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

1997 Fiscal Year Final Research Report Summary

• Project/Area Number: 08672557
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 医薬分子機能学
• Research Institution: Osaka University
• Principal Investigator: MIZOGUCHI Tadashi Bio-Functional Engineering, Faculty of Pharmaceutical Sciences, Osaka University, Professor, 薬学部, 教授 (00028838)
• Project Period (FY): 1996 – 1997
• Keywords: Oxidative stress / Cultured cells / IEC-6 cells / Thioredoxin / Thioredoxin reductase / Small intestine / Bovine / Vitamin E

Research Abstract

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.