| Summary: | 博士 === 國立臺灣海洋大學 === 食品科學系 === 103 === Abstract
Arsenic (As), one of most abundant element in the earth, has been recognized as a strong toxic material and leads people to a variety of diseases such as neurodegenerative disease and vascular disease. As is widely existed in the environment such as industrial waste, fungicide, ground water and As contamination is a global issue and caused a huge impact on human health. For instance, As exposure results in loss of short-term memory and causes damage to endothelial cells which is concerned as a risk factor of dementia. Indeed, there are 20-30% patients of dementia associated with pathological changes of blood vessels in their brain. To provide a protective agent in the aspect of food supplement, the study proposed to examine whether taurine, an amino acid containing sulfur group and rich in seafood, possesses neuro-protective effect against As-induced neurotoxicity both in vitro and vivo.
The first part of research used mouse lymph node endothelial SVEC4-10 cells as cell model to investigate preventive effects of taurine against As-induced vasculopathy and inflammatory response. Within As-treated SVEC4-10 cells, taurine could effectively reduce oxidative stress and improve redox parameters and mitochondrial function, recover ATP production and reduce ER stress and ratio of anti-apoptotic protein Bcl-2 / pro-apoptotic protein Bax, activity of caspase 3, 8, 9, which suggested that taurine ceased As-induced endothelial dysfunction and its protective mechanisms. In addition, vascular disease is also recognized as a chronic inflammatory response. Under As-induced inflammation in SVEC4-10 endothelial cells, taurine significantly reduced oxidative stress and inhibited NF-B activation as well as its downstream inflammatory genes, interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-), monocyte chemoattractant protein (MCP-1) and recovered endothelial nitric oxide synthase (eNOS) expression, which avoided further inflammation in endothelial cells.
The second part of the research used mouse neuroblastoma N2A cells as a model to explore preventive mechanism of taurine against As-induced arrest of neuronal differentiation and protection from the damage of synapse in post-differentiated N2A cells. The results showed that the As exposure disrupted neuronal differentiation process through increasing intracellular oxidative stress, protein carbonyl levels and reducing total thiol content, SOD and catalase activity. Furthermore, our results found that As would reduce peroxisome proliferator activated receptor gamma (PPAR-) expression and inhibit its downstream target gene, neuron-specific class III β-tubulin (Tuj-1) expression, which led to abnormal growth of synapses. Similar trends were observed in other neuronal differentiated genes such as microtube associated protein-2 (MAP-2) and synapsin-I. In the parallel experiment, As exposure also reduced MMP, integrity of mitochondrial DNA (mtDNA) and disrupted ATP synthesis, which resulted in lack of energy supply during neuronal differentiation. Impaired mitochondrial metabolism caused excessive generation of reactive oxygen molecules and exacerbated oxidative stress. Exposure to As in the differentiation process induced 78 kDa glucose-regulated protein (GRP78), and CEBP homologous protein (CHOP) expression as well as interfered intracellular calcium homeostasis. Similarly, As would destroy neurites of post-differentiated N2A cells, accompanied oxidative stress and mitochondrial dysfunction. Additional taurine would efficiently resumed neuronal differentiation and protected intergrity of neurite through counteracting these deleterious effect in As-treated differentiating and differentiated N2A cells.
The third part of the research content used ICR mice and human neuroblastoma SH-SY5Y cells as cell and animal model to explore protective mechanisms of taurine against As-induced neurotoxicity. The following experiments in cell model showed that As exposure caused increased oxidative stress and protein carbonyl levels, and reduced total thiol content, superoxide dismutase (SOD) and catalase activity, which led to cell cycle arrest in SH-SY5Y cells. As exposure decreased mitochondrial membrane potential (MMP) and induced the mitochondria-dependent apoptosis, included increase of pro-apoptotic protein Bax / Bcl-2 anti-apoptotic proteins ratio and caspases 3, 9 activity in SH-SY5Y cells. In addition, As exposure can cause endoplasmic reticulum stress (ER stress) and interfered intracellular calcium homeostasis, which resulted in caspase 8 activation and mitochondrial dysfunction. On the other hand, As disturbed phosphorylation of nuclear factor kappa B (nuclear transcriptor kappa B, NF-B) and inhibited its downstream targeted gene, hypoxia induced protein 1 alpha (hypoxia inducible factor 1 alpha, HIF-1) expression. Meanwhile, As inhibited neurotrophic related gene expression, such as n-myc gene downstream gene regulation 4 (NDRG-4), brain derived neurotrophic factor (BDNF) and sirtuin-1 (SIRT-1). It is worthy to note that NDRG-4 and BDNF expression is memory-related gene and both deficient in the brains of Alzheimer's disease (AD) patients. This phenomenon explained why As exposure causes memory loss and provide correlation between As exposure and AD. Co-treatment with taurine could prevent As-induced SH-SY5Y cell cycle arrest and apoptosis, mitochondria and ER-dependent apoptosis, intracellular redox imbalance and significant response neurotrophic gene expression. As-treated cells expressing BDNF and SIRT-1 gene in taurine had no effect on these parameters. Pre-treatment of taurine (100 mg/ kg b.w./ day) for 5 days significantly reduced damage of DG area in hippocampus of As-treated ICR-mice (10 mg/ kg b. w./day; 2 day), which elucidated bioactivity of taurine against acute As-intoxication in animal model.
As exposure interfered process of neuronal differentiation and caused matured neurons and endothelial cells death. Also, As induced inflammatory response in endothelial cells, which showed As exposure only cause neuronal degeneration but also cerebrovascular disease, which led to insufficient blood work. Morover, cross-talking of inflammatory cytokine between neurons and endothelial cells collaborated to accelerate the development of neurological symptoms in the micro-environment of brain. Since oxidative stress, mitochondrial dysfunction and ER stress usually coexisted in neuropathology environment, taurine can inhibit As-induced neuronal death and arrest of differentiation, vascular dysfunction and inflammation (as seen in above indicators). The results of the study elucidated the protective mechanism of taurine against As-induced neurological degeneration. In addition, the results provided possible correlation between As exposure and AD dand clarified further evidence of neurotoxic mechanism of As in neurologic disorders and protective roles of dietary taurine against As intoxication.
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