Berberine alleviates dextran sodium sulfate-induced colitis by improving intestinal barrier function and reducing inflammation and oxidative stress

Abstract

Berberine has demonstrated efficacy in alleviating experimental colitis in vivo and in vitro. However, the anti-colitis mechanisms of berberine that enable it to promote intestinal barrier function in vivo remain unclear. The present study aimed to evaluate the effect of berberine on intestinal epithelial barrier function, expression of tight junction proteins and the levels of inflammatory and oxidative stress factors in the intestinal mucosa of dextran sulfate sodium (DSS)-induced colitis mice. Berberine (100 mg/kg) was administered for five days to mice with established colitis, induced by administration of DSS (3% w/v) for six days. Intestinal barrier function and the presence of proinflammatory factors, oxidative stress and active signaling pathways in the colon were determined principally by western blotting and reverse transcription-quantitative polymerase chain reaction. It was observed that berberine reduced weight loss, shortening of the colon and colon damage in DSS-colitis mice. In addition, berberine significantly inhibited the increase of fluorescein isothiocyanate-dextran in serum and the decrease of zonula occluden-1 (also known as tight junction protein-1), occludin and epithelial cadherin expression in colonic tissue, relative to a DSS-treated control group. Berberine also significantly inhibited the expression of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α mRNA and phosphorylation of signal transducer and activator of transcription 3. Furthermore, berberine reduced the levels of myeloperoxidase and increased the levels of superoxide dismutase and catalase in colon and serum samples relative to the control group. The expression of cluster of differentiation 68 in the colon of colitis mice was also reduced by berberine. Collectively, these data suggest that berberine alleviates colitis principally by improving intestinal barrier function and promoting anti-inflammatory and antioxidative stress responses. In turn these effects inhibit macrophage infiltration into the colon and thus may be central to the anti-colitis activity of berberine.

Keywords: berberine; dextran sulfate sodium; inflammation; intestinal barrier function; tight junction protein; ulcerative colitis.

Figure 1.

Effects of berberine on body weight, colon length and histological evaluation in DSS-induced colitis mice. Mice were administered with a 3% DSS solution for six days (days 1 to 6). BER (100 mg/kg) was then administered for five days (days 7 to 11) to the DSS+BER group. Control mice received water alone. On day 12, (A) body weight and (B) colon length were measured. (C) Colon damage was subsequently assessed by hematoxylin and eosin staining of paraffin-embedded colon sections. Images shown are representative of 10 mice in each group. Right column images are magnifications of the highlighted regions in left column images. (D) Colon injury scores were allocated following histological examination. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01 vs. DSS group, ^##P<0.01 vs. control group; n=10. DSS, dextran sulfate sodium; BER, berberine; Con, control.

Figure 2.

Effects of berberine on intestinal barrier function in DSS-colitis mice. (A) Levels of FITC-dextran in the serum of DSS-colitis mice administered with FITC-dextran were measured as an indicator of intestinal permeability. n=10. (B) Homogenates of colonic tissue were analyzed by western blotting to detect the expression of tight junction-associated proteins (ZO-1, E-cadherin and occludin). Representative blots from 4 samples of three independent experiments are shown and β-actin was used as a loading control. Levels of (C) ZO-1, (D) E-cadherin and (E) occludin expression were subsequently quantified. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01 vs. DSS group, ^#P<0.05 and ^##P<0.01 vs. control group; n=10 DSS, dextran sulfate sodium; FITC, fluorescein isothiocyanate; ZO-1, zonula occluden-1; E-cadherin, epithelial cadherin; BER, berberine; Con, control.

Figure 3.

Effects of berberine on proinflammatory factor mRNA expression and STAT3 phosphorylation in the colon of DSS-colitis mice. Colonic tissue was analyzed by reverse transcription-quantitative polymerase chain reaction to measure the levels of (A) IL-1β, (B) IL-6 and (C) TNF-α mRNA. Levels of proinflammatory factor expression in DSS and DSS+BER mice were measured relative to untreated control mice. (D) Levels of p-STAT3 were determined by western blot analysis. Representative blots of three independent experiments are shown and β-actin was used as a loading control. *P<0.05 vs. DSS group, ^#P<0.05 and ^##P<0.01 vs. control group; n=4. STAT3, signal transducer and activator of transcription 3; DSS, dextran sulfate sodium; IL, interleukin; TNF-α, tumor necrosis factor-α BER, berberine; Con, control; p-, phosphorylated.

Figure 4.

Effect of berberine on the oxidative stress response in DSS-colitis mice. As indicators of oxidative stress, levels of (A) MPO, (B) SOD and (C) CAT in the serum of DSS-colitis mice were measured by enzyme activity assays. Colonic tissues were also analyzed to determine the levels of (D) MPO, (E) SOD and (F) CAT. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01 vs. DSS group, ^#P<0.05 and ^##P<0.01 vs. control group; n=10. DSS, dextran sulfate sodium; MPO, myeloperoxidase; SOD, superoxide dismutase; CAT, catalase; BER, berberine; Con, control.

Figure 5.

Effects of berberine on CD68-positive macrophages in the colon of DSS-colitis mice. Paraffin-embedded colon tissues were analyzed by immunofluorescence to detect macrophage infiltration in DSS-colitis mice. Expression of CD68 was used as a marker of macrophages and monocytes (green staining; white arrows). Cell nuclei were stained with DAPI (blue staining). Images are representative of 10 mice in each group. CD68, Cluster of Differentiation 68; DSS, dextran sulfate sodium; BER, berberine; Con, control.