The efficacy of dietary curcumin in TNBS colitis varies in BALB/c and SJL/J mouse strains. Although the exact mechanism underlying these differences is unclear, the results suggest that the therapeutic value of dietary curcumin may differ depending on the nature of immune dysregulation in IBD.
Extracts from Boswellia serrata have been reported to have anti-inflammatory activity, primarily via boswellic acid-mediated inhibition of leukotriene synthesis. In three small clinical trials, boswellia was shown to improve symptoms of ulcerative colitis and Crohn's disease, and because of its alleged safety, boswellia was considered superior over mesalazine in terms of a benefit-risk evaluation. The goal of this study was to evaluate the effectiveness of boswellia extracts in controlled settings of dextran sulfate- or trinitrobenzene sulfonic acid-induced colitis in mice. Our results suggest that boswellia is ineffective in ameliorating colitis in these models. Moreover, individual boswellic acids were demonstrated to increase the basal and IL-1beta-stimulated NF-kappaB activity in intestinal epithelial cells in vitro as well as reverse proliferative effects of IL-1beta. We also observed hepatotoxic effect of boswellia with pronounced hepatomegaly and steatosis. Hepatotoxity and increased lipid accumulation in response to boswellia were further confirmed in vitro in HepG2 cells with fluorescent Nile red binding/resazurin reduction assay and by confocal microscopy. Microarray analyses of hepatic gene expression demonstrated dysregulation of a number of genes, including a large group of lipid metabolism-related genes, and detoxifying enzymes, a response consistent with that to hepatotoxic xenobiotics. In summary, boswellia does not ameliorate symptoms of colitis in chemically induced murine models and, in higher doses, may become hepatotoxic. Potential implications of prolonged and uncontrolled intake of boswellia as an herbal supplement in inflammatory bowel disease and other inflammatory conditions should be considered in future clinical trials with this botanical.
Sodium butyrate (NaB) stimulates sodium and water absorption by inducing colonic Na(+)/H(+) exchange. NaB induces Na(+)/H(+) exchanger (NHE)3 activity and protein and mRNA expression both in vivo and in vitro. Our previously published observations indicated that this induction is Ser/Thr kinase dependent and that NaB-responsive elements were localized within -320/-34 bp of the rat NHE3 promoter. Here we further delineate the mechanism of NaB-mediated NHE3 gene transcription. Transient and stable transfection of Caco-2 cells with NHE3 gene reporter constructs identified Sp binding site SpB at position -58/-55 nt as critical for NaB-mediated induction. Gel mobility shift (GMSA) and DNA affinity precipitation assays indicated NaB-induced binding of Sp3 and decreased binding of Sp1 to SpB element. While no changes in expression of Sp1 or Sp3 were noted, NaB induced phosphorylation of Sp1 and acetylation of Sp3. Sp3 was a more potent inducer of NHE3 gene transcription, which suggested that change in balance, favoring binding of Sp3 to the SpB site, would result in significant increase in NHE3 promoter activity. Small interfering RNA studies in Caco-2 cells and data from NaB-treated SL2 cells used as a reconstitution model confirmed this hypothesis. In addition to the SpB site, which played a permissive role, an upstream novel butyrate response element located at -196/-175 nt was necessary for maximal induction. GMSA identified a protein-DNA complex with a -196/-175 nt probe; this interaction was not affected by NaB treatment, thus suggesting that in response to NaB Sp3 binding to site SpB precedes and results in recruitment of the putative factor to this upstream site.
ated using EMSA, immunohistochemistry, Western blot and luciferase promoter assays. Results: 1) TNF-␣ causes a decrease in TJ epithelial permeability across CaCo-2 monolayers. 2) TNF-␣ causes translocation of the p65 subunit of NF-B from the cytoplasm to the nucleus, an increase in DNA binding to an NF-B consensus oligonucleotide and activation of an NF-B responsive promoter. 3) TNF-␣ causes down-regulation of Occludin protein expression, the key component of the extra-cellular TJ barrier. 4) Prednisolone (1-10µM) and Dexamethasone (0.25µM) pre-treatment completely inhibited the TNF-␣ induced decrease in CaCo-2 TJ permeability. 5) GC treatment did not affect TNF-␣ induced NF-B nuclear translocation, DNA binding or NF-B responsive promoter activation. 6) GC inhibited the TNF-␣ induced down-regulation of Occludin protein expression. Conclusion: Prednisolone inhibits the increase in CaCo-2 TJ permeability caused by TNF-␣. The mechanism of prednisolone action involved inhibition of the TNF-␣ induced decrease in TJ protein expression without altering NF-B activation. Future studies will elucidate the mechanism of GC action on TNF-␣ responsive tight junction proteins.
SCFAs, and especially butyrate (NaB), stimulate sodium and water absorption by inducing colonic Na+/H+ exchange (NHE). NaB induces NHE3 activity, protein and mRNA expression both in vivo and in vitro. Our previously published observations indicated that this induction is PKA-dependent and NaB-responsive elements were localized within -320 /-34 bp of the NHE3 promoter. Here, we further delineate the mechanism of NaB-mediated NHE3 gene transcription. Transient transfection of Caco-2 cells with reporter constructs containing -320/+58 nt of the NHE gene promoter identified Sp binding site located at position -58-55 nt (SpB) as critical for NaB-mediated induction. Gel mobility shift assay (GMSA) with or without neutralizing antibodies for Sp1 or Sp3 indicated increased binding of Sp3 to SpB element, with no concomitant changes in Sp1 or Sp3 protein expression in the nuclei of NaB-treated Caco-2 cells. We also demonstrated that in SL-2 Drosophila cells transfected with HA-tagged Sp1 or Sp3 cDNA, Sp3 was a much stronger inducer of NHE3 gene transcription than Sp1, which suggests that even a small change in balance, favoring binding of Sp3 to SpB site might result in significant increase in NHE3 promoter activity. Also, siRNA experiments demonstrated that knock down of Sp3 expression in Caco-2 cells significantly blunted the response of NHE3 promoter to NaB. We have also found that p300 was not involved in the induction of NHE3 promoter by NaB, since its overexpression, expression a p300 mutant lacking the histone deacetylase domain, or overexpression of E1A oncogene remained without effect on NaB-stimulated NHE3 promoter activity. Surprisingly, a construct containing -118/+58 nt conferred only partial responsiveness to NaB. Similarly, -320/-33 nt of the NHE3 promoter placed upstream of a heterologous transcription start site was stimulated stronger that -118/-33 nt region suggesting an element upstream of the SpB site, crucial for a full response to NaB. This element was further narrowed down to a -196/-175 nt region. Although GMSA identified an unknown protein/DNA complex with -196/-175 nt probe, it's intensity was not affected by treatment of cells with NaB. This may suggest either post-translational modification leading to a higher transactivation potency of the putative factor, or a possibility that binding of Sp3 to SpB site has to precede and perhaps result in recruitment of the putative factor to site -196/-175.
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