Mie Mochizuki

Transcription Factor Nrf2 Regulates Inflammation by Mediating the Effect of 15-Deoxy-Δ12,14-Prostaglandin J2

ABSTRACT Activated macrophages express high levels of Nrf2, a transcription factor that positively regulates the gene expression of antioxidant and detoxication enzymes. In this study, we examined how Nrf2 contributes to the anti-inflammatory process. As a model system of acute inflammation, we administered carrageenan to induce pleurisy and found that in Nrf2-deficient mice, tissue invasion by neutrophils persisted during inflammation and the recruitment of macrophages was delayed. Using an antibody against 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), it was observed that macrophages from pleural lavage accumulate 15d-PGJ2. We show that in mouse peritoneal macrophages 15d-PGJ2 can activate Nrf2 by forming adducts with Keap1, resulting in an Nrf2-dependent induction of heme oxygenase 1 and peroxiredoxin I (PrxI) gene expression. Administration of the cyclooxygenase 2 inhibitor NS-398 to mice with carrageenan-induced pleurisy caused persistence of neutrophil recruitment and, in macrophages, attenuated the 15d-PGJ2 accumulation and PrxI expression. Administration of 15d-PGJ2 into the pleural space of NS-398-treated wild-type mice largely counteracted both the decrease in PrxI and persistence of neutrophil recruitment. In contrast, these changes did not occur in the Nrf2-deficient mice. These results demonstrate that Nrf2 regulates the inflammation process downstream of 15d-PGJ2 by orchestrating the recruitment of inflammatory cells and regulating the gene expression within those cells.

Pathogenic factors of glucose intolerance in obese Japanese adolescents with type 2 diabetes.

We attempted to identify the pathogenic factors involved in the progression to type 2 diabetes in obese Japanese adolescents. Subjects included 18 nondiabetic obese adolescents, 12 obese adolescents with type 2 diabetes on diet therapy, 10 obese adolescents with type 2 diabetes manifesting ketosis at onset or with a history of treatment with hypoglycemic agents, and 26 non-obese adolescent control subjects. The first-phase insulin response (FPIR), glucose disappearance constant (Kg), glucose effectiveness (Sg), and insulin sensitivity (S(I)) were obtained using an insulin-modified frequently sampled intravenous glucose tolerance test (FSIGT) and a minimal model analysis. The disposition index (DI, by FPIR x S(I)) was determined to assess any endogenous insulin effect. The results showed that Kg was decreased significantly (P = .0006) with the progression to severe diabetes in the obese groups. Although S(I) and Sg did not differ significantly among the 3 obese groups, both parameters were significantly lower in each obese group versus the non-obese controls. As a result of the significant decrease in FPIR (P < .0001), the DI decreased (P = .0006) with the progression to severe diabetes in the obese groups. In conclusion, an early manifestation of type 2 diabetes with occasional ketosis at onset may result from beta-cell dysfunction to glucose stimulation. This finding is demonstrated by the relatively low FPIR to decreased S(I) in obese Japanese adolescents, as well as the low Sg as a synergic role in glucose intolerance. The present findings from a Japanese population for pathogenic factors aside from obesity may help us to gain a better understanding of the progression to adolescent, early-onset, obese type 2 diabetes and its severity.

Ebselen suppresses late airway responses and airway inflammation in guinea pigs

Although ebselen, a seleno-organic compound, inhibits inflammation in various animal models, its efficacy as an anti-asthma drug remains to be clarified. In this study, we investigated the inhibitory effect of ebselen on a guinea pig asthma model. Ebselen was orally administered at dosages of 1-20 mg/kg 2 h before an ovalbumin (OA) challenge, and then airway responses, airway inflammation, the generation of superoxide, H(2)O(2), and nitrotyrosine, and the induction of inducible nitric oxide synthase (iNOS) were evaluated. Sensitized animals challenged with OA aerosol showed dual airflow limitations, i.e., immediate and late airway responses (IAR and LAR). Ebselen significantly inhibited LAR at dosages greater than 10 mg/kg, but did not inhibit IAR at any dosage. Bronchoalveolar lavage (BAL) examination showed that airway inflammation was significantly suppressed by ebselen at 10 mg/kg. The generation of superoxide and H(2)O(2) occurred on endothelial cells of LAR bronchi, and was inhibited by 10 mg/kg of ebselen. Superoxide generation was inhibited by diphenyleneiodonium chloride (DPI), a NAD(P)H oxidase inhibitor, but not by allopurinol, a xanthine oxidase inhibitor. Immunoreactivities for iNOS and nitrotyrosine were also observed on endothelial cells of LAR bronchi and were abolished in ebselen-treated animals. The present findings suggest that ebselen can be applied as a new therapeutic agent for asthma. The possible mechanisms by which ebselen inhibits LAR likely involve suppression of oxidant formation and iNOS induction in endothelial cells.

Anti-inflammatory activity of creatine supplementation in endothelial cells in vitro

Creatine (CR) supplementation augments muscle strength in skeletal muscle cells by increasing intracellular energy pools. However, the effect of CR supplementation on endothelial cells remains to be clarified. In this study, we investigated whether CR supplementation had any anti‐inflammatory activity against human pulmonary endothelial cells in culture. We confirmed that supplementation with 0.5 mM CR significantly increased both intracellular CR and phosphocreatine (PC) through a CR transporter while keeping intracellular ATP levels constant independent of CR supplementation and a CR transporter antagonist. In the assay system of endothelial permeability, supplementation with 5 mM CR significantly suppressed the endothelial permeability induced by serotonin and H2O2. In cell adhesion experiments, supplementation with 5 mM CR significantly suppressed neutrophil adhesion to endothelial cells. In the measurement of adhesion molecules, CR supplementation with more than 0.5 mM CR significantly inhibited the expressions of ICAM‐1 and E‐selectin on endothelial cells, and the inhibition was significantly suppressed by an adenosine A2A receptor antagonist. The present study suggests that CR supplementation has anti‐inflammatory activities against endothelial cells.

Treatment with α-Galactosylceramide Attenuates the Development of Bleomycin-Induced Pulmonary Fibrosis

Pulmonary fibrosis is an end-stage disorder for which efficacious therapeutic options are not readily available. Although its pathogenesis is poorly understood, pulmonary fibrosis occurs as a result of various inflammations. NKT cells modulate inflammation because of their ability to produce large amounts of cytokines by stimulation with their glycolipid ligand. In the present study, we investigated the effects of α-galactosylceramide (α-GalCer), a selective NKT cell ligand, on the development of bleomycin-induced pulmonary fibrosis. Treatment of mice with α-GalCer prolonged their survival under bleomycin administration by attenuating the development of pulmonary fibrosis. The protective effects of α-GalCer were associated with an increase in the pulmonary level of IFN-γ and a decrease in the pulmonary level of fibrogenic cytokines such as TGF-β and connective tissue growth factor. The initial pulmonary inflammation caused by bleomycin was also attenuated by α-GalCer with the reduction of the macrophage inflammatory protein-2 level. The protective effects of α-GalCer were markedly reduced in mice lacking NKT cells or as a result of treatment with anti-IFN-γ Ab. These results suggest that α-GalCer suppresses bleomycin-induced acute pulmonary inflammation and thus attenuates the development of pulmonary fibrosis possibly by regulating several cytokine levels.

Ebselen Decreases Ozone-Induced Pulmonary Inflammation in Rats

Abstract. We studied the effects of ebselen on rat lung inflammatory responses against ozone exposure. Rats were treated with ebselen every 12 h from 1 h before a single 4-h exposure to 2 ppm ozone. Treatment with ebselen (10 mg/kg) significantly decreased pulmonary inflammation as indicated by the albumin concentration and the number of neutrophils in the bronchoalveolar lavage fluid 18 h after the ozone exposure. Although treatment with ebselen did not alter the macrophage expression of inducible nitric oxide synthase after the ozone exposure, it did markedly inhibit the nitration reaction of tyrosine residues, suggesting that ebselen scavenges peroxynitrite during ozone-induced pulmonary inflammation. Treatment with ebselen also enhanced the pulmonary expression of both copper, zinc, and manganous superoxide dismutases at the same time point. These enzymes may also contribute to a decrease in the formation of peroxynitrite by lowering the concentration of superoxide. Thus, ebselen represents a useful compound for protecting against certain acute lung injuries by modulating the oxidant-related inflammatory process.

Association of angiotensin-converting enzyme gene polymorphism with lipid profiles in children and adolescents with insulin-dependent diabetes mellitus.

We attempted to clarify the association between angiotensin-converting enzyme (ACE) gene polymorphism and the other predictive factors for macroangiopathy in children and adolescents with uncomplicated insulin-dependent diabetes mellitus (IDDM). Sixty-three patients were divided into 3 groups according to the ACE genotypes. The lipid profiles were evaluated according to ACE genotypes. The level of lipoprotein(a) (Lp(a)) in the II genotype was significantly lower than that in groups with the D allele. Lp(a) significantly correlated with apo B/apo A-I (p < 0.001, r = 0.63) and atherogenic index (AI = (total cholesterol – high-density lipoprotein cholesterol)/high-density lipoprotein cholesterol; p = 0.004, r = 0.36). We suggest that the D allele may affect the level of Lp(a) and the other lipid profiles in IDDM.

S-carboxymethylcysteine inhibits neutrophil activation mediated by N-formyl-methionyl-leucyl-phenylalanine

In this study, the possible mechanisms of action for the inhibitory effects of S-carboxymethylcysteine on the activation of human neutrophils by N-formyl-methionyl-leucyl-phenylalanine (FMLP) were investigated. Preincubation of neutrophils with more than 10 microg/ml of S-carboxymethylcysteine was found to impair neutrophil chemotactic activity toward FMLP, and to inhibit FMLP-mediated neutrophil adherence to pulmonary vascular endothelial cells. Preincubation of neutrophils with 10 and 100 microg/ml of S-carboxymethylcysteine decreased in the production of inositol 1,4,5-triphosphate (IP(3)) and diacylglycerol in neutrophils stimulated with FMLP, respectively. Preincubation of neutrophils with S-carboxymethylcysteine did not affect the cellular cyclic AMP (cAMP) levels in neutrophils stimulated with FMLP. S-carboxymethylcysteine inhibited the enzymatic activity of phosphatidyl inositol-specific phospholipase C in vitro in a concentration-dependent manner. These findings indicate that S-carboxymethylcysteine attenuates FMLP-stimulated neutrophil activation at least in part by inhibiting phosphatidyl inositol-specific phospholipase C-mediated signal transduction.

Promoter activity of human tissue inhibitor of metalloproteinase 2 gene with novel single nucleotide polymorphisms

Objective:u2003 The single nucleotide polymorphism (SNP) −418Gu2003>u2003C in the TIMP2 gene promoter region has been shown to be associated with in chronic obstructive pulmonary disease (COPD). The purpose of this study was to search for novel single nucleotide polymorphism (SNP) in the TIMP2 promoter region around the −418Gu2003>u2003C locus, and to investigate whether any of these SNP, including −418Gu2003>u2003C, had an influence on TIMP2 transcription activity.

The association of Ala45Thr polymorphism in NeuroD with child-onset Type 1a diabetes in Japanese

Recently Iwata et al. reported that the polymorphism in NeuroD exon 2(Ala45Thr) was associated with adult-onset Type 1 diabetes in Japanese. Furthermore, the mutations in the NeuroD as a regulator of insulin transcription have been reported to result in Type 2 diabetes. We, therefore, aimed to clarify the role of this Ala45Thr polymorphism in the susceptibility to Type 1a, immune-mediated, diabetes of child-onset Japanese patients. Eighty patients with child-onset Type 1 diabetes were examined along with 121 non-diabetic subjects as the controls. The polymorphism in Ala45Thr was defined using the PCR-RFLP method. The GAD Ab, IA-2 Ab, HLA-DRB1 genotypes and residual beta-cell function at 3 years from onset were evaluated in relation to the difference in this polymorphism. The frequency of the Ala45Thr heterozygotes was significantly higher in the Type 1 diabetic patients than in the controls (21.3 versus 9.9%, P=0.0252). The frequency of loss of beta-cell function was higher in heterozygotes patients than in wild type homozygotes patients (P=0.0112). Type 1 diabetic patients with DRB1*0901 allele showed a significantly higher frequency, 27.9%, of the Ala45Thr variant than the controls (P=0.0041). In conclusion, the Ala45Thr polymorphism contributes to the risk of development of, and to the early deterioration of beta-cell function, in Type 1a diabetes among the Japanese population.