Bergenin, Acting as an Agonist of PPAR?, Ameliorates Experimental Colitis in Mice through Improving Expression of SIRT1, and Therefore Inhibiting NF-?B-Mediated Macrophage Activation.
ABSTRACT: Bergenin, isolated from the herb of Saxifraga stolonifera Curt. (Hu-Er-Cao), has anti-inflammatory, antitussive and wound healing activities. The aim of the present study was to identify the effect of bergenin on experimental colitis, and explored the related mechanisms. Our results showed that oral administration of bergenin remarkably alleviated disease symptoms of mice with dextran sulfate sodium (DSS)-induced colitis, evidenced by reduced DAI scores, shortening of colon length, MPO activity and pathologic abnormalities in colons. Bergenin obviously inhibited the mRNA and protein expressions of IL-6 and TNF-? in colon tissues, but not that of mucosal barrier-associated proteins occludin, E-cadherin and MUC-2. In vitro, bergenin significantly inhibited the expressions of IL-6 and TNF-? as well as nuclear translocation and DNA binding activity of NF-?B-p65 in lipopolysaccharide (LPS)-stimulated peritoneal macrophages and RAW264.7 cells, which was almost reversed by addition of PPAR? antagonist GW9662 and siPPAR?. Subsequently, bergenin was identified as a PPAR? agonist. It could enter into macrophages, bind with PPAR?, promote nuclear translocation and transcriptional activity of PPAR?, and increase mRNA expressions of CD36, LPL and ap2. In addition, bergenin significantly up-regulated expression of SIRT1, inhibited acetylation of NF-?B-p65 and increased association NF-?B-p65 and I?B?. Finally, the correlation between activation of PPAR? and attenuation of colitis, inhibition of IL-6 and TNF-? expressions, NF-?B-p65 acetylation and nuclear translocation, and up-regulation of SIRT1 expression by bergenin was validated in mice with DSS-induced colitis and/or LPS-stimulated macrophages. In summary, bergenin could ameliorate colitis in mice through inhibiting the activation of macrophages via regulating PPAR?/SIRT1/NF-?B-p65 pathway. The findings can provide evidence for the further development of bergenin as an anti-UC drug, and offer a paradigm for the recognization of anti-UC mechanisms of compound with similar structure occurring in traditional Chinese medicines.
Project description:P21-activated kinases (PAKs) are multifunctional effectors of Rho GTPases with both kinase and scaffolding activity. Here, we investigated the effects of inflammation on PAK1 signaling and its role in colitis-driven carcinogenesis. PAK1 and p-PAK1 (Thr423) were assessed by immunohistochemistry, immunofluorescence, and Western blot. C57BL6/J wildtype mice were treated with a single intraperitoneal TNF? injection. Small intestinal organoids from these mice and from PAK1-KO mice were cultured with TNF?. NF-?B and PPAR? were analyzed upon PAK1 overexpression and silencing for transcriptional/translational regulation. PAK1 expression and activation was increased on the luminal intestinal epithelial surface in inflammatory bowel disease and colitis-associated cancer. PAK1 was phosphorylated upon treatment with IFN?, IL-1?, and TNF?. In vivo, mice administered with TNF? showed increased p-PAK1 in intestinal villi, which was associated with nuclear p65 and NF-?B activation. p65 nuclear translocation downstream of TNF? was strongly inhibited in PAK1-KO small intestinal organoids. PAK1 overexpression induced a PAK1-p65 interaction as visualized by co-immunoprecipitation, nuclear translocation, and increased NF-?B transactivation, all of which were impeded by kinase-dead PAK1. Moreover, PAK1 overexpression downregulated PPAR? and mesalamine recovered PPAR? through PAK1 inhibition. On the other hand PAK1 silencing inhibited NF-?B, which was recovered using BADGE, a PPAR? antagonist. Altogether these data demonstrate that PAK1 overexpression and activation in inflammation and colitis-associated cancer promote NF-?B activity via suppression of PPAR? in intestinal epithelial cells.
Project description:NF-κB-mediated inflammatory phenotypic switching of vascular smooth muscle cells (VSMCs) plays a central role in atherosclerosis and neointimal formation. However, little is known about the roles of circRNAs in the regulation of NF-κB signaling. Here, we identify the involvement of circ-Sirt1 that was one of transcripts of SIRT1 host gene in VSMC inflammatory response and neointimal hyperplasia. First, in the cytoplasm, circ-Sirt1 directly interacts with and sequesters NF-κB p65 from nuclear translocation induced by TNF-α in a sequence-dependent manner. The inhibitory complex of circ-Sirt1-NF-κB p65 is not dependent on IκBα. Second, circ-Sirt1 binds to miR-132/212 that interferes with SIRT1 mRNA, and facilitates the expression of host gene SIRT1. Increased SIRT1 results in deacetylation and inactivation of the nuclear NF-κB p65. These findings illustrate that circ-Sirt1 is a novel non-coding RNA regulator of VSMC phenotype.
Project description:Macrophages are crucially involved in the pathogenesis of rheumatoid arthritis (RA). Macrophages of the M1 phenotype act as pro-inflammatory mediators in synovium, whereas those of the M2 phenotype suppress inflammation and promote tissue repair. SIRT1 is a class 3 histone deacetylase with anti-inflammatory characteristics. However, the role played by SIRT1 in macrophage polarization has not been defined in RA. We investigated whether SIRT1 exerts anti-inflammatory effects by modulating M1/M2 polarization in macrophages from RA patients. In this study, SIRT1 activation promoted the phosphorylation of an adenosine monophosphate-activated protein kinase (AMPK) ?/acetyl-CoA carboxylase in macrophages exposed to interleukin (IL)-4, and that this resulted in the expressions of M2 genes, including MDC, Fc?RII, MrC1, and IL-10, at high levels. Furthermore, these expressions were inhibited by sirtinol (an inhibitor of SIRT1) and compound C (an inhibitor of AMPK). Moreover, SIRT1 activation downregulated LPS/interferon ?-mediated NF-?B activity by inhibiting p65 acetylation and the expression of M1 genes, such as CCL2, iNOS, IL-12 p35, and IL-12 p40. Macrophages from SIRT1 transgenic (Tg)-mice exhibited enhanced polarization of M2 phenotype macrophages and reduced polarization of M1 phenotype macrophages. In line with these observations, SIRT1-Tg mice showed less histological signs of arthritis, that is, lower TNF? and IL-1? expressions and less severe arthritis in the knee joints, compared to wild-type mice. Taken together, the study shows activation of SIRT1/AMPK? signaling exerts anti-inflammatory activities by regulating M1/M2 polarization, and thereby reduces inflammatory responses in RA. Furthermore, it suggests that SIRT1 signaling be viewed as a therapeutic target in RA.
Project description:Bergenin is a C-glucoside of 4-O-methyl gallic acid isolated from several medicinal plants and has multiple biological activities. The aim of this study was to assess the potential usefulness of bergenin in hyperuricemia. We found that bergenin reduced serum urate levels in hyperuricemia mice by promoting renal and gut uric acid excretion. Bergenin treatment increased Abcg2 expression both in the kidneys and intestine, while the expression of Slc2a9 was suppressed in the kidney and increased in the intestine. Moreover, bergenin induced ABCG2 expression in HK-2 and Caco-2 cells, as well as SLC2A9 in Caco-2 cells, via the activation of PPAR?. Nevertheless, bergenin suppressed SLC2A9 expression in HK-2 cells by inhibiting the nuclear translocation of p53. Furthermore, bergenin decreased the serum levels of IL-6, IL-1?, and TNF-? in hyperuricemia mice, and promoted a polarization shift from the M1 to M2 phenotype in RAW264.7 cells. In conclusion, these findings provide evidence supporting the further development of bergenin as a novel therapeutic strategy for hyperuricemia.
Project description:Selective expression of dominant negative (DN) peroxisome proliferator-activated receptor ? (PPAR?) in vascular smooth muscle cells (SMC) results in hypertension, atherosclerosis, and increased nuclear factor-?B (NF-?B) target gene expression. Mesenteric SMC were cultured from mice designed to conditionally express wild-type (WT) or DN-PPAR? in response to Cre recombinase to determine how SMC PPAR? regulates expression of NF-?B target inflammatory genes. SMC-specific overexpression of WT-PPAR? or agonist-induced activation of endogenous PPAR? blunted tumor necrosis factor ? (TNF-?)-induced NF-?B target gene expression and activity of an NF-?B-responsive promoter. TNF-?-induced gene expression responses were enhanced by DN-PPAR? in SMC. Although expression of NF-?B p65 was unchanged, nuclear export of p65 was accelerated by WT-PPAR? and prevented by DN-PPAR? in SMC. Leptomycin B, a nuclear export inhibitor, blocked p65 nuclear export and inhibited the anti-inflammatory action of PPAR?. Consistent with a role in facilitating p65 nuclear export, WT-PPAR? coimmunoprecipitated with p65, and WT-PPAR? was also exported from the nucleus after TNF-? treatment. Conversely, DN-PPAR? does not bind to p65 and was retained in the nucleus after TNF-? treatment. Transgenic mice expressing WT-PPAR? or DN-PPAR? specifically in SMC (S-WT or S-DN) were bred with mice expressing luciferase controlled by an NF-?B-responsive promoter to assess effects on NF-?B activity in whole tissue. TNF-?-induced NF-?B activity was decreased in aorta and carotid artery from S-WT but was increased in vessels from S-DN mice. We conclude that SMC PPAR? blunts expression of proinflammatory genes by inhibition of NF-?B activity through a mechanism promoting nuclear export of p65, which is abolished by DN mutation in PPAR?.
Project description:Microglial activation is involved in a variety of neurological disorders, and overactivated microglial cells can secrete large amount of proinflammatory factors and induce neuron death. Therefore, reducing microglial activation is believed to be useful in treating the disorders. In this study, we used 10 ng/ml lipopolysaccharide plus 10 U/ml interferon γ (LPS/IFNγ) to induce N9 microglial activation and explored resveratrol- (RSV-) induced effects on microglial activation and the underlying mechanism. We found that LPS/IFNγ exposure for 24 h increased inducible nitric oxide synthase (iNOS) and nuclear factor κB (NF-κB) p65 subunit expressions in the cells and enhanced tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) releases from the cells. RSV of 25 μM reduced the iNOS and NF-κB p65 subunit expressions and the proinflammatory factors' releases; the knockdown of silent information regulator factor 2-related enzyme 1 (SIRT1) or suppressor of cytokine signaling 1 (SOCS1) by using the small interfering RNA, however, significantly abolished the RSV-induced effects on iNOS and NF-κB p65 subunit expressions and the proinflammatory factors' releases. These findings showed that microglial SIRT1-SOCS1 pathway may mediate the RSV-induced inhibition of microglial activation in the LPS/IFNγ-treated N9 microglia.
Project description:Sirtuin 1 (Sirt1) is a deacetylase that regulates many cellular processes in the liver, and so far its role in endotoxemic liver injury is elusive. So we conditionally inactivate Sirt1 in murine hepatocytes to determine its role in d-galactosamine (GalN)/lipopolysaccharide (LPS)-induced liver damage, which is a well-established experimental model mimicking septic liver injury and fulminant hepatitis. Ablation of Sirt1 shows remarkable protection against GalN/LPS-induced liver injury, which is a result of enhanced NF-?B response because knockdown of RelA/p65 negates the protective effect of Sirt1 knockout. Mechanistically, NF-?B p65 is maintained in a hyperacetylated, DNA-binding competent state in tumor necrosis factor-? (TNF-?)-challenged albumin-Cre+ (AlbCre+) hepatocytes. Transfection of hepatocytes with a recombinant acetylated p65 expression construct replicates the protection afforded by Sirt1 knockout. Transfection of AlbCre+ hepatocytes with a recombinant wild-type Sirt1 construct, rather than a deacetylase-defective one, compromises NF-?B activation and resensitizes hepatocytes to TNF-?-induced apoptosis. Taken together, our results demonstrate that Sirt1 deacetylates p65 and compromises NF-?B activity in hepatocytes when confronted with LPS/TNF-? stimulation, leading to increased susceptibility to endotoxemic injury. These findings identify a possible protein effector to maneuver the hepatic NF-?B signaling pathway under inflammatory circumstances and a feasible way to increase hepatocellular resistance to endotoxin/TNF-? toxicity.
Project description:BACKGROUND: Different from three clonal lineages of Toxoplasma gondii in North America and Europe, the genotype China 1 is predominantly prevalent in China. However, there are different virulent isolates within China 1, such as virulent TgCtwh3 and avirulent TgCtwh6, and little is known about differences in macrophage activation between them. The objective of this study focused on cytokine production, phenotype and markers of activated macrophages, and correlated signaling pathway induced by the two isolates. METHODS: Adherent peritoneal macrophages (termed Wh3-M? and Wh6-M?, respectively) harvested from infected mice were cultured for detection of Nitric Oxide and arginase activity, and activated markers on Wh3-M?/Wh6-M? were determined by flow cytometry. In in vitro experiments, the levels of IL-12p40 and TNF-? were measured using ELISA kits, and mRNA expressions of IL-12p40, TNF-?, iNOS, Arg-1 and Ym1 were assayed by real-time PCR. To confirm the activation state of NF-kB p65 in infected cells stained by IF, protein levels of iNOS, Arg-1, Ym1, nuclear NF-?B p65, and phosphorylation of STAT6/STAT3/I?B? were evaluated by Western Blotting. A one-way ANOVA test was used to compare differences among multiple groups. RESULTS: The result revealed that contrary to the virulent TgCtwh3, the less virulent TgCtwh6 isolate induced a significant increase in IL-12p40 and TNF-?. Although both isolates down-regulated CD80, CD86 and MHCII molecule expression on macrophages, TgCtwh3 promoted up-regulation of PD-L2 and CD206. Wh6-M? generated a high level of NO whereas Wh3-M? up-regulated Ym1 and arginase expression at transcriptional and protein levels. In terms of signaling pathway, TgCtwh3 induced phospho-STAT6, conversely, TgCtWh6 led to NF-?B p65 activation. CONCLUSIONS: The virulent TgCtwh3 isolate induced macrophages to polarize toward alternatively activated cells with STAT6 phosphorylation, whereas the less virulent TgCtwh6 elicited the development of classically activated macrophages with nuclear translocation of NF-?B p65. This discrepancy suggests that it is necessary to thoroughly analyze the genotype of TgCtwh3 and TgCtwh6, and to further study other effector molecules that contribute to the macrophage polarization in T. gondii.
Project description:BACKGROUND AND PURPOSE: We have recently reported that phytosteryl ferulates isolated from rice bran inhibit nuclear factor-kappaB (NF-kappaB) activity in macrophages. In the present study, we investigated the effect of gamma-oryzanol (gamma-ORZ), a mixture of phytosteryl ferulates, cycloartenyl ferulate (CAF), one of the components of gamma-ORZ, and ferulic acid (FA), a possible metabolite of gamma-ORZ in vivo, on a model of colitis in mice. EXPERIMENTAL APPROACH: We induced colitis with dextran sulphate sodium (DSS) in mice and monitored disease activity index (DAI), histopathology score, tissue myeloperoxidase (MPO) activity, mRNA expressions of cytokines and COX-2, colon length, antioxidant potency and NF-kappaB activity in colitis tissue. KEY RESULTS: Both DAI and histopathology score revealed that DSS induced a severe mucosal colitis, with a marked increase in the thickness of the muscle layer, distortion and loss of crypts, depletion of goblet cells and infiltration of macrophages, granulocytes and lymphocytes. MPO activity, pro-inflammatory cytokines and COX-2 levels, NF-kappaB p65 nuclear translocation and inhibitory protein of nuclear factor-kappaB-alpha degradation levels were significantly increased in DSS-induced colitis tissues. gamma-ORZ (50 mg kg(-1) day(-1) p.o.) markedly inhibited these inflammatory reactions and CAF had a similar potency. In vitro assay demonstrated that gamma-ORZ and CAF had strong antioxidant effects comparable to those of alpha-tocopherol. CONCLUSIONS AND IMPLICATIONS: Phytosteryl ferulates could be new potential therapeutic and/or preventive agents for gastrointestinal inflammatory diseases. Their anti-inflammatory effect could be mediated by inhibition of NF-kappaB activity, which was at least partly due to the antioxidant effect of the FA moiety in the structure of phytosteryl ferulates.
Project description:BACKGROUND:Chronic inflammation is one of the important mediators of colitis-related colon cancer (CRC). Abundant mast cells (MCs) were observed in the tumor microenvironment and mediators released upon MC activation play an important role in the process of chronic inflammation. Previously, we found that activation of intestine mucosal MCs recruited and modulated the inflammatory CD11b(+)Gr1(+) cells to promote the CRC development. In the current study we investigated the effects of Vam3, a resveratrol dimer with potent anti-inflammatory effects, on CRC development. METHODS:RBL-2H3 cells, a basophilic leukemia cell line, were pretreated with 2.5 or 5 µM Vam3 and then stimulated with dinitrophenol-conjugated bovine serum albumin (DNP-BSA) plus lipopolysaccharide (LPS). The MC degranulation was determined by measuring ?-hexosaminidase release. Generation of TNF-? and IL-6 in RBL-2H3 cells or in peritoneal macrophages was determined by ELISA and real-time qPCR. NF-?B p65 and phospho-NF-?B p65 expression was determined by Western blotting. NF-?B activity in RAW264.7 cells was determined by luciferase reporter assay. CRC was induced in C57BL/6 mice by intraperitoneal injection of azoxymethane (AOM), followed by oral exposure to dextran sodium sulfate (DSS). Vam3 at 50 mg/kg, or disodium cromoglycate (DSCG, MC stabilizer) at 100 mg/kg, or vehicle were administrated to the mice 4 weeks after DSS withdrawal. Levels of TNF-?, IL-6, and mouse MC protease-1 were determined by ELISA. Infiltration of CD11b(+)Gr1(+) cells was determined by flow cytometry analysis. One-way ANOVA was used to compare difference between groups. RESULTS:Pretreatment with Vam3 significantly inhibited RBL-2H3 cell degranulation and inflammatory cytokine production from RBL-2H3 cells and from peritoneal macrophages. After Vam3 treatment, NF-?B activity in RAW264.7 cells, and expressions of phospho-NF-?B p65 in RBL-2H3 cells and in peritoneal macrophages were significantly down-regulated. In the AOM plus DSS-induced CRC murine model, the Vam3 and DSCG-treated mice had less tumor numbers than those treated with vehicle. Expression of phospho-NF-?B p65, production of inflammatory cytokines, and infiltration of MCs and CD11b(+)Gr1(+) cells were attenuated in the Vam3-treated mice. CONCLUSION:Vam3 treatment could attenuate the CRC development. This effect may be due to its inhibition on NF-?B signaling pathway in MCs and macrophages of the inflamed intestines.