Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/51577
標題: S-Adenosylhomocysteine (SAH): enhancement of apoptosis,beta-amyloid formation and beta-amyliod-induced DNA damage in BV-2 cells
S-腺核苷同半胱胺酸促進BV-2細胞細胞凋亡,類澱粉斑形成及類澱粉班所誘導DNA傷害
作者: 林鴻琦
Lin, Hung-Chi
關鍵字: S-腺核苷;S-adenosylhomocysteine;同半胱胺酸;阿茲海默症;細胞凋亡;類澱粉斑形成;DNA;Alzheimer's disease;apoptosis;amyloid formation;DNA repair enzyme
出版社: 食品暨應用生物科技學系所
摘要: 
Alzheimer's disease (AD) is the most common disease in the elderly over 65 years old. Epidemiological studies have demonstrated that several factors are involved in AD, including oxidative damage and wound in brain. The mechanism of AD may involve in neuron cell apoptosis, DNA damage and the aggregation of Aβin brain. Homocysteine (Hcy) and S-adenosylhomocysteien (SAH) are produced during methionine metabolism. The plasma Hcy concentration was positively correlated with the severity of AD and cerebrovascular disease (CVD). Recent studies have shown that SAH is highly correlated with Hcy-related diseases. However, it is unclear what roles of SAH plays in these diseases. We here examined our hypothesis that SAH may play a role in AD. The mechanism may involve in apoptosis of BV-2 cells, amyloid formation and DNA damage. We found that an incubation of BV-2 cells with 1 mM Hcy, 1 μM SAH and 100 μM Ado (SAH+Hcy+Ado) led to marked apoptosis of BV-2 cells, as evidenced by several markers of apoptosis. A synergistic effect of SAH+Hcy+Ado on apoptosis was obtained, as calculated using the data of Annexin V-positive cells. This combination markedly induced intracellular levels of reactive oxygen species (ROS) starting at 6 h and significantly decreased the mitochondrial potential starting at 12 h. The combination significantly elevated caspase-9 and caspase-3 activities at 24 and 48 h. The combination also induced hypomethylation (at 24 and 48 h), as indicated by significantly decreased 5-methyldeoxycytidine levels and SAM/SAH ratios. Pre-incubation of cells with α-tocopherol (30 μM) reduced the increase of ROS (at 6 h) and significantly restored cell viability (at 24 and 48 h) in the SAH+Hcy+Ado group.
Amyloid peptide plays an important role in Alzheimer's disease (AD), which may cause AD by formation of tangle in brain. The present results showed that SAH increased A beta
protein formation in a concentration-dependent manner (10-500 nM), and this effect of SAH was accompanied by significantly increased expression of APP and PS1 proteins, although SAH only significantly increased the expression of BACE1 at the highest concentration used (500 nM). SAH (500nM) markedly induced hypomethylation of APP and PS1 gene promoters. Incubation of cells with 5'-azc (20 uM), also an inhibitor of DNA methyltransferases enhanced A beta protein expression and APP and PS1 gene promoters hypomethylation. By contrast, pre-incubation of cells with betaine (1.0 mM), 30 min followed by incubation with SAH (500 nM) or 5'-azc (20 uM) for 24 h markedly prevented the expression of beta-amyloid protein (by 50%, P < 0.05) and the gene promoter hypomethylation of APP and PS1.
Another possible mechanism in causeing of Alzheimer's disease is DNA damage. In our previous study, we found that SAH may inhibit the DNA repair capacity and induce miniscripataion to induce DNA damage of BV-2 cell. In addition, amyloid has been reported to induce DNA damage through production rexative oxygen species (ROS). Here, we examined the mechanism in DNA damage of BV-2 cells. We analyze DNA damage by using comet assay, and we found that SAH may dose-dependently increase DNA damage, further we pre-incubated with BACE1 specific inhibitor (beta-secretase inhibitor IV) for 30 min, prevented the formation of amyloid, DAN damage and generaion of ROS. Furthermore, we analyzed 8-oxo-dG content by using HPLC and we also found that SAH may dose-dependently increase 8-oxo-dG content. We then found that SAH can inhibit OGG1 protein and mRNA expression. Furthermore, SAH at the hihest concentration (500 nM) may induce OGG1promoter hypomethylation. By contrast, pre-incubation of cells with betaine (1.0 mM), 30 min followed by incubation with SAH (500 nM) for 24 h markedly increased the expression of OGG1 protein (by 50%, P < 0.05) and prevernted promoter hypomethylation of OGG1.
In summary, this dissertation research suggest that SAH may be a risk factor for Alzheimer' s disease. Our results suggest that the effect of SAH may involve a complex pathway of AD including: (1) SAH, Hcy and Ado syngerstically induce apoptosis of BV-2 cells. (2) SAH can promote the formation of amyloid peptide, and (3) SAH can enhance DNA damage of BV-2 cells by inhibition of OGG1 activity and amyloid-induced production of ROS.

阿茲海默症(Alzheimer’s disease, AD)是一種常發生在65歲年老的人身上的疾病,流行病學調查顯示,阿茲海默症發生的病因很多,如氧化壓力、腦部受到創傷等,而發生病因之機制上分為神經細胞細胞凋亡DAN傷害及類澱粉斑堆積。同半胱胺酸(Hcy)及S-腺核苷同半胱胺酸(SAH均為甲硫胺酸之代謝的產物,血漿中的Hcy跟AD與心血管疾病(cerebrovascular disease , CVD)呈現正相關;而最近研究也指出SAH與Hcy相關疾病中扮演重要角色,但是SAH在此中扮演何種角色及其發生可能機制,目前尚不清楚。我們提出假說為SAH是造成AD的制病因子之一,可能機制為造成細胞凋亡、類澱粉斑形成與DNA傷害等。
以SAH(1μM), Hcy (1 mM)及腺核苷(adenosine, Ado) (100 μM)加入神經膠細胞 (BV-2)共同培養,發現顯著地造成BV-2細胞細胞凋亡。我們利用凋亡細胞數來計算SAH+Hcy+Ado三者間是具有有協同作用。SAH+Hcy+Ado在六小時會促使胞內ROS開始增加,且在12小時會顯著抑制粒線體膜電位。另外SAH+Hcy+Ado亦會分別在24及48小時顯著增加caspase-3及caspase-9活性,及測定SAM/SAH比例及5-mdc含量所得知,會促使低甲基化情況發生。我們也預培養維生素E (α-tocopherol) 30分鐘,結果顯示alpha-tocopherol可以減少SAH+Hcy+Ado所產生的ROS及讓細胞數減少死亡(24及48小時)。
類澱粉班會在AD腦部中會產生纏結,是此AD重要病徵之一。 結果顯示SAH會呈現劑量(10、50、100、500 nM)與時間效應(12、24、48 h)地促使類澱粉斑形成。已知類澱粉斑是由類澱粉斑前趨蛋白(amliod--beta precursor protein, APP)經過剪切酵素切割所形成之產物,結果顯示SAH會促使剪切酵素(presilin 1, PS1)蛋白質含量與mRNA表現,但只有在高濃度(500 nM)之下稍微會促進另一剪切酵素(beta-site amyloid precursor protein cleaving enzyme 1, BACE1)蛋白表現,同時SAH亦會促使類澱粉前趨蛋白(APP)蛋白質表現。另有研究指出低甲基化會造成胞內PS1與APP表現,因此我們進一步探討BV-2細胞之PS1與APP promoter的甲基化的狀況,結果顯示SAH在高濃度下,的確會造成BV-2細胞此兩種基因promoter低甲基化。我們也添加5’-azc (20 uM) 一種甲基轉移脢抑制劑,去證實SAH是否真的會造成BV-2細胞之低甲基化。由結果發現,處理5’-azc之後會造成類澱粉斑形成且亦會造成胞內PS1與APP基因promoter低甲基化,另外若BV-2細胞先betaine (一種可以提供甲基的物質) (1mM)預培養30分鐘,發現SAH所誘發類澱粉斑會顯著低減少,同時也會恢復SAH所造成PS1與APP低甲基化情況。
另一個會造成AD的因素是神經細胞之DNA傷害。之前我們研究室發現SAH會抑制DNA修復能力,而造成DNA傷害擴大。由於類澱粉斑也有研究指出會產生ROS而造成DNA傷害,因此我們探討了SAH造成DNA傷害的可能機制。我們以彗星電泳法去觀察DNA傷害,發現SAH(10-500 nM)會成劑量關係造成BV-2細胞的DNA傷害。我們預培養BACE1抑制劑(beta-secretase inhibitor IV) 處理30分鐘之後,結果顯示可以減少類澱粉斑形成與DNA傷害。我們進一步以HPLC去觀察胞內8-oxo-G含量產生,結果顯示SAH呈劑量關係造成8-oxo-G產生。接著我們分析了OGG1蛋白表現,發現SAH會呈劑量關係(10-500 nM) 抑制BV-2細胞之OGG1蛋白及 mRNA表現。進一步探討SAH對OGG1 promoter影響時,發現在高濃度之下(500 nM)會造成OGG1 promoter 低甲基化。而在添加5’-azc (20 uM)之後亦和SAH一樣會造成BV-2細胞 OGG1 promoter低甲基化。當BV-2細胞先以betaine (1mM)預培養30分鐘,會恢復SAH所造成OGG1promoter低甲基化的程度。
本論文證實: SAH會協同Hcy及Ado造成BV-2細胞凋亡; 此外以SAH單獨處理會造成BV2細胞之DNA傷害,而此結果可能與SAH促進類澱粉斑形成而增加胞內ROS及抑制DNA修復酵素OGG1有關。這些結果也說明SAH可能會是導致阿茲海默症形成的一個致病因子。
URI: http://hdl.handle.net/11455/51577
Appears in Collections:食品暨應用生物科技學系

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