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    標題: 山奈酚及鼠李檸檬素保護 PC12 細胞對抗氧化傷害
    Kaempferol and rhamnocitrin protect PC12 cells against oxidative stress
    作者: 洪靖婷
    Jing-Ting Hong
    貢獻者: 吳明娟
    嘉南藥理科技大學:生物科技研究所
    關鍵字: 山奈酚
    鼠李檸檬素
    MAPK路徑
    HO-1
    MAPK pathway
    rhamnocitrin
    kaempferol
    日期: 2008
    上傳時間: 2008-12-29 15:21:20 (UTC+8)
    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,因此在預防保健或治療上,可透過補充或投予具抗氧化、抗發炎的膳食或藥物。山奈酚 (kaempferol) 和鼠李檸檬素 (rhamnocitrin, kaempferol 7-O-methylether) 是植物中含量極高的黃酮醇及其甲基衍生物,具高抗氧化及抗發炎活性。本論文之目的在探討山奈酚 (kaempferol) 和鼠李檸檬素 (rhamnocitrin) 在體外清除自由基的能力,並進一步以老鼠腎上腺嗜鉻細胞瘤 PC12 細胞為模式探討此兩個化合物在細胞缺乏血清及添加H2O2的逆境中保護的幼臚峊i能參與的分子機轉。
    試管實驗顯示山奈酚 (kaempferol) 和鼠李檸檬素 (rhamnocitrin) 皆具有清除 xanthine/xanthine oxidase 產生的超氧自由基之能力,且前者的清除能力約為後者的十倍。兩者亦能有效清除ABTS•+ 及Fenton reaction 產生的氫氧自由基。細胞培養實驗顯示,山奈酚 (kaempferol) 能抵禦PC12 細胞在無血清環境所誘導的細胞凋亡,因而提高其細胞存活率。山奈酚(kaempferol) 和鼠李檸檬素 (rhamnocitrin) 皆明顯的降低PC12 細胞受到 H2O2 所誘導產生的活性氧(ROS),因而減少細胞毒性。血紅素加氧酶(heme oxygenase-1, HO-1) 的誘導與PC12細胞抵抗各種逆境相關。以RT-Q-PCR分析發現山奈酚 (kaempferol) 和鼠李檸檬素 (rhamnocitrin) 能顯著誘導HO-1的mRNA表現。添加HO-1的酵素活性抑制劑 Zinc protoporphyrin (Znpp) 後,山奈酚 (kaempferol) 與鼠李檸檬素 (rhamnocitrin) 保護 PC12 細胞抵抗H2O2氧化傷害的能力亦隨之降低。
    目前已知缺乏血清會造成PC12細胞的絲裂原活化蛋白激酶路徑 [Mitogen-activated protein kinases (MAPKs) pathway] 的extracellular signal-regulated kinase 磷酸化程度下降;然而,缺乏血清或添加H2O2皆會誘導c-Jun NH2-terminal kinase (JNK) 及p38的磷酸化。在極低血清的培養基中,添加山奈酚 (kaempferol) 可強烈且持續的誘導 ERK1/2 磷酸化,同時也可降低p38的磷酸化,因此具有使細胞生長、增生且避免細胞凋亡的能力。鼠李檸檬素 (rhamnocitrin) 雖只能短暫性的誘導 ERK1/2 的磷酸化,但是可有效且持續降低p38 的磷酸化,因此亦有增加PC12細胞對抗氧化壓力的能力。
    Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially neural diseases. One of the effective ways to prevent the reactive oxygen species (ROS) mediated cellular injury is dietary or pharmaceutical augmentation of free radical scavengers. Our aim of research is to investigate the free radical scavenging activities and the protective effects and mechanisms of kaempferol and rhamnocitrin (kaempferol-7-methyl ether) on oxidative damage in rat pheochromocytoma PC12 cells induced by serum deprivation and hydrogen peroxide.
    Kaempferol and rhamnocitrin strongly scavenged ABST free radicals, xanthine/xanthine oxidase-generated superoxide radicals and Fenton reaction-generated hydroxyl radicals in vitro. Cell culture experiment revealed that kaempferol (40-80 M) reduced PC12 cell death caused by serum deprivation as determined by MTT. Flow cytometry further demonstrated that kaempferol decreased the ratio of sub-G0/G1 indicating it has anti-apoptotic effect. Kaempferol and rhamnocitrin (40-80 M) significantly decreased H2O2-induced cytotoxicity. Concomitantly, intracellular generation of reactive oxygen species (ROS) in response to H2O2 was also diminished by these two compounds, suggesting a ROS-scavenging activity of kaempferol and rhamnocitrin by attenuating intracellular H2O2 level in PC12 cells.
    Induction of heme oxygenase-1 (HO-1) expression has been associated with adaptive cytoprotection against a wide array of toxic insults in PC12 cells. Kaempferol and rhamocitrin significantly induced HO-1 gene expression as determined by RT-Q-PCR. Addition of a HO-1 competative inhibitor, Zinc protoporphyrin (Znpp), reduced the protective effects of kaempferol and rhamocitrin on H2O2-treated PC12 cells.
    It has been demonstrated that serum deprivation results in decreased phosphorylation of extracellular signal-regulated kinase (ERK); while both serum deprivation and H2O2 induce phosphorylations of c-Jun NH2-terminal kinase (JNK) and p38, of the family of mitogen-activated protein kinases (MAPKs). Western blotting demonstred that kaempferol induced sustained ERK phosphorylation, the activation of which induced neuronal proliferation and suppressed apoptosis when PC12 cells were cultured in serum-free medium. On the other hand, rhamnocitrin stimulated only transient ERK cascade. Both kaempferol and rhamnocitrin were able to persistently attenuate serum-deprivation induced p38 phosphorylation, the activation of which would cause cell death. These results demonstrate that kaempferol and rhamnocitrin can directly scavenge H2O2, induce HO-1 expression and regulate MAPK signal transduction, thereby protecting PC12 cells from oxidative-stress-induced cell death.
    關聯: 校內校外均不公開
    Appears in Collections:[生物科技系(所)] 博碩士論文

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