KMUP-1促進高血壓鼠之NO/cGMP及抑制低氧型肺高壓之ROCK/VEGF訊息途徑

KMUP-1 Potentiates NO/cGMP Signaling Pathway in Hypertension and Inhibits ROCK/VEGF Signaling Pathway in Hypoxic Pulmonary Arterial Hypertension

王怡雅(Yi-Ya Wang)

指導教授 ： 陳英俊

高雄醫學大學/醫學院/藥理學科研究所/碩士(2008年)

摘要

自發性高血壓會損害內皮系統功能，使得血管收縮造成血壓上升。在正常的內皮細胞中，若活化endothelial nitric oxide synthase (eNOS)，soluble guanylyl cyclase (sGC)，以及protein kinase G (PKG) 表現，會導致血管平滑肌舒張，促使血壓下降。實驗中所使用的KMUP-1是以xanthine及piperazine為基本骨架的化學衍生合成物；先前文獻已證實，KMUP-1會活化NO/ sGC/ cGMP路徑，促使血管舒張。    本實驗主要選取8週大的自發性高血壓大鼠 (Spontaneously hypertensive rat；SHR) 為實驗動物，並以Wistar-Kyoto (WKY) 大鼠做為SHR大鼠的對照組別，將實驗動物分成五組：(1) W1 (WKY 第一組：WKY對照組，未給予藥物治療)，(2) W2 [WKY 第二組：給予KMUP-1 (10 mg/kg) 治療]，(3) S1 (SHR 第一組：SHR對照組，未給予藥物治療)，(4) S2 [SHR 第二組：給予KMUP-1 (10 mg/kg) 治療]，(5) S3 [SHR 第三組：給予KMUP-1 (30 mg/kg) 治療]。在持續給予KMUP-1治療28天內，每週定時使用尾動脈壓測量法測量大鼠全身血壓，進而探討KMUP-1對於WKY、SHR大鼠血壓的影響，並瞭解KMUP-1對於高血壓的治療效果。在給予KMUP-1 28天治療後，取下大鼠主動脈進行西方點墨法分析，討論高血壓中eNOS、sGCα1、PKG的蛋白質表現，以及給予KMUP-1治療後的差異。    實驗結果顯示WKY、SHR大鼠血壓的表現，的確會隨著週齡的增長而有所增加。比較WKY與SHR大鼠的血壓表現，SHR大鼠血壓明顯高於WKY大鼠。此外，結果指出給予KMUP-1 (10 mg/kg) 不會顯著影響WKY大鼠血壓。然而，給予KMUP-1 (10 mg/kg，30 mg/kg) 顯著改善SHR大鼠的血壓表現。關於大鼠主動脈蛋白質表現方面，在給予KMUP-1治療後，無論是WKY或SHR大鼠主動脈中eNOS、sGCα1、PKG蛋白質表現皆顯著增加。綜觀以上得到本篇結論為：KMUP-1可以透過活化NO/cGMP路徑，達到降低自發性高血壓大鼠的血壓，未來可建議做為治療高血壓的藥物。長期暴露在低氧環境下，會造成心血管循環功能及構造上的傷害。而造成心肺血管病理性的改變，包含內皮傷害、血管增生以及血管收縮。低氧會降低endothelial nitric oxide synthase (eNOS) 表現，也會活化Rho kinase (ROCK)，血管內皮生長因子 (vascular endothelial growth factor；VEGF) 表現，使肺血管收縮、血管增生，已證實會誘導產生肺動脈高血壓。在正常內皮的eNOS，soluble guanylyl cyclase (sGC) 和protein kinase G (PKG) 蛋白質表現，會使肺動脈血管舒張、減少肺動脈壓及減少血管增生。先前文獻指出，本實驗所使用的KMUP-1屬於xanthine、piperazine所衍生出的化學合成物，會活化NO/ sGC/ cGMP路徑，進而使血管達到舒張效用。    本研究中，將實驗動物Wistar大鼠分成四組：(1) 正常組別；(2)在低氧環境 (10% O2) 21天；(3) 在低氧環境 (10% O2) 21天，同時給予藥物KMUP-1 (5 mg/kg/day) 治療；(4) 在低氧環境下 (10% O2) 21天，同時給予藥物Sildenafil (5 mg/kg/day) 治療。低氧環境下21天後，測量其平均動脈壓、平均肺動脈壓、及心跳速率。犧牲後的大鼠，取下其右肺及心臟，以Hematoxylin-Eosin染色法，來探討肺動脈相對血管壁壁厚，及心臟右心室肥大情形。此外，透過西方點墨法及免疫組織染色，討論與血管收縮作用相關的蛋白質改變。實驗結果指出，低氧環境會增加大鼠的肺動脈壓、增加肺動脈血管的相對壁厚、以及使右心室肥大。而在分子機轉上，低氧環境的確會使eNOS、sGCα1、PKG蛋白質表現減少；ROCK II、VEGF蛋白質表現增加。然而，透過KMUP-1 (5 mg/kg/day) 或Sildenafil (5 mg/kg/day) 治療，可以改善低氧對於大鼠所造成的肺動脈壓上升、肺動脈血管管壁增厚、右心室肥大，及蛋白質表現的改變。因此，本實驗結果證實KMUP-1可透過活化eNOS、sGCα1、PKG表現，進而改變大鼠在低氧環境下所造成的肺血管收縮，促使肺動脈壓下降，達到改善肺動脈高血壓的病理狀況。未來可建議做為低氧所誘導肺動脈高血壓的用藥。

關鍵字

高血壓 ； 肺動脈高血壓

並列摘要

The endothelial dysfunction resulting in vessel contraction observed in hypertension appears to be a consequence of high blood pressure. In normal endothelial cell, activation of endothelial nitric oxide synthase (eNOS), soluble guanylyl cyclase (sGC) and protein kinase G (PKG) resulted in vasodilation and anti-hypertension. Our previous studies have demonstrated that KMUP-1, a unique xanthine and piperazine derivative, activated the NO/ sGC/ cGMP pathway, and could lead to vascular relaxation. We used 8 week-old Spontaneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) rat for our experimental model. In the present study, the experimental rats were subdivided into five groups：(1) W1 (WKY group 1：control), (2) W2 [WKY group 2：treating with KMUP-1 (10 mg/kg)], (3) S1 (SHR group 1：control), (4) S2 [SHR group 2：treating with KMUP-1 (10 mg/kg)], (5) S3 [SHR group 3：treating with KMUP-1 (30 mg/kg)]. During 28 days of treatments, systolic blood pressure (SBP) was measured weekly to confirm whether KMUP-1 could ameliorate SBP in SHR. Furthermore, we used aorta to check eNOS, sGCα1, PKG protein expression by Western blotting.Our results showed that SBP of SHR elevated more than that of WKY with age. KMUP-1 (10 mg/kg) did not significantly decrease SBP of WKY. However, SBP of SHR by treating with KMUP-1 (10 mg/kg, 30 mg/kg) was significantly decreased as compared with SHR control. Moreover, eNOS, sGCα1 and PKG protein expression in SHR or WKY aorta by treating with KMUP-1 were significantly increased. In conclusion, KMUP-1 could active NO/cGMP pathway to improve SBP of SHR, suggesting that KMUP-1 could be a potential drug for hypertension.    Hypoxia exposure induced impairment in the structure and function of cardiopulmonary circulation. The pathological changes of cardiopulmonary arteries included endothelial injury, vessel remodeling, and contraction. It has been confirmed that hypoxia promoted downregulation of endothelial nitric oxide synthase (eNOS), upregulation of Rho kinase (ROCK) and vascular endothelial growth factor (VEGF) expression resulting in vascular contraction and remodeling to induce pulmonary arterial hypertension. Furthermore, activation of eNOS, soluble guanylyl cyclase (sGC), and protein kinase G (PKG) protein expression resulted in pulmonary arterial vasodilation and anti-remodeling. Previous studies have demonstrated that KMUP-1, a unique xanthine and piperazine derivative, activated the NO/sGC/cGMP pathway, and could lead to vascular relaxation. In the present study, the Wistar rats were subdivided into four groups：(1) Normoxia, (2) Hypoxia (10% O2) for 21 days, (3) Hypoxia (10% O2) + KMUP-1 (5 mg/kg/day) for 21 days, (4) Hypoxia (10% O2) + Sildenafil (5 mg/kg/day) for 21 days. After 21 days of hypoxia, we measured pulmonary arterial pressure to evaluate the development of pulmonary arterial hypertension. Through method of Hematoxylin-Eosin staining, we investigated wall thickness of pulmonary artery and right ventricular hypertrophy. Moreover, molecular mechanism was analyzed by Western blotting and immunohistochemistry.Our findings indicated that hypoxia could increase pulmonary arterial pressure, wall thickness ratio of pulmonary artery, and right ventricular hypertrophy as well as downregulate eNOS, sGCα1 and PKG protein expression whereas upregulate ROCK II and VEGF protein expression in molecular mechanism. However, the above effects could be reversed by treating with KMUP-1 or Sildenafil. In conclusion, our study confirmed that KMUP-1 is involved in the expression of eNOS/sGCα1/PKG signaling pathway resulting in vessel relaxation and may be useful for the improvement of hypoxia-induced pulmonary arterial hypertension in the future.

並列關鍵字

hypertension ； pulmonary arterial hypertension