Abstract：
Mucopolysaccharidosis type II (MPS II) is an inborn error of the metabolism resulting from several possible mutations in the gene coding for iduronate-2-sulfatase (IDS), which leads to a great clinical heterogeneity presented by these patients. Many studies demonstrate the involvement of oxidative stress in the pathogenesis of inborn errors of metabolism, and mitochondrial dysfunction and oxidative stress can be related since most of reactive oxygen species come from mitochondria. Cellular models have been used to study different diseases and are useful in biochemical research to investigate them in a new promising way. The aim of this study is to develop a heterozygous cellular model for MPS II and analyze parameters of oxidative stress and mitochondrial dysfunction and investigate the in vitro effect of genistein and coenzyme Q10 on these parameters for a better understanding of the pathophysiology of this disease. The HP18 cells (heterozygous c.261_266del6/c.259_261del3) showed almost null results in the activity of the IDS enzyme and presented accumulation of glycosaminoglycans (GAGs), allowing the characterization of this knockout cellular model by MPS II gene editing. An increase in the production of reactive species was demonstrated (p < .05 compared with WT vehicle group) and genistein at concentrations of 25 and 50 µm decreased in vitro its production (p < .05 compared with HP18 vehicle group), but there was no effect of coenzyme Q10 in this parameter. There was a tendency for lysosomal pH change in HP18 cells in comparison to WT group and none of the antioxidants tested demonstrated any effect on this parameter. There was no increase in the activity of the antioxidant enzymes superoxide dismutase and catalase and oxidative damage to DNA in HP18 cells in comparison to WT group and neither genistein nor coenzyme q10 had any effect on these parameters. Regarding mitochondrial membrane potential, genistein induced mitochondrial depolarization in both concentrations tested (p < .05 compared with HP18 vehicle group and compared with WT vehicle group) and incubation with coenzyme Q10 demonstrated no effect on this parameter. In conclusion, it is hypothesized that our cellular model could be compared with a milder MPS II phenotype, given that the accumulation of GAGs in lysosomes is not as expressive as another cellular model for MPS II presented in the literature. Therefore, it is reasonable to expect that there is no mitochondrial depolarization and no DNA damage, since there is less lysosomal impairment, as well as less redox imbalance.
摘要：
II型粘多糖贮积症（MPSII）是由于编码艾杜糖醛酸-2-硫酸酯酶（IDS）的基因中的几种可能突变而导致的先天性代谢错误，这导致了这些患者呈现的巨大临床异质性。许多研究表明，氧化应激参与了先天性代谢错误的发病机理，线粒体功能障碍和氧化应激可能相关，因为大多数活性氧来自线粒体。细胞模型已用于研究不同的疾病，并可用于生化研究，以一种新的有希望的方式研究它们。本研究的目的是建立MPSII的杂合细胞模型，分析氧化应激和线粒体功能障碍的参数，并研究金雀异黄素和辅酶Q10对这些参数的体外作用，以更好地了解该疾病的病理生理学。HP18细胞（杂合子c.261_266del6/c.259_261del3）在IDS酶的活性方面几乎没有结果，并呈现糖胺聚糖（GAG）的积累，允许通过MPSII基因编辑来表征这种敲除细胞模型。证明了反应性物种的产生增加（与WT媒介物组相比，p<0.05），并且在25和50µm浓度的染料木黄酮在体外减少了其产生（与HP18媒介物组相比，p<0.05），但辅酶Q10对该参数没有影响。与WT组相比,HP18细胞中存在溶酶体pH变化的趋势,并且所测试的抗氧化剂均未显示对该参数的任何影响。与WT组相比，HP18细胞中抗氧化酶超氧化物歧化酶和过氧化氢酶的活性以及对DNA的氧化损伤没有增加，染料木素和辅酶q10对这些参数都没有任何影响。关于线粒体膜电位，在两种测试浓度(与HP18媒介物组和与WT媒介物组相比,p<.05)和用辅酶Q10孵育表明对该参数没有影响。总之,假设我们的细胞模型可以与较温和的MPSII表型进行比较，鉴于溶酶体中GAG的积累不如文献中提出的MPSII的另一种细胞模型具有表达力。因此,可以合理地预期没有线粒体去极化和DNA损伤，因为溶酶体损伤较少，以及较少的氧化还原失衡。
