Jingjing Zhang

Protective effect of naringenin against experimental colitis via suppression of Toll-like receptor 4/NF-κB signalling

Naringenin, one of the most abundant flavonoids in citrus, grapefruits and tomatoes, has been used as a traditional anti-inflammatory agent for centuries. However, the molecular mechanism of naringenin in intestinal inflammation remains unknown so far. The present study investigated a molecular basis for the protective effect of naringenin in dextran sulphate sodium-induced murine colitis. Pre-administration of naringenin significantly reduced the severity of colitis and resulted in down-regulation of pro-inflammatory mediators (inducible NO synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), cyclo-oxygenase-2 (Cox2), TNF-α and IL-6 mRNA) in the colon mucosa. The decline in the production of pro-inflammatory cytokines, specifically TNF-α and IL-6, correlated with a decrease in mucosal Toll-like receptor 4 (TLR4) mRNA and protein. Phospho-NF-κB p65 protein was significantly decreased, which correlated with a similar decrease in phospho-IκBα protein. Consistent with the in vivo results, naringenin exposure blocked lipopolysaccharide-stimulated nuclear translocation of NF-κB p65 in mouse macrophage RAW264.7 cells. In addition, in vitro NF-κB reporter assays performed on human colonic HT-29 cells exposed to naringenin demonstrated a significant inhibition of TNF-α-induced NF-κB luciferase expression. Thus, for the first time, the present study indicates that targeted inhibition of the TLR4/NF-κB signalling pathway might be an important mechanism for naringenin in abrogating experimental colitis.

Chrysin Ameliorates Chemically Induced Colitis in the Mouse through Modulation of a PXR/NF-κB Signaling Pathway

Targeted activation of pregnane X receptor (PXR) in recent years has become a therapeutic strategy for inflammatory bowel disease. Chrysin is a naturally occurring flavonoid with anti-inflammation activity. The current study investigated the role of chrysin as a putative mouse PXR agonist in preventing experimental colitis. Pre-administration of chrysin ameliorated inflammatory symptoms in mouse models of colitis (dextran sodium sulfate– and 2,4,6-trinitrobenzene sulfonic acid–induced) and resulted in down-regulation of nuclear transcription factor κB (NF-κB) target genes (inducible NO synthase, intercellular adhesion molecule-1, monocyte chemotactic protein-1, cyclooxygenase 2, tumor necrosis factor-α, and interleukin 6) in the colon mucosa. Chrysin inhibited the phosphorylation/degradation of inhibitor κBα (IκBα), which correlated with the decrease in the activity of myeloperoxidase and the levels of tumor necrosis factor–α and interleukin 6 in the colon. Consistent with the in vivo results, chrysin blocked lipopolysaccharide -stimulated nuclear translocation of NF-κB p65 in mouse macrophage RAW264.7. Furthermore, chrysin dose-dependently activated human/mouse PXR in reporter gene assays and up-regulated xenobiotic detoxification genes in the colon mucosa, but not in the liver. Silencing of PXR by RNA interference demonstrated necessity of PXR in mediating chrysin’s ability to induce xenobiotic detoxification genes and NF-κB inactivation. The repression of NF-κB transcription activity by chrysin was confirmed by in vitro PXR transduction. These findings suggest that the effect of chrysin in preventing chemically induced colitis is mediated in large part by a PXR/NF-κB pathway. The data also suggest that chrysin or chrysin-like flavonoids could be further developed as intestine-specific PXR activators.

Mangiferin attenuates the symptoms of dextran sulfate sodium-induced colitis in mice via NF-κB and MAPK signaling inactivation

Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disorder of the gastrointestinal (GI) tract, and currently no curative treatment is available. Mangiferin, a natural glucosylxanthone mainly from the fruit, leaves and stem bark of a mango tree, has a strong anti-inflammatory activity. We sought to investigate whether mangiferin attenuates inflammation in a mouse model of chemically induced IBD. Pre-administration of mangiferin significantly attenuated dextran sulfate sodium (DSS)-induced body weight loss, diarrhea, colon shortening and histological injury, which correlated with the decline in the activity of myeloperoxidase (MPO) and the level of tumor necrosis factor-α (TNF-α) in the colon. DSS-induced degradation of inhibitory κBα (IκBα) and the phosphorylation of nuclear factor-kappa B (NF-κB) p65 as well as the mRNA expression of pro-inflammatory mediators (inducible NO synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), TNF-α, interleukin-1β (IL-1β) and IL-6) in the colon were also downregulated by mangiferin treatment. Additionally, the phosphorylation/activation of DSS-induced mitogen-activated protein kinase (MAPK) proteins was also inhibited by mangiferin treatment. In accordance with the in vivo results, mangiferin exposure blocked TNF-α-stimulated nuclear translocation of NF-κB in RAW264.7 mouse macrophage cells. Transient transfection gene reporter assay performed in TNF-α-stimulated HT-29 human colorectal adenocarcinoma cells indicated that mangiferin inhibits NF-κB transcriptional activity in a dose-dependent manner. The current study clearly demonstrates a protective role for mangiferin in experimental IBD through NF-κB and MAPK signaling inhibition. Since mangiferin is a natural compound with little toxicity, the results may contribute to the effective utilization of mangiferin in the treatment of human IBD.

Paeoniflorin abrogates DSS-induced colitis via a TLR4-dependent pathway

Paeonia lactiflora Pall is one of the most well-known herbs in China, Korea, and Japan for more than 1,200 years. Paeoniflorin, the major bioactive component of peony root, has recently been reported to have anticolitic activity. However, the underlying molecular mechanism is unclear. The present study was to explore the possible mechanism of paeoniflorin in attenuating dextran sulfate sodium (DSS)-induced colitis. Pre- and coadministration of paeoniflorin significantly reduced the severity of colitis and resulted in downregulation of several inflammatory parameters in the colon, including the activity of myeloperoxidase (MPO), the levels of TNF-α and IL-6, and the mRNA expression of proinflammatory mediators (MCP-1, Cox2, IFN-γ, TNF-α, IL-6, and IL-17). The decline in the activation of NF-κB p65, ERK, JNK, and p38 MAPK correlated with a decrease in mucosal Toll-like receptor 4 (TLR4) but not TLR2 or TLR5 expression. In accordance with the in vivo results, paeoniflorin downregulated TLR4 expression, blocked nuclear translocation of NF-κB p65, and reduced the production of IL-6 in LPS-stimulated mouse macrophage RAW264.7 cells. Transient transfection assay performed in LPS-stimulated human colon cancer HT-29 cells indicated that paeoniflorin inhibits NF-κB transcriptional activity in a dose-dependent manner. TLR4 knockdown and overexpression experiments demonstrated a requirement for TLR4 in paeoniflorin-mediated downregulation of inflammatory cytokines. Thus, for the first time, the present study indicates that paeoniflorin abrogates DSS-induced colitis via decreasing the expression of TLR4 and suppressing the activation of NF-κB and MAPK pathways.

Plant flavonol isorhamnetin attenuates chemically induced inflammatory bowel disease via a PXR-dependent pathway

Isorhamnetin is an O-methylated flavonol present in fruit and vegetables. We recently reported the identification of isorhamnetin as an activator of the human pregnane X receptor (PXR), a known target for abrogating inflammation in inflammatory bowel disease (IBD). The current study investigated the role of isorhamnetin as a putative mouse PXR activator in ameliorating chemically induced IBD. Using two different models (ulcerative colitis like and Crohns disease like) of experimental IBD in mice, we demonstrated that isorhamnetin abrogated inflammation through inhibiting the activity of myeloperoxidase, the levels of TNF-α and IL-6, the mRNA expression of proinflammatory mediators (iNOS, ICAM-1, COX2, TNF-α, IL-2 and IL-6) and the phosphorylation of IκBα and NF-κB p65. PXR gene overexpression inhibited NF-κB luciferase activity, and the inhibition was potentiated by isorhamnetin treatment. PXR knockdown by siRNA demonstrated the necessity for PXR in isorhamnetin-mediated up-regulation of xenobiotic metabolism genes. Ligand pocket-filling mutants (S247W/C284W and S247W/C284W/S208W) of human PXR weakened the effect of isorhamnetin on PXR activation. Molecular docking studies and time-resolved fluorescence resonance energy transfer competitive binding assays confirmed the ligand (isorhamnetin)-binding affinity. These results clearly demonstrated the ameliorating effect of isorhamnetin on experimental IBD via PXR-mediated up-regulation of xenobiotic metabolism and down-regulation of NF-κB signaling. The novel findings may contribute to the effective utilization of isorhamnetin or its derivatives as a PXR ligand in the treatment of human IBD.

Notoginsenoside R1 Attenuates Experimental Inflammatory Bowel Disease via Pregnane X Receptor Activation

Notoginsenoside R1 (R1) is the main bioactive component in Panax notoginseng, an old herb medicine widely used in Asian countries in the treatment of microcirculatory diseases. However, little is known about the effect of R1 on inflammatory bowel disease (IBD). The present study demonstrated that R1 alleviated the severity of dextran sulfate sodium–induced colitis in mice by decreasing the activity of myeloperoxidase, the production of cytokines, the expression of proinflammatory genes, and the phosphorylation of IκB kinase, IκBα, and p65 in the colon. Further studies indicated that R1 dose-dependently activated human/mouse pregnane X receptor (PXR), a known target for decreasing inflammation in IBD, and upregulated the expression of genes involved in xenobiotic metabolism in colorectal cells and the colon. Ligand pocket–filling mutant (S247W/C284W or S247W/C284W/S208W) of the human PXR abrogated the effect of R1 on PXR activation. Time-resolved fluorescence resonance energy transfer PXR competitive binding assay confirmed R1 (ligand) binding affinity. In addition, PXR overexpression inhibited nuclear factor-κB (NF-κB)–luciferase activity, which was potentiated by R1 treatment. PXR knockdown by small interfering RNA demonstrated the necessity of PXR in R1-induced upregulation of the expression of xenobiotic-metabolizing enzymes and downregulation of NF-κB activity. Finally, the anti-inflammatory effect of R1 was confirmed in trinitrobenzene sulfonic acid–induced colitis in mice. These findings suggest that R1 attenuates experimental IBD possibly via the activation of intestinal PXR signaling.

The anti-inflammatory effect and potential mechanism of cardamonin in DSS-induced colitis

Cardamonin is a naturally occurring chalcone with strong anti-inflammatory activity. However, the direct effect of cardamonin on intestinal inflammation remains elusive. In the present study, we found that cardamonin markedly ameliorated dextran sulfate sodium-induced mouse body weight loss, diarrhea, colon shortening, spleen swelling, and histological damage, which correlated with a decline in the activity of myeloperoxidase and the production of nitric oxide, tumor necrosis factor-α and interleukin-6 in the colon. The upregulation of toll-like receptor 4 after dextran sulfate sodium treatment was associated with an increase in the activation of myeloid differentiation factor 88, interleukin-1 receptor-associated kinase-1, nuclear factor-κB (NF-κB) p65, inhibitor κBα, and inhibitor κB kinase-α/β, as well as the mitogen-activated protein kinase molecules of extracellular signal-regulated kinase and c-Jun NH2-terminal kinase, and this upregulation was reversed by cardamonin administration. Moreover, cardamonin blocked the nuclear translocation of NF-κB p65, inhibited NF-κB-luciferase activity, and downregulated NF-κB target genes expression. The present study clearly demonstrates a beneficial effect of cardamonin on experimental inflammatory bowel disease via a mechanism associated with suppression of toll-like receptor 4 expression and inactivation of NF-κB and mitogen-activated protein kinase pathways. This study may give insight into the further evaluation of the therapeutic potential of cardamonin or its derivatives for human inflammatory bowel disease.

Baicalein ameliorates TNBS-induced colitis by suppressing TLR4/MyD88 signaling cascade and NLRP3 inflammasome activation in mice

Baicalein (5,6,7-trihydroxyflavone), a predominant bioactive component isolated from the root of Scutellaria baicalensis Georgi, has established potent anti-inflammatory activity via multi-targeted mechanisms. However, little is known about the effect of baicalein on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, which shares pathology related to human Crohn’s disease (CD). The present study demonstrated that baicalein alleviated the severity of TNBS-induced colitis in mice by decreasing the activity of myeloperoxidase (MPO) and the expression of pro-inflammatory mediators. The decline in the activation of nuclear factor-kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) correlated with a decrease in the expression of mucosal toll-like receptor 4 (TLR4) and its adaptor myeloid differentiation factor 88 (MyD88). In vitro, baicalein down-regulated the TLR4/MyD88 signaling cascades (NF-κB and MAPKs) in lipopolysaccharide (LPS)-stimulated macrophages. At the upstream level, baicalein bound to the hydrophobic region of the myeloid differentiation protein-2 (MD-2) pocket and inhibited the formation of the LPS-induced MD-2/TLR4 complex. Furthermore, baicalein reduced NOD-like receptor 3 (NLRP3) inflammasome activation and downstream interleukin-1β expression in a dose-dependent manner. Our study provided evidence for the first time that baicalein attenuated TNBS-induced colitis, at least in part, via inhibition of TLR4/MyD88 signaling cascade as well as inactivation of NLRP3 inflammasome.

Anti-Inflammatory Effects of Fargesin on Chemically Induced Inflammatory Bowel Disease in Mice

Fargesin is a bioactive lignan from Flos Magnoliae, an herb widely used in the treatment of allergic rhinitis, sinusitis, and headache in Asia. We sought to investigate whether fargesin ameliorates experimental inflammatory bowel disease (IBD) in mice. Oral administration of fargesin significantly attenuated the symptoms of dextran sulfate sodium (DSS)-induced colitis in mice by decreasing the inflammatory infiltration and myeloperoxidase (MPO) activity, reducing tumor necrosis factor (TNF)-α secretion, and inhibiting nitric oxide (NO) production in colitis mice. The degradation of inhibitory κBα (IκBα), phosphorylation of p65, and mRNA expression of nuclear factor κB (NF-κB) target genes were inhibited by fargesin treatment in the colon of the colitis mice. In vitro, fargesin blocked the nuclear translocation of p-p65, downregulated the protein levels of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), and dose-dependently inhibited the activity of NF-κB-luciferase in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Taken together, for the first time, the current study demonstrated the anti-inflammatory effects of fargesin on chemically induced IBD might be associated with NF-κB signaling suppression. The findings may contribute to the development of therapies for human IBD by using fargesin or its derivatives.