| Objective: To establish a monocrotaline-induced rat pulmonary hypertension model, explore the effect of activin receptor-like kinase1(ALK-1) signaling in the pathogenesis of pulmonary hypertension, and observe the role and potential mechanism of N-acetylcysteine in preventing and treating pulmonary hypertension of this model.Methods: Thirty-two healthy male Wistar rats were randomly divided into normal control group, pulmonary hypertension model group, low-dose acetylcysteine(100mg/kg/d) treatment group and high-dose acetylcysteine(500mg/kg/d) treatment group, with 8 in each. The monocrotaline(60mg/Kg) was injected subcutaneously at a time in the model group and in the treatment groups respectively. From the day of monocrotaline administration, the treatment groups underwent intragastric administration of 100mg/kg/d and 500mg/kg/d acetylcysteine, respectively; the same volume of saline was intragastrically administrated in the control group and the model group once a day. The end of the experiment was achieved after six weeks. The general conditions and survival rates of all groups were observed. After intervention finished in each group, the mean pulmonary arterial pressure(m PAP) and mean right ventricular pressure(m RVP) of rats were determined; the right ventricular hypertrophy index [RV/(LV+S)] was calculated; the structural changes in the pulmonary arterioles were observed using HE staining; the percentage of pulmonary artery wall thickness to vascular external diameter(WT%), the percentage of wall area to vessel area(WA%), and vascular inflammation score were determined using image analysis system to reflect pulmonary vascular reconstruction conditions. The expression of ALK-1 and Smad1 in pulmonary arterioles as well as the distribution of proliferating cell nuclear antigen(PCNA) of pulmonary vascular smooth muscle cells were detected using immunohistochemical staining; the apoptosis of pulmonary vascular smooth muscle cells were detected using TUNEL staining, with mean absorbance calculated, respectively. The expressions of ALK-1 and Smad1 proteins in lung tissue were detected using Western Blotting technique.Result: 1. Survival situation: At the experiment end after 6 weeks, the survival rates in the control group, the model group, the low-dose group and the high-dose group were 100%, 75%, 88% and 100%, respectively. Compared with the model group, the survival rates in the low-dose and the high-dose treatment groups were significantly higher(P<0.05).2.Hemodynamic determination, morphological observation of pulmonary arteriloes, and determination of right ventricular hypertrophy index: In the model group, m RVP, m PAP and RV/(LV+S) were significantly higher(P<0.01), the pulmonary artery wall was significantly thickened with lumen stenosis, the degree of inflammation, WA% and WT% were significantly higher(P<0.01), when compared with the control group. In the treatment groups, all of m RVP, m PAP, RV/(LV+S), degree of inflammation, WA% and WT% were significantly decreased(P <0.05), the pulmonary artery wall thickening and stenosis degrees were significantly reduced(P <0.05), when compared with the model group; more significant reduction was reported in the high-dose treatment group. In comparison of the low-dose and the high-dose treatment groups, there was no difference in m RVP and m PAP(P> 0.05), while the differences in RV/(LV+S), WA% and WT% were statistically significant(P <0.05) but had not been restored to the levels of the control group(P<0.01).3. Expression of ALK-1 and Smad1 proteins in pulmonary arterioles: The expression of ALK-1 and Smad1 proteins in the model group was more significant than the control group(P<0.01); the expressions in low-dose and high-dose treatment groups were reduced compared with the model group(P<0.05), and the differences between different dosages were statistically significant(P<0.05), but they had not yet been restored to the levels of the control group(P<0.05).4. Proliferation and apoptosis of smooth muscle cells: The mean absorbance of PCNA in the model group was significantly higher than the control group(P<0.01), while it was reduced in the low-dose and the high-dose treatment groups when compared with the model group(P<0.05); the differences between different dosages were significant(P<0.05), but they had not yet been restored to the level of the control group(P<0.01). The mean absorbance of TUNEL in the model group was significantly lower than the control group(P<0.01), while that in the high-dose treatment group was significantly higher than the model group(P<0.05); the differences between low-dose and high-dose treatment groups were statistically significant(P<0.05), but they had not been elevated to the level of the control group(P<0.01).5. Expression levels of ALK-1 and Smad1 proteins in lung tissue: The expression levels of ALK-1 and Smad1 proteins in the model group were significantly higher than the control group(P <0.01); the expressions in low-dose and high-dose treatment groups were reduced compared with the model group(P<0.05); the differences between different dosages were statistically significant(P<0.05), but they had not yet been restored to the levels of the control group(P<0.01).Conclusion: ALK-1 and Smad1 participate in the initiation of monocrotaline-induced rat pulmonary hypertension, and both of them are involved in pulmonary hypertension and pulmonary vascular remodeling process; the proliferation and apoptosis of pulmonary vascular smooth muscle cells are two important mechanisms for the formation of pulmonary hypertension. Acetylcysteine can significantly inhibit the contents of ALK-1 and Smad1 proteins in lung tissue and pulmonary arterioles of rats with pulmonary hypertension, lower smooth muscle cell proliferation, and reduce pulmonary vascular remodeling and right ventricular hypertrophy, thereby relieving the established pulmonary hypertension, which partially exhibits the action mechanism of acetylcysteine in reducing pulmonaryhypertension from different levels. |
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