| Summary: | 博士 === 中山醫學大學 === 生物化學研究所 === 91 === 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.
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