| Summary: | 碩士 === 高雄醫學大學 === 醫學研究所 === 91 === Excessive ethanol consumption may increase the production of reactive oxygen species (ROS) and free radicals, which results in the damage of cells and organs. Antioxidants, however, can decrease the effects of ethanol-induced oxidative damage. In this study, we evaluated the effects of melatonin and whey protein concentrate （WPC）on ethanol-induced oxidative stress in PC12 cell line. Melatonin, a hormone secreted from the pineal gland, acts as maintaining circadian rhythms, as scavenging directly free radicals and as stimulating several antioxidative enzymes. Many studies demonstrated that mice fed with WPC exhibited a marked increase in the production of glutathione (GSH). GSH is of major significance in cellular antioxidant system because it participates directly in the destruction of reactive oxygen species and modulates the immunity of cells and promotes the effect of anticancer drug on cancer treatment. In this study, we adopted a pheochromocytoma (PC12) cell line as an in vitro neuronal cell model to establish the lethal concentration of 50%（LC50）as an in vitro model of evaluation through the treatment with different concentrations of ethanol and durations. After the addition of different levels of WPC and melatonin for 24 hours, the cell culture was treated with a ethanol solution of 150 mM for 4 hours and the parameter including the cell viability, the percentage of lactate dehydrogenase released（% LDH released）, GSH level, the activities of antioxidative enzymes〔superoxide dismutase （SOD）, glutathione reductase（GRx）〕and protein carbonyl were assayed. 1. The effects of melatonin：The results showed that the supplement of 10μM melatonin could elevate the cell viability and decrease the ethanol-induced cytotoxicity of PC12 cell line in ethanol-treated group as compared with those of non- melatonin. In the level of GSH, the results showed that the melatonin could promote the production of GSH in PC12 cell line. In addition, the activity of SOD of PC12 cell line showed nonsignificant difference after the treatment of melatonin. Nevertheless, the activity of GRx of PC12 cell line showed lower activity after 1000μM melatonin treatment. The finding of lower activity of GRx may be explained that did not need more the function of GRx which could reduce the GSSG（oxidized GSH） to GSH, because the high concentration of melatonin neutalize the attack of free radical. In the level of protein carbonyl, the results showed that melatonin, in a moderate concentration（10μM）, could decrease the level of protein carbonyl in PC12 cell line after acute ethanol exposure. 2. The effects of WPC：The results showed that the supplement of WPC could not elevate the cell viability, but could decrease the ethanol-induced cytotoxicity of PC12 cell line in ethanol-treated group as compared with those of non- WPC. In the level of GSH, the results showed that the WPC could promote the production of GSH in PC12 cell line. The elevated activity of GRx, however, occurred after supplement of higher concentration of WPC, which may be one of pathway of GSH production.
In conclusion, we showed that melatonin and WPC, in a moderate concentration, could be a protective agent to alleviate ethanol-induced oxidative stress in PC 12 cell line.
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