花生四烯酸在大白鼠胚胎大腦皮質神經細胞與大白鼠腎臟髓質嗜鉻
腫瘤細胞之神經毒性作用
The neurotoxicity of arachidonic acid on the primary neuronal cell culture and PC12 cells
中文摘要
花生四烯酸 (arachidonic acid)是許多重要不飽和脂肪酸之一，它是細胞膜之 主要成分之一，也是重要的二級訊息傳遞物質，參與了許多生理及病理現象。過 去的研究指出，在海馬迴神經細胞培養上,高劑量之 AA 其經由 lipoxygenase pathway 所產生的代謝會造成細胞死亡，由此可知 AA 在中樞神經細胞毒性上 扮演了重要的角色。而本實驗室過去也在大腦皮質神經細胞培養上發現相似之情 形，但是 lipoxygenase 抑制劑並無法完全阻斷 AA 的毒性。為了更加了解 AA 對神經細胞生存與發育的影響，與產生毒性作用的機轉和造成細胞死亡的方式，
我們使用大白鼠胚胎大腦皮質細胞與 PC12 細胞來探討 AA 造成細胞毒性的機 制，與是否走向 apoptosis。結果發現，在大白鼠腦部皮質神經細胞體外培養 3-5 天 AA 並不會透過活化 protein kinase C 與 cannabinoid receptor 產生 毒性作用，顯然 AA 是經由 cycloxygenase，lipoxygenase，或 epoxygenase 其中一個 pathway 產生毒性，而在體外培養 12-15 天其部分毒性可能有透過 活化 protein kinase C 的途徑。又 AA 會代謝成 anandamide(ANA)，我們想 知道 ANA 是否牽涉在 AA 的毒性機制中。而結果顯示 ANA 會造成神經毒性作 用，在 DIV3─5 天細胞上顯然並不是經由活化 protein kinase C 或
cannabinoid receptor 之途徑，然而在 DIV12─15 天，抑制掉
cycloxygenase，lipoxygenase，與 epoxygenase pathway 有增加毒性之 現象。而抑制掉 cannabinoid receptor 或合併抑制 cycloxygenase，
lipoxygenase，epoxygenase pathway 與 PKC 或 cannabinoid receptor 也是使毒性增加。
在 AA 對 PC12 細胞的影響方面，結果顯示在沒有神經生長因子 (NGF) 的狀態 下，AA 會造成細胞毒性作用(EC50=191.9±17.97)，劑量愈高毒性愈大且使 細胞數減少(EC50=67.07±6.804)。而 TUNEL assay 也證實 AA 毒性作用會 造成細胞走向 apoptosis 的趨勢。
[3H]thymidine incorporation 的數據顯示，AA 300mM 顯著抑制細胞 之[3H]thymidine incorporation，而 1-100mM 之 AA 則增加
[3Hthymidine incorporation。因此，1-100mM 之 AA 可以促進細胞之 DNA synthesis，也就是說 1-100mM 之 AA 可能可以促進細胞增生的作用。
在給予 NGF 之 PC12 細胞上，加入各濃度 AA 處理後發現除了 AA1mM 之外，
均隨劑量增加造成細胞死亡程度、走向 apoptosis 之比例增加(EC50=216.6±
15.15)。
對於以 NGF 合併 AA 處理 PC12 細胞七天後，可以增加由 NGF 所誘發趨向分 化細胞比例的這種現象，可能是由於細胞繼續複製增生的能力減少，而細胞趨向 分化的能力增加，或是已分化完全的細胞進行 apoptosis 的速率降低所導致。
而在給予 NGF 之 PC12 細胞上，AA 並無法影響 PC12 細胞 neurite
outgrowth，原因可能是 AA 可以促進分化之 initiation process，但卻比較無 法影響分化之 maturation of neurite framework。這與過去本實驗室發現 naloxone 對細胞之影響類似。
英文摘要
Polyunsaturated fatty acids are integral structural components of membrane phospholipids, where they play an important role in maintaining the structural and functional characteristics of bilayer cell membranes. Arachidonic acid (AA) is one of the most important fatty acids in biological systems including the brain, since it can serve as a precursor of a number of biologically active substances such as
prostaglandins, leukotrines and thromboxanes. AA acts as an intracytoplasma second messenger in a variety of physiological or pathological condition, including the hypoxia-ischemia neuronal degeneration. Previous studies indicated that high concentration of AA produce overt neuronal cell death in culture and this toxic effect could be partly inhibited by lipoxygenase inhibitor. Our previous study also shows the similar data on primary neural cell culture, suggesting that the metabolites of AA in this pathway may be hazardous for neurons. Recently, anandamide has been identified as a brain AA derivative that actives cannabinoid receptors and protein kinase C. We further explore the mechanism underlying the AA-mediated toxicity using primary neuron cell culture and explore the effect of AA on cell survival, proliferation, and differentiation on pheochromocytoma PC12 cells. Mixed type cortical neuronal cultures were derived from E15-17 embryos of Spraque-Dawley rat embryos. On DIV3 and DIV12 cells were co-application of AA and various inhibitors for 2 hours. LDH in the culture medium were measured 24 hours after the treatment to serve as a quantification assay of cell death. Our results show that combine treatment with aspirin (cycloxygenase inhibitor), nordihydroguaiaretic acid (lipoxygenase inhibitor) and miconazole (epoxygenase inhibitor) could partly inhibit the AA induced toxicity in DIV3 cells but had no effect on DIV12 cells. Treatment with H7 (protein kinase C inhibitor) or AM281 (cannabinoid receptor antagonist) had no effect on AA or ANA induced toxicity on DIV3 cells. However,H7 partly decreased AA toxicity on DIV12 cells and no effect on DIV3 cells. We further determined the toxic effect of AA on the differentiated PC12 cell induced by nerve growth
factor(NGF). AA concentration-dependent produced cell death. Tunnel assay studies
showed that the AA-induced cell death including apoptosis. On the other hand, low concentration of AA (1mM) increased the induction of differentiation of PC12 cell by NGF stimulation but had no effect on the neurite growth of PC12 cells.
