| Abstract: | 天然的抗氧劑中具有抗動脈粥狀硬化形成的有Vit E、Vit C、beta-carotene，這些已經很清楚能使LDL氧化情形減少或減低氧化型LDL被噬入的作用。在先前的研究中，我們已經發現原兒茶酸 (PCA) 和洛神花水萃取物 (HSE) 對於氧化型LDL所表現的負電荷增加 (lipogel electrophoresis) 及脂肪酸氧化 (TBARS) 有抑制氧化的作用，在本實驗中我們以PCA和HSE進一步探討其對抗LDL氧化的作用強弱。我們以10 mM CuSO4與100 mg protein/ml 的LDL在37℃下共同培養 12小時誘發總膽固醇量降解，當加入不同劑量的PCA或HSE同時作用，我們發現0.1 mg/ml 的HSE使總膽固醇降解的現象被回復18.15%， 而0.5 mg/ml 的HSE則回復了67.25%；PCA則是隨著濃度的增加，總膽固醇降解也出現逐漸被回復的現象，在0.05 mg/ml 的濃度下總膽固醇降解的現象約被回復74.96 %。而在分析OxLDL片段化的情形時，我們發現隨著HSE的濃度增加，ApoB片段化的現象逐漸被回復，在0.5 mg/ml 的濃度下ApoB片段化的現象被回復近乎 100%；而PCA則在0.03及0.05 mg/ml 的濃度下ApoB片段化的現象皆約被回復100 %。藉此，我們推測PCA與HSE的抗LDL氧化的能力，應可以在預防動脈粥狀硬化的發生上扮演一個重要的角色。
由於第一部分的研究中已經發現原兒茶酸 (PCA)和洛神花水萃取物 (HSE) 具有抑制LDL氧化的作用，因此在本部分中我們將巨噬細胞共同處理原兒茶酸或洛神花水萃取物以及100 mg/ml氧化型LDL (OxLDL)，藉此來觀察細胞的變化。我們發現OxLDL會引起程度嚴重的細胞死亡，而此種死亡經過 Leukostat染色及DNA 片段化 (TUNEL assay) 的測試後，證實此種細胞死亡為程式性死亡 (apoptosis)。當處理不同劑量的PCA或HSE，則發現細胞死亡的情形可隨著PCA或HSE的濃度增加而呈現良好的抑制，在 1.0 mM的PCA或 0.5 mg/ml HSE所處理的組別甚至可使細胞型態與控制組一致。而在apoptosis的指標測定包括 PARP 蛋白斷裂及Caspase-3 蛋白活性，可發現 PCA或HSE可依劑量增高而使PARP 蛋白斷裂被抑制至 100% 或 93%；另外在 Caspase-3 蛋白活性的測定上，則發現 PCA或HSE可抑制74.2% 或72.3%。另外，我們也利用 migration assay 發現不管是PCA或HSE除了可以直接抑制OxLDL的被巨噬細胞攝入之外，也可能與巨噬細胞自己有某種交互作用，藉此使巨噬細胞不因OxLDL的存在而死亡。在前人研究中指出巨噬細胞吞噬 OxLDL後將可進一步將其分解，倘若細胞吞噬 OxLDL後即死亡，將無法分解 OxLDL；且大量死亡的巨噬細胞可能加速脂肪條塊 (fatty streak) 的形成而演變為動脈粥狀瘤。由於這些結果，我們推測PCA或HSE應可保護巨噬細胞在攝入 OxLDL 後免於死亡，如此將可使細胞能充分分解 OxLDL，藉此將可減緩動脈粥狀硬化的進程而達到預防動脈粥狀硬化的作用。
在上述機轉的探討方面，我們首先探討細胞在處理過OxLDL及抗氧化劑後，細胞內有關細胞凋亡蛋白的變化。細胞粒線體的電位差在處理過OxLDL後下降為控制組的27.4%，而處理HSE和PCA的組別則於最高劑量時可使膜電位恢復為100% 及104%。而此時因為氧化壓力而誘發粒線體膜蛋白、MAPK蛋白及P53改變，致使細胞趨向於凋謝死亡。然而，處理HSE或PCA的組別 (特別是高濃度組) 則可改變這些蛋白的表達以使細胞趨向生存。另一方面，我們也測定細胞內外的維生素E及自由基的含量，以及HSE或PCA對LDL於細胞內外氧化抑制的情形，結果發現高濃度的HSE與PCA分別可以減緩細胞內外LDL氧化的程度，而這種抑制LDL氧化程度的作用應是來自於增加細胞內維生素E的含量與有限程度的減緩自由基的危害而達成。綜合本研究結果推論，這兩種抗氧化劑在胞內外均具有減低氧化壓力的能力，而這種作用將可減緩OxLDL造成巨噬細胞死亡的情形。在這種情形下，HSE及PCA將可運用這種機制以達到減緩動脈粥狀損傷的作用，而這正是HSE及PCA在預防心血管疾病上的一種重要的效能。
The natural antioxidants, such as Vit E, Vit C and beta-carotene, has been found to possess the ability to avoid LDL oxidation or reduce oxidized LDL (oxLDL) uptaken by macrophage; and furthermore, they were able to prevent the formation of atherosclerosis. In our previous studies, we explored that both protocatechuic acid (PCA) and the aqueous extract of its original plant, Hibiscus Sabdariffa (HSE), showed strong inhibitory effects on the negative charge increasing of protein moiety in oxLDL (lipogel electrophoresis) and on the lipid peroxidation of lipid moiety in oxLDL (TBARS). At present, we further investigate the effects of PCA and HSE on LDL oxidation。We incubated 10 mM CuSO4 and 100 mg protein/ml of LDL at 37℃ for 12 hrs to induce the degradation of cholesterol. When cotreating with various concentrations of PCA or HSE, the results showed that 0.1 mg/ml of HSE recover the degradation of cholesterol by 18.15% and 0.5 mg/ml of HSE by 67.25% compared to native LDL. On the other side, the more concentrated PCA showed more significant recovery of cholesterol degradation. At the concentration of 0.05 mg/ml, the degradation of cholesterol was recovered by 74.96 %. On the investigation of ApoB fragmentation, HSE recovered the fragmentation gradually depending on the increasing concentrations. Moreover than, the ApoB fragmentation recovered almost by 100% at the concentration of 0.5 mg/ml. When cotreating with PCA, the similar situation also occurred. At the concentration of 0.03 and 0.05 mg/ml, ApoB fragmentation was recovered by 100 %. Taken above together, we suggested that PCA or HSE should play an important role on preventing atherosclerosis via inhibiting LDL oxidation.
In part I, we found that protocatechuic acid (PCA) and the aqueous extract of Hibiscus Sabdariffa (HSE) possessed the abilities to inhibit LDL oxidation. Because of that, in this study, OxLDL was added to RAW264.7 macrophages to further investigate the effects of PCA or HSE. When treating with 100 mg/ml OxLDL, the cell was induced severe death. Under the demonstrations of Leukostat stain and DNA fragmentation (TUNEL assay), we proved that the cell death was apoptosis. When co-treating with PCA or HSE, the apoptosis was able to reduce in a dose-dependent. At the concentration of 1.0 mM PCA or 0.5 mg/ml, the cell morphology showed almost same as the control. We further detect PARP protein cleavage and Caspase-3 activity to confirm the inhibitory effects of PCA or HSE on macrophage apoptosis. The levels of PARP protein cleavage was able to decrease to 100% by 1.0 mM PCA and 93% by 0.5 mg/ml HSE; and Caspase-3 activity was also inhibited by 74.2% or 72.3% when co-treating with 1.0 mM PCA or 0.5 mg/ml HSE. In migration assay, we found that the inhibitory effect of PCA or HSE attributed to reduce the uptake of OxLDL by macrophage, and some interactions between PCA/HSE and macrophage. Some researchers pointed that the OxLDL-laden macrophages possessed the ability to digest OxLDL. However, when macrophage uptaking too much OxLDL, they would die, and they would not digest OxLDL. Furthermore, the abundant death macrophages could accelerate the formation of fatty streak to process to atheroma. According to these results, we suggested that PCA or HSE was able to protect macrophage from dead when uptaking OxLDL; and that could make macrophage to digest OxLDL and further decelerate atherosclerosis. Under the circumstance, PCA and HSE were able to prevent atherosclerosis.
According to the above, we observed the inhibitory effects of HSE and PCA on OxLDL-induced macrophage apoptosis. Based on that, we further investigated the mechanisms here. Firstly, we surveyed the changes of apoptotic proteins, such as mitochondrial proteins, MAPK proteins and P53. When treating with OxLDL in macrophages, the mitochondrial potent lowered to 27.4% compared to control (the group of native LDL). Interestingly, the group co-treating with HSE and PCA in highest concentrations recovered the mitochondrial potent to 100% and 104%. At this moment, the mitochondrial proteins, MAPK proteins and P53 were changed because of the extreme oxidative stress, and more over than, this stress would make cell to trigger to apoptosis. However, co-treated with HSE or PCA (especially the groups in high concentrations) would change the expressions of these proteins and make cell survive. In addition to, we also detected the contents of vitamin E and free radicals existing in cell inside and in media. By the way, either in cell inside or in media, we examined the inhibitory level of OxLDL by co-treating HSE or PCA. We found that high concentrations of HSE and PCA were able to decelerate the level of LDL oxidation, and the inhibitory effects could be contributed by increasing vitamin E and by decreasing the content of free radicals. Taken the above together, these two antioxidants possessed the abilities to reduce oxidative stress in either cell inside or environment (media). These inhibitory effects would slow down the macrophage apoptosis induced by OxLDL. Under the circumstance, HSE and PCA were able to prevent the blood vessel from forming atherosclerotic lesions, and further prevent vascular diseases. |
