Bamboo Shoot and Artemisia capillaris Extract Mixture Ameliorates Dextran Sodium Sulfate-Induced Colitis

Abstract

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract and is characterized by recurrent chronic inflammation and mucosal damage of the gastrointestinal tract. Recent studies have demonstrated that bamboo shoot (BS) and Artemisia capillaris (AC) extracts enhance anti-inflammatory effects in various disease models. However, it is uncertain whether there is a synergistic protective effect of BS and AC in dextran sodium sulfate (DSS)-induced colitis. In the current study, we tested the combined effects of BS and AC extracts (BA) on colitis using in vivo and in vitro models. Compared with control mice, oral administration of DSS exacerbated colon length and increased the disease activity index (DAI) and histological damage. In DSS-induced colitis, treatment with BA significantly alleviated DSS-induced symptoms such as colon shortening, DAI, histological damage, and colonic pro-inflammatory marker expression compared to single extracts (BS or AC) treatment. Furthermore, we found BA treatment attenuated the ROS generation, F-actin formation, and RhoA activity compared with the single extract (BS or AC) treatment in DSS-treated cell lines. Collectively, these findings suggest that BA treatment has a positive synergistic protective effect on colonic inflammation compared with single extracts, it may be a highly effective complementary natural extract mixture for the prevention or treatment of IBD.

Keywords: Artemisia capillaris; bamboo shoot; colitis; dextran sodium sulfate; inflammatory bowel disease.

Figure 1

Effects of the combined ratio of BA extract mixtures on body weight loss, stool constancy, bloody stool, and DAI score. C57BL/6N (Male) mice were administrated with 2.5% DSS in drinking water (ad libitum) and treated with or without BA (BS:AC; 0:100, 30:70, 50:50, 70:30, 100:0; 250 mg/kg/day; p.o). (A) Body weight, (B) Stool consistency score, and (C) Bloody stool score was measured daily during the experimental period. (D) The disease activity index (DAI) was scored once daily for 5 days. Each data represents the percentage or mean ± SD. Statistical data were obtained by two-way ANOVA with Dunnett’s post-hoc test (n = 5 per group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CON; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. DSS; ^& p < 0.05, ^&& p < 0.01 vs. DSS + BA (50:50)). Detailed statistical differences were provided in Supplementary Figure S1.

Figure 2

Effects of the ratio of BA extract mixtures on colon shortening in DSS-induced colitis. (A,B) A representative photograph of colon tissues in each group is shown. The data represent the percentage or mean ± SD. Statistical differences were determined by one-way ANOVA with Tukey’s post-hoc test (n = 5 per group, ** p < 0.01 vs. CON; ^# p < 0.05, ^## p < 0.01 vs. DSS).

Figure 3

Effects of the ratio of BA extract mixtures on the histological score in DSS-induced colitis. (A) Representative images of Swiss roll mounts of colons were stained with H&E (left panels). Enlarged images (right panels) show magnified views of the boxed areas in the left panels. (B) Histological score in DSS-induced mice was estimated. Values are the mean ± SD (n = 5–6 per group, **** p < 0.0001 vs. CON; ^# p < 0.05 vs. DSS) significances were determined using one-way ANOVA with Tukey’s post-hoc test.

Figure 4

Effect of BA on pro-inflammatory mediator expression DSS-induced colitis. (A) Protein expression of pro-inflammatory mediators (P-STAT3, COX2, and iNOS) in mice colon from control, 2.5% DSS-treated, and 2.5% DSS with 250 mg/kg/day BA (BS:AC, 50:50) groups on 3 or 6 days. (B) The intensities of the bands in each panel were quantified for each group, and the values are expressed as the mean ± SD of three independent experiments. The data were obtained from three mice from each group. (C,D) ELISA using either phosphorylated-NF-κB p65 or total-NF-κB p65 antibody was used to measure the ratio of phospho-NF-κB p65/total-NF-κB p65 in HT-29 and Caco2 cells (control, 2% DSS-treated, and 2% DSS-treated with 200 μg/mL BA (BS:AC, 50:50) for 24 h). The phosphorylation levels were normalized against the control group and presented as the % of control. Statistical differences were determined by one-way ANOVA with Tukey’s post-hoc test (n = 3 per group, n.s.: not significant; ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. CON; ^# p < 0.05, ^### p < 0.001, ^#### p < 0.0001 vs. DSS).

Figure 5

Effect of BA on ROS generation in DSS-treated cells. (A,B) Reduced ROS generation was shown by DCFH-DA staining in 2% DSS-treated HT-29 and Ca-co2 cells incubated with or without 200 μg/mL BS (BS:AC, 100:0), AC (BS:AC, 0:100), and BA (BS:AC, 50:50) analyzed by fluorescence microscopy (upper panels). All pictures are representative of multiple images from three independent experiments (scale bars: 100 μm). Quantification of ROS level in percentage DCFH-DA fluorescence in DSS-treated HT-29 and Caco2 cells incubated with 200 μg/mL BS, AC, and BA for 24 h. Fluorescence levels were performed as described (Section 2.10). The bar graph illustrates ROS generation normalized to and statistically compared to each group (bottom panels). Statistical differences were determined by one-way ANOVA with Tukey’s post-hoc test (n = 3 per group, **** p < 0.0001 vs. CON; ^# p < 0.05, ^#### p < 0.0001 vs. DSS; ^& p < 0.05 vs. BS).

Figure 6

Effect of BA on F-actin formation and RhoA activity in DSS-treated cell lines. (A,B) Immunocytochemical staining for F-actin in 2% DSS-treated HT-29 and Caco2 cells with or without 200 μg/mL BS (BS:AC, 100:0), AC (BS:AC, 0:100), and BA (BS:AC, 50:50) for 24 h. Cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.2% Triton X-100 in PBS. F-actin was stained with Alexa Fluor 488-phalloidin. All pictures are representative of multiple images from three independent experiments (scale bars: 50 μm). Measurement of F-actin level performed as described (Section 2.11). Bar graph illustrates F-actin formation normalized to and statistically compared to each group (n = 5–8 per group **** p < 0.0001 vs. CON; ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 vs. DSS; ^& p < 0.05 vs. AC, right panels). (C,D) Measurement of RhoA-GTP by G-LISA RhoA activation assay in DSS-treated cells with or without BS, AC, and BA. The values were expressed as the mean ± SD of three independent experiments. Statistical differences were determined by one-way ANOVA with Tukey’s post-hoc test (n = 3 per group, **** p < 0.0001 vs. CON; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. DSS).