Translated Abstract
Back ground:Kashin–Beck disease (KBD) is a chronic, endemic osteochondropathy, which is mainly distributed in the area ranging from the northeastern to the southwestern China. The etiology of the disease is unknown, although 3 major environmental hypotheses have been proposed in the past 150 years: endemic selenium deficiency, serious cereal contamination by mycotoxin-producing fungi, and high humic acid levels in drinking water. The disease is manifested as degradation of the matrix, cell necrosis mainly in the articular and growth plate cartilage, which can result in growth retardation, secondary osteoarthrosis, and disability in advanced stages. The chondrocytes are the only cell type present in mature cartilage. They produce and maintain the cartilaginous matrix, and are responsible for repairing the damaged tissue.Mitochondria are membrane enclosed organelles found in most eukaryotic cells. They are semi-autonomous organelles with their own genetic systems under control of the nuclear genome. They play an essential role in many cellular events: oxidization of a wide range of metabolic intermediates, generation of most of the cell’s energy supply in the form of adenosine triphosphate (ATP), and modulation of ionic homeostasis. In addition, mitochondria serve as both a source of and a target for free radicals and mediate apoptosis. It is possible that a mitochondrial dysfunction leads to a signal cascade linked to programmed cell death, or apoptosis.Previous studies found that mitochondrial injury was involved in articular chondrocytes of Kashin-Beck disease. Morphological changes of mitochondria were also detected when normal articular chondrocytes were exposed to mycotoxins or low selenium, suspected risk factors of Kashin-Beck disease. Selenium could partly block the apoptosis of chondrocytes induced by T-2 toxin. However, the effect of mitochondria on the pathogenesis of Kashin- Beck disease and the relationship between mitochondria and the suspected risk factors have not yet been systematically studied.Objectives:1. To establish general methods of mitochondrial research through the study of cell models with mitochondrial dysfunction.2. To evaluate mitochondrial dysfunction, oxidative stress and mitochondria-mediated apoptosis in adult KBD chondrocytes.3. To demonstrate that the mitochondrial dysfunction similar to KBD can be induced by T-2 toxin, and selenium can partly block this process.4. To explore the role of mitochondria in pathogenesis of KBD Explain the relationship between mitochondria and the suspected risk factors T-2 toxin and low selenium Effectively promote the study of “suspected environmental risk factors - mitochondrial dysfunction - articular chondrocytes death” pathway in KBD and find a way to block it Finally achieve the purpose of early diagnosis, prevention and treatment of KBD.Methods:1. The LL/2S cell lines, who partly restored mitochondrial function, were selected by the method of culturing the LL/2 wild-type cell lines with mitochondrial dysfunction in galactose medium 2-4 weeks. The mtDNA of all cell lines was sequenced, and their mitochondrial function was predicted and detected. We established the methods of mitochondrial research in vitro cultured cell lines through this study.2. Mitochondrial function and oxidative stress in KBD articular chondrocytes were evaluated by the methods we established. Apoptotic cell death was evaluated by analyzing the cytochrome c release from mitochondria to the cytosol, caspase-9 and caspase-3 activities, and the apoptosis rate of articular chondrocytes. The correlation between age of donors and mitochondrial function was also analyzed.3. Human articular chondrocytes cultured in vitro were treated with T-2 toxin with different concentrations (0 ng/ml, 1 ng/ml, 10 ng/ml, 20 ng/ml and 100 ng/ml) for 5 days. The effects of T-2 toxin on chondrocytes mitochondrial dysfunction, oxidative stress and mitochondria- mediated apoptosis were investigated. We have also examined the inhibitory effects of selenium on chondrocyte mitochondrial dysfunction induced by T-2 toxin.Results:1. G14756A mutation in mtDNA was detected in galactose selected LL/2S cell lines, which resulted in a G204E amino acid change in cytochrome b. The change in G204E amino acid caused change of cytochrome b conformational and increases of electron transport chain dynamics, thereby improving the respiratory chain complex III function and the whole mitochondrial function as follow: reduction of non-coupled cell respiration, increase of complex III dependent oxygen consumption and protein synthesis, increase of mitochondrial membrane potential and cellular ATP content, decrease of reactive oxygen species (ROS) in cell and lactic acid in cell culture medium.2. Activities of complexes II, III, IV and V were reduced in KBD articular chondrocytes compared with cells from normal controls. However, the mitochondrial mass was increased in KBD samples. Cultured KBD chondrocytes had a reduction of cellular ATP content and contained a higher proportion of cells with de-energized mitochondria. Mitochondrial cytochrome c release and caspase-9 and 3 activation were also observed. The percentages of positive apoptotic chondrocytes from the KBD group stained by Hoechst 33258 and Annexin V/PI for flow cytometry exhibited higher levels than that of the healthy controls. Multiple regression analysis showed no significant relationship between age of donors and mitochondrial function in either normal or KBD chondrocytes.3. T-2 toxin decreased chondrocytes viabilities and increased apoptosis in concentration-and time-dependent manners. Meantime, complexes III, IV and V activities, mitochondrial membrane potential and the cellular ATP levels were significantly reduced following T-2 toxin administration at 5 days. Furthermore, levels of intracellular ROS were activated by T-2 toxin. Mitochondrial cytochrome c release and caspase-9 and 3 activation were also observed. Interestingly, selenium can partly blocked T-2 toxin-induced mitochondrial dysfunction, oxidative stress and chondrocytes apoptosis.Conclusion:1. ROS plays an important role in the complex III injury-induced mitochondrial dysfunction G14756A mutation in mtDNA can improve mitochondrial function through regulation of cellular ROS.2. Mitochondrial dyfunction, oxidative stress and mitochondria-mediate apoptosis were involved in the pathophysiology of KBD No significant correlation between mitochondrial function and age of donors was found in either normal or KBD chondrocytes.3. T-2 toxin exoposed to normal chondrocytes can cause mitochondrial dysfunction which is similar to KBD Selenium can partly block T-2 toxin -induced mitochondrial dysfunction.4. The “oxidative stress - mitochondrial dysfunction - chondrocytes apoptosis” pathway may play a key role in the pathogenesis of KBD, and selenium could prevent the occurrence of KBD through this pathway.
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