Sham Group(P<0.01).7. Effects of simvastatin pretreatment on expression of ROCK1 and ROCK2 protein and the level of phosphorylated MYPT1 in the lungs tissues:The expression of ROCK1 and ROCK2 protein of lung tissues at every time point in PTE group were significantly higher than that of Sham group(P<0.01). Compared with PTE Group,the expression of ROCK1 and ROCK2 protein of lung tissue at every time point in Statins Group decreased slightly(P>0.05).The level of phosphorylated MYPT1 of lung tissues at every time point in PTE group was significantly higher than that of Sham group(P<0.01).Compared with PTE Group,the level of phophorylated MYPT1 of lung tissue at every time point in Statins Group decreased significantly(P<0.05), but it was still higher than that of Sham Group(P<0.05).Conclusion1. Pretreatment with simavastatin decreased RVSP and mPAP, improved hypoxemia and endothelial dysfunction in the PTE rats.2. The plasma levels of IL-6 and TNF-αwas not increased at first 24h after APE.3. Pretreatment with simavastatin attenuated APE-induced pulmonary hypertension by mechanisms that result in attenuated increases in plasma and lung activated MMP-2 and MMP-9 after APE.4. Lung Rho kinase protein expression and activity were increased in the PTE rats, pretreatment with simavastatin attenuated APE-induced pulmonary hypertension and the severity of hypoxemia in the PTE rats through mechanisms that involving attenuation of lung Rho kinase activity after APE.