豬腦酒精萃取物對過氧化氫誘導PC12神經細胞損傷之保護作用

Protective effects of swine brain ethanol extracts on H2O2-induced damages in PC12 cells

施彥合(Yen-Ho Shih)

指導教授 ： 陳億乘

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201700501

摘要

目前認為失智症的發生和氧化壓力造成腦損傷有很大的關聯性。PC12具有神經細胞之特性，是一株適合用以誘導氧化壓力上升和模擬失智症產生的細胞。磷脂質為神經細胞之重要化合物，高齡和失智症患者其腦內所含之磷脂質皆較年青人和正常人少。動物副產品中亦含有高量磷脂質，已有研究說明補充磷脂質具有改善老鼠之學習和記憶力之能力。
第一部份的結果顯示，經酒精萃取後，在動物副產物中，豬腦擁有最高之產率。以HPLC分析發現酒精萃取物中豬腦酒精萃取物(swine brain phospholipid, SBEE)亦有最適當之磷脂質組成份，同時也擁有萃取時間和方便性較佳之優點，因此選用豬腦酒精萃取物(SBEE)進行第二部份之實驗。確認PC12細胞型態為具有神經細胞特性之細胞株後，將0.031、0.125、0.5、2 mg/mL之SBEE對PC12細胞處理6小時後測定細胞存活率和乳酸脫氫酶釋放量，發現並不會對細胞造成毒性，但可以減緩和150 μM H2O2 處理下6小時之細胞毒性(p<0.05)，而在細胞型態中亦有發現SBEE處理具有減緩150 μM H2O2所造成PC12細胞型態破裂之情形。更進一步測定了細胞的氧化狀態指標TBARS、reduced GSH量和TEAC值以及抗氧化酵素SOD、CAT和GPx，皆可以發現SBEE具有能力減緩150 μM H2O2所造成之氧化壓力(p<0.05)。細胞發炎激素TNF-α、IL-6和IL-1β以及nitrite分泌量也在添加SBEE之組別中顯著下降(p<0.05)。在促細胞凋亡基因表現的部份亦可使iNOS、Bax、Cytochrome c、Caspase9、Caspase8和Caspase3之表現量下降(p<0.05)，同時使Bcl-2之表現量有上升的趨勢。綜觀上述，動物副產物中以豬腦進行酒精萃取具有較佳之產率、萃取效益和磷脂質組成，在SBEE處理下PC12細胞具有對抗氧化壓力之能力，亦可減緩發炎激素之釋放和使細胞凋亡相關基因之表現量降低，而這也顯示於細胞存活率和細胞型態之結果。

關鍵字

氧化壓力 ； 失智症 ； H2O2 ； PC12細胞 ； 抗氧化 ； 抗發炎 ； 抗細胞凋亡

並列摘要

The dementia is not only high incident with cerebral cortical atrophy as well as oxidative-induced damage but also coupled with the phospholipid loss in the brain. It was supposed that daily phospholipid supplementation could enhance the membrane stability of brain neuron cells, thus reducing the dementia occurrence. In comparison with other animal byproducts, brain, liver, and heart tissues are rich in phospholipids. Hence, the objectives of this study were to find out a suitable phospholipid source from animal byproducts and investigate if the extracted phospholipids own protection against oxidative stress via a neuron-like cell, PC12 cell.
The results showed that swine brain has the highest yield and phospholipid contents (p<0.05), compared to broiler brain and liver, and so are processing convenience, shorter processing time, and higher slaughtered amount. In the second part of the experiment, there were no (p<0.05) differences among viability and lactate dehydrogenase release of PC12 cells after treating 0.031, 0.125, 0.5, 2, and 4 mg/mL swine brain ethanol extraction (SBEE), but SBEE ameliorated (p<0.05) them of PC12 cells treated with 150 μM H2O2. Meanwhile, the morphological observation showed the similar results. Furthermore, TBARS, reduced GSH, and TEAC values of PC12 cells treated 150 μM H2O not;2 were improved (p<0.05) by adding SBEE, and so were the antioxidant enzyme (SOD, CAT, and GPx) activities (p<0.05). Moreover, the SBEE addition decreased (p<0.05) the inflammatory cytokines, i.e. TNFα, IL-6, IL-1β, and nitrite level of PC12 cells treated with 150 μM H2O2. Regarding apoptosis-related gene expressions of PC12 cells, the SBEE downregulated (p<0.05) iNOS, Bax, Caspase3, Caspase8, Caspase9, and Cytochrome c expressions in PC12 cells treated with 150 μM H2O2, but the Bcl-2 expression was upregulated (p<0.05).
In conclusion, swine brain can be a suitable candidate for the phospholipid extraction with the higher yield and phospholipid content. Furthermore, SBEE also alleviates oxidative damage, reduces the inflammation cytokine, and decreases the apoptosis-related gene expressions, thus ameliorating viability and lactate dehydrogenase release in PC12 cells treated with 150 μM H2O not;2. Altogether, our SBEE can be a potential healthy ingredient for neuron cells against oxidative stress in the niche market.

並列關鍵字

anti-apoptosis ； anti-inflammation ； antioxidative ； dementia ； H2O2 ； oxidative stress ； PC12 cells ； phospholipids

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