氧化苦参碱舒张肺动脉抑制肺动脉高压的机制及其联合用药研究

Oxymatrine is one of alkaloid derived from medicinal plant such as Sophora flavescens Ait and Sophora alopecuoides L. Our previous study showed that oxymatrine attenuated monocrotaline-induced pulmonary hypertension via the regulation of pulmonary ET-1/sGC pathway, but its effects on ET-1-induced pulmonary arterial contraction is still unclear. In the present study, with the combined methods of pharmacology, molecular biology and biochemistry, the protective effects of oxymatrine on pulmonary arterial function during the development of pulmonary hypertension are to be investigated, and the relaxation effects of oxymatrine and its metabolite matrine on ET-1-constricted rat pulmonary arterial rings are to be estimated. To elucidate the mechanism action of oxymatrine on pulmonary arterial rings, NO synthase inhibitor, calcium and PKA inhibitor are to be used, the expression of pulmonary arterial eNOS, sGC, cGMP, Rho A, ROCK are to be measured, and the phosphorylation of pulmonary arterial myosin light chain kinase levels are to be evaluated. Furthermore, to provide new therapeutic options for the treatment of pulmonary arterial hypertension, the hypothesis that combination of oxymatrine with ET-1 receptor antagonist or phosphodiesterase inhibitor may act synergistically to relax ET-1-constricted pulmonary arteries are also to be tested.

氧化苦参碱是苦参和苦豆子等药用植物中的主要生物碱之一，我们前期研究结果表明，氧化苦参碱通过调控肺脏ET-1/sGC等信号通路抑制大鼠肺动脉高压，而其对肺动脉高压时ET-1所致肺动脉收缩功能的影响及机制尚未阐明。本项目拟综合应用药理学、分子生物学和生物化学等方法与技术手段，研究氧化苦参碱对野百合碱致大鼠肺动脉高压形成过程中肺动脉舒缩功能的保护作用；研究氧化苦参碱及其体内代谢产物苦参碱对ET-1所致离体肺动脉收缩的抑制作用等。重点观察NO合成酶抑制剂、钙离子和PKA抑制剂等对氧化苦参碱舒张肺动脉作用的影响；肺动脉中影响NO生成及信号转导的eNOS、sGC、cGMP、RhoA和ROCK等含量变化；肺动脉肌球蛋白轻链激酶磷酸化水平等，并在此基础上，进一步开展氧化苦参碱与ET-1受体阻断药或PDE抑制剂联合用药后协同作用的研究等,为今后氧化苦参碱联合用药防治肺动脉高压方案的制定提供实验依据。

目的：本研究探讨了氧化苦参碱联合他达那非或安倍生坦对野百合碱致大鼠肺动脉高压的抑制作用及其机制。方法：（1）雄性Sprague-Dawley 大鼠，按60 mg/kg/只背部皮下注射野百合碱建立肺动脉高压模型。氧化苦参碱联合他达那非或安倍生坦于野百合碱皮下注射后1hr灌胃给予，给药共28天。实验结束后，大鼠经麻醉后进行血流动力学测定；取血清、肺和右心室等组织，采用生物化学、病理、酶联免疫和Western blot等方法进行检测；（2）雄性Sprague-Dawley 大鼠麻醉后，采用压力-容积系统测定法，研究氧化苦参碱和苦参碱腹腔注射给药对心脏舒缩性能和心肌能量代谢的影响。结果：氧化苦参碱（25或50 mg/kg）+ 他达那非（10 mg/kg）和氧化苦参碱（25或50 mg/kg）+ 安倍生坦（10 mg/kg）均可抑制野百合碱所致肺动脉高压大鼠右心室收缩压（RVSP）的升高、肺脏和右心室质量的增加、右心室肥厚指数的升高，并改善右心室功能等。氧化苦参碱联合他达那非或安倍生坦抑制野百合碱致大鼠肺动脉高压的机制与增加肺组织中eNOS、GSK3α、GSK3β和ROCK1的表达，降低MMP9表达有关；氧化苦参碱联合他达那非或安倍生坦抑制野百合碱致肺动脉高压大鼠右心室肥厚的作用均与降低右心室组织中RhoA、ROCK1、ROCK2、MMP9和COX-2的表达有关。此外，氧化苦参碱（200 mg/kg）及其代谢产物苦参碱（50或100 mg/kg）可升高前负荷补充搏功、收缩末期弹性、压力导数的最大值dP/dtmax与舒张期容积关系和V0等负荷非依赖性心脏收缩性指标，提高心室效率并降低压力-容积面积、心脏潜能和心室-动脉耦合效率等心肌能量代谢参数。结论：氧化苦参碱联合他达那非或安倍生坦对野百合碱致大鼠肺动脉高压具有一定协同抑制作用，其机制与不同程度调控肺脏和右心室组织中eNOS/GSK、Rho A/ROCK和前列腺素（PG）/环氧酶（COX）等信号通路有关。此外，氧化苦参碱及其代谢产物苦参碱对整体心脏具有加强大鼠心室收缩性能，并相应协调改善心肌能量代谢的作用。