Fermented Pueraria Lobata extract ameliorates dextran sulfate sodium-induced colitis by reducing pro-inflammatory cytokines and recovering intestinal barrier function

Abstract

Inflammatory bowel disease is a chronic inflammatory disorder occurring in the gastrointestinal track. However, the efficacy of current therapeutic strategies has been limited and accompanied by side effects. In order to eliminate the limitations, herbal medicines have recently been developed for treatment of IBD. Peuraria Lobata (Peuraria L.) is one of the traditional herbal medicines that have anti-inflammatory effects. Bioavailability of Peuraria L., which is rich in isoflavones, is lower than that of their fermented forms. In this study, we generated fermented Peuraria L. extracts (fPue) and investigated the role of fPue in inflammation and intestinal barrier function in vitro and in vivo. As the mice or intestinal epithelial cells were treated with DSS/fPue, mRNA expression of pro-inflammatory cytokines was reduced and the architecture and expression of tight junction proteins were recovered, compared to the DSS-treated group. In summary, fPue treatment resulted in amelioration of DSS-induced inflammation in the colon, and the disrupted intestinal barrier was recovered as the expression and architecture of tight junction proteins were retrieved. These results suggest that use of fPue could be a new therapeutic strategy for treatment of IBD.

Keywords: IBD; Pueraria Lobata; herbal medicines; inflammation; intestinal barrier.

Figure 1. Anti-inflammatory effect of Peuraria L. extract and its fermented form. A. Western blot analysis for phosphorylation of NF-κB in Raw264.7 cells after treatment of Pue or fPue with LPS. B. RT-PCR for transcriptional expression levels of pro-inflammatory cytokines in Raw264.7 cells after treatment of Pue or fPue with LPS. Pue, Pueraria Lobata aqueous extract; fPue, fermented form of Pueraria Lobata aqueous extract. Intensity of each band in western blot and RT-PCR results was quantified by using ImageJ and normalized to GAPDH (A) and β-actin (B).

Figure 2. Effect of fPue on DSS-induced colitis in mice. A. Schedule for administration of 5% DSS and fPue. B. Assessment of disease activity index (DAI) by fecal analysis. C. Histological evaluation of DSS-induced colitis according to degree of epithelial cell loss, crypt damage, and infiltration of inflammatory cells. D. Histological figures of colon stained by H&E. (a, mucosa; b, submucosa; c, muscularis; d, crypt) E. Histological figures to observe goblet cell loss by Alcian Blue staining. Numerical data were expressed as mean±standard deviation (n=7, ^*P<0.05, ^**P<0.01, ^***P<0.001). DSS, dextran sulfate sodium; fPue, fermented form of Pueraria Lobata aqueous extract; H&E, hematoxylene and eosin.

Figure 3. Infiltration of macrophages in colonic mucosa and submucosa. A. Immunofluorescence assay for F4/80, using frozen colon section (bar=50 µm). B. Quantification of number of F4/80-possitive cells per field. C. Transcriptional expression levels of pro-inflammatory cytokines, using colon tissue. Numerical data were expressed as mean ± standard deviation (n=7, ^*P<0.05, ^**P<0.01, ^***P<0.001). DSS, dextran sulfate sodium; fPue, fermented form of Pueraria Lobata aqueous extract; HPF, high power field.

Figure 4. Effect of fPue on recovery of colonic epithelial barrier. A. Immunofluorescence assay for tight junction proteins, ZO-1, Claudin-1, and Occludin, on frozen colon tissue. B. Blood agar assay, using cells isolated from mesenteric lymph nodes. C. Representative figures for blood agar assay. Numerical data were expressed as mean ± standard deviation (n=7, ^*P<0.05, ^**P<0.01, ^***P<0.001). DSS, dextran sulfate sodium; fPue, fermented form of Pueraria Lobata aqueous extract.

Figure 5. Evaluating a direct effect of fPue on recovery of tight junction proteins, using Caco-2 cells. A. Western blot analysis for expression of tight junction proteins, ZO-1 and Occludin after treatment of fPue with 2% DSS. B. Immunofluorescence assay for ZO-1 and Occludin on Caco-2 cells. DSS, dextran sulfate sodium; fPue, fermented form of Pueraria Lobata aqueous extract.