Immunosuppressant FK506 enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity.

Immunosuppressants FK506 and cyclosporin A inhibit neurotoxicity of N-methyl-D-aspartate in primary cortical cultures, while having no effect on quisqualate- and kainate-mediated neurotoxicity. Rapamycin completely reverses the neuroprotective effect of FK506. Both FK506 and cyclosporin A inhibit NMDA-elicited/nitric oxide-mediated increases in cGMP levels in cortical cultures. FK506 has no effect on sodium nitroprusside-induced increases in cGMP. In a stably transfected human kidney 293 cell line overexpressing the gene encoding nitric oxide synthase [L-arginine, NADPH:oxygen oxidoreductase (nitric oxide-forming), EC 1.14.13.39], FK506 inhibits the calcium ionophore A23187, stimulated increases in nitrite (a breakdown product of nitric oxide), and potentiates phorbol ester-mediated inhibition of nitrite formation. FK506-mediated inhibition of nitric oxide formation is completely reversed by rapamycin. Calcineurin dephosphorylates protein kinase C-mediated phosphorylation of nitric oxide synthase. FK506 prevents the calcineurin-mediated dephosphorylation of nitric oxide synthase and thereby diminishes the enzyme''s catalytic activity. These data establish nitric oxide synthase as a calcineurin substrate. Nitric oxide synthase catalytic activity is regulated by the phosphorylation state of the enzyme. Enhanced phosphorylation of nitric oxide synthase diminishes catalytic activity, and dephosphorylation (through activation of calcineurin) enhances catalytic activity. The neuroprotective effect of FK506 and cyclosporin A presumably involves the inhibition of calcineurin, preventing the dephosphorylation of nitric oxide synthase and its subsequent activation.     

Cytokine Expression of Microscopic Colitis Including Interleukin-17

Background/Aims

Microscopic colitis is characterized by chronic watery diarrhea with specific pathological changes that can be diagnosed by microscopic examination. We performed immunohistochemical analysis of proinflammatory cytokines to investigate the pathogenic mechanism of microscopic colitis.

Methods

This study consisted of six patients with lymphocytic colitis, six patients with collagenous colitis, and six patients with functional diarrhea but normal pathology. We performed an immunohistochemical analysis of the colonic mucosal biopsies to assess the expression of cyclo-oxygenase-2, interleukin-17, nuclear factor-κB, interferon-γ, inducible nitric oxide synthase, and tumor necrosis factor-α. We compared the quantity score of immunohistochemical staining among the groups.

Results

The microscopic colitis group showed significantly higher expression of cyclo-oxygenase-2, interleukin-17, nuclear factor-κB, and interferon-γ compared with the control group. Cytokine expression was similar between collagenous colitis and lymphocytic colitis. However, the expression of cyclo-oxygenase-2 was higher in collagenous colitis.

Conclusions

Proinflammatory cytokines, including interleukin-17 and interferon-γ, are highly expressed in microscopic colitis. The expression of cyclo-oxygenase-2 was higher in collagenous colitis than in lymphocytic colitis. This study is the first on interleukin-17 expression in microscopic colitis patients.     

Abnormal modulation of cell protective systems in response to ischemic/reperfusion injury is important in the development of mouse sickle cell hepatopathy

Background

Sickle cell disease, a genetic red cell disorder inherited in an autosomal recessive manner, occurs throughout the world. Hepatic dysfunction and liver damage may be present in sickle cell disease, but the pathogenesis of these conditions is only partially understood.

Design and Methods

Transgenic mice with sickle cell disease (SAD mice) and wild-type mice were exposed to an ischemic/reperfusion stress. The following parameters were evaluated: hematologic profile, transaminase and bilirubin levels, liver histopathology, and mRNA levels of nuclear factor-κB p65, endothelial nitric oxide synthase, inducible nitric oxide synthase, heme oxygenase-1 and phosphodiesterase-1, -2, -3, and -4 genes in hepatocytes obtained by laser-capture microdissection. Immunoblotting was used to analyze the expression of the following proteins: nuclear factor-κB p65 and phospho-nuclear factor-κB p65, heme oxygenase-1, biliverdin reductase, heat shock protein-70, heat shock protein-27 and peroxiredoxin-6. A subgroup of SAD mice was treated with the phosphodiesterase-4 inhibitor rolipram (30 mg/Kg/day by gavage) during the ischemic/reperfusion protocol.

Results

In SAD mice the ischemic/reperfusion stress induced liver damage compatible with sickle cell disease hepatopathy, which was associated with: (i) lack of hypoxia-induced nuclear factor-κB p65 activation; (ii) imbalance in the endothelial/inducible nitric oxide synthase response to ischemic/reperfusion stress; (iii) lack of hypoxia-induced increased expression of heme oxygenase-1/biliverdin reductase paralleled by a compensatory increased expression of heat shock proteins 70 and 27 and peroxiredoxin-6; and (iv) up-regulation of the phosphodiesterase-1, -2, -3, and -4 genes. In SAD mice the phosphodiesterase-4 inhibitor rolipram attenuated the ischemic/reperfusion-related microcirculatory dysfunction, reduced the inflammatory cell infiltration and induced the heme oxygenase-1/biliverdin reductase cytoprotective systems.

Conclusions

In SAD mice, sickle cell hepatopathy is associated with perturbed nuclear factor-κB p65 signaling with an imbalance of endothelial/inducible nitric oxide synthase levels, lack of heme oxygenase-1/biliverdin reductase expression and up-regulation of two novel cytoprotective systems: heat shock protein-27 and peroxiredoxin-6.     

Lethal hepatic apoptosis mediated by tumor necrosis factor receptor, unlike Fas-mediated apoptosis, requires hepatocyte sensitization in mice

Background/Aims: Tumor necrosis factor α (TNF-α) and Fas ligand are apoptotic cell-death mediators that act by binding to their responsive receptors. The aims of this study were to assess the differences between liver cell deaths induced by TNF-α and anti-Fas antibody, and to investigate the mechanism by which GalN sensitizes the hepatocyte to injury by TNF-α.Methods: TNF-α or anti-Fas antibody was injected into BALB/c mice sensitized or unsensitized by D-galactosamine (GalN). Liver injury was assessed biochemically and histologically. The expressions of TNF receptor (TNFR)1 and TNFR2 mRNA in the liver were determined by Northern blot analysis. Nuclear factor-κB (NF-κB) DNA binding activity was determined by gel shift assay.Results: In GalN-sensitized mice, hepatocyte apoptosis and liver failure were observed after TNF-α injection, but neither occurred in unsensitized mice. Microscopically, GalN preceding TNF-α caused massive hemorrhagic liver damage with fragmented hepatocyte nuclei resembling effects of anti-Fas antibody, but GalN largely failed to sensitize to injury by this antibody. TNFR1 mRNA expression in the liver was upregulated within 3 h after GalN administration, and anti-TNFR1 antibody protected GalN-sensitized mice from hepatotoxic effects of TNF-α. GalN treatment failed to affect TNF-α-induced NF-κB activation.Conclusions: Unlike Fas-related apoptosis, TNFR-mediated apoptosis requires hepatocyte sensitization involving TNFR1 upregulation.     

Chronic Treatment with FK506 Increases p70 S6 Kinase Activity Associated with Reduced Nitric Oxide Synthase Activity in Rabbit Hearts

FK506, an immunosuppressant, modulates phosphorylation of nitric oxide (NO) synthase, and induces cardiac hypertrophy in clinical settings. Having recently reported that chronic treatment with an inhibitor of NO synthase induces cardiac hypertrophy associated with the activation of 70-kD S6 kinase (p70^S6K), which plays an important role in cardiac hypertrophy by regulating protein synthesis, we investigated the effects of chronic administration of FK506 on NO synthase and p70^S6K activities in hearts. Twenty rabbits were divided into four groups: untreated rabbits, those treated with low-dose FK506 (0.10 mg/kg), those treated with medium-dose FK506 (0.20 mg/kg), and those treated with high-dose FK506 (0.40 mg/kg). FK506 was administered intravenously twice a day. After 4 weeks of treatment with FK506, calcium-dependent NO synthase activity in myocardium in the high-dose FK506 group was lower (P < 0.05) than in the untreated group. p70^S6K activity in myocardium in the high-dose group was higher (P < 0.05) than in the untreated group. There was a significant (P < 0.05) inverse correlation between NO synthase and p70^S6K activities in myocardium. However, the endothelial-dependent vasodilation of aortic rings or plasma levels of NO metabolites during experimental protocols did not differ among the groups studied. These findings suggest that chronic treatment of FK506 activates p70^S6K and reduces NO synthase activity in rabbit hearts. Reduced NO synthase and/or activated p70^S6K activities in hearts might contribute to the cardiac hypertrophy observed in some patients receiving FK506.     

Effects of baicalin in CD4 + CD29 + T cell subsets of ulcerative colitis patients

AIM: To evaluate the role of baicalin in ulcerative colitis (UC) with regard to the CD4⁺CD29⁺ T helper cell, its surface markers and serum inflammatory cytokines.METHODS: Flow cytometry was used to detect the percentage of CD4⁺CD29⁺ cells in patients with UC. Real time polymerase chain reaction was used to detect expression of GATA-3, forkhead box P3, T-box expressed in T cells (T-bet), and retinoic acid-related orphan nuclear hormone receptor C (RORC). Western blotting was used to analyze expression of nuclear factor-κB (NF-κB) p65, phosphorylation of NF-κB (p-NF-κB) p65, STAT4, p-STAT4, STAT6 and p-STAT6. The concentrations of interferon-γ (IFN-γ), interleukin (IL)-4, IL-5, IL-6, IL-10 and TGF-β in serum were determined by ELISA assay.RESULTS: The percentages of CD4⁺CD29⁺ T cells were lower in treatment with 40 and 20 μmol/L baicalin than in the treatment of no baicalin. Treatment with 40 or 20 μmol/L baicalin significantly upregulated expression of IL-4, TGF-β1 and IL-10, increased p-STAT6/STAT6 ratio, but downregulated expression of IFN-γ, IL-5, IL-6, RORC, Foxp3 and T-bet, and decreased ratios of T-bet/GATA-3, p-STAT4/STAT4 and p-NF-κB/NF-κB compared to the treatment of no baicalin.CONCLUSION: The results indicate that baicalin regulates immune balance and relieves the ulcerative colitis-induced inflammation reaction by promoting proliferation of CD4⁺CD29⁺ cells and modulating immunosuppressive pathways.     

Nardostachys jatamansi extract protects against cytokine-induced ��-cell damage and streptozotocin-induced diabetes

AIM: To investigate the anti-diabetogenic mechanism of Nardostachys jatamansi extract (NJE).METHODS: Mice were injected with streptozotocin via a tail vein to induce diabetes. Rat insulinoma RINm5F cells and isolated rat islets were treated with interleukin-1β and interferon-γ to induce cytotoxicity.RESULTS: Treatment of mice with streptozotocin resulted in hyperglycemia and hypoinsulinemia, which was confirmed by immunohistochemical staining of the islets. The diabetogenic effects of streptozotocin were completely abolished when mice were pretreated with NJE. Inhibition of streptozotocin-induced hyperglycemia by NJE was mediated by suppression of nuclear factor (NF)-κB activation. In addition, NJE protected against cytokine-mediated cytotoxicity. Incubation of RINm5F cells and islets with NJE resulted in a significant reduction in cytokine-induced NF-κB activation and downstream events, inducible nitric oxide synthase expression and nitric oxide production. The protective effect of NJE was further demonstrated by the normal insulin secretion of cytokine-treated islets in response to glucose.CONCLUSION: NJE provided resistance to pancreatic β-cell damage from cytokine or streptozotocin treatment. The β-cell protective effect of NJE is mediated by suppressing NF-κB activation.     

Asiatic acid inhibits cardiac hypertrophy by blocking interleukin-1β-activated nuclear factor-κB signaling in vitro and in vivo

Background

Activated interleukin (IL)-1β signaling pathway is closely associated with pathological cardiac hypertrophy. This study investigated whether asiatic acid (AA) could inhibit IL-1β-related hypertrophic signaling, and thus suppressing the development of cardiac hypertrophy.

Methods

Transverse aortic constriction (TAC) induced cardiac hypertrophy in C57BL/6 mice and cultured neonatal cardiac myocytes stimulated with IL-1β were used to evaluate the role of AA in cardiac hypertrophy. The expression of atrial natriuretic peptide (ANP) was evaluated by quantitative polymerase chain reaction (qPCR) and the nuclear factor (NF)-κB binding activity was measured by electrophoretic mobility shift assays (EMSA).

Results

AA pretreatment significantly attenuated the IL-1β-induced hypertrophic response of cardiomyocytes as reflected by reduction in the cardiomyocyte surface area and the inhibition of ANP mRNA expression. The protective effect of AA on IL-1β-stimulated cardiomyocytes was associated with the reduction of NF-κB binding activity. In addition, AA prevented TAC-induced cardiac hypertrophy in vivo. It was found that AA markedly reduced the excessive expression of IL-1β and ANP, and inhibited the activation of NF-κB in the hypertrophic myocardium.

Conclusions

Our data suggest that AA may be a novel therapeutic agent for cardiac hypertrophy. The inhibition of IL-1β-activated NF-κB signaling may be the mechanism through which AA prevents cardiac hypertrophy.     

FK506 attenuates developing and established joint inflammation and suppresses interleukin 6 and nitric oxide expression in bacterial cell wall induced polyarthritis

OBJECTIVE: To determine the efficacy of therapeutic administration of FK506 (Tacrolimus) in suppressing developing and established joint inflammation, proinflammatory cytokine expression, and nitric oxide (NO) production in peptidoglycan-polysaccharide (PG/PS) induced experimental polyarthritis in rats. METHODS: Chronic joint inflammation was induced by intraperitoneal injection of PG/PS, and joint inflammation was quantified using arthritis index and paw volume. Serum and joint levels of interleukin 6 (IL-6) were measured by bioassay and Western blot analysis respectively, and serum levels of NO production were determined by the Griess procedure and the expression of the inducible isoform of nitric oxide synthase (i-NOS) in the joints was determined by Western blot analysis. RESULTS: Arthritis induced by PG/PS is biphasic, progressing through an initial acute phase and a remission phase, which is followed by a persistent chronic phase. Daily administration of FK506 initiated during the remission phase significantly attenuated the onset and development of chronic joint inflammation. We observed a significant reduction in joint inflammation and swelling, an apparent suppression of pannus development, and minimal erosive damage to the articular cartilage and subchondral bone. Fully established chronic joint inflammation was also ameliorated by daily administration of FK506. Joint swelling and inflammation was significantly reduced by 5 days post-treatment with FK506 and the erosive activity associated with the pannus appeared diminished. The elevated expression of IL-6 and NO characteristic of chronic joint inflammation in the serum and in joint tissue was significantly reduced by FK506 treatment. CONCLUSION: Therapeutic administration of FK506 has a profound antiinflammatory effect on the development of the chronic, erosive arthritis induced by PG/PS. This attenuation in joint inflammation was associated with suppression of IL-6 and NO production systemically and locally in the joints. Our data suggest that FK506 may be effective in the treatment of chronic joint inflammation associated with rheumatoid arthritis.     

Inducible nitric oxide synthase gene promoter polymorphism is associated with increased gastric mRNA expression of inducible nitric oxide synthase and increased risk of gastric carcinoma

BACKGROUND AND AIMS: Stimulation of inducible nitric oxide synthase gene expression by Helicobacter pylori, with subsequent overproduction of nitric oxide, has been implicated in gastric carcinogenesis. We investigated whether inducible nitric oxide synthase promoter gene polymorphisms are associated with (a) inducible nitric oxide synthase mRNA expression in the gastric mucosa, and (b) the risk of gastric carcinoma. MATERIALS AND METHODS: The relationship between gastric inducible nitric oxide synthase mRNA expression and inducible nitric oxide synthase promoter polymorphisms (CCTTT repeat polymorphism and -2445 C-->G SNP) was examined in 74 H. pylori-infected patients with gastric cancer, peptic ulcer, or functional dyspepsia. In a case-control study the prevalence of the polymorphisms was examined in H. pylori-infected gastric carcinomas (n=77) and noncancerous controls (n=154). RESULTS: Inducible nitric oxide synthase mRNA levels were significantly higher in long CCTTT repeat (either allele>11) carriers than in short ones (P=0.015). Multivariate regression analysis showed that inducible nitric oxide synthase mRNA expression was significantly linked to long CCTTT repeat and gastric cancer (P=0.026), but not to -2445 C-->G SNP and other parameters. The case-control study showed that long CCTTT repeat carriers had an increased risk of gastric cancer with an odds ratio of 2.0 (P=0.021). -2445 C-->G SNP was not associated with the risk. CONCLUSIONS: Helicobacter pylori induces higher inducible nitric oxide synthase mRNA expression in carriers of long CCTTT repeats of inducible nitric oxide synthase promoter, and this polymorphism is associated with an increased risk of gastric carcinoma.     

Diallyl Trisulfide Inhibits Tumor Necrosis Factor-α Expression in Inflammed Mucosa of Ulcerative Colitis

The present study aimed to investigate the effect of diallyl trisulfide (DATS) on tumor necrosis factor (TNF)-α expression in inflammed mucosa of ulcerative colitis and its possible mechanism. Colonic biopsies from ulcerative colitis were treated with 0, 1, 5, and 10 μ M DATS for 24 hr. Lactate dehydrogenase (LDH) activities and concentrations of TNF-α in supernatants were measured. mRNA expressions of TNF-α in biopsies were analyzed by RT-PCR. The expression of NF-κ B P65 in tissues was studied by immunohistochemistry. Concentrations of TNF-α in supernatants of biopsies treated with 5 and 10 μ M DATS were lower than those of untreated biopsies. There were fewer lamina propria mononuclear cells whose NF-κ B P65 expression in nuclei was positive, and less mRNA expression of TNF-α in biopsies treated with 10 μ M DATS than in untreated biopsies. There were no differences in LDH activities in supernatants between tissues treated with DATS and untreated tissues. DATS could downregulate TNF-α production and inhibit NF-κ B activation in lamina propria mononuclear cells of inflammed mucosa, without any effect on the viability of colonic tussue cells.     

Peroxynitrite formation during rat hepatic allograft rejection

The role of nitric oxide (NO) on tissue injury of hepatic allografts during rejection remains controversial. We investigated inducible nitric oxide synthase (iNOS) expression and formation of peroxynitrite in ACI rat liver grafts implanted in recipients. Animals were divided into four experimental groups: group I, isografts; group II, untreated hepatic allografts; group III, allografts treated with FK506; and group IV, allografts pretreated with donor-specific blood transfusion (DST). Serum nitrite/nitrate, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) concentrations increased significantly in group II rats after transplantation but were significantly lower in groups I, III, and IV. The numbers of macrophages that reacted with an antimacrophage iNOS monoclonal antibody as well as iNOS messenger RNA (mRNA) levels in liver specimens were also much lower in groups I, III, and IV as compared with group II. Immunostaining and Western blot analysis showed prominent tissue nitrotyrosine expression in untreated hepatic allografts, but not in allografts treated with FK506 or donor-specific blood. These results suggest that one of the mechanisms by which production of NO results in injury in rat hepatic allografts may be because of its reaction with superoxide to form peroxynitrite.     

Therapeutic effect of a hydroxynaphthoquinone fraction on dextran sulfate sodium-induced ulcerative colitis

AIM: To evaluate the therapeutic effect of hydroxynaphthoquinone mixture (HM) on dextran sulfate sodium (DSS)-induced colitis and explore the underlying mechanisms.METHODS: BALB/c mice received 3.5% DSS for 6 d to induce ulcerative colitis. Groups of mice were orally administered HM 3.5, 7 and 14 mg/kg and mesalazine 200 mg/kg per day for 7 d. During the experiment, clinical signs and body weight, stool consistency and visible fecal blood were monitored and recorded daily. A disease activity index score was calculated for each animal. At the conclusion of the experiment, the colonic histopathological lesions were evaluated. Myeloperoxidase (MPO) activity and tumor necrosis factor-α (TNF-α) levels were determined. Protein expression levels of TNF-α, nuclear factor-κB (NF-κB) p65, inhibitor of κB (IκB) and phosphorylation of IκB (p-IκB) were analyzed by Western blot analysis.RESULTS: Administration of 3.5% DSS for 6 d successfully induced acute colitis associated with soft stool, diarrhea, rectal bleeding, and colon shortening, as well as a loss of body weight. Administration of HM effectively attenuated the severity of colonic mucosa injury. For histopathological analysis, HM treatment improved histological alterations and lowered pathological scores compared with the DSS only group. This manifested as a reduction in the extent of colon injury and inflammatory cell infiltration, as well as the degree of mucosal destruction. In addition, HM at doses of 7 and 14 mg/kg significantly decreased MPO activity in colonic tissue (0.98 ± 0.22 U/g vs 1.32 ± 0.24 U/g, 0.89 ± 0.37 U/g vs 1.32 ± 0.24 U/g tissue, P < 0.05) and serum TNF-α levels (68.78 ± 7.34 ng/L vs 88.98 ± 17.79 ng/L, 64.13 ± 14.13 ng/L vs 88.98 ± 17.79 ng/L, P < 0.05). Furthermore, HM down-regulated the expression of TNF-α, NF-κB p65 and p-IκBα in colonic tissue while up-regulating IκBα protein expression. These results suggest that the significant anti-inflammatory effect of HM may be attributable to its inhibition of TNF-α production and NF-κB activation.CONCLUSION: HM had a favorable therapeutic effect on DSS-induced ulcerative colitis, supporting its further development and clinical application in inflammatory bowel disease.     

内毒素诱导产生的一氧化氮对体外肝细胞的损伤作用

目的 探讨NO在LPS所致肝损害中的作用。方法 采用LPS刺激体外肝细胞、Kupffer细胞或两者混合培养,观察NO产生情况及培养肝细胞形态改变。结果 LPS刺激后,各级培养上清中NO产物水平均显著升高,尤其Kupffer细胞单独培养及肝细胞/Kuppffer细胞混合培养组升高更加显著,分别约为对照组的10倍和8倍,并且混合培养组中肝细胞形态出现明显改变,贴壁肝细胞变小、变园,核内染色质致密、结块,部分染色质边积,核膜皱折变形。而加用iNOS抑制剂氨基胍则培养上清中NO产物水平明显降低(约降低60％～65％),肝细胞形态未见明显改变。结论 通过诱导肝细胞、尤其Kupffer细胞产生大量NO而介导肝细胞损伤可能是LPS所致肝损害的重要机理之一。     

Nitric oxide and TNF-α trigger colonic inflammation and carcinogenesis in Helicobacter hepaticus-infected, Rag2-deficient mice

Recombinase-activating gene-2-deficient (Rag2^−/−) mice lacking functional lymphocytes provide a useful model of chronic inflammatory bowel disease-emulating events in human colon cancer. Infection of Rag2^−/− mice with Helicobacter hepaticus led to accumulation of macrophages and neutrophils in the colon, a process temporally related to up-regulation of tissue inducible nitric oxide synthase (iNOS) expression at the site of infection and increased nitric oxide (NO) production, as evidenced by urinary excretion of nitrate. Progressive development of increasingly severe inflammation, hyperplasia, dysplasia, and cancer accompanied these changes. Concurrent administration of an iNOS inhibitor prevented NO production and abrogated epithelial pathology and inhibited the onset of cancer. The presence of Gr-1⁺ neutrophils and elevated tumor necrosis factor-α (TNF-α) expression in colon were required for increased iNOS expression and cancer, whereas interleukin-10 (IL-10) down-regulated TNF-α and iNOS expression and suppressed cancer. Anti-inflammatory CD4⁺ regulatory lymphocytes also down-regulated iNOS and reduced cancer formation. Collectively, these results confirm essential roles for inflammation, increased TNF-α expression, and elevated NO production in colon carcinogenesis.