法舒地尔可减轻野百合碱诱导的大鼠肺动脉高压

Fasudil reverses monocrotaline-induced pulmonary hypertension in rats

目的探讨法舒地尔对野百合碱诱导的大鼠肺动脉高压的疗效及其机制。方法健康雄性Sprague—DaMey大鼠56只，随机数字法分为正常对照4周组（N4）8只、模型对照4周组（M4）8只、正常对照8周组（N8）8只、模型对照8周组（M8）16只、法舒地尔8周组（F8）16只。采用一次性皮下注射1％野百合碱50mg／kg建立Sprague—Dawley大鼠肺动脉高压模型。F8组于造模第4周末开始予腹腔内注射法舒地尔（15mg·kg-1·d-1）治疗4周，正常对照组和模型组注射生理盐水。干预结束后经右心导管法测量右心室收缩压（Rvsp）、平均肺动脉压力（mPAP）和右心室肥厚指数[RV／（LV＋S）]；HE染色观察肺小动脉50—150μm血管厚度与血管外径之比（WT％）和管壁面积占血管总面积的百分比（WA％）；逆转录聚合酶链式反应（RT—PCR）法检测Rho相关激酶．1（ROCK-1）mRNA在肺组织的表达。蛋白免疫印迹（Westernblot）法检测ROCK—1、肌球蛋白磷酸酶靶蛋白-1（MYPT-1）、磷酸化肌球蛋白磷酸酶靶蛋白-1（p-MYPT-1）蛋白在肺组织中的表达情况。结果56只大鼠41只存活至药物干预结束，其中N4、M4、N8各组8只全部存活，M8组存活5只（5／16），F8组存活12只（12／16）。M4组大鼠RVSP、mPAP、RV／（LV＋S）、WT％和WA％均显著高于N4组，分别为（62．25±3．24）比（31．334-2．35）mmHg（1mmHg=0．133kPa）、（36．38±2．31）比（20．32±1．81）mmHg、0．5648±0．0580比0．3458±0．0455、（25．63±5．35）％比（13．38±3．45）％和（60．36±2．51）％比（38．42±2．84）％（P均〈0．01）。而F8组大鼠RVSP、mPAP、RV／（LV＋S）、WT％和wA％则低于M8组，分另0为（54．64±4．11）比（67．37±4．68）mmHg、（26．25±2．32）比（39．83±1．83）mmHg、0．3985±0．0210比0．7600±0．0341、（15．64±2．81）％比（28．26±4．38）％和（40．35±2．82）％比（68．83±1．63）％（P均〈0．05），ROCK．1mRNA基因表达亦低于M8组（1．2139±0．1778比1．6839±0．3251，P〈0．01），ROCK-1蛋自在肺组织的表达亦较M8组低（P〈0．01），相应的下游通路因子MYPT-1、p-MYPT-1蛋白在肺组织的表达均亦较M8组低（P均〈0．01）。结论法舒地尔可减轻野百合碱诱导的大鼠肺动脉高压。

Objective To observe the effects and related mechanisms of fasudil on monocrotalineinduced pulmonary arterial hypertension （PAH） in rats. Methods A total of 56 healthy male Sprague- Dawley rats were randomly divided into 5 groups ： 4 weeks control group （ N4）, 4 weeks PAH group （ M4）, 8 weeks control group （NS） , 8 weeks PAH group （MS） , 8 weeks PAH and fasudil group （ F8 ）. PAH was induced by subcutaneous injection of monocrotaline （50 mg/kg ）. Animals in F8 group received intraperitoneal injection of fasudil hydrochloride （ 15 mg - kg--1 d -1 ） from the end of the 4th week to the end of the 8th week. Rats in control groups and PAH groups received equal volume saline injection. Polyethylene catheters were inserted into the RV through the jugular vein for fight ventricular systolic pressure （RVSP） and mean pulmonary artery pressure （mPAP） measurements after various treatment protocols. RV hypertrophy index [ RV/（LV ＋ S）] was also measured. Arteries of 50 to 150 μm were evaluated for the median wall thickness and wall area by HE staining as follows： percent wall thickness （WT%） = [ （medial thickness × 2/external diameter）] ×100 and percent wall area （WA%） = （wall area/total area） × 100% . The mRNA expression of ROCK-1 in lung tissue was analyzed by reverse transcription-polymerase chain reaction （RT-PCR）. The protein expressions of ROCK-1 and MYPT-1 in lung tissue were analyzed by Western blot and MYPT-1 phosphorylation, respectively. Results Forty-one rats survived and mortality rate was zero in N4, N8 and M4 groups. Survival rate was significantly higher in 178 group compared to M8 group （75.00% vs. 31.25%, P〈0.05）. At the end of the 4th week, RVSP [（62.25±3.24） vs. （31.33±2.35） mm Hg（1 mm Hg=0.133 kPa）], mPAP [ （ 36. 38 ± 2. 31） vs. （20. 32± 1.81 ） mm Hg], [ RV/（ LV ＋ S） ] （0. 5648 ± 0. 0580 vs. 0. 3458 ±0. 0455 ）, WT% [ （25.63 ± 5.35）% vs. （ 13.38 ±3.45） % ] ,WA% [ （60. 36 ±2. 51 ） % vs. （38.42 ±2. 84） % 1 were all significantly higher in M4 group than in N4 group （ all P 〈 0. 01 ）. RVSP [ （ 54. 64 ±4. 11 ） vs. （ 67.37 ± 4. 68 ） mm Hg ], mPAP [ （26. 25 ±2. 32） vs. （39.83 ±1.83） mm Hg], and markedly relieve [ RV/（LV ＋S） ] （0. 3985± 0.0210 vs. 0.7600±0.0341）, WT% [（15.64 ±2.81）% vs. （28.26±4.38）%],WA% [（40.35± 2. 82） % vs. （68.83 ± 1.63 ） % ] were all significantly lower in F8 group than in M8 group （ all P 〈 0.05 ） while the expression of ROCK-1 mRNA （ 1. 2139 ± 0. 1778 vs. 1. 6839 ± 0. 3251, P 〈 0.01 ）, and the protein expression of ROCK-1 and MYPT-1 as well as the extent of MYPT-1 phosphorylation were all downregualted in F8 group compared to M8 group （ all P 〈 0. 01 ）. Conelus｜ons Fasudil can effectively reverse the MCT-indueed PAH in rats via downregulating ROCK-1 and MYPT-1.