| ObjectiveIt aimed at using the rat PH(pulmonary hypertension) model built through ascending aorta constriction to, on one hand, investigate the pathomechanism of how PH relating to left heart diseases formed, the change of transcriptinal level of Rho A/ROCK signaling pathway and the influence of its activity on the remodeling of pulmonary vascular induced by PH relating to left heart diseases; on the other hand, investigate the interactive relationship between Rho A/ROCK signaling pathway and other vaso-active substances(NO, ET-1)as well as the function and mechanism of ROCK inhibitor, fasudil, in the remodeling of pulmonary vascular induced by PH relating to left heart diseases.MethodRandomly divided 40 SD rats at 3-4 ages with 90-100 g into comparison group( group G: n=10), ascending aorta constriction group( group P: n=10), fasudil treatment after ascending aorta constriction group( group PF: n=10) and fasudil group( group F :n=10).For group P, adopted ascending aorta constriction to build rat PH model; For group C,carried out sham-operation(Apart from the Titanium clip which was fixed on the mediastinal tissue near vessel instead of ascending aorta, other operations were all same as group P); For group PF,carried out intraperitoneal injection of 30mg/kg fasudil everyday one week after the operation( group F shared the same time.), while, for group ‘C and group P, carried out intraperitoneal injection of the same volume of physiological saline as placebo at their respective time points for administration. Injected for 4 weeks in succession. If any rat died from severe illness during the experiment, used the one with same condition and processing to fill the gap immediately. 60 days after model building, carried out hemodynamics( right ventricular systolic pressure and average pulmonary artery pressure) examination for the rats in all groups after weighting and comparing their weight. Used PBS to carry out lungs lavage until they became white. Fixed left lung tissue on 4% of paraform to carry out pathological section so that the pathomorphological changes of lung tissue could be observed.Used Image-Pro Plus 6.0 to measure the diameter and pipe wall of pulmonary arterioles so that the thickening degree( the ratio of the thickness of pulmonary arterioles and the diameter of blood vessels) of arterioles’ middle layer could be calculated. Took out the heart, weighted RV and LV+S respectively, and calculated the thickness index of RV /LV+S.The tissue fluid of right lung was —80℃after freezing, C-freezing was used for RT-PCR method to examine gene expression(ROCK m RNA, Rho A m RNA, ET-A receptor m RNA and ET-B receptor m RNA). Before executing the rats, adapted eyeball blood sampling method to reserve 5-10 ml of nun-anticoagulative blood from each of them. Collected serum Elisa kits for concentration examination of NO and ET-1, please follow the instructions of respective kits for concrete steps.Results1. Testing results of right ventricular systolic pressure and average pulmonary artery pressure as well as weight changes of rats in each group.Compared with group C and F, the right ventricular systolic pressure of group P rose significantly(From 26.02±4.10 mm Hg and 24.35±4.06 mm Hg to 56.48±8.93 mm Hg) and average pulmonary artery pressure also rose significantly( from 15.69±2.86 mm Hg and 15.91±1.68 mm Hg to 28.98±1.82 mm Hg respectively), both of which had statistical significance( P<0.01); Compared with group P, both of the right ventricular systolic pressure and average pulmonary artery pressure of group PF reduced significantly( from 56.4±8.93 mm Hg to 39.03±1.52 mm Hg and from 28.98±1.82 mm Hg to 25.25±0.81 mm Hg respectively), both of which had statistical significance( P<0.05).Before hemodynamics examination, compared the weight of rats in each group( The primary weight has 90-100g). The results showed that, Compared with group C and group F the weight of rats in group P reduced significantly( from 461.50±16.50 g and 454.38±29.88 g to 593.11±65.01g), which difference had statistical significance( P<0.01); Compared with group P, the weight of the rats in group PF rose significantly( from 393.11±63.01 g to 463.00±72.75g), which difference had statistical significance( P<0.01).2. The comparison of the pathomorphological changes of lung tissue and the thickness index of ventricle.Compared with group C and F, the middle layer of pulmonary arterioles of the rats in group P thickened significantly( that is, the ratio of the thickness of pulmonary arterioles and the diameter of blood vessels changed from 0.17±0.04 and 0.06±0.01 to 0.39±0.07),which difference had statistical significance( P<0.01); Compared with group P, the arteries’ middle layer of group PF became thiner significantly( that is, the ratio of the thickness of pulmonary arterioles and the diameter of blood vessels changed from 0.39±0.07 to 0.19±0.03), which difference had statistical significance( P<0.01).The radio of the wet weight of RV and LV+S was the thickness index of ventricle( RV/LV+S). The results showed that compared with group C and F, the RV/LV+S of group P reduced significantly( from 0.29±0.04 and 0.29±0.03 to 0.21±0.03), which difference had statistical significance( P<0.01); Compared with group P, the ventricle thickness index of group PF had no significant difference( 0.24±0.02 and 0.21±0.03, P>0.05),while compared with group C and F, the ventricle thickness index of group PF reduced significantly( from 0.29±0.04 and 0.29±0.03 to 0.24±0.02), which difference had statistical significance( P<0.05).3.The testing results of the expression of ROCK m RNA, Rho A m RNA, ET-A receptor m RNA and ET-B receptor m RNA.Compared with group C and F, the relative expression quantity of Rock m RNA, Rho A m RNA, ET-A receptor m RNA of group P rose significantly, which difference had statistical significance( P<0.05), while that of ET-B receptor m RNA had no significant change( P>0.05). Compared with group P, the relative expression quantity of ROCK m RNA, ET-A receptor m RNA reduced significantly, which difference had statistical significance( P<0.05), while that of Rho A m RNA and ET-B receptor m RNA had no significant change( P>0.05).4. The testing results of the concentration of NO and ET-1 in serumCompared with group C and F, the concentration of NO of group P reduced significantly( from 73.58±12.31μM and 57.13±8.04μM, to 38.68±2.80μM), while that of ET-1 rose significantly( from 0.63±0.18pg/ml and 0.66±0.22pg/ml to 2.83±0.25pg/ml), which difference had statistical significance( P<0.01); Compared with group P, the concentration of NO of group PF rose significantly(from 38.68±2.80μMto 105.22±11.04μM), while that of ET-1 reduced significantly( from 2.83±0.25pg/ml to 1.30±0.23pg/ml), which difference had statistical significance( P<0.01).Conclusions1. The abnormal activation of Rho A / ROCK signaling pathway played on important role in the appearance and development of PH relating to left heart diseases, related to the regulation of NO and ET-1 expression and may participate in the vessel remodeling of pulmonary arterioles.2. The specific inhibitor of ROCK fasudil, may reduce the pulmonary circulating pressure of rat PH( include by ascending aorta constriction) model through regulating up NO and down ET-1 indirectly so that the pulmonary vascular remodeling could be improved. |
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