四氢生物蝶呤对高血压左室舒张功能的影响及机制

Heart failure with normal left ventricular ejection fraction (HFNEF) is one consequence of hypertension and is caused by impaired cardiac diastolic relaxation. Nitric oxide (NO) is a known modulator of cardiac relaxation. Hypertension can lead to a reduction of NO in cardiac cells, in part because NO synthase (NOS) becomes uncoupled when oxidative depletion of its cofactor tetrahydrobiopterin (BH4) occurs. NOS activity could arise by supplement of BH4. Guanosine triphosphate cyclohydrolase-1 (GTPCH-1) is the rate-limiting enzyme when BH4 oxidated to dihydrobioptein (BH2) which competitive inhibit BH4. This reaction can be reversed by dihydrofolate reductase (DHFR). We hypothesize that nicotinamide-adenine dinucleotide phosphate (NADPH) activity, as well as the expression of DHFR and GTPCH-1 will lead to uncoupled NOS and diastolic dysfunction by mediating the generation of BH4. We also suppose that S-glutathionylation in cadiac cells can neutralize ventricular diastolic dysfunction through reversion of cardiac NOS uncoupling.This study will be performed in a desoxycorticosterone acetate (DOCA)-salt hypertensive mice model(male mice with unilateral nephrectomy, with subcutaneous implantation of a controlled-release deoxycorticosterone acetate pellet, and given 1% saline to drink are mildly hypertensive and have diastolic dysfunction in the absence of systolic dysfunction).All DOCA mice are radimized postoperative when they have evidence of diastolic dysfuction,to either 5mg or 10mg of BH4 for 7 days. Left ventricular diastolic function is evaluated by pulsed-wave tissue Doppler and invasive pressure-volume measurements.Cardiac ventricular myocytes are isolated and analyzed from the hearts of DOCA-salt mice and from age matched controls using a modified enzymatic digest protocol.NADPH activity in cardiac tissue and cells will be measured using western blot analysis.We will assay neuronal NOS and endothelial NOS monomers with SDS-PAGE western blot analysis. Cardiac superoxide anion, BH4 and BH2 will be measured with a dihydroethidium-based highperformance liquid chromatography assay. The expression of DHFR and GTPCH-1 will be measured using a enzyme immunoassay. Furthermore, we will detect the concentration of oxidized glutathione/reduced glutathione in cardiac tissues and cells using a fluorescence labeled enzyme-linked immunosorbent assay,to prove the effect of glutathionylation modifications for myocardium diastolic dysfunction and its association with NOS. We intend to prove cardiac oxidation, independently of vascular changes, can lead to uncoupled cardiac NOS and diastolic dysfunction. BH4 may represent a possible treatment for diastolic dysfunction since it involve in the NOS uncoupling. Our study also intend to domenstrate the role of DHFR, GTPCH-1 and glutathionylation modifications in mechanism of HFNEF to insight the future studys about the potential targets for revising cadiac distiolic dysfunction in hypertension.

高血压致心脏损害早期为左室射血分数正常心力衰竭(HFNEF)，机制不明且疗效不佳。前期研究发现HFNEF与一氧化氮合酶(NOS)解偶联有关，其辅助因子四氢生物蝶呤(BH4)有一定疗效。我们假设还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)、二氢叶酸还原酶（DHFR）和鸟苷三磷酸环化水解酶-1(GTPCH-1)降低可通过BH4途径促NOS解偶联；转录后谷胱甘肽氧化修饰可逆转NOS解偶联,延缓HFNEF。本研究用高血压致HFNEF小鼠模型(DOCA盐敏感小鼠)，两种剂量BH4干预。组织多普勒及有创压力容积法评价左室舒张功能。Western-blot等法测定心肌组织一氧化氮、NOS与NADPH；高效液相色谱等法评价BH4及代谢产物。酶免疫法检测DHFR和GTPCH-1表达,并测定心肌还原及氧化型谷胱甘肽含量。进一步阐明高血压致HFNEF机理及外源性BH4通过抑制NOS解偶联改善心脏舒张功能的机制。

本项目研究四氢生物蝶呤（BH4）对醋酸脱氧皮质酮（DOCA）-盐型高血压小鼠左心室舒张功能及蛋白激酶C ε（PKC ε）表达的影响，及其与磷脂酰肌醇3激酶/磷酸化蛋白激酶B(PI3K/p-Akt)信号通路的关系。以期为探索左室舒张性心功能不全的治疗提供理论依据。研究采用10周龄雄性C57BL/6小鼠随机分为手术组与对照组。手术组小鼠切除左侧肾脏后并在颈部皮下埋入醋酸脱氧皮质酮（DOCA）药片，术后以普通饲料及1.05% 氯化钠（NaCl）溶液饲养，至术后14天，随机分为DOCA组DOCA+BH4组，并分别用生理盐水（每天每只0.1mL/10g）和2.5%的BH4溶液（每天每只0.1mL/10g）灌胃7天。分别进行以下实验测定：1.鼠尾动脉压测定小鼠收缩压（SBP）及舒张压（DBP）。2.应用彩色多普勒超声心动图二维成像与组织多普勒成像技术测定各组小鼠结构与心功能参数。3.血流动力学测定。所有亚组均于术后22天时进行取材，所取组织均行以下实验测定：酶联免疫吸附实验测定环磷酸鸟苷（cGMP）、丙二醛(MDA)以及超氧化物歧化酶(SOD)；高效液相色谱法(HPLC)测定心肌组织中BH4及二氢生物蝶呤(BH2)的含量；放免法测定血浆血管紧张素II（Ang II）含量；实时荧光定量聚合酶链反应测定心肌组织中α-SA、TGFβ1、Prkce及β-MHC的含量；免疫组化测定VEGF、PLB、PKC-zeta、Serca2、BCL-2、PLB及Bax；Western-blot测定eNOS与PKCε的表达。结果显示：高血压时，体内NOS解偶联、氧化应激水平升高、RAAS系统激活，心肌细胞肥厚增生，肌纤维排列紊乱，心肌顺应性下降，这些均可能导致高血压左室舒张功能不全的发生；左室舒张功能不全可以先于收缩功能不全出现；体内PKC ε表达减少，给予外源性BH4干预后PKC ε表达增加；外源性BH4可以通过PKC ε途径改善左室舒张功能不全，其具体机制可能与PKC ε能增加NO表达、降低体内氧化应激水平及抑制RAAS活性有关。此外，BH4可以改善高血压引起的左室舒张功能不全，降低心肌组织的氧化应激，对高血压患者早期引起的心功能损害具有保护作用。而此作用可能与PI3K/Akt信号有关系，至于BH4通过其他信号通路介导影响心肌组织中NO的水平的表达，而改善心室的舒张功能，尚需要进一步研究证实。