| Abstract: | 在心臟血管疾病中，動脈粥狀硬化（Atherosclerosis）是一種系統性的疾病，為主要的致死原因。根據研究證據顯示，許多的因素參與在動脈粥狀硬化的致病過程。而關於動脈粥狀硬化的眾多推論中，低密度脂蛋白（LDL）受到的氧化修飾作用（oxidative modification）及血管內膜（intima）平滑肌細胞的增生作用，被視為是早期動脈粥狀硬化的致病原因。如有物質能保護LDL不受氧化修飾影響並能抑制血管平滑肌細胞的增生，則能有效預防動脈粥狀硬化的發生。類黃酮素（Flavonoids）為自然界常見的多酚化合物（polyphenolic compounds），廣泛的存在植物、蔬果中，具有很強的抗氧化能力。因此本研究之目的在於探討桑葉水萃取物（MLE），對於抗LDL氧化及抑制血管平滑肌細胞增生的影響。
從銅離子誘導LDL進行體外氧化實驗的模式中發現，添加桑葉水萃取物後，LDL受到氧化修飾所造成的Apo B衍蛋白斷裂現象及LDL蛋白表面電荷改變的程度明顯降低，而脂質過氧化產物MDA的生成量亦減少，且清除1,1-diphenyl-2-picrylhy drazyl（DPPH）自由基的作用佳。因此證實桑葉水萃取物具有抗LDL氧化的能力。而在抑制血管平滑肌細胞增生的實驗方面，過去本實驗室已證實桑葉水萃取物（MLE）會誘導血管平滑肌細胞進行凋謝死亡（apoptosis）。在較低的桑葉水萃取物（MLE）劑量下又是怎樣的作用機轉？因此本論文將著重在桑葉萃取物是否能抑制血管平滑肌細胞細胞週期（cell cycle）的進行與細胞移動（migration）的能力？首先，在不誘發細胞凋亡的劑量下（1~1.5 mg/ml），桑葉水萃取物（MLE）能有效的抑制細胞的生長；flow cytometry 分析下也證實血管平滑肌細胞在處理桑葉水萃取物（MLE：1 mg/ml）之後S 與G2/M phase明顯減少，而G1 phase大量的增加。在西方墨點法的分析方面， cyclinD1和CDK4蛋白以及phospho-p53（p-p53）、p27、p21、p16表現有增加的情形，而其他cyclinA、cyclinE、CDK2、phospho-Rb（p-Rb）等蛋白表現都呈現降低趨勢；免疫沉澱法（Immunoprecipitation）的分析中証實過多的cyclinD1和CDK4實際結合情形隨著萃取物的劑量增加而減少，表示萃取物係透過干擾動脈平滑肌細胞細胞週期進行到S phase的機轉進而抑制細胞的增生。另外，傷口癒合（Wound healing assay）與Boyden chamber migration assay實驗中得知桑葉水萃取物（MLE）亦能抑制細胞轉移，在MMP2/9的分析以及西方墨點法分析small GTPase、phospho-FAK（p-FAK）、NF-κB等蛋白，皆呈現遞減的趨勢。
綜合上述結果顯示，桑葉萃取物同時兼具抗LDL氧化及抑制血管平滑肌細胞異常增生與轉移的作用。推測其具有保護心臟血管的功能，可作為自然保健產品的開發。
Atherosclerosis is the leading cause of death in the cardiovascular disease. It is a systemic disease, a huge number of studies have revealed several risk factors involving in the progression of atherosclerosis. In early atherogenesis, oxidative modification hypothesis of atherosclerosis proposes that LDL oxidation and proliferation of vascular smooth muscle cell (VSMC) in the intima plays a causative role. If agents that can prevent LDL oxidation and proliferation of VSMC, it could possibly attenuate the development of atherosclerosis.
Flavonoids are a group of naturally occurring polyphenolic compounds ubiquitously found in plants, fruits and vegetables. It has shown potential antioxidative effects. In this study, we evaluated the effects of mulberry leaf extracts (MLE) to inbibit LDL oxidation and VSMC proliferation.
In the Cu2+-induced LDL oxidation model, we observed that MLE were able to apparently reduce the Apo B fragmentation, relative electrophoretic mobility (EM) and the products of lipid peroxide-MDA formation. MLE possessed the best ability of DPPH radical scavenging. Taken together, MLE showed a strong potency to inhibit the LDL oxidation induced by Cu2+. In our previous studies, in cultured cells experiments, have demonstrated that MLE could induce VSMC apoptosis. Nevertheless, does the lower dose of MLE effect In this study, we focus on that is MLE able to inhibit VSMC on cell cycle progression and migration First, MLE effectively inhibit VSMC growth on the dose of 1~1.5 mg/ml MLE which can’t induce cell apoptosis. In the experiments of flow cytometry assay, we also evaluated the proportion of S and G2/M phase of cell cycle were decreased, but G1 phase were increased abundantly on treatment VSMC with 1.0 mg/ml MLE. The western blot have revealed that cyclinD1, CDK4, p-p53, p27, p21, p16 were increased and cyclinA, cyclinE, CDK2, p-Rb were reduced. In the experiments of immunoprecipitation, we analyzed attenuation of cyclinD/CDK4 complexes following the advancement of MLE dose. It means MLE inhibited cell proliferation by interferencing the mechanism of VSMC cell cycle progress to S phase. In addition, the best ability of MLE inhibited VSMC migration by wound healing assay and Boyden chamber assay. The expression of MMP-9, MMP-2, small GTPase, p-FAK and NF-κB were decrease progressively by gelatin zymography assay and westerb blotting.
In conclusion, mulberry leaf extracts (MLE) possess ability to inhibit LDL oxidation and abnormal proliferation of vascular smooth muscle cell. Therefore, it is suggestive the mulberry leaves that may have multiple benefical effects on cardiovascular health, and the potential clinical application of these fascinating natural substance. |
