Rho激酶抑制剂对先天性膈疝大鼠模型胎仔肺血管重构的作用

Effects of Rho-kinase inhibitor on lung vascular remodeling in fetal rats with congenital diaphragmatic hernia

摘要:

目的：探讨先天性膈疝（congenital diaphragmatic hernia，CDH）大鼠模型胎仔肺Rho激酶的表达及其抑制剂法舒地尔对肺血管重构的影响。方法：将定时配种成功的SD雌鼠随机分为对照组（5只）、膈疝组（6只）和法舒地尔干预组（6只）。分别给予膈疝组和干预组除草醚（100 mg/只）一次性灌胃建立CDH动物模型，对照组灌等量橄榄油。孕16.5 d给予干预组腹腔注射法舒地尔（15 mg/kg，1次/d，连续３ d），对照组和膈疝组予等量生理盐水腹腔注射。定时剖宫产取出胎肺，光镜下进行肺血管重构观察，采用RT-PCR法和Western blot法观察肺组织Rho激酶表达变化；Western blot法检测Rho激酶底物肌球蛋白磷酸酶的磷酸化状态。结果：膈疝组肺小动脉管壁厚度占血管外径百分比（WT%）和肺小动脉中膜厚度百分比（MT%）均明显高于对照组[（26.88±2.41） vs. （13.50±1.45）；（25.59±2.57） vs. （16.47±2.07），P=0.000]；管腔面积与血管总面积百分比（LA%）明显低于对照组[（39.22±2.23） vs. （61.20±3.23），P=0.000]。法舒地尔干预组WT%、MT%显著低于膈疝组[（15.38±2.14） vs. （26.88±2.41）；（17.69±1.79） vs. （25.59±2.57），P=0.000]，LA%明显高于膈疝组[（58.67±3.06） vs. （39.22±2.23），P=0.000]。与对照组相比，膈疝组Rho激酶mRNA和蛋白质表达明显升高，肌球蛋白磷酸酶磷酸化水平也明显增高（P=0.000）。法舒地尔干预后Rho激酶mRNA和蛋白、肌球蛋白磷酸酶磷酸化水平均较膈疝组明显降低（P<0.01）。结论：先天性膈疝胎仔肺内存在肺血管重构，Rho/Rho激酶信号通路参与此过程，产前给予法舒地尔明显改善CDH胎仔的肺血管重构。

Abstract:

Objective：To investigate the expression of Rho kinase in fetal rats with congenital diaphragmatic hernia（CDH），and to explore the effect of Rho kinase inhibitor fasudil on lung vascular remodeling. Methods：Timed-pregnant SD rats were randomly divided into control group（5 rats），CDH group（6 rats） and fasudil intervention group（6 rats）. Nitrofen of 100 mg was given to by gavage CDH and intervention group for establishing CDH models and olive oil of the same dose was given to the control group. At day 16.5 of gestation，fasudil（15 mg/kg，qd，for 3 consecutive days） was given via intraperitoneal injection to the intervention group，while Ns was given to by the same way control and CDH group. All fetal rats were delivered by cesarean in fixed time. The small pulmonary arterial morphologic changes in all groups were analyzed with microscopy. The gene and protein expression of Rho kinase were respec－tively examined by RT-PCR and Western blot；the phosphory－lation of myosin binding subunit（MBS） of myosin phosphatase-a substrate of Rho kinase was detected by Western blot as a marker of the activated kinase. Results：Morphometry investiga－tion showed that the ratio of small pulmonary artery wall thick－ness to vascular external diameter（WT%） and the medium thickness percentage（MT%） were significantly increased in CDH group than those in control group[（26.88±2.41） vs. （13.50±1.45）；（25.59±2.57） vs. （16.47±2.07），P=0.000]. The ratio of small pulmonary vascular lumen area to the total area（LA%） in the CDH group was markedly lower than that in the control group[（39.22±2.23） vs. （61.20±3.23），P=0.000]. However，compared with those in the CDH group，WT% and MT% in the intervention group were significantly lower [（15.38±2.14） vs. （26.88±2.41）；（17.69±1.79） vs. （25.59±2.57），P=0.000] and LA% was higher[（58.67±3.06） vs. （39.22±2.23），P=0.000]. The expression of Rho kinase mRNA and Rho kinase protein was significantly higher in the CDH group than in the control group（P=0.000）. The phosphorylation of MBS was also significantly higher in the CDH group（P=0.000）. Compared with those in the CDH group，expressions of Rho kinase mRNA and protein and the phosphorylation of MBS were significantly reduced in the fasudil intervention group（all P<0.01）. Conclusion：Lung vascular remodeling is confirmed in fetal rats with CDH and Rho/Rho kinase signaling pathway may be involved in. Prenatal fasudil treatment may obviously improve the lung vascular remodeling in fetal rats with CDH.