Casuarinin suppresses TARC/CCL17 and MDC/CCL22 production via blockade of NF-κB and STAT1 activation in HaCaT cells

Casuarinin is a naturally occurring tannin that is isolated from the leaves of Hippophae rhamnoides. It has been shown to have anti-oxidant, anti-cancer, anti-viral, and anti-inflammatory activities. The aim of this study was to investigate the possible mechanism by which casuarinin inhibits TNF-α/IFN-γ-induced Th2 chemokines expression in the human keratinocytes cell line HaCaT. We found that casuarinin suppressed TNF-α/IFN-γ-induced expression of TARC and MDC mRNA and protein in HaCaT cells. Casuarinin significantly inhibited TNF-α/IFN-γ-induced activation of NF-κB, STAT1, and p38 MAPK. Furthermore, we observed that p38 MAPK contributes to inhibition of TNF-α/IFN-γ-induced TARC and MDC production by blocking NF-κB and STAT1 activation in HaCaT cells. Taken together, these results suggest that casuarinin may exert anti-inflammatory responses by suppressing TNF-α/IFN-γ-induced expression of TARC and MDC via blockage of p38 MAPK activation and subsequent activation of NF-κB and STAT1. We propose that it could therefore be used as a therapeutic agent against inflammatory skin diseases.     

Anti-inflammatory activity of myricetin from Diospyros lotus through suppression of NF-κB and STAT1 activation and Nrf2-mediated HO-1 induction in lipopolysaccharide-stimulated RAW264.7 macrophages

Diospyros lotus is traditionally used for the treatment of diabetes, diarrhea, tumor, and hypertension. The purpose of this study was to investigate the anti-inflammatory effect and underlying molecular mechanisms of myricetin in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Myricetin dose-dependently suppressed the production of pro-inflammatory mediators (NO, iNOS, PGE₂, and COX-2) in LPS-stimulated RAW264.7 macrophages. Myricetin administration decreased the production of NO, iNOS, TNF-α, IL-6, and IL-12 in mice. Myricetin decreased NF-κB activation by suppressing the degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Moreover, myricetin attenuated the phosphorylation of STAT1 and the production of IFN-β in LPS-stimulated RAW264.7 macrophages. Furthermore, myricetin induced the expression of HO-1 through Nrf2 translocation. In conclusion, these results suggest that myricetin inhibits the production of pro-inflammatory mediators through the suppression of NF-κB and STAT1 activation and induction of Nrf2-mediated HO-1 expression in LPS-stimulated RAW264.7 macrophages.     

Suppression of iNOS expression by fucoidan is mediated by regulation of p38 MAPK,JAK/STAT,AP-1 and IRF-1, and depends on up-regulation of scavenger receptor B1 expression in TNF-α- and IFN-γ-stimulated C6 glioma cells

In neurodegenerative disorders, activated glial cells overproduce nitric oxide (NO), which causes neurotoxicity. Inducible NO synthase (iNOS) is a potential therapeutic target in neurodegenerative diseases. Here, we examined the action of fucoidan, a high-molecular-weight sulfated polysaccharide, on tumor necrosis factor-α (TNF-α)- and interferon-γ (IFN-γ)-induced NO production in C6 glioma cells. Fucoidan suppressed TNF-α- and IFN-γ-induced NO production and iNOS expression. In addition, fucoidan inhibited TNF-α- and IFN-γ-induced AP-1, IRF-1, JAK/STAT and p38 mitogen-activated protein kinase (MAPK) activation and induced scavenger receptor B1 (SR-B1) expression. Blocking of SR-B1 did not reverse the inhibitory effect of fucoidan on TNF-α- and IFN-γ- stimulated NO production. However, inhibition of SR-B1 expression by siRNA increased iNOS expression and p38 phosphorylation in TNF-α- and IFN-γ-stimulated C6 cells.Overall, p38 MAPK, AP-1, JAK/STAT and IRF-1 play an important role in the inhibitory effect of fucoidan on TNF-α- and IFN-γ-stimulated NO production, and intracellular SR-B1 expression may be related to the inhibition of iNOS expression by fucoidan via regulation of p38 phosphorylation. The present results also suggest that fucoidan could be a potential therapeutic agent for treating inflammatory-related neuronal injury in neurological disorders.     

Picrasma quassioides inhibits LPS- and IFN-γ-stimulated nitric oxide production and inflammatory response in RAW264.7 macrophage cells

The present study was designed to investigate the anti-inflammatory effects of Picrasma quassioides (P. quassioides) in lipopolysaccharide (LPS)- and interferon (IFN)-γ-stimulated RAW 264.7 cells. P. quassioides has been used as a traditional medicine for the treatment of gastro-enteritis, eczema, and snakebite. P. quassioides significantly decreased LPS- and IFN-γ-stimulated nitric oxide (NO) production in a concentration-dependent manner. Real-time PCR or Western blotting confirmed that the expression of the extra-cellular signal-regulated kinase (ERK), p42 and, p38 mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) mediated MAPK signaling pathways in LPS- and IFN-γ-stimulated RAW264.7 cells. Mechanistic studies revealed the activities of nuclear factor kappa B (NF-κB). As P. quassioides regulated the gene expression of iNOS and COX-2 in RAW264.7 cells, it might be a promising agent for the prevention and/or treatment of various inflammatory diseases.     

Sodium butyrate inhibits interferon-gamma induced indoleamine 2,3-dioxygenase expression via STAT1 in nasopharyngeal carcinoma cells

Aims

Indoleamine 2,3-dioxygenase (IDO) inhibits T-cell proliferation by catalyzing the conversion of l-tryptophan to l-kynurenine. IDO-induced immune tolerance weakens the clinical outcomes of immunotherapies. Sodium butyrate (NaB), one of the histone deacetylase inhibitors (HDACIs), has potential anti-tumor effects. Our previous studies revealed that NaB could inhibit IFN-γ induced IDO expression in nasopharyngeal carcinoma cells, CNE2. In the present study, we aim to investigate to the mechanism of NaB interfering with the interferon-gamma (IFN-γ)-mediated IDO expression signaling transduction.

Main methods

IDO expression and STAT1 phosphorylation in CNE2 cells were analyzed by western blotting and STAT1 acetylation was evaluated by immunoprecipitation. STAT1 nuclear translocation and NF-κB activity were detected by transient transfection and reporter gene assay.

Key findings

We found that NaB inhibited IFN-γ-induced IDO expression in CNE2 cells via decreasing phosphorylation and nuclear translocation of STAT1, but not via down-regulation of IFN-γ-receptor (IFNGR). Immunoprecipitation assays revealed that NaB increased STAT1 acetylation. Furthermore, NaB elevated the activity of NF-κB in CNE2 cells, and blocking the NF-κB activity had no effect on the IFN-γ-induced IDO expression.

Significance

These results suggest that NaB inhibited IFN-γ-induced IDO expression via STAT1 increased acetylation, decreased phosphorylation, and reduced nuclear translocation. These provided new evidence for the anti-tumor action of NaB and potential drug targets to reduce the IDO-induced immune tolerance.     

Inhibition of lipopolysaccharide-induced inducible nitric oxide synthase expression by endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is induced in infectious and inflammatory conditions, but its role in inflammatory responses still remains elusive. In this study we found tunicamycin (TM) and brefeldin A (BFA), two ER stressors, could attenuate lipopolysaccharide (LPS)-elicited inducible nitric oxide synthase (iNOS) gene expression in murine RAW264.7 macrophages, and this effect was not resulting from the effects on IKK or MAPKs activation. However, ER stressors could block NF-κB binding to the iNOS promoter in late-phase signaling evoked by LPS. Results indicated that inhibition of RelB nuclear translocation and p300 expression are involved in the anti-inflammatory actions of ER stressors. We also found that ER stressors could block LPS- and IFN (α, β, and γ)-mediated STAT1 phosphorylation. Our results suggest that activation of MKP-1 via a Ca/calmodulin/calcineurin pathway accounts for the inhibitory effect of ER stressors on IFN signaling. MKP-1 was downregulated by IFN-γ and is a newly identified protein phosphatase targeting STAT1. Taken together, these results indicate that multiple mechanisms are involved in the inhibition of LPS-induced iNOS gene expression by ER stressors. These include downregulation of RelB and p300, upregulation of MKP-1, and inhibition of the JAK/STAT signaling pathway.     

Effects of physiological quercetin metabolites on interleukin-1β-induced inducible NOS expression

Cytokines released by inflammatory cells around the pancreatic islets are implicated in the pathogenesis of diabetes mellitus. Specifically, interleukin-1β (IL-1β) is known to be involved in islet β-cell damage by activation of nuclear factor-κB (NF-κB)-mediated inducible nitric oxide synthase (iNOS) gene expression. Though most flavonoids are shown to have various beneficial effects, little is known about the anti-inflammatory effects of their metabolites. Therefore, we investigated the effects of quercetin and its metabolites quercetin 3'-sulfate, quercetin 3-glucuronide and isorhamnetin 3-glucuronide on IL-1β-stimulated iNOS gene expression in RINm5F β-cells. The nitrite level, iNOS protein and its mRNA expression levels and iNOS promoter activity were measured. In addition, IκBα protein phosphorylation, nuclear translocation of nuclear factor-κB (NF-κB) and NF-κB DNA binding activity were determined. Adenosine 5'-triphosphate disodium salt-induced insulin release was also measured. Quercetin significantly reduced IL-1β-induced nitrite production, iNOS protein and its mRNA expression levels, and it also inhibited IL-1β-induced IκBα phosphorylation, NF-κB activation and iNOS promoter activity. Additionally, quercetin significantly restored the inhibition of insulin secretion by IL-1β. Meanwhile, quercetin metabolites did not show any effect on IL-1β-induced iNOS gene expression and also on insulin secretion. Therefore, in terms of iNOS expression mechanism, dietary ingestion of quercetin is unlikely to show anti-inflammatory effects in rat islet β-cells exposed to IL-1β.     

Suppression of thymus- and activation-regulated chemokine (TARC/CCL17) production by 1,2,3,4,6-penta-O-galloyl-β-d-glucose via blockade of NF-κB and STAT1 activation in the HaCaT cells

Keratinocytes, one of major cell types in the skin, can be induced by TNF-α and IFN-γ to express thymus- and activation-regulated chemokine (TARC/CCL17), which is considered to be a pivotal mediator in the inflammatory responses during the development of inflammatory skin diseases, such as atopic dermatitis (AD). In this study, we examined the effect of 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG), isolated from the barks of Juglans mandshurica, on TNF-α/IFN-γ induced CCL17 expression in the human keratinocyte cell line HaCaT. Pretreatment of HaCaT cells with PGG suppressed TNF-α/IFN-γ-induced protein and mRNA expression of CCL17. PGG significantly inhibited TNF-α/IFN-γ-induced NF-κB activation as well as STAT1 activation. Furthermore, pretreatment with PGG resulted in significant reduction in expression of CXCL9, 10, and 11 in the HaCaT cells treated with IFN-γ. These results suggest that PGG may exert anti-inflammatory responses by suppressing TNF-α and/or IFN-γ-induced activation of NF-κB and STAT1 in the keratinocytes and might be a useful tool in therapy of skin inflammatory diseases.