Indigo Naturalis ameliorates murine dextran sodium sulfate-induced colitis via aryl hydrocarbon receptor activation.

BACKGROUND: Indigo Naturalis (IN) is used as a traditional herbal medicine for ulcerative colitis (UC). However, the mechanisms of action of IN have not been clarified. We aimed to evaluate the efficacy of IN for ameliorating colonic inflammation. We further investigated the mechanisms of action of IN. METHODS: Colitis severity was assessed in dextran sodium sulfate-induced colitis and trinitrobenzene sulfonic acid-induced colitis models with or without the oral administration of IN or indigo, which is a known major component of IN. Colonic lamina propria (LP) mononuclear cells isolated from IN-treated mice were analyzed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry. LP and splenic mononuclear cells cultured in vitro with IN or indigo were also analyzed. The role of the candidate receptor for indigo, the aryl hydrocarbon receptor (AhR), was analyzed using Ahr-deficient mice. RESULTS: Colitis severity was significantly ameliorated in the IN and indigo treatment groups compared with the control group. The mRNA expression levels of interleukin (Il)-10 and Il-22 in the LP lymphocytes were increased by IN treatment. The treatment of splenocytes with IN or indigo increased the expression of anti-inflammatory cytokines and resulted in the expansion of IL-10-producing CD4+ T cells and IL-22-producing CD3-RORγt+ cells, but not CD4+Foxp3+ regulatory T cells. The amelioration of colitis by IN or indigo was abrogated in Ahr-deficient mice, in association with diminished regulatory cytokine production. CONCLUSIONS: IN and indigo ameliorated murine colitis through AhR signaling activation, suggesting that AhR could be a promising therapeutic target for UC.

Aryl hydrocarbon receptor regulates pancreatic IL-22 production and protects mice from acute pancreatitis.

BACKGROUND & AIMS: The type of immune response during development of acute pancreatitis (AP) determines disease severity. Pancreatic epithelial cells express the interleukin (IL)-22 receptor A1 (IL-22RA1). The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that regulates expression of IL-22. We investigated sources and role of IL-22 in the pancreas, along with the effects of AhR activation on IL-22 expression and AP progression in mice. METHODS: We analyzed the effects of recombinant IL-22, a monoclonal antibody against IL-22, and agonists and antagonists of AhR in mice with AP (induced with caerulein or a choline-deficient diet supplemented with DL-ethionine) and control mice. We also analyzed transgenic mice with AhR deficiency (AhR(d) and AhR(-/-) mice). RESULTS: CD4(+) T cells were the main source of IL-22 in pancreatic tissues from healthy mice. During development of AP, numbers of IL-22(+) CD4(+) T cells were reduced, whereas IL-22RA1 was up-regulated. Consistent with high levels of IL-22RA1 expression, pancreatic acinar cells responded to IL-22 signaling via signal transducers and activators of transcription 3; administration of IL-22 reduced AP and associated lung injury in mice. AhR was required for production of IL-22 and protected mice from AP. Mice that did not respond to AhR activation developed AP, but administration of IL-22 reduced AP; blockade of IL-22 reversed the ability of activated AhR to protect against AP. CONCLUSIONS: AhR activation protects mice from AP by inducing expression of IL-22. AhR therefore mediates interactions between pancreatic leukocytes and epithelial cells and might be developed as a therapeutic target.

Crambene, a bioactive nitrile derived from glucosinolate hydrolysis, acts via the antioxidant response element to upregulate quinone reductase alone or synergistically with indole-3-carbinol.

Epidemiological studies show that cruciferous vegetables play a role in dietary protection against cancers. The protective effects of crucifers are thought to be associated with secondary metabolites termed glucosinolates, the hydrolysis products of which upregulate hepatic detoxification enzymes. Crambene, a nitrile product of the glucosinolate progoitrin, increases hepatic quinone reductase (QR) when included in the diet of animals. Here we evaluate the mechanism of upregulation of detoxification enzymes by crambene. The regulatory region of the QR gene contains two response elements, the antioxidant response element (ARE) and the xenobiotic response element (XRE), that respond to glucosinolate hydrolysis products. We compared upregulation of QR mRNA expression by crambene in wild-type and Ah receptor-deficient mouse hepatoma cell lines. Both cell lines showed a similar increase in QR mRNA, suggesting that the Ah receptor-dependent XRE pathway is not required for crambene to act. Transient transfection of HepG2 cells with reporter constructs containing portions of the 5' regulatory region of the rat QR gene confirmed this, revealing that crambene significantly activated ARE, but not XRE, in a dose-dependent manner. Furthermore, both indole-3-carbinol (I3C) and I3C acid condensates (I3C-A) activated the ARE for QR gene expression whereas only I3C-A activated the XRE at the concentrations studied. In addition, co-treatment with crambene and I3C-A caused synergistic increases in QR transcriptional activity and mRNA levels in HepG2 cells. Based on these findings, we propose that synergistic upregulation of QR is due to co-activation of the ARE and the XRE by crambene and I3C-A.