- 學位論文
桑椹萃取物抑制低密度脂蛋白氧化，與抑制血管平滑肌細胞 轉移及增生作用
Mulberry extracts inhibit LDL oxidation, vascular smooth muscle cell migration and proliferation.
摘要
近年來，科學家在追尋抗氧化、抗衰老的過程中，發現在一些高等植物中所含之多酚化合物，具有抗氧化作用，眾多研究證實藉由抗氧化作用可用來抑制LDL的氧化修飾，是一種有效預防動脈粥狀硬化發生的方法。心血管疾病與癌症為現代人生命健康的二大殺手，而心血管疾病中動脈粥狀硬化（Atherosclerosis）是主要的致死原因。而關於動脈粥狀硬化的眾多推論中，1.低密度脂蛋白（LDL）在血管內膜（intima）受到的氧化修飾作用（oxidative modification）、2.動脈平滑肌細胞受血管內膜中發炎因子的刺激而從血管中層 (media) 轉移到血管內膜（intima）及3.血管內膜（intima）動脈平滑肌細胞過度的增生作用，被視為是早期動脈粥狀硬化的一些致病原因。如有天然物質能保護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。 第一部分：首先分離桑椹水萃取物（MWEs）及其中所含之多酚化合物（MPEs）探討其抗氧化與抑制泡沫細胞（foam cells）形成的能力。本研究證據顯示桑椹葉與桑椹果粒中含有豐富類黃酮素與酚酸，具有抗氧化作用。從銅離子誘導LDL進行體外氧化實驗的模式中發現，添加桑椹果粒萃取物後，LDL受到氧化修飾所造成的Apo B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，且具有清除1,1-diphenyl-2-picrylhy drazyl（DPPH）自由基的作用；而這樣的現象在多酚萃取物的一組更為明顯，更加確認了這些萃取物具有強的抗氧化能力。除此之外；在本實驗中証實桑椹萃取物能有效抑制巨噬細胞scavenger receptors（CD36、SR-A）的表達，進而達到抑制泡沫細胞的形成。 第二部分：此本研究之目的在於探討桑椹果粒的水萃取物（MWEs）、多酚萃取物（MPEs），誘導血管平滑肌細胞（VSMC）程序性凋亡（apoptosis）的影響、並觀察是否能抑制血管平滑肌細胞轉移到血管內膜（intima）。在細胞實驗方面，發現桑椹萃取物會誘導過度增生狀態的VSMC凋謝死亡（apoptosis）。從western blot的結果顯示：與凋謝死亡相關的蛋白（apoptotic-related proteins）如：p-p53、Myc、Bax、Fas、FasL、cytochrome c、caspase 3,8,9、MAPK family（p-SEK、p38、p-JNK、p-Jun），在桑椹萃取物處理後的細胞中呈現增加的趨勢。相對的抗凋謝死亡的相關蛋白（anti-apoptotic related proteins）Bcl-2、Mcl-1、PI3K、Akt、NF-κB等，表現量則都明顯下降。推測桑椹萃取物主要在於抑制anti-apoptotic related proteins的表達進而誘發細胞產生apoptosis。 第三部分：利用較低的劑量來探討桑椹萃取物對VSMC增生的影響，Flow cytometry分析結果發現，VSMC受萃取物的影響下，細胞週期有停滯於G1 phase的現象。在西方墨點法（Western blotting）的分析方面，phosspho-p53（p-p53）、p27、p21、p16表現皆有增加的情形；透過免疫沉澱法（Immunoprecipitation）分析得知cyclin D1/CDK4、cyclin A、E/CDK2及p53/Mdm2蛋白實際結合情形隨著萃取物處理時間增加而減少。最後證實，桑椹萃取物可藉由iNOS誘導血管平滑肌細胞NO產生並活化AMPK，藉此機轉而誘導VSMC細胞週期停滯。 第四部分：利用不影響生長的劑量觀察桑椹萃取物對於VSMC移動能力的影響；傷口癒合（wound healing assay）、Boyden chamber分析的結果中，都發現明顯抑制的現象，進一步用Zymography assay、western blot、EMSA（Electrophoretic mobility shift assay）分析，證實是桑椹萃取物透過降低NF-κB轉錄活性進而降低Matrix metalloproteinases-2（MMP-2）基因的表現；另外FAK、RhoA、Rac-1、Cdc42、Ras調節細胞移動與細胞骨架組裝的蛋白，其蛋白表現也受到抑制。最後在Ras轉殖的一連串實驗中證實，桑椹萃取物可透過iNOS與AMPK的活化造成Ras蛋白的分解，進而達到抑制VSMC移動的能力。 第五部分：利用Ras轉職誘導VSMC老化的模式中，發現桑椹多酚萃取物能有效抑制老化指標β-Galactosidase的活性，並恢復老化VSMC細胞週期的運行。最後證實桑椹萃取物是透過AMPK活化而抑制VSMC中Ras的過度表現，而達到減緩Ras所誘導的細胞老化。表示桑椹萃取物透過抑制VSMC細胞老化的現象，可以減少血管斑塊不穩定與剝離的機會。 綜合上述結果顯示，桑椹萃取物（MWEs與MPEs）同時兼具抗LDL氧化及抑制血管平滑肌細胞增生的作用、而在極低的劑量下就能成功的預防血管平滑肌細胞的轉移與老化。推測其具有良好保護心臟血管的功能，可作為將來自然保健產品的開發與應用。
並列摘要
Atherosclerosis, a disease occurring in arteries, is one of the primary causes of heart diseases and is often the cause of death. Previous studies have shown that atherosclerosis is closely r elated to oxidative low-density lipoprotein dk3u3d. The oxidative modification hypothesis proposes that low density lipoprotein (LDL) oxidation and foam cell formation. Mulberry, a local edible fruit of Morus alba L., is used effectively in traditional medicines against fever, hepatic damage, rheumatic arthritis and hypertension. But few studies have investigated in detail the mechanism and effects of the mulberry fruit against atherosclerosis. In recent studies, the oxidative modification of LDL plays a key role in the pathogenesis of atherosclerosis. In this study, we evaluated two extracts, MWEs (mulberry water extracts) and MPEs (mulberry polyphenol-rich extracts ), which exhibited antioxidative ability in vitro. The antioxidative activity of the mulberry extracts on LDL oxidation was defined by relative electrophoretic mobility (REM), fragmentation of Apo B, thiobarbituric acid reaction substances (TBARS), and radical scavenging assay. Our results showed that low doses of MPEs were able to reduce the REM, Apo B fragmentation, and MDA formation in Cu2+-mediacted LDL oxidation model. MWEs and MPEs also had strong ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging. Further, we demonstrated that mulberry extracts could inhibit the foam cell formation and inhibit the expression of the macrophages scavenger receptors (CD36, SR-A) expression induced by oxLDL. Mulberry extracts showed strong potency for scavenging radical, and inhibiting the LDL oxidation and foam cell formation. We have demonstrated that the mulberry water extracts (MWEs) can effectively inhibit LDL oxidation and reduce the development of atherosclerosis in cholesterol-fed rabbits, suggesting that these extracts may prevent atherosclerosis by reducing early atherogenesis. The mechanisms by which mulberry extracts reduce the development of atherosclerosis are not yet fully understood. In these experiments, MWEs and MPEs could inhibit the migration and proliferation of ASMCs (A7r5 cells) by down regulation of Ras/PI3K/Akt pathway. MWEs and MPEs caused cell cycle arrest by inducing AMPK activation and attenuating of cyclin D/CDK and p53/Mdm2 complexes. At highly concentration of the extracts, MWEs and MPEs induced apoptosis by down regulation anti-apoptosis proteins strongly.In conclusion, we not only evaluated the strong antioxidative activity of the polyphenol extracts, but also observed MWEs and MPEs can inhibit proliferation and migration of ASMCs by decreace the transcription activity of NF-κB, FAK, small GTPase, and Ras protein, which were facilitated by integrin receptor. Therefore, it is suggested that the mulberry could be a healthy food to prevent individuals from atherosclerosis .