Abstract
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.
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Abbreviations
- AhR:
-
Aryl hydrocarbon receptor
- CD:
-
Crohn’s disease
- CECs:
-
Colonic epithelial cells
- DMSO:
-
Dimethyl sulfoxide
- DSS:
-
Dextran sodium sulfate
- Foxp3:
-
Forkhead box p3
- IBD:
-
Inflammatory bowel disease
- ILC:
-
Innate lymphoid cells
- IN:
-
Indigo Naturalis
- LP:
-
Lamina propria
- MLN:
-
Mesenteric lymph node
- RORγ:
-
RAR-related orphan receptor gamma
- TCDD:
-
2,3,7,8-Tetrachlorodibenzo-p-dioxin
- TNBS:
-
Trinitrobenzene sulfonic acid
- Tr1 cells:
-
Type 1 regulatory T cells
- UC:
-
Ulcerative colitis
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Acknowledgements
Supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (Grant No. 26460969).
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535_2016_1292_MOESM3_ESM.tif
Fig. S1. Flow cytometric analysis of the LP mononuclear cells of mice treated with IN. Similar populations of CD4-IL-10+cells (A) and CD3+IL-22+cells (B) were observed in IN-treated and control mice. A representative figure for flow cytometric analysis is shown on the left side (A). Shaded area isotype control, solid line IN, dotted line control. The data are expressed as the mean + standard error of 3 mice per group (TIFF 785 kb)
535_2016_1292_MOESM4_ESM.tif
Fig. S2. Cytokine expression profiles of the MLN mononuclear cells of mice treated with IN. Cytokine expression levels in the MLN cells isolated from mice with DSS-induced colitis were not significantly different between the high-dose IN treatment (IN) and control groups. The expression of Il-22 mRNA was undetectable in both groups. The measurements are expressed as the mean + standard error of 5 mice. *p < 0.05 (TIFF 985 kb)
535_2016_1292_MOESM5_ESM.tif
Fig. S3. Cytokine expression profile of the splenic mononuclear cells in mice treated with IN. Cytokine expression levels in the splenocytes isolated from mice with DSS-induced colitis were not significantly different between the high-dose IN treatment (IN) and control groups. The expression of Il-22 was undetectable in both groups. The measurements are expressed as the mean + standard error of 5 mice. *p < 0.05 (TIFF 990 kb)
535_2016_1292_MOESM6_ESM.tif
Fig. S4. Cytokine expression profile in LP mononuclear cells in Ahr-deficient mice treated with or without IN. There were no significant differences between the IN and control groups. The data are expressed as the mean + standard error of 4 mice per group (TIFF 838 kb)
535_2016_1292_MOESM7_ESM.tif
Fig. S5. AHR expression was elevated in the inflamed mucosa of UC patients. The expression levels of AHR mRNA in uninflamed mucosa (Mayo endoscopic score of 0) and inflamed mucosa (Mayo endoscopic score: 1–3) were compared in 10 UC patients. *p < 0.05 (TIFF 712 kb)
535_2016_1292_MOESM8_ESM.tif
Fig. S6. Expressions of STAT3 and phosphorylated (p)-STAT3 in CECs of mice treated with IN or vehicle control. Mice had DSS-induced colitis that was treated with IN or vehicle control. CECs were isolated from the colon by gentle stirring in the medium followed by Percoll gradient. Western blotting of the cellular lysates was performed by using anti-STAT3, anti-phospho-STAT3, and anti-β-actin antibodies. There was no apparent difference in the expression of p-STAT3 between the IN-treated and control groups. The data are representative of three independent experiments (TIFF 909 kb)
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Kawai, S., Iijima, H., Shinzaki, S. et al. Indigo Naturalis ameliorates murine dextran sodium sulfate-induced colitis via aryl hydrocarbon receptor activation. J Gastroenterol 52, 904–919 (2017). https://doi.org/10.1007/s00535-016-1292-z
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DOI: https://doi.org/10.1007/s00535-016-1292-z