Effects of Intestinal Microflora on Superoxide Dismutase Activity in the Mouse Cecum

Abstract

Abstract:In the antioxidant defense system, superoxide dismutase (SOD) catalyzes the breakdown of superoxide into hydrogen peroxide and oxygen. In the cecum, the influence of intestinal microflora on SOD activity is unknown. In this study, we used germ-free (GF) mice to examine the effect of intestinal microflora on SOD activity in the cecum, and SOD activity was compared between GF and conventional (CV) mice. The activity of CuZnSOD and MnSOD was determined using the SOD Assay Kit-WST. Expressions of CuZnSOD mRNA and protein were determined by real-time PCR and western blot analyses, respectively. The activities of CuZnSOD and MnSOD were significantly higher in the ceca of GF IQI and FVB/N strain mice than in CV mice (P<0.01-0.05). The gene expressions of CuZnSOD mRNA in the ceca of GF mice were significantly higher than those in CV mice (P<0.05), and CuZnSOD protein expression showed similar tendencies. Consistent with the abovementioned results, the total SOD activity in conventionalized mice decreased to the level of total SOD activity observed in the ceca of CV mice. Furthermore, no differences between GF and CV mice were observed in the SOD activities in the liver and thymus. Our results suggest that the antioxidant defense system in the mouse cecum is influenced by the intestinal microflora that downregulate SOD activity.

Upregulation of Superoxide Dismutase Activity in the Intestinal Tract Mucosa of Germ-Free Mice

“…These findings are in agreement with the results of our previous report in the whole cecal body [7]. Furthermore, we previously reported that the total SOD activity in the ceca of CVz mice was significantly lower than in the ceca of GF mice [7]. In addition, we confirmed that total SOD activity in the cecal mucosa of pseudo-GF mice treated with antibiotics in CV mice (1,500 U/ml penicillin-G and 2 mg/ml streptomycin sulfate for 4 days) was significantly higher than that of CV mice (data not shown), although it was a preliminary study.…”

“…We also found that CuZnSOD mRNA expression showed similar tendencies with respect to SOD activities in the intestinal mucosa. These findings are in agreement with the results of our previous report in the whole cecal body [7]. Furthermore, we previously reported that the total SOD activity in the ceca of CVz mice was significantly lower than in the ceca of GF mice [7].…”

“…The reaction product was subjected to quantitative real-time polymerase chain reaction (PCR) performed following the instructions for the 7500 Real-time PCR system (Applied Biosystems, Foster City, CA, U.S.A.). After cDNA denaturation at 95°C for 30 sec, PCR was performed according to the following thermal cycling protocol: 95°C for 5 sec and 60°C for 34 sec in 20 µl buffer containing SYBR Premix Ex Taq (Takara Bio Inc.) and 0.8 µM each of forward CuZnSOD (5'-GGGTTC-CACGTCCATCAGT-3') and reverse CuZnSOD (5'-CA-CACGATCTTCAATGGACAC-3') primers [7]. The cDNA sequence was obtained from GenBank (accession number, NM_011434), and forward and reverse primers were designed to span different exons to avoid genomic DNA amplification.…”

“…Consistent with these results, the total SOD activity in conventionalized (CVz) mice decreased to the level of total SOD activity observed in the ceca of CV mice. These results suggested that the antioxidant defense system in the mouse cecum is influenced by intestinal microflora, which downregulates SOD activity [7]. However, because we measured SOD activity in the whole body of the cecum in the aforementioned study, it was unclear whether upregulation of SOD activity in the cecum of GF mice occurs only in the mucosa or in other cecal tissues as well.…”

“…We recently reported that total SOD, CuZnSOD and MnSOD activities in the ceca of germ-free (GF) mice were significantly higher than those in the ceca of conventional (CV) mice [7]. CuZnSOD mRNA expression in the ceca of GF mice was significantly higher than that in CV mice, and CuZnSOD protein expression showed similar tendencies.…”