| IntroductionPulmonary arterial hypertension (PAH) is a common disease with a sustained elevation in pulmonary artery pressure (PAP), which may lead ultimately to right ventricular failure. The pathobiology of PAH includes pulmonary vasoconstriction, vascular remodelling, inflammation and microthrombosis. PAH has a complex causes, in which chronic obstructive pulmonary disease (COPD), hypoxia, chronic bronchial inflammation and portal hypertension are frequently encountered. Several vasoactive factors are considered to be involved in the development of pulmonary hypertension, including endothelin, prostacyclin,5-hydroxytryptamine (5-HT), thromboxane A2 (TXA2), platelet-derived growth factor, epidermal growth factor and inflammatory cytokines (TNF-α, IL-1, IL-6, MCP-1 et al). Yet, the pathogenesis of PAH is still not quite clear.In the recent years, phosphodiesterase-5 (PDE-5) inhibitors have been used for the therapy of PAH, and the pathway of NO-cGMP was considered to be involved. Sildenafil is a selective PDE-5 inhibitor of cyclic guanosine monophosphate (cGMP)-specific inhibitor, producing an inhibition on the hydrolytic breakdown of cGMP. PDE 5-inhibitors cause vasodilation in various vascular beds by increasing intracellular cGMP levels in vascular smooth muscle. And the cGMP activate the cGMP-dependent protein kinase (PKG) signaling pathway, leading to the opening of potassium ion channels opening and the calcium ion decreased that induced vasodilation of vascular smooth muscle. PDE-5 inhibitors produce the effect activating the NO-cGMP pathway, but the NO-cGMP pathway can induce the increase of the reactive oxygen species (ROS). ROS play an essential role in signal transduction and physiologic regulation of vascular function in the settings of chronic hypertension, inflammation, or acute injury, ROS may trigger signaling events that further exacerbate smooth muscle hypercontractility and vascular remodelling associated with hypertension and endothelial barrier dysfunction associated with acute lung injury and pulmonary edema. It is conflicted with the effect of sildenafil in treatment in PAH. There are more than ten articles about the effect of sildenafil on monocrotaline induced pulmonary hypertension in rats, the dose of sildenafil changed from 100μg/kg/d to100 mg/kg/d,and it is 1000 fold difference in dose. The time of the administration is also different. Therefore, this study want to provide experimental evidence for PAH accompany with inflammation by investigating the effect of PDE-5 inhibitor (sildenafil, yonkenafil) on monocrotaline (MCT) induced pulmonary hypertension in rats.The content of the present study was including (1) the effect of PDE-5 inhibitor (sildenafil, yonkenafil) on isolated rat pulmonary artery (PA) rings; (2) the effect of PDE-5 inhibitors (sildenafil, yonkenafil) on monocrotaline induced pulmonary hypertension in rats.Methods1. The effect of PDE-5 inhibitor (sildenafil, yonkenafil) on isolated rat pulmonary artery ringsPhenylephrine(PE) or KCl was added to the preparation to obtain a tonic contraction, then different dose of sildenafil or yonkenafil was gradually added to the preparation to obtain Cumulative Concentration-Response Curves(CCRC). The pEC50 values for sildenafil and yonkenafil were determined as-log of the molar concentration to produce 50% of the maximal relaxation in phenylephrine or KCl-contracted pulmonary artery rings. The other part of rings were pretreated with sildenafil or yonkenafil, then PE or KCl was added to the preparation to investigate the inhibition of sildenafil or yonkenafil on PE or KCl-induced contraction.2. Establishment of Monocrotaline-induced'inflammatory'pulmonary hypertension in rats.To investigate the preventive activity of PDE5 inhibitor, rats were administrated with different dose of sildenafil and yonkenafil by intragastric lavadge 3 weeks after the injection of MCT. The control group was injected vehicles and administrated with distilled water. Then pulmonary haemodynamic measurement, right ventricular index and lung tissue morphological investigation were performed. To investigate the therapeutic activity of PDE5 inhibitor, two weeks after the injection of MCT, rats were administrated with different dose of sildenafil and yonkenafil by intragastric lavadge for two weeks. The variables measured in the therapeutic study were the same as those in the preventive study. Statistics were made to compare these indexes in different groups, and investigate the effect of PDE-5 on MCT-induced PAH in rats.3. Statistical analysisAll data are expressed as mean±SD. Statistical analyses were performed by one-way ANOVA. A value of P<0.05 was considered statistically significant.Results1. Effect of sildenafil and yonkenfil on PA rings precontracted with phenylephrineThe relaxant effect of sildenafil on PA rings precontracted with phenylephrine occurred at the cumulative concentration of sildenafil reached 3 X 10-9 mol/L, the percentage of vasodilation was 0.78%±2.37%. When the cumulative concentration of sildenafil reached 10-4mol/L, the percentage of vasodilation is 84.27%±6.77%. The pEC50 values for sildenafil was 5.1±0.32. While the relaxant effect of yonkenafil on PA rings precontracted with phenylephrine occurred at the cumulative concentration of yonkenafil reached 10-10 mol/L, the percentage of vasodilation was 0.88%±2.15%. When the cumulative concentration of yonkenafil reached 10-4mol/L, the percentage of vasodilation was 78.88%±8.59%. The pEC50 values for sildenafil was 5.24±0.29.2. Effect of sildenafil and yonkenfil on PA rings precontracted with KClThe relaxant effect of sildenafil and yonkenafil on PA rings precontracted with KCl occurred at the cumulative concentration reached 10-6 mol/L, the percentage of vasodilation was 1.21%±1.35%,0.42%±1.03%respectively. When the cumulative concentration reached 10-4 mol/L, the percentage of vasodilation for sildenafil and yonkenafil were 79.38%±5.48%,74.90%±9.96%respectively. The pEC50 values for sildenafil and yonkenafil were 4.49%±0.11%,4.35%±0.11%respectively.3. The inhibition of pretreatment with sildenafil and yonkenfil on PA rings contracted with phenylephrineThe inhibition of pretreatment with sildenafil and yonkenfil on PA rings contracted with phenylephrine was not significant at 10-6 mol/L. At 10-5 mol/L, the inhibition of pretreatment with sildenafil and yonkenfil was significant, the percentage of vasoconstriction was 28.94%±18.77%,56.84%±15.88%(P＜0.05) respectively. At 10-4 mol/L, the percentage of vasoconstriction was 0.54%±1.21%,11.37%±6.10% (P＜0.05) respectively.4. The inhibition of pretreatment with sildenafil and yonkenfil on PA rings contracted with KClThe inhibition of pretreatment with sildenafil and yonkenfil on PA rings contracted with KCl was not significant at 10-6 mol/L. At 10-5 mol/L, the inhibition of pretreatment with sildenafil and yonkenfil was significant, the percentage of vasoconstriction was 59.91%±16.45%,59.24%±26.53%(P<0.05) respectively. At 10-4 mol/L, the percentage of vasoconstriction was 11.21%±8.62%,11.24%±7.51% (P＜0.05) respectively.5. Effect of sildenafil and yonkenfil on MCT rats in preventive studyThree weeks after MCT injection, the chronic'inflammatory' PHT model in rats induced by MCT was successfully established. Mean pulmonary artery pressure was elevated in MCT group compared with control group from 15.70±1.98 mmHg to 24.79±6.40 mmHg (P< 0.01). Sildenafil (7.5 mg/kg/d) and yonkenfil (7.5 mg/kg/d, 15 mg/kg/d) decreased MCT-induced pulmonary arterial pressure which decreased from 24.79±6.40 mmHg to 24.22±7.34 mmHg,23.28±4.79 mmHg and 23.67±6.97 mmHg, respectively. But there was no significance compared wit MCT group.Right ventricular index was increased by MCT from 0.293±0.026 to 0.380±0.095 (P< 0.01 vs control). Right ventricular index was reduced from 0.380±0.095 to 0.354±0.051 by sildenafil (7.5 mg/kg/d), and 0.360±0.072,0.362±0.062,0.365±0.057 by yonkenafil (3.75 mg/kg/d,7.5 mg/kg/d,15 mg/kg/d), respectively, but there was also no significance compared with MCT group.The thickness of pulmonary arterial wall was elevated in MCT group compared with control group from 50.90%±11.62% to 79.24%±9.50%(P< 0.05). Compared with MCT group, the thickness of pulmonary arterial wall in sildenafil and yonkenfil (7.5 mg/kg/d,15 mg/kg/d) groups were decreased from 79.24%±9.50%to 66.14%±16.80%,67.07%±10.55%and 68.11%±14.92%(P< 0.05 vs MCT), respectively. The thickness of the yonkenafil (3.75 mg/kg/d) group was 74.03%±13.49%, but there was also no significance compared with MCT group.6. Effect of sildenafil and yonkenfil on survival rate in MCT rats in therapeutic studyThe survival rate of Control group was 100%, the survival rate of MCT group was 63.6%, the survival rate of sildenafil and yonkenafil (25 mg/kg/d,50 mg/kg/d,100 mg/kg/d) was 72.7%,80.0%,81.8%and 81.8%respectively. The survival rate of sildensfil or yonkenafil was higher than MCT group, but there was no significance by Kaplan-Meier survival analysis.7. Effect of sildenafil and yonkenfil on MCT rats in therapeutic studyFour weeks after MCT injection, mean pulmonary artery pressure was elevated in MCT group compared with control group from 15.00±3.07 mmHg to 30.00±1.67 mmHg (P< 0.01). Sildenafil (50 mg/kg/d) and yonkenfil (50 mg/kg/d) decreased MCT-induced pulmonary arterial hypertension; PAP was decreased from 30.00±1.67 mmHg to 27.57±5.16 mmHg and 28.14±7.17 mmHg, respectively. But there was no significance compared with MCT group.Right ventricular index was increased by MCT from 0.278±0.023 to 0.519±0.117 (P<0.01 vs control). Right ventricular index was reduced from 0.519±0.117 to 0.491±0.079 by sildenafil (50 mg/kg/d), and 0.490±0.117 by yonkenafil (50 mg/kg/d), but there was also no significance compared with MCT group.The thickness of pulmonary arterial wall was elevated in MCT group compared with control group from 58.62%±9.68% to 81.47%±12.04%(P< 0.05). The thickness of pulmonary arterial wall in sildenafil (50 mg/kg/d) and yonkenfil(25 mg/kg/d,100 mg/kg/d) groups were 79.14%±10.95%,74.82%±10.75%and 86.36%±14.58%respectively, but there was also no significance compared with MCT group. The thickness of the yonkenafil (50 mg/kg/d) group was decreased from 81.47%±12.04%to 70.93%±14.58%(P< 0.05 vs MCT).Conclusion1 PDE-5 inhibitors, sildenafil and yonkenfil produced significant relaxant effect on vasoconstriction of isolated rat pulmonary artery rings induced by phenylephrine or KCl.2 Sildenafil and yonkenfil have mild but not significant amelioration of PAH on MCT rats in both preventive and therapeutic administration.3 Based on the present study, it is suggested that PDE-5 inhibitors, sildenafil and yonkenfil are not suitable for patients with pulmonary hypertension accomponied inflammation.
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