Rumex japonicus Houtt. alleviates dextran sulfate sodium-induced colitis by protecting tight junctions in mice

Abstract

Background: Rumex japonicus Houtt. (RJ) is widely distributed in Korea, Japan, and China. The root of RJ has traditionally been used to treat constipation, jaundice, hematemesis, dysfunctional uterine bleeding, and gastrointestinal diseases. According to recent studies, plants of the genus Rumex have beneficial functionalities such as anti-microbial, antioxidative, and anti-inflammatory effects. Inflammatory bowel disease, including Crohn's disease and ulcerative colitis, is a chronic inflammatory disease characterized by an abnormal immune response and epithelial barrier dysfunction. This study evaluates the protective effect of RJ against dextran sulfate sodium (DSS)-induced colitis.

Methods: Male 8-week-old C57BL/6 N mice were treated with methanolic extract of RJ for 14 days, and DSS-induced groups were administered 2.5% DSS for last 7 days. After sacrifice, the length and weight of the colon were measured, and colon sections were subjected to H&E staining, immunohistochemistry and Western blotting to investigate the changes of inflammatory cytokines, tight junction and apoptosis-related factors.

Results: The colon of DSS-treated mouse was significantly shorter and heavier than the normal mouse. Moreover, DSS exposure induced an increase of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, occludin, zonula occludens-1, p21, p53 and Bcl-2, and decreased the expressions of IL-10, claudin-2 and cleaved caspase-3 in the colon tissue. These DSS-induced changes were inhibited by RJ treatment.

Conclusion: Our results indicate that RJ effectively suppresses DSS-induced colitis by protecting tight junction connections in the colonic tissue. We therefore infer that RJ has the potential as a medicine or ingredient for treating colitis.

Keywords: Colitis; Cytokine; Inflammation; Rumex japonicas; Tight junction.

Fig. 1

Effects of dextran sulfate sodium and Rumex japonicus Houtt. on body weight, colonic length, and colon weight/length ratio. (A) Dextran sulfate sodium (DSS) significantly decreases body weights, regardless of Rumex japonicus Houtt. (RJ) treatment. (B) Treatment with DSS results in decreased colon length, which is prevented by RJ treatment. (C) DSS significantly increases colonic weight/length ratio, and RJ significantly inhibits the edematous change. Values are the means ± SD (n = 6). *p < 0.05 and ***p < 0.001 versus normal group, #p < 0.05, ##p < 0.01, and ###p < 0.001 versus DSS group, $$$p < 0.001 versus RJ group.

Fig. 2

Changes in inflammatory cytokines in the colon of DSS- or RJ-treated mouse. Changes in inflammatory cytokines were confirmed by Western blotting (A). DSS treatment significantly increases the levels of TNF-α (B), IL-1β (C) and IL-6 (D) and decreases the level of IL-10 (E) in the colon, which suppressed by RJ treatment. Values are the means ± SD (n = 3). **p < 0.01 and ***p < 0.001 versus normal group, ##p < 0.01 and ###p < 0.001 versus DSS group, $$p < 0.01 versus 5-ASA group. TNF, tumor necrosis factor. IL, interleukin.

Fig. 3

Histological changes and expressions of tight junction-related proteins in the colon. (A) Hematoxylin and eosin (H&E) staining and immunohistochemical staining of occludin and ZO-1 in the colon. RJ suppresses the DSS-induced erosive lesions, crypt loss, and severely transformed cells in the mucous layers. Moreover, RJ suppresses the DSS-induced decrease expressions of occludin and ZO-1 in the colon. (B) Confirmation of changes in occludin, ZO-1, and claudin-2 expressions in the colon. RJ treatment significantly prevents the DSS-induced decrease of occludin (C) and ZO-1 (D), and increase of claudin-2 (E) in the colon. Values are the means ± SD (n = 3). Scale bar indicates 5 µm in H&E staining, and 50 µm in immunohistochemistry staining. *p < 0.05, **p < 0.01, and ***p < 0.001 versus normal group, ###p < 0.001 versus DSS group. ZO, zonula occludens.

Fig. 4

Changes in apoptosis-related proteins in the colon. Changes in apoptosis-related proteins were confirmed by Western blotting (A). DSS induces the decrease of Bcl-2 (B) and increase of cleaved caspase-3 (C) in the colon, whereas RJ alleviates these changes. DSS also induces alterations in factors of colitis-associated colorectal cancer, namely, increased levels of COX-2 (D) and decreased levels of p21 (E) and p53 (F). These changes are also suppressed by RJ. Values are the means ± SD (n = 3). **p < 0.01 and ***p < 0.001 versus normal group, #p < 0.05, ##p < 0.01, and ###p < 0.001 versus DSS group. COX, cyclooxygenase.