| [Backgroud]Pulmonary hypertension (PH) is a common pathophysiologic syndrome causedby a variety of disorders and is characterized by a progressive increase in pulmonaryvascular resistance and pulmonary artery pressure. This severe condition leads toright heart failure and death. Therefore, PH is a major health concern withconsiderable morbidity and mortality. It has been proved recently that inflammatorymechanisms could play a part in the genesis or progression of PH. Simvastatin is3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors and has been shown toexert the immunosuppressive and anti-inflammatory effects on systemic vascularwall function. To determine the possible prevention of simvastatin on pulmonaryhypertension, we investigated the protective effects of simvastatin on monocrotalineinduced pulmonary hypertension with focus on anti-inflammation.[Objective]1. To investigate the protection of simvastatin on monocrotaline-inducedpulmonary hypertension.2. To study the change of lung interleukin 6, tumor necrosisαand monocytechemotactic protein 1 in rats injected with monocrotaline, and explore themechanism of simvastatin protection.[Methods]1. 32 male Sprague-Dawley rats were randomly divided into four groups: Group C(n=8) were injected subcutaneously with sodium chloride. Group S (n=8) wereinjected subcutaneously with sodium chloride and received with simvastatin(2mg/kg) by daily gavage from day 1st to day 21st. Group P (n=8) were injected subcutaneously with monocrotaline (80mg/kg). Group PS were injectedsubcutaneously with monocrotaline (80mg/kg) and received simvastatin(2mg/kg) by daily gavage from day 1st to day 21st2. Right ventricular systolic pressure (RVSP) and mean pulmonary arterial pressure(mPAP) were detected by right heart catheter. The right ventricle (RV) wasseparated from the left ventricle (LV) and septum (S), and weighed. Rightventriclular hypertrophy index was caculated.3. Histopathology changes of pulmonary arterioles were evaluated by hematoxylinand eosin staining, and perivascular inflammation was scored with subjectivescale of 0(no) to 4(severe) in a blind manner.4. Pulmonary interleukin 6, tumor necrosis factorαand monocyte chemotacticprotein 1 were tested with ELISA5. Data were analyzed with the analysis of variance and the t test. Statisticalsignificance was assigned for P＜0.05.[Results]1. Effects of simvastatin on general condition of rats injected with monocrotalineMean bodyweight of pulmonary hypertension rats(group P) was significantlylower than in the three other group (group P 273±8.80g,group C 325±7.64g,group S 326±11.36g, group PS 306±10.63g, p＜0.05). From the 14th day afterMCT injection, rats became weak, lethargic and presented a dull coat. Comparedwith group C, mean bodyweight of monocrotaline-injected rats treated withsimvastatin (group PS) was no significant difference. Until the day of sacrifice, ratsof group PS were spontaneously active, feeding and presented with a normal coat.There was no significant difference between group C and group S.2. Effects of simvastatin on hemodynamics and right ventricular hypertrophyIn normal rats(Group C), mean pulmonary artery pressure(mPAP),rightventricular systolic pressure(RVSP) and right ventricular hypertrophy index(RVHI)were 20.05±3.96mmHg, 44.67±8.62mmHg and 0.27±0.04. mPAP, RVSP andRVHI of Group P increased significantly (34.37±9.16mmHg, 87.63±16.63mmHg, 0.39±0.06, p＜0.01). Simvastatin normalized mPAP and RVSP (23.25±7.32mmHg,55.12±11.90mmHg) and decreased RVHI (0.32±0.04) significantly. Between groupC and group S, significant differences of mPAP, RVSP and RVHI were notobserved(p＞0.05).3. Effects of simvastatin on lung vascular morphology of rats injected withmonocrotalineProminent medial wall hypertrophy, smooth muscle proliferation in muscularpulmonary arteries and muscularized arterioles were evident from rats injected withMCT (group P). Compared to group C, arterial wall area/vessel area(W/V), arterialwall thickness/vessel external diameter(T/D) of group P increased significantly(55.93±8.56% vs 40.77±4.93%, 36.77±5.54% vs 30.15±3.47%, p＜0.01). Simvastatinprevented the increase of W/V and T/D in rats injected with MCT (44.19±6.13%,32.49±4.48%, p＜0.05). No differences in vascular morphology were observedbetween group C and group S.4. Effects of simvastatin on perivascular inflammation of rats injected withmonocrotalineMacrophages significantly increased in alveoli and primarily mononuclear cellsinfiltrated around the arterioles in group P, moreover, the perivascular inflammationscale of group P was 3.40±0.65. Simvastatin significantly reduced perivascularinflammation scales in group PS, when compared to group P (2.19±0.81 vs3.40±0.65,p＜0.05).There is no significant difference between group C and group S.5. Effects of simvastatin on lung IL-6, TNF-αand MCP-1 level of rats injected withmonocrotalineLung IL-6, TNF-αand MCP-1 level of rats injected with monocrotaline aresignificantly higher than group C (764.56±178.83pg/ml vs 59.50±26.46pg/ml,446.82±85.86pg/ml vs 37.95±11.48pg/ml,4427.86±757.51pg/ml vs 32.23±7.24pg/ml,p＜0.001). Although simvastatin alone did not affect these inflammatory factors, itsignificantly inhibited the level of IL-6, TNF-αand MCP-1 in rats injected withMCT (264.25±127.29pg/ml, 179.37±91.32pg/ml, 696.62±211.24pg/ml, p＜0.01) andlung MCP-1 level near to normal level. [Conclusions]1. Simvastatin could delay the increase in pulmonary artery pressure and rightventricular systolic pressure, and improve hemodynamic parameters inpulmonary hypertension rats induced with monocrotaline.2. Simvastatin could delay the remodeling of pulmonary vessels and inhibit rightventricular hypertrophy in pulmonary hypertension rats induced withmonocrotaline.3. Simvastatin exerts protection on monocrotaline-induced pulmonary hypertensionpartly by inhibiting the pervascular inflammation and the increase in lung IL-6,TNF-αand MCP-1. [Backgroud]The recent development of therapy, such as prostacyclin analogues, phosphodiesterase inhibitors, and endothelin receptor antagonist, represents a significant advance in the management of pulmonary hypertension(PH). However, a limitation of current therapies for PH is that they are mostly aimed at vasodilation, rather than against the proliferation response of pulmonary vessels, moreover, the therapeutic effects of these agents are limited by their short-life lives, expensive and only partially effectives in some patients. Simvastatin, inhibitors of HMG-CoA reductase, confers substantial cardiovascular benefits that independent its capacity to lower serum cholesterol. Several reports recently have showed that simvastatn attenuated vessel remodeling of pulmonary hypertension, however, the detailed mechanisms have not yet been clarified. Heme oxygenase (HO-1) is the rate-limiting enzyme in the degradation of heme. HO-1 induction had been shown to have vasodilatory, anti-inflammation, and antiapoptotic properties. An increased in HO-1 prevented hypoxia-induced pulmonary hypertension. Concomitantly, a lack of HO-1 expression is associated with organized mural thrombi and subsequent pulmonary hypertension in response to hypoxia.We investigated the effect of simvastatin on established severe pulmonary hypertension induced by moncrotaline and explored its effect on HO-1.[Objective]1. To investigate the effect of simvastatin on established severe pulmonary hypertension rats induced by monocrotaline.2. To study the effect of simvastatin on lung eNOS and HO-1 protein and explore the mechanism of simvastatin on monocrotaline-induced pulmonary hypertension.[Methods] 1. 48 male Sprague-Dawley rats were randomly divided into five groups. Group C (n=8): sodium chloride was given by subcutaneous injection. Group S(n=8) : sodium chloride was given by subcutaneous injection, and simvastatin 2mg/kg, was given daily by gavage from day 21 to day 35. Group P(n=12): monocrotaline(80mg/kg) was given by subcutaneous injection. Group PS(n=8): rnonocrotaline(80mg/kg) was given by intraperitoneal injection, and simvastatin (2mg/kg) was given daily by gavage from day 21 to day 35. Group PSZ(n=12): monocrotaline(80mg/kg) was given by intraperitoneal injection, and simvastatin (2mg/kg) was given daily by gavage from day 21 to day 35, moreover, Znpp(20μmol/kg) was given by subcutaneous injection on day 21 and day 28.2. Mean pulmonary arterial pressure (mPAP) and right ventricular systolic pressure(RVSP) were detected by right heart catheter. The right ventricular free wall and left ventricular plus interventricular septal weight were weighed.3. Histopathology changes of small intrapulmonary arteries were evaluated by hematoxylin and eosin staining.4. Immunohistochemical analysis was used to investigate the expression and location of HO-1. The eNOS and HO-1 protein content in lung tissue were determined by western blot.5. Data were analyzed with the analysis of variance and the / test. Statistical significance was assigned for P<0.05.[Results]1. Effects of simvastatin on general condition of rats.Mean bodyweight of pulmonary hypertension rats(group P) was significantly lower than in the three other group (group P 317±16. 62g , group C 377±14. 55g , group S 370±12.43g, group PS 345±16.01g, p<0.05) , and the rats became weak, lethargic and their peeling became dull. 5 rats died between the 21th and the 35th and mortality was 42%. Two or three days before their death, breathing of difficult and muzzle cyanosis was oberseved in some rats. Autopsy showed pleura and abdominal effusion, hemorrhagic zones on lungs and liver, which appeared augmented and cyanosed, right ventricular distention. Compared with group P, mean bodyweight of monocrotaline-injected rats treated with simvastatin (group PS) increased significantly (p<0.05). Until the day of sacrifice, rats of group PS were spontaneously active, feeding and presented with a normal coat. Mean bodyweight of group PSZ lower than that of group PS and mortality was 25%. There was no significant difference between group C and group S.2. Effects of simvastatin on hemodynamics and right ventricular hypertrophymPAP, RVSP and RVHI of group P increased significantly, compared with group C(65.87±15.51mmHgvsl9.25±3.20mmHg, 91.00±12.83mmHgvs42.50±4.37 mmHg, 0.53±0.06vs0.24±0.03, p<0.001) . Simvastatin normalized RVSP and decreased mPAP and RVHI significantly (35.63±5.10mmHg vs65.78±15.51mmHg, 57.13±4.79 mmHg vs42.50±4.37mmHg, 0.33±0.05vs0.53±0.06, p<0.05). Effects of simvastatin was partly reduced in rats injected with Znpp(group PSZ) (52.88±17.45mmHg vs 35.63±5.10mmHg, 71.63±14.71 mmHg vs 57.13±4.79mmHg, 0.51±0.05 vs O.33±O.O5, p<0.05). Between group C and group S, mPAP, RVSP and RVHI were not significantly different(p>0.05).3. Effects of simvastatin on vascular morphologyProminent medial wall hypertrophy, smooth muscle proliferation in muscular pulmonary and muscularized arterioles are evident from pulmonary hypertension rats(group P). Compared to group C, arterial wall area/vessel area(W/V), arterial wall thickness/vessel external diameter(T/D) of group P increased significantly (65.92±7.19% vs 41.26±6.25%, 52.00±5.53% vs 29.13±2.47%, p<0.001 ) . Simvastatin partially reversed the increase of W/V and T/D in rats injected with MCT (50.78±9.03% vs 65.92±7.19%, 43.75±4.23% vs 52.00±5.35%, p<0.01) . Effects of simvastatin was partly reduced in rats injected with Znpp(group PSZ) (p<0.05). No differences in vascular morphology were observed between group C and group S.4. Effects of simvastatin on lung HO-1 and eNOS proteinIn group C and group S, intensity of HO-1 immunohistochemical staining in pulmonary artery endothelial cell(EC), smooth muscle cell(SMC) and alveolar macrophage(AM)was weakly positive. In group P, intensity of HO-1 immunohistochemical staining was positive in AM, but weakly positive in modeling pulmonary artery EC and SMC. Treatment of simvastatin resulted in a significantly increased intensity of HO-1 staining in EC, SMC and AM. Znpp significantly inhibited the intensity of HO-1 immunohistochemical staining in AM, EC and SMC.The level of lung HO-1 protein expression in group P was higher than that in group C. Simvastatin increased significantly HO-1 protein expression in the rats injected with monocrotaline, which was totally inhibited by Znpp(p<0.05). Compared with group C, MCT was associated with a significant decreased expression of eNOS in whole lung protein (p<0.01). Simvastatin treatment restored significantly the expression of eNOS protein comparted to group P. Compared to group PS, Znpp did not affect the expression of eNOS protein. HO-1 protein and eNOS protein in the lung were no significantly different between group C and group S.[Conclusions]1. Simvastatin could attenuate established pulmonary artery hypertension and right ventricular hypertrophy , and improve pulmonary artery remodeling in rats injected with monocrotaline.2. Simvastatin could restore lung eNOS protein and increase HO-1 protein of pulmonary artery endothelial cell and smooth muscle cell in rats injected with monocrotaline.3. Znic protoporphyrin, chemical inhibitor of HO, partly blocks the beneficial effects of simvastatin on monocrotaline-induced pulmonary hypertension. The result suggests that HO-1 is required for lung vascular effects of simvastatin.
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