三氧化二砷 (As2O3) 是一個有毒的化合物質，可以通過腦血管障壁並且導致許多神經系統的干擾疾病，例如多發性神經炎及腦電波圖異常等。過去已有研究指出，三氧化二砷是藉由細胞自我吞噬反應的發生使得神經膠質瘤細胞株產生死亡。然而其機制仍不清楚。硫辛酸（α-lipoic acid）已被認為是一種有效的抗氧化物。藉由處理硫辛酸可以提升腦內細胞的麩胱苷肽含量，並且保護因為暴露三氧化二砷所造成的硫氫基減少。而硫辛酸對於三氧化二砷所誘發的神經毒性的保護機制尚未被研究過。本研究的目的主要是想了解三氧化二砷對於神經細胞所誘發的毒性機制以及前處理硫辛酸的保護機制。實驗以細胞模式進行，細胞前處理硫辛酸再處理三氧化二砷，利用流式細胞儀觀察細胞凋亡的發生，利用acridine orange染色看細胞自我吞噬反應的典型產物酸性小胞器(AVO)發生，利用西方點墨法分析細胞自我吞噬反應或細胞凋亡的上游調控蛋白的改變。研究中發現，神經膠質瘤細胞處理三氧化二砷之後產生細胞自我吞噬反應。同時, Hsp70、Bcl-2、 PI3K及 p-42/44 MAPK表現增加。然而p53, p-Akt(Ser)表現下降。而前處理硫辛酸可以預防三氧化二砷對於神經膠質瘤癌細胞所造成的毒性影響及細胞內總硫氫基的含量減少。此外，前處理特定的AKT抑制劑LY294002，可以更進一步的增加三氧化二砷誘發膠質瘤癌細胞的細胞自我吞噬反應，然而，前處理MAPK的特定抑制劑 U0126不會影響三氧化二砷誘發神經膠質瘤細胞的細胞自我吞噬反應。綜合以上兩點，三氧化二砷誘使神經膠質瘤細胞的細胞自我吞噬死亡可能是藉由AKT訊息調控路徑。前處理硫辛酸可以顯著的減少三氧化二砷誘使的細胞毒性及細胞自我吞噬細胞死亡。

Arsenic trioxide (As2O3) is a toxic compound; it could cross the blood-brain barrier and cause many nervous system disturbances such as polyneuropathy, EEG abnormalities, etc. It has been reported that As2O3 led to autophagic cell death in malignant glioma cells. However, the mechanisms of autophagy in glioma cells are not clear. α-lipoic acid (LA) has been shown to be a potent antioxidant. Intracellular glutathione levels in brain could be elevated by treatment of LA which protects the sulfhydryl groups in the reduced form when exposed to arsenic. The aim of this study is to investigate the mechanisms of arsenic trioxide-induced toxicity and the preventive potency of LA in glioma cells. Glioma cells were pretreated with LA followed by treatment of As2O3. To detect and quantify apoptotic cells, flow cytometry analysis was performed. To observe the expressions of acidic vesicular organelle (AVO) which is characteristic of autophagy, cells were stained with acridine orange. To analyze the expression of up-stream regulated proteins related to autophagy or apoptosis, western blotting was performed. Our results indicated that after treated with As2O3, autophagic but not apoptotic cell death was induced in glioma cells. Meanwhile, Hsp70、Bcl-2、PI3K and p-42/44 MAPK expression were increased. Whereas, p53, p-Akt(Ser) expression were reduced. Pretreatment of LA could prevent the toxic effects induced and cellular total SH group reduced by arsenic trioxide in glioma cells. In addition, pretreated with LY294002, a specific inhibitor of AKT, further enhanced the As2O3-induced autophagic effect on glioma cells. However, pretreated with U0126, a specific inhibitor of MAPK, didn’t affect the autophagic effect of glioma cells induced by As2O3. Taken together, As2O3-induced glioma cell autophagic cell death might through AKT regulated signaling pathway. Pretreated with LA could dramatically attenuate the cytotoxicity and autophagic cell death induced by As2O3.

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