Protective effect of dihydropteridine reductase against oxidative stress is abolished with A278C mutation

Abstract

Objective

To evaluate the antioxidation of dihydrobiopterin reductase and to explore the effect of A278C mutation of the quinoid dihydropteridine reductase (QDPR) gene on its antioxidant activity.

Methods

First, plasmids with different genes (wild and mutant QDPR) were constructed. After gene sequencing, they were transfected into human kidney cells (HEK293T). Then, the intracellular production of reactive oxygen species (ROS) and tetrahydrobiopterin (BH4) was detected after cells were harvested. Activations of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), glutathione peroxidase 3 (GPX3), and superoxide dismutase 1 (SOD1) were analyzed to observe the oxidative stress after transfection. The expression of the neuronal nitric oxide synthase (nNOS) gene was analyzed by semiquantitative reverse-transcription polymerase chain reaction (RT-PCR). We also detected the activation of transforming growth factor β1 (TGF-β1) by enzyme-linked immunosorbent assay (ELISA) to observe the connection of TGF-β1 and oxidative stress.

Results

The exogenous wild-type QDPR significantly decreased the expression of nNOS, NOX4, and TGF-β1 and induced the expression of SOD1 and GPX3, but the mutated QDPR lost this function and resulted in excessive ROS production. Our data also suggested that the influence on the level of BH4 had no significant difference between mutated and the wild-type QDPR transfection.

Conclusions

Wild-type QDPR played an important role in protecting against oxidative stress, but mutant QDPR failed to have these beneficial effects.

中文概要

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 (nNOS)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nNOS、 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。