花青素配基對大鼠神經膠C6細胞於缺氧時之保護

Abstract

腦缺血時，因為氧氣供應不足，會進一步形成腦缺氧狀態。在缺氧環境中，人體腦部容易產生過多的自由基，形成氧化壓力，並導致腦細胞受傷甚至死亡。 花青素是一群能夠對抗體內過多自由基的天然成份，並有增強血管彈性、增加循環、預防心血管病變等功效，其結構由花青素配基（anthocyanidin）和不同糖類組合而成。本實驗利用低亞硫酸鈉（Na2S2O4）建立細胞缺氧模式，並使用5種酚酸（沒食子酸、咖啡酸、香豆酸、肉桂酸、阿魏酸）、3種多酚（兒茶素、檞皮素、芸香素）、5種花青素配基（天竹葵素、花翠素、牡丹素、矢車菊素與錦葵素）於缺氧環境下對大鼠神經膠細胞株C6之保護效果。分別將處於指數生長期的神經膠C6細胞分別與酚酸、多酚和花青素配基預培養24小時，接著加入Na2S2O4並置於厭氧箱（1％ O2、5％ CO2、94％ N2）中缺氧培養30分鐘後，以MTT試驗計算細胞存活率並測量細胞抗氧化酵素：超氧化物歧化酶（Superoxide Dismutase, SOD）及觸酶（Catalase, CAT）活性與穀胱甘胺酸（Glutathione, GSH）濃度。 實驗結果顯示，以酚酸或多酚與神經膠C6細胞預培養的組別，在有氧培養下，對於神經膠C6細胞並無明顯毒性（濃度3.125 mg/L 至50 mg/L）；但在缺氧損傷下，其細胞存活率均下降，與無添加的控制組並無差異，僅有50 mg/L與100 mg/L咖啡酸具有較顯著的保護效果，顯示使用的酚酸與多酚對於神經膠C6細胞對於缺氧所造成的損傷並無保護效果。而高濃度（100 mg/L與200 mg/L）的花青素配基，與神經膠C6細胞預培養24小時後造成細胞數目的減少；在缺氧損傷下，以濃度25 mg/L的花青素配基與神經膠C6細胞預培養24小時後，細胞存活率和有氧控制組相比均高於74％，尤以delphnidin可維持最高存活率（達92％），而未加入花青素配基預培養的實驗組別缺氧培養後細胞存活率降至47％至59％，可知缺氧損傷確實可對細胞以產生損傷甚至死亡的效果。 另在缺氧損傷後，與花青素配基預培養的細胞中，其過氧化氫酶活性與穀胱甘胺酸濃度均高於未與花青素配基預培養。而與malvidin、peonidin和pelargonidin預培養24小時的組別在缺氧模式下，其神經膠C6細胞內SOD活性高於未與花青素配基預培養的組別。顯見以花青素配基與神經膠C6細胞預培養，可提升抗氧化酵素等的活性，達到神經保護的效果。

Hypoxia happens during various pathophysiologic conditions such as ischemia and promotes the generation of reactive oxygen species (ROS). The increase in ROS levels may lead to the occurrence of oxidative stress. Oxidative stress has long been associated with the development of brain cell damage and neurodegenerative disorders. Anthocyanins occur widely in plants. They are well-known free radical scavengers against oxidative damage in the cell and are reported as a potential chempreventive agent. The anthocyanidins (or aglycons) are the basic structures of the anthocyanins. The anthocyanins consist of an anthocyanidin and various sugar moiety. The present study investigated the neuroprotective effect of phenolic acid (including gallic acid, caffeic acid, coumaric acid, cinnamic acid, ferulic acid) and polyphenol (including catechin, quercetin, rutin) and anthocyanidins (including cyanidin, delphinidin, malvidin, pelargonidin and peonidin) against hypoxia in C6 glial cells. The cells were incubated with a medium containing phenolic acid, polyphenol, anthocynidin in normoxia condition first, and then with a medium containing sodium dithionite (Na2S2O4) in an anaerobic incubator for the hypoxia treatment. Exponentially-growing cells were pre-incubation with each of the phenolic acids, polyphenol and anthocyanidins for 24 hour. A 200 μL aliquot of 5 mM Na2S2O4 was added to each well for the generation of hypoxia stress. The plates were then incubated at 37°C for 30 min in an anaerobic incubator with 1% O2, 5% CO2, and 94% N2 atmosphere. Methylthiazole tetrazolium test and the evaluation of antioxidant enzymes activities and glutathione concentration were performed on the treated cells. Pre-incubation of the cells with phenolic acid or polyphenol at concentration from 3.125 mg/L to 50 mg/L did not cause cell toxicity. The viability of cells pre-incubated with a phenolic acid or a polyphenol was reduced in the hypoxia treatment significantly. Pre-incubation of the cells with caffeic acid at 50 and 100 mg/L concentration may improve the viability in the hypoxia treatment. The concentration of an anthocyanidin up to 100-200 mg/L may cause cell toxicity. Generally, at least 74% of the C6 glial cells pre-incubated with an anthocyanidin at 25 mg/L survived the hypoxia treatment. Among the five anthocyanidins tested, delphinidin at 25 mg/L concentration showed the highest protective effect on C6 cells against the hypoxia treatment, by retaining more than 92% of the cell viability as compared with a survival rate in between 47~59% for the hypoxia control that was pre-incubated without an anthocyanidin. The cells pre-incubated with any among the five anthocyanidins showed higher catalase activity and glutathione concentration after the hypoxia treatment as compared with the corresponding samples without the pre-incubation with anthocyanidin. The cells pre-incubated with malvidin, pelargonidin or peonidin also showed higher superoxide dismutase activities. The results of this study justify further research for the development of anthocyanidins into neuroprotective food ingredients against hypoxia injury.