| Summary: | 碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 100 === Mitochondria are the main source of the reactive oxygen species (ROS) in mammalian cells. High levels of ROS are considered to play an important role in aging and diverse aging-related diseases. Accumulated evidence indicates that mitochondrial dysfunction-elicited ROS is a key contributor to -cell dysfunction in the pathogenesis of type 2 diabetes mellitus (T2DM). Sirt3 is a member of the mammalian sirtuin family of NAD+-dependent protein deacetylases. Sirt3 plays an important role in regulating mitochondrial function. Sirt3 defect has been reported to associate with aging and aging-related diseases, including aging-related hearing loss and cardiac hypertrophy. Moreover, recent studies have confirmed that these aging-related diseases and diabetes are accompanied by overproduction of ROS in affected tissues. The aim of this study was to elucidate the role of Sirt3 in the effect of ROS overproduction on human cells with mitochondrial dysfunction. This study was divided into two parts. I first used menadione to induce overproduction of ROS. By treatment of 143B cells with 10 M menadione for 24 hours, the cells produced large amounts of superoxide anions. Excess production of ROS significantly decreased the Sirt3 protein expression level. By pre-treatment of cells with 1 mM N-acetyl cysteine (NAC) for 2 hours, the menadione-induced ROS was inhibited by NAC. Besides, the menadione-induced decrease of Sirt3 expression was recovered. Similar results were obtained by treatement of the cells with oligomycin A. I then used the Seahorse XF-24 extracellular flux analyzer to analyze mitochondrial respiration. The results showed that after menadione treatment of 143B cells, the mitochondrial respiration was significantly decreased. However, with NAC pretreatment, the mitochondrial respiration was restored.
In the second part of this study, I used human cells with mitochondrial dysfunction and high ROS levels induced by pathogenic mtDNA mutations. The results showed a significant decrease of the mRNA and protein expression levels of Sirt3 in cybrids harboring 4977 bp deletion and A3243G mutation of mtDNA, respectively. I then used NAC to treat cybrids harboring 4977 bp deletion of mtDNA. The results showed that the ROS levels were decreased and the Sirt3 protein levels were increased by NAC treatment. On the other hand, I used skin fibroblasts from MELAS patients and normal skin fibroblast to investigate the difference in the Sirt3 expression. We found the Sirt3 protein level and mitochondrial respiration of skin fibroblasts of the patients were lower than those of normal skin fibroblasts. Moreover, I used 10-50 M resveratrol (RSV) to alter the Sirt3 protein level in cancer cell lines and skin fibroblasts. The results showed that RSV treatment could increase the Sirt3 level and mitochondrial respiration in the skin fibroblasts from both patients and normal subjects.
Taken the results together, I suggest that ROS overproduction due to mitochondrial dysfunction significantly decreases the expression of Sirt3, which can be reversed by antioxidants such as NAC and RSV in human cells. These findings provide novel information for us to better understand the molecular mechinesm of pathogenesis of mitochondrial disease caused by mtDNA mutations.
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