In our flow cytometry analysis (Fig
In our flow cytometry analysis (Fig. Nrf2, may function to increase resistance to oxidative stress in (14). In addition to safety conferred by Nrf2-dependent ARE-driven genes, Nrf2 is also directly involved in apoptosis signaling pathways. One study actually implies that Nrf2 is a substrate for caspase-3-like proteases (15), and another shows that Nrf2 inhibits Fas-mediated apoptosis pathway (16). Moreover, Nrf2 is an important effector of PERK-mediated cell survival (17) and regulates the level of sensitivity of death receptor signals (18). These data suggest that the Nrf2-ARE pathway promotes cell survival by modulating both cellular antioxidant potentials and apoptosis signaling pathways. Although Nrf2 is definitely indicated widely and is important for cellular antioxidant potential, Nrf2 knockout mice develop and grow normally (5). Small Nrf2-/- mice are not anemic (5), whereas targeted disruption of either NF-E2 or Nrf1 (binding factors of locus control region of -globin) resulted in anemia (19, 20). Interestingly, we observed indicators of anemia in aged Nrf2-/- mice offered by splenomegaly and spleen toxicity. Because anemia can reactivate splenic extramedullary hematopoiesis and consequently induce splenomegaly, we hypothesized that aged Nrf2-/- mice suffer from anemia. We also regarded as that erythrocytes might be a sensitive indication of oxidative stress as adult erythrocytes lack an adaptive response to external stimuli due to insufficient genetic material. This study, Tropanserin consequently, was designed to investigate the part of Nrf2 in erythrocyte maintenance, and the mechanism by which Nrf2-/- mice develop anemia. Materials and Methods Mice. Nrf2-/- Tropanserin mice were generated as explained earlier (5). Mice were killed by CO2, and blood was collected into EDTA-coated tubes for the hematological analysis and erythrocytes morphology exam (Wright’s staining). Animals were perfused with PBS, Tropanserin and organs were weighed and freezing. Cytotoxicity. Spleens were sectioned (10 m), fixed (4% paraformaldehyde, 20 min), and stained for terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (Roche Molecular Biochemicals) and cyclooxygenase-2 (Santa Cruz Biotechnology) (6, 7). For cytotoxicity, main splenocytes were prepared as explained by Gal DNA polymerase (Promega). PCR primers specific to each gene are as follows: Nrf2, 5-TCTCCTCGCTGGAAAAAGAA-3 and 5-AATGTGCTGGCTGTGCTTTA-3; HO-1, 5-TACACATCCAAGCCGAGAAT-3, and 5-GTTCCTCTGTCAGCATCACC-3; NQO1, 5-CATTCTGAAAGGCTGGTTTGA-3 and 5-CTAGCTTTGATCTGGTTGTCAG-3; GST A4, 5-GCCAAGTACCCTTGGTTGAA-3 and 5-CAATCCTGACCACCTCAACA-3; GCLM, 5-ACCTGGCCTCCTGCTGTGTG-3 and 5-GGTCGGTGAGCTGTGGGTGT-3; GCLC, 5-ACAAGCACCCCCGCTTCGGT-3 and 5-CTCCAGGCCTCTCTCCTCCC-3; ferritin light chain, 5-GTGGAAGCTGCCGTGAAC-3 and 5-CAGTCTGCGCTGGTTGTG-3; ferritin heavy chain, 5-AAGTGCGCCAGAACTACCAC-3 and 5-TCTTGCGTAAGTTGGTCACG-3; thioredoxin reductase-1, 5-GGGAGAAAAAGGTCGTCTA-3 and 5-ACATTGGTCTGCTCTTCATC-3; peroxiredoxin 1, 5-TGCCAGATGGACAATTCAAA-3 and 5-CAGCTGGACACACTTCACCA-3; and -actin, 5-AGAGCATAGCCCTCGTAGAT-3 and 5-CCCAGAGCAAGAGAGGTATC-3. Hemolysis. Blood was collected into EDTA-coated tubes and centrifuged (600 = 7). S, spleen; K, kidney; L, liver; b, body. (= 6). Each data pub or point represents imply SE. *, 0.05 by Student’s test. Open in a separate windows Fig. 2. Decreased expression levels of Nrf2-dependent ARE-driven genes in Nrf2-/- mice. Whole cell components (spleens) were used for Western blot analysis of HO-1 (= 3). (hemolysis, erythrocytes were incubated with H2O2 (37C, 12 h). After centrifugation (600 = 4). *, 0.05 by Student’s test. Open in a separate windows Fig. 4. Immune-mediated hemolytic anemia resulting from oxidative damages. (and = 5). (and Rabbit polyclonal to BNIP2 in and in = 4). Nrf2-/- mice showed decreased total GSH (= 4). *, 0.05 by Student’s test. Glutathione (GSH) and H2O2 Levels. Total GSH levels were identified from serum by measuring the reaction of GSH and dithionitrobenzoic acid coupled to the recycling.