動脈粥狀硬化對於冠狀動脈疾病及中風是一潛在機制，此多發性因子疾病的主要表徵乃是慢性發炎。在動脈粥狀硬化的發展病程中，平滑肌細胞會由收縮態轉變為合成態，並有細胞增生及移行的情形。小分子G 蛋白RhoA 及其下游作用分子Rho 激酶在平滑肌收縮的調節扮演重要角色。亦有報告指出，由RhoA 所調控的肌動蛋白聚合作用會使得平滑肌細胞分化標記的基因表現增加。目前已有研究指出，Rho 激酶與動脈粥狀硬化的發展有關。在動脈粥狀硬化的發展病程中，Rho 蛋白激酶的活性是否增加，目前為止並不十分清楚，因此本實驗的目的在探討Rho 蛋白激酶的抑制對apoE 基因剔除小鼠動脈粥狀硬化的病程發展與收縮力的影響。
本實驗採用apoE 基因剔除母鼠，共分為二組，分別為：一、實驗組，每天給予腹腔注射Rho 激酶抑制劑Y27632 (12.5mg/kg)。二、對照組，每天給予腹腔注射生理食鹽水。兩組動物皆餵食含有0.15%膽固醇的高脂飼料，經過4 週、10 週及20 週後將其犧牲。利用Oil Red-O 染色方法來分析胸主動脈的動脈粥狀硬化粥瘤損傷區域，並進行影像量化分析。在4 週、10 週的Y27632 處理組與對
照組相比，是沒有差異的。在4 週為1.3 ± 0.38% 與1.0 ± 0.36 %；在10 週為5.6 ± 1.78 % 與8.7 ± 2.48%；但在20 週的Y27632 處理組較對照組，有顯著減少(p<0.05)。在20 週為14.5 ± 2.10% 與23.7 ± 2.53%。將H＆E 染色的組織切片，定量分析的結果顯示， 20 週的實驗組內膜層的面積較對照組明顯地減少(p<0.05)，而在4 週及10 週的處理組，兩組之間並無顯著差異。顯示長期處理
Rho 激酶抑制劑Y27632，能有效抑制動脈粥狀硬化的形成及血管內膜層的增厚。為偵測Rho 激酶的抑制對於動脈粥狀硬化組成細胞的影響，我們利用免疫組織染色，染具巨噬細胞專一性的標記蛋白macrophage-specific marker F4/80，發炎反應相關因子NFκB p65 subunit,以及平滑肌細胞的標記蛋白Smooth muscle specific α-actin。結果發現，在偵測巨噬細胞及發炎反應相關因子NFκB p65 的表現方面，相同時間的實驗組與對照組相比較，實驗組標定巨噬細胞及NFκB p65染色的程度較對照組來的淡。在偵測平滑肌細胞專一性標誌蛋白smooth musclespecific α-actin 方面，在20 週處理組，實驗組較對照組的α−actin 表現程度則高。
- 4 -
此外我們藉由給予α1−adrenergic 受體致活劑(receptor agonist) phenylephrine 或thromboxane A2 類似物(analogue) U46619 刺激主動脈環引起收縮反應，來探討長期處理Rho 激酶抑制劑Y27632 對主動脈收縮力的影響。另外以乙醯膽鹼(acetylcholine, ACh)所誘發之血管舒張反應，探討Rho 激酶的抑制對主動脈舒張的影響。我們的實驗結果發現，經過20 週的實驗組，由U46619 所引起之主動
脈收縮曲線較對照組明顯右移，其血管收縮力顯著地受抑制(P<0.05)；但由phenylephrine 所引起之主動脈收縮曲線，在兩組之間則無差異。而在血管舒張曲線方面，同時間的實驗組與對照組相比較，則無差異。另外，為偵測Rho 激酶的抑制對於發炎反應的影響，進行血清中與脂肪相關的生化數值分析及介白素-6(Interleukin-6, IL-6)的測定。總膽固醇(TC)、三酸甘油脂(TG)、高密度脂蛋白(HDL-c)、低密度脂蛋白(LDL-c)、極低密度脂蛋白(VLDL)的濃度值在實驗組或對照組，隨著年齡增長皆有穩定性減少的趨勢。實驗組較對照組的生化數值似乎來得高，但兩組間並無差異。血清中促發炎的細胞素IL-6 的濃度，在20 週實驗組的濃度明顯地低於對照組。顯示長期處理Rho 激酶抑制劑Y27632，能有效抑制發炎反應。
由我們的實驗結果顯示，Rho 蛋白激酶的抑制可減緩ApoE 基因剔除母鼠動脈粥狀硬化的病程發展及發炎反應，並選擇性地抑制平滑肌細胞的收縮力。

- 1 -
Atherosclerosis, the underlying mechanism for coronary artery disease
and stroke, is a multifactorial disorder characterized by chronic
inflammation. An important hallmark of atherosclerosis is the increased
proliferation and migration of smooth muscle cells (SMCs) associated
with the transformation from contractile to synthetic phenotype. The
changed SMC phenotype is characterized by altered expression levels of
differentiation markers. The small GTPase RhoA and its downstream
effector Rho kinase are thought to be key players in the regulation of
smooth muscle contraction. It was recently reported that RhoA-mediated
actin polymerization played a major role in regulating gene expression of
SMC differentiation markers. Rho kinase was recently reported to be
involved in the development of atherosclerosis. Whether Rho kinase
activation is increased during atherogenesis remains unknown. This study
was designed to investigate the effects of long-term Rho kinase inhibition
on atherosclerosis development and contraction-relaxation profile of
apoE-deficient mice. ApoE-deficient mice were fed with cholesterol
-containing diet for 4, 10 or 20 weeks with or without daily peritoneal
injection of a Rho kinase inhibitor Y-27632 (12.5 mg/kg). Atherosclerotic
lesion area of thoracic aorta assessed by Oil red-O staining was not
different between Y27632-treated and saline control groups after 4-week
(1.3 ± 0.38% vs. 1.0 ± 0.36 % for Y27632-treated and control,
respectively) or 10-week treatments (5.6 ± 1.78 % vs. 8.7 ± 2.48%), but
was significantly smaller after the 20-week Y27632 treatments (14.5 ±
2.10% vs. 23.7 ± 2.53%, p<0.05). Effects of Y27632 treatment on
inflammatory responses and smooth muscle cell phenotypes were
investigated with immunostaining of a macrophage-specific marker F4/80,- 2 -
anti-αsmooth muscle actin and anti- NFκΒ p65 subunit. Macrophage
infiltration and NFκΒ expression were significantly decreased following
a 20-week Y27632 treatment. Contraction profiles of aortic rings
stimulated with two vasoconstrictors, α1-adrenoceptor agonist
phenylephrine and thromboxane A2 mimetic U46619, and relaxation
induced by acetylcholine were examined. After 20 weeks of Y27632
treatment, U46619-stimulated contraction was shifted to the right
compared to the control while that of phenylephrine was not different
between two groups. No difference in relaxation profile was detected in
two groups throughtout the treatment period. Lipid profiles and serum
concentrations of proinflammatory cytokine interleuken-6 (IL-6) were
analyzed. The concentrations of total cholesterol (TG), high density
lipoprotein (HDL-c), low density lipoprotein (LDL-c) and very low
density lipoprotein (VLDL-c) in Y27632 treatment and control groups
both decreased with aging, no difference in lipid profiles was detected
between two groups. In contrast, serum concentrations of IL-6 was
significantly decreased after 20-week Y27632 treatment compared to the
control. These results demonstrate that long-term inhibition of Rho kinase
decreases atherosclerosis progression and inflammatory responses and
modulate smooth muscle contractility in ApoE-deficient mice.

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