低密度脂蛋白(low density lipoprotein ; LDL)的氧化會增加動脈粥狀硬化的發生機率，先前的文獻證實copper具有氧化LDL的能力，它能造成濃度相關的LDL氧化狀況，如增加LDL的electrophoretic mobility (增加LDL的陰電性)，亦有文獻證明nitric oxide donor-sodium nitroprusside有讓LDL負電荷增加的能力。而天然抗氧化劑中具有抗動脈硬化形成的有Vit E 、Vit C和beta-carotene， 這些已經被清楚知道能使LDL氧化情形減少，也就是使relative electrophoretic mobilities (REM)減少與thiobarbituric acid-reactive substances (TBARS)減少且呈dose-dependent現象。在本篇論文中，我們以原兒茶酸(Protocatechuic acid; PCA)和馬栗樹皮素(Esculetin; ECT)這兩種天然抗氧化劑為主要題材，以REM及TBARS來檢測其對氧化型LDL的作用為何？我們利用Beckman lipo gel測得PCA與ECT對於氧化型LDL的REM有很明顯的抑制現象，由TBARS也證實有很強的抗氧化作用。更進一步利用巨噬細胞試驗，亦得到一致的結果，也就是PCA與ECT能抑制LDL的氧化進而減少動脈粥狀硬化產生的機率。而另一部份，我們針對此二種抗氧化劑抑制氧化型LDL的特性而進行機轉的探討，在機轉的探討上，則著重於訊息傳遞蛋白表現的探討，結果發現氧化型LDL作用時會引起細胞內Akt及Bcl-2蛋白減少表現，如此的結果應會使細胞進行apoptosis (細胞程序性死亡)，這也是現今引起動脈粥狀硬化的學說之一：氧化型LDL引起細胞死亡，而死亡細胞會在血管壁上堆積，長期的堆積結果造成動脈粥狀硬化。而加入PCA後，細胞內Akt及Bcl-2蛋白表現量會隨劑量增加而增加，因此在PCA預防心血管疾病的機轉可能是：PCA會降低氧化型LDL引起的細胞死亡，藉此減低死亡細胞在血管壁上的堆積，進而減少動脈粥狀硬化的發生。 The oxidation of low-density lipoprotein (LDL) could increase the incidence of atherosclerosis. In previous studies showed that copper and nitroprusside possessed the ability to oxidize LDL in dose-dependent condition. They increased the negative charge existing in LDL and increased the electrophoretic mobility. On the other side, many antioxidants were observed to lower the formation of atherosclerosis such as Vit.E, Vit C and -carotene. These antioxidants reduced the relative electrophoretic mobilities (REM) and thiobarbituric acid-reactive substances (TBARS) induced by oxidative LDL in dose-depentently. In this thesis, we used both protocatechuic acid and esculetin to define the antioxidative activity in oxidative LDL by REM and TBARS. The data had showed that both of the antioxidants possessed strong antioxidative activity in oxidative LDL. Furthermore, the RAW264.7 macrophage cells were used to detect the effect of PCA in oxidative LDL-existed. The same results showed that PCA could inhibit the oxidative LDL. In the other part, we would like to discover the antioxidative mechanism of PCA to reduce oxidative LDL. In the results showed that oxidative LDL could induced the reduction of Bcl-2, Akt and some proteins working in MAPK signaling pathway. These reduced proteins could increase cell apoptosis, and death cell could accumulate on the vessel wall to progress atherosclerosis. When PCA pretreating, Akt, Bcl-2 and MAPK proteins increased in dose-dependently. Taken together, the mechanism of atherosclerosis prevented by PCA may be concluded that PCA reduced the apoptosis induced by oxidative LDL and lowered the progression of atherosclerosis.
