Degradation of the Transcription Factors NF-kappaB, STAT3, and STAT5 Is Involved in Entamoeba histolytica-Induced Cell Death in Caco-2 Colonic Epithelial Cells

Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-kappaB (p65) in Caco-2 cells. However, IkappaB, an inhibitor of NF-kappaB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-kappaB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-kappaB and STATs in colonic epithelial cells, which ultimately accelerates cell death.

Effect of chinese traditional herb Epimedium grandiflorum C.Morren and its extract Icariin on osteoarthritis via suppressing NF-kB pathway.

Osteoarthritis (OA), which is also called degenerative arthritis, is the leading cause of disabilities in the old people. The Chinese traditional herb Epimedium grandiflorum had long been found to attenuate osteoarthritis process, but the detailed mechanism was not clear. To study the mechanisms of E. grandiflorum in the treatment of osteoarthritis, rabbit osteoarthritis model combined with D-galactose was used. After different treatments for 10 weeks, cartilage sections were analyzed by immunohistochemistry for uPA, uPAR and PAI expression level. E. grandiflorum could significantly attenuate OA condition and decrease uPA, uPAR and PAI expression. The extract of E. grandiflorum, icariin also had a similar effect when compared with E. grandiflorum treatment alone. Rabbit chondrocytes were further isolated to be stimulated by TNFα combined with different reagents treatment. Here, icariin treatment significantly reduced nuclear factor kappa B NF-B (P65) activity, decreased uPA expression level and increased IBα protein level. The results indicated that E. grandiflorum and its extract icariin could attenuate OA condition, reduce the expression of uPA and uPAR and increase PAI in experimental rabbit model and this effect may be conducted by suppressing NF-kB activity by increasing IkBα level.

The role of gut-liver axis in the restriction of intrauterine growth in a model of experimental gastroschisis / O papel do eixo intestino-fígado na restrição de crescimento intra-uterino em um modelo de gastrosquise experimental

PURPOSE: To evaluate the intrauterine growth restriction (IUGR) by the expression of IR-β, IRS-1, IRS-2, IGF-IRβ and Ikappaβ in experimental model of gastroschisis. METHODS: Pregnant rats at 18.5 days of gestation were submitted to surgery to create experimental fetal gastroschisis (term = 22 days) were divided in three groups: gastroschisis (G), control (C) and sham (S). Fetuses were evaluated for body weight (BW), intestinal (IW), liver (LW) and their relations IW/BW and LW/BW. IR-β and IGF-IRβ receptors, IRS-1 and IRS-2 substrates and Ikappaβ protein were analyzed by western blotting. RESULTS: BW was lower in G, the IW and IW / BW were greater than C and S (p<0.05) groups. The liver showed no differences between groups. In fetuses with gastroschisis, compared with control fetuses, the expression of IGF-IRβ (p<0.001) and Ikappaβ (p<0.001) increased in the liver and intestine, as well as IR-β (p<0.001) which decreased in both. In contrast to the intestine, IRS-1 (p<0.001) increased in the liver and IRS-2 decreased (p<0.01). CONCLUSION: The axis of the intestine liver has an important role in inflammation, with consequent changes in the metabolic pathway of glucose can contribute to the IUGR in fetuses with gastroschisis.

OBJETIVO: Avaliar a restrição de crescimento intra-uterino (RCIU) pela expressão de IR-β, IRS-1, IRS-2, IGF-IRβ e a via inflamatória do Ikappaβ no modelo de gastrosquise experimental. MÉTODOS: Ratas grávidas com 18,5 dias de gestação foram submetidas a cirurgia experimental para criar gastrosquise fetal (termo = 22 dias) e os fetos foram divididos em três grupos: gastrosquise (G), controle (C) e sham (S). Os fetos foram avaliados quanto ao peso corporal (BW), intestinal (IW), fígado (LW) e suas relações IW/BW e LW/BW. Os receptores IR-β e IGF-IRβ, os substratos IRS-1 e IRS-2 e a proteína Ikappaβ foram analisados por western blotting. RESULTADOS: O BW de G foi menor, o IW e IW/BW foram superiores a C e S (p < 0.05). O fígado não apresentou diferenças entre os grupos. Nos fetos com gastrosquise, quando comparados com fetos controles, a expressão de IGF-IRβ (p<0.001) e Ikappaβ (p<0.001) aumentou no fígado e intestino, assim como IR-β (p<0.001) que diminuiu em ambos. Inversamente ao intestino, IRS-1 (p<0.001) aumentou no fígado e IRS-2 diminuiu (p<0.01). CONCLUSÃO: O eixo do intestino fígado tem um papel importante na inflamação, com consequentes alterações na via metabólica de glicose que pode contribuir para a RCIU em fetos com gastrosquise.

Anti-inflammatory Mechanism of Lactobacillus rhamnosus GG in Lipopolysaccharide-stimulated HT-29 Cell / 대한소화기학회지

BACKGROUND/AIMS: Probiotics are live non-pathogenic organisms that belong to the resident microflora, and confer health benefits by multiple mechanisms. Lactobacillus rhamnosus GG (LGG) is one of the probiotic bacteria that ameliorates intestinal injury and inflammation caused by various stimuli. We aimed to evaluate the anti-inflammatory effect and mechanism of LGG in lipopolysaccharide (LPS)-stimulated HT-29 cells. METHODS: HT-29 cells were stimulated with interleukin (IL)-1beta (2 ng/mL), tumor necrosis factor (TNF)-alpha (20 ng/mL), and LPS (20 microg/mL) in the presence or absence of LGG (107-109 colony forming units/mL). Production of the pro-inflammatory chemokine IL-8 was measured by ELISA and semi-quantitative PCR. Transcriptional activity of NF-kappaB-responsive gene was evaluated by luciferase assay with reporter gene. Toll-like receptor 4 (TLR4) mRNA expression was assessed by semi-quantitative PCR. The IkappaBalpha degradation was evaluated by western blot and intranuclear translocation of NF-kappaB was determined by western blot and immunofluorescence. RESULTS: LGG did not affect the viability of HT-29 cells. Pretreatment of HT-29 cells with LGG significantly blocked TNF-alpha, and LPS induced IL-8 activation at both mRNA and protein level (p<0.05). Pretreatment of HT-29 cells with LGG attenuated LPS-induced NF-kappaB nuclear translocation and also blocked LPS-induced IkappaBalpha degradation. LGG also down-regulated TLR4 mRNA activated by LPS. CONCLUSIONS: LGG attenuates LPS induced inflammation, and this may be associated with TLR4/NF-kappaB down-regulation.

Toll-Like Receptor 4 Signaling is Involved in IgA-Stimulated Mesangial Cell Activation

PURPOSE: Deposition of polymeric IgA1 in the kidney mesangium is the hallmark of IgA nephropathy, but the molecular mechanisms of IgA-mediated mesangial responses and inflammatory injuries remain poorly understood. We hypothesize that Toll-like receptor 4 (TLR4) is involved in IgA-induced mesangial cell activation. MATERIALS AND METHODS: Mouse mesangial cells were stimulated with lipopolysaccharide (LPS) (1 microg/mL), IgA (20 microg/mL), or both, and TLR4 expression was measured by real time RT-PCR and Western blot. Intracellular responses to LPS or IgA were assessed by Western blot for ERK1/2, JNK, p38 MAP kinases (MAPKs), Ikappa-Balpha degradation and fibronectin secretion. MCP-1 secretion was assessed by ELISA. Small interfering RNA (siRNA) of TLR4 was used to confirm that the effects were caused by TLR4 activity. RESULTS: LPS- or IgA-treatment upregulated the levels of TLR4 mRNA and protein in cultured MMC at 24 h. LPS and IgA induced rapid phosphorylation of MAPKs, but degradation of Ikappa-Balpha was observed only in LPS-treated MMC. LPS, but not IgA, induced increased secretion of MCP-1 and fibronectin at 24 h or 48 h. Combined LPS and IgA treatment did not cause additional increases in TLR4 mRNA and protein levels or Ikappa-Balpha degradation, and MCP-1 and fibronectin secretions were less than with LPS alone. LPS- or IgA-induced TLR4 protein levels and MAPK activation were inhibited by transfection with TLR4 siRNA. CONCLUSION: These results indicate that the activation of MAPKs and MCP-1 secretion are mediated by TLR4, at least in part, in IgA-treated mesangial cells. TLR4 is involved in mesangial cell injury by induction of pro-inflammatory cytokines in IgA nephropathy.

Coptidis rhizoma extract protects against cytokine-induced death of pancreatic beta-cells through suppression of NF-kappa B activation

We demonstrated previously that Coptidis rhizoma extract (CRE) prevented S-nitroso-N-acetylpenicillamine-induced apoptotic cell death via the inhibition of mitochondrial membrane potential disruption and cytochrome c release in RINm5F (RIN) rat insulinoma cells. In this study, the preventive effects of CRE against cytokine-induced beta-cell death was assessed. Cytokines generated by immune cells infiltrating pancreatic islets are crucial mediators of beta-cell destruction in insulin-dependent diabetes mellitus. The treatment of RIN cells with IL-1beta and IFN-gamma resulted in a reduction of cell viability. CRE completely protected IL-1beta and IFN-gamma-mediated cell death in a concentration-dependent manner. Incubation with CRE induced a significant suppression of IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding which correlated well with reduced levels of the iNOS mRNA and protein. The molecular mechanism by which CRE inhibited iNOS gene expression appeared to involve the inhibition of NF-kappa B activation. The IL-1beta and IFN-gamma-stimulated RIN cells showed increases in NF-kappa B binding activity and p65 subunit levels in nucleus, and IkappaBalpha degradation in cytosol compared to unstimulated cells. Furthermore, the protective effects of CRE were verified via the observation of reduced NO generation and iNOS expression, and normal insulin-secretion responses to glucose in IL-1beta and IFN-gamma-treated islets.

Corn silk induces nitric oxide synthase in murine macrophages

Corn silk has been purified as an anticoagulant previously and the active component is a polysaccharide with a molecular mass of 135 kDa. It activates murine macrophages to induce nitric oxide synthase (NOS) and generate substantial amounts of NO in time and dose-dependent manners. It was detectable first at 15 h after stimulation by corn silk, peaked at 24 h, and undetectable by 48 h. Induction of NOS is inhibited by pyrolidine dithiocarbamate (PDTC) and genistein, an inhibitor of nuclear factor kappa B (NF-kappaB) and tyrosine kinase, respectively, indicating that iNOS stimulated by corn silk is associated with tyrosine kinase and NF-kappaB signaling pathways. IkappaB-alpha degradation was detectible at 10 min, and the level was restored at 120 min after treatment of corn silk. Corn silk induced nuclear translocation of NF-kappaB by phosphorylation and degradation of IkappaB-alpha.

Nitric oxide suppresses inducible nitric oxide synthase expression by inhibiting post-translational modification of I kappa B

The expression of inducible nitric oxide synthase (iNOS) is a critical factor in both normal physiological functions and the pathogenesis of disease. This study was undertaken to determine the molecular mechanism by which nitric oxide (NO) exerts negative feedback regulation on iNOS gene expression. Isolated rat hepatocytes stimulated with cytokines exhibited a marked increase in NO production as well as iNOS mRNA and protein levels, which were significantly reduced by pretreatment of the NO donors S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and V-PYRRO/NO. This effect of SNAP was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO2-, or NO3- did not suppress the cytokine-induced NO production. Moreover, LPS/ IFN-gamma-stimulated RAW264.7 cells, which produce endogenous NO, expressed lower levels of iNOS, IL-1beta, IL-6 and TNF-alpha mRNAs, without changes in their mRNA half-lives, than those in the presence of the iNOS inhibitor NG-monomethyl- L-arginine. The iNOS gene transcription rate exhibited an 18-fold increase after cytokine stimulation, which was significantly inhibited by SNAP pretreatment. SNAP also blocked cytokine- induced increase in NF-kappa B activation, iNOS promoter activity, nuclear translocation of cytosolic NF-kappa B p65 subunit, and I kappa B alpha degradation, which correlated with its inhibitory effect on phosphorylation and ubiquitination of I kappa B. These data indicate that NO down-regulates iNOS gene expression and NO production by inhibiting the post-translational processes of I kappa B alpha thereby preventing NF-kappa B activation. These results identify a novel negative feedback mechanism whereby NO down-regulates iNOS gene expression.