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目的:评估二氢生物蝶呤还原酶(QDPR)的抗氧化作用,并初步探讨QDPR基因A278C位点突变对其抗氧化作用的影响。创新点:首次在体外实验中发现QDPR有抗氧化作用,且此作用在A278C位点突变后减弱。方法:我们构建了野生型和突变型QDPR质粒,且分别转染至人胚肾293细胞中(HEK293T)。实验可分为以下三组:空白质粒对照组、野生型QDPR组和突变型QDPR组。三天后收集细胞观察活性氧(ROS)和四氢生物蝶呤(BH4)的表达量,使用免疫印迹的方法检测烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)、谷胱甘肽过氧化物酶3(GPX3)和超氧化物歧化酶1(SOD1)的蛋白表达水平。用半定量逆转录-聚合酶链反应(RT-PCR)方法分析神经型一氧化氮合成酶(n NOS)基因的表达。用酶联免疫吸附测定(ELISA)试剂盒检测转化生长因子-β1(TGF-β1)的活性。结论:本实验中野生型QDPR可以显著降低n NOS、NOX4和TGF-β1的水平,同时提高SOD1和GPX3表达。但当QDPR发生位点突变后没有观察到上述现象,并且突变型会导致ROS过量产生。我们的数据还表明,野生型和突变型QDPR对BH4含量的影响无显著差异。综上所述,QDPR有抗氧化作用,但A278C位点突变后会影响QDPR的抗氧化功能。
OBJECTIVE: To evaluate the antioxidant effect of dihydrobiopterin reductase (QDPR) and to investigate the effect of A278C site mutation of QDPR gene on its anti-oxidative activity. Innovation: The first in vitro experiments found that QDPR have antioxidant effects, and this role in the A278C site mutation weakened. Methods: We constructed both wild-type and mutant QDPR plasmids and transfected them into human embryonic kidney 293 cells (HEK293T) respectively. The experiment can be divided into the following three groups: blank plasmid control group, wild-type QDPR group and mutant QDPR group. After three days, the cells were collected to observe the expression levels of reactive oxygen species (ROS) and tetrahydrobiopterin (BH4). Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and glutathione Oxidase 3 (GPX3) and superoxide dismutase 1 (SOD1) protein expression levels. The expression of neuronal nitric oxide synthase (nNOS) gene was analyzed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The activity of transforming growth factor-β1 (TGF-β1) was detected by enzyme-linked immunosorbent assay (ELISA) kit. CONCLUSION: Wild-type QDPR can significantly reduce the levels of nNOS, NOX4 and TGF-β1 and increase the expression of SOD1 and GPX3 in this experiment. However, this phenomenon was not observed when QDPR was mutated at the site, and the mutated form resulted in ROS overproduction. Our data also show that there is no significant difference in the effect of wild type and mutant QDPR on BH4 content. In summary, QDPR has antioxidant effects, but A278C site mutation will affect the QDPR antioxidant function.