Indirubin, a Constituent of the Chinese Herbal Medicine Qing-Dai, Attenuates Dextran Sulfate Sodium-induced Murine Colitis

Abstract

Background: Indirubin, a constituent of the Chinese herbal medicine "Qing-Dai," has anti-cancer and anti-inflammatory activities. We aimed to evaluate the efficacy of indirubin for ameliorating colonic inflammation in a mouse model of inflammatory bowel disease.

Methods: Mice with dextran sulfate sodium (DSS)-induced acute and chronic colitis were treated with indirubin in their diet. Clinical and histologic changes were evaluated. In addition, colon levels of interleukin-6, a critical pro-inflammatory mediator, was detected by enzyme-linked immunosorbent assay.

Results: In the model of acute colitis, indirubin treatment improved the loss of body weight. Histology of colonic tissue revealed that indirubin treatment improved the histology grading of colitis (P = 0.02), the extent of submucosal fibrosis (P = 0.018), the number of mucosal toluidine blue-positive cells (P = 0.004) and colon length (P = 0.01). In the model of chronic colitis, indirubin treatment had no significant effect on pathologic findings except for colon length (P = 0.003). However, indirubin administration significantly reduced colon levels of interleukin-6 in the chronic-colitis model (P = 0.001).

Conclusion: Our study clearly showed that oral intake of indirubin can improve murine DSS-induced colitis (which mimics human inflammatory bowel disease).

Keywords: Chinese herbal medicine; DSS-induced colitis model; Qing-Dai; indirubin; inflammatory bowel disease.

Fig. 1.

Chemical structures of indirubin (A) and indigo (B).

Fig. 2.

Experimental protocols for DSS-induced acute and chronic colitis experiments. (A) In acute-colitis studies, mice were subjected to one, 6-day DSS cycle. Mice were fed a normal or indirubin-supplemented diet, commencing 5 days before the start of the experiment and continuing until the end of the experiment. Mice were killed on day-6 (arrow). (B) For the study of chronic colitis, mice were subjected to three consecutive DSS cycles. Each cycle consisted of a DSS-administration period followed by a recovery period in which water was administrated. Mice were fed a normal or indirubin-supplemented diet commencing 5 days before the start of the experiment and continuing until the end of the third DSS cycle. All surviving mice were killed at the completion of the third DSS cycle (arrow). DSS, dextran sulfate sodium.

Fig. 3.

Intracellular granules of mast cell were stained with toluidine blue (arrow). It shows metachromasia and shows reddish purple color. Bar = 0.2 mm.

Fig. 4.

Changes (0, baseline) in body weight (mean ± SE) of mice receiving water (control), DSS, or DSS and indirubin in (A) acute colitis and (B) chronic colitis. DSS, dextran sulfate sodium; N.S, not significant.

Fig. 5.

Effect of indirubin on the length of the large intestine of mice with DSS-induced colitis. (A) Acute-colitis model on day-6. (B) Chronic-colitis model on day-42. The large intestine was isolated, and its length measured. Median scores are indicated by horizontal lines. DSS, dextran sulfate sodium; N.S, not significant.

Fig. 6.

Histology (H&E stain) of colonic samples taken from mice not given DSS or indirubin (control) (A), given DSS and indirubin (B) or given DSS but not indirubin (C). Compared with that of control, the colons of DSS-induced colitis mice showed: complete destruction of epithelial architecture with loss of crypts and epithelial integrity; submucosal fibrosis and edema; intense infiltration of inflammatory cells in all layers. Indirubin treatment attenuated morphologic damage but showed mild infiltration of inflammatory cells. Bar = 1 mm. DSS, dextran sulfate sodium.

Fig. 7.

Histology score of colitis in mice not given DSS or indirubin (control), given DSS but not indirubin, or given DSS and indirubin. Mice were administered indirubin (0.05 μg/g/day, p.o.) in their diet. (A) Acute-colitis model on day-6. (B) Chronic-colitis model on day-42. The large intestine was isolated, and morphologic analyses undertaken. The histopathologic severity of colitis was calculated. DSS, dextran sulfate sodium; N.S, not significant; p.o., per os.

Fig. 8.

Effects of indirubin treatment on colonic cytokine expression in DSS-induced colitis. (A) Acute-colitis model on day-6. (B) Chronic-colitis model on day-42. Mice were killed. A rectum sample (5 mm × 5 mm) was excised and homogenized. Tissue content of IL-6 was measured by ELISA specific for mouse IL-6. Median scores are indicated by horizontal lines. DSS, dextran sulfate sodium; ELISA, Enzyme-linked immunosorbent assay; N.S, not significant.