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

Abstract

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.