Prevention against oxidative stress of the small intestine by its endogeneous thioredoxin

采用热处理、硫酸铵分馏、pH5.2乙酸处理、阴离子交换柱层析、Sephacry S-100凝胶过滤、CM-Sepharose柱层析等标准方法纯化牛小肠硫氧还蛋白和硫氧还蛋白还原酶。在ADP-Sepharose之后，用亲和层析法纯化了牛小肠硫氧还蛋白配体Sepharose 4B。从MTT试验和乳酸脱氢酶的释放判断，过氧化氢引起小肠上皮细胞系IEC-6的细胞毒性。硫氧还蛋白还原酶活性和SH含量随H_2O_2浓度的增加呈剂量依赖性降低，但在低H_2O_2浓度下硫氧还蛋白活性升高。此外，硫氧还蛋白活性的增加在氧化应激的初始阶段是时间依赖性的。逆转录聚合酶链反应（RT-PCR）结果显示，在0.25 mMH_2O_2下，IEC-6细胞mRNA含量呈时间依赖性增加。这些结果表明，细胞氧化休克导致硫氧还蛋白活性增加，而硫氧还蛋白参与了抗氧化应激的防御机制。

Thioredoxin and thioredoxin reductase were purified from bovine small intestine according to standard methods, including heat treatment, ammonium sulfate fractionation, acetic acid treatment at pH5.2, anion-exchange column chromatography, Sephacry S-100 gel-filtration, and CM-Sepharose column chromatography. Thioredoxin reductase was finally purified on affinity chromatography of bovine small intestine thioredoxin-ligand Sepharose 4B following ADP-Sepharose. Hydrogen peroxide caused cytotoxicity of the small intestine epitherial cell line, IEC-6, as judged from an MTT assay and the release of lactate dehydrogenase. Thioredoxin reductase activity and SH content decreased dose-dependently with H_2O_2, but thioredoxin activity increased at low H_2O_2 concentrations. In addition, the increase in thioredoxin activity was time-dependent during the initial stages of oxidative stress. Reverse transcription-polymerase chain reaction (RT-PCR) amplification also showed that the mRNA content in IEC-6 cells increased time-dependently at 0.25 mMH_2O_2. These results indicate that cellular oxidative shock causes an increase in the activity of thioredoxin, which is involved in the defense mechanism against oxidative stress.