| 摘要: | 神經退化性疾病之主要致病理論乃與氧化壓力、發炎及神經損傷有關。文獻顯示柑橘的多甲氧基類黃酮 (polymethoxylated flavone, PMF) 川陳皮素 (nobiletin, NOB) 的代謝產物3',4'-dihydroxy-5,6,7,8-tetramethoxyflavone (DTF) 具高抗氧化及抗發炎活性。本研究之目的在探討DTF在體外清除自由基的能力並進一步利用大鼠腎上腺嗜咯細胞瘤 PC12 細胞培養在無血清，或含過氧化氫的培養基中，探討DTF對氧化損傷的保護功效及可能的分子機轉。
試管呈色實驗結果顯示DTF清除脂溶性DPPH• 自由基的能力遠高於DL--tocopherol，清除水溶性ABTS•+自由基的能力則與Trolox相當。相對的，其原型化合物NOB則完全無清除自由基的活性。DTF及NOB皆具保護PC12細胞抵抗無血清逆境造成的細胞凋亡的能力，並提高細胞存活率。DTF能顯著增加細胞內的 GSH的量，並誘導ARE-Nrf2的活化及下游Phase II抗氧化基因 GCLC、HO-1 的表現；相反的，NOB則無此功能。NOB及DTF 都會大量誘導ERK及Akt的磷酸化，但在誘導p38的磷酸化皆無太大改變。因此NOB的抗氧化活性可能與ERK及 PI3K/Akt的訊息傳遞路徑有關 ; 代謝物DTF的抗氧化機轉則更為複雜且多樣，可能與清除自由基的能力、ARE-Nrf2及下游的Phase II解毒基因的表現及ERK及 PI3K/Akt的訊息傳遞路徑皆有關。DTF及NOB皆能保護PC12細胞在無血清條件下受到H2O2 所引起的氧化傷害，前者效果尤佳。此結果可能與DTF具清除胞內ROS的能力及誘導Phase II 蛋白的表現有關。
Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially neural diseases. Our aim of research is to investigate the protective effect and mechanism of the metabolite of nobiletin, 3',4'-dihydroxy-5,6,7,8-tetramethoxyflavone (DTF, 3',4'-didemethylnobiletin) on oxidative damage in rat pheochromocytoma PC12 cells induced by limited serum supply and hydrogen peroxide (H2O2) in PC12 cells. It was found that DTF significantly scavenged DPPH• and ABTS•+ free radicals; while the mother compound, NOB, lacked the activity. In PC12 cells, DTF attenuated serum withdrawal-induced apoptosis, increased GSH level and diminished intracellular generation of reactive oxygen species (ROS) in response to H2O2. DTF significantly induced heme oxygenase HO-1 and glutamate-cysteine ligase catalytic subunit (GCLC) expression and enhanced nuclear factor E2-related factor 2 (Nrf2) activity, which serves as an transcription factor for HO-1 promoter and its singnaling is important for GCLC transcription. Addition of zinc protoporphyrin (Znpp), a HO-1 competitive inhibitor, significantly attenuated the protective effect of DTF in both serum-deprivation and H2O2-treated cells, indicating the vital role of HO-1 in cell resistance to oxidative injury. While investigating upstream signaling pathways, we observed that nobiletin and DTF induced sustained extracellular signal-regulated protein kinase 1/2 (ERK1/2) and modest activation of PI3K/Akt in PC12 cells grown in serum-free medium. Addition of U0126, a highly selective inhibitor of MEK1/2, which is upstream of ERK1/2, significantly decreased the cell viability, indicating possible involvement of ERK singaling in cytoprotection. Taking together, the above findings suggest that DTF can augment cellular antioxidant defense capacity through upregulation of HO-1 and GCLC expression as well as PI3K/Akt and MAPK signals. |
