Resveratrol alleviates intestinal mucosal barrier dysfunction in dextran sulfate sodium-induced colitis mice by enhancing autophagy

Abstract

Background: Intestinal mucosal barrier dysfunction plays an important role in the pathogenesis of ulcerative colitis (UC). Recent studies have revealed that impaired autophagy is associated with intestinal mucosal dysfunction in the mucosa of colitis mice. Resveratrol exerts anti-inflammatory functions by regulating autophagy.

Aim: To investigate the effect and mechanism of resveratrol on protecting the integrity of the intestinal mucosal barrier and anti-inflammation in dextran sulfate sodium (DSS)-induced ulcerative colitis mice.

Methods: Male C57BL/6 mice were divided into four groups: negative control group, DSS model group, DSS + resveratrol group, and DSS + 5-aminosalicylic acid group. The severity of colitis was assessed by the disease activity index, serum inflammatory cytokines were detected by enzyme-linked immunosorbent assay. Colon tissues were stained with haematoxylin and eosin, and mucosal damage was evaluated by mean histological score. The expression of occludin and ZO-1 in colon tissue was evaluated using immunohistochemical analysis. In addition, the expression of autophagy-related genes was determined using reverse transcription-polymerase chain reaction and Western-blot, and morphology of autophagy was observed by transmission electron microscopy.

Results: The resveratrol treatment group showed a 1.72-fold decrease in disease activity index scores and 1.42, 3.81, and 1.65-fold decrease in the production of the inflammatory cytokine tumor necrosis factor-α, interleukin-6 and interleukin-1β, respectively, in DSS-induced colitis mice compared with DSS group (P < 0.05). The expressions of the tight junction proteins occludin and ZO-1 in DSS model group were decreased, and were increased in resveratrol-treated colitis group. Resveratrol also increased the levels of LC3B (by 1.39-fold compared with DSS group) and Beclin-1 (by 1.49-fold compared with DSS group) (P < 0.05), as well as the number of autophagosomes, which implies that the resveratrol may alleviate intestinal mucosal barrier dysfunction in DSS-induced UC mice by enhancing autophagy.

Conclusion: Resveratrol treatment decreased the expression of inflammatory factors, increased the expression of tight junction proteins and alleviated UC intestinal mucosal barrier dysfunction; this effect may be achieved by enhancing autophagy in intestinal epithelial cells.

Keywords: Autophagy; Intestinal inflammation; Intestinal mucosal barrier;Dextran sulfate sodium-induced colitis; Resveratrol; Ulcerative colitis.

Figure 1

Chemical structures of resveratrol and 5-aminosalicylic acid, and timeline of in vivo experiment. A: The chemical structures of resveratrol; B: The chemical structures of 5-aminosalicylic acid; and C: Timeline of in vivo experiment. Dextran sulfate sodium (DSS) group was induced by two cycles of intake of drinking water containing 3% DSS for 7 d and normal drinking water for 7 d. Other groups were also treated by two cycles, first received DSS by drinking water for 7 d, followed by treatment of 5-aminosalicylic acid or resveratrol by gavage for 7 d. DSS: Dextran sulfate sodium; 5-ASA: 5-aminosalicylic acid; RES: Resveratrol.

Figure 2

Clinical evaluation of dextran sulfate sodium-induced chronic colitis by disease activity index, body mass loss and colon length (n = 12 for the control group, n = 9 for disease activity index group, n = 11 for dextran sulfate sodium + 5-aminosalicylic acid group, n = 12 for dextran sulfate sodium + resveratrol treatment group). A: The disease activity index score was reduced in the resveratrol treated group compared with the dextran sulfate sodium group (^aP < 0.05); B and C: Resveratrol treatment increased the body mass and colon length of dextran sulfate sodium-induced colitis mice. Body mass loss was calculated as (detected body mass - initial mass)/initial mass. DSS: Dextran sulfate sodium; 5-ASA: 5-aminosalicylic acid; RES: Resveratrol.

Figure 3

Inflammatory cytokine expression in the dextran sulfate sodium-induced chronic colitis determined by enzyme-linked immunosorbent assay. The levels of interleukin-1β, interleukin-6 and tumor necrosis factor-α were decreased in the resveratrol treatment group compared with the dextran sulfate sodium group. ^aP < 0.05. TNF: Tumor necrosis factor; DSS: Dextran sulfate sodium; 5-ASA: 5-aminosalicylic acid; RES: Resveratrol.

Figure 4

Histological staining showed colitis induced dysfunction. A: Dextran sulfate sodium group showed significant colonic mucosal damages, crypt depletion, infiltration of inflammatory cells into the mucosa and submucosa, loss of epithelial barrier. Resveratrol treatment group and 5-aminosalicylic acid could alleviate colitis-induced intestinal mucosal barrier dysfunction; and B: The histological score of resveratrol treatment group was lower than the dextran sulfate sodium group. ^aP < 0.05. DSS: Dextran sulfate sodium; 5-ASA: 5-aminosalicylic acid; RES: Resveratrol.

Figure 5

Immunohistochemical staining of ZO-1 and occludin. Expressions of ZO-1 and occludin were higher in resveratrol treatment than in the dextran sulfate sodium group and 5-aminosalicylic acid treated group. Negative control: antibody was replaced by phosphate buffer saline. DSS: Dextran sulfate sodium; 5-ASA: 5-aminosalicylic acid; RES: Resveratrol.

Figure 6

Expression levels of Beclin-1 and LC3B in dextran sulfate sodium induced chronic colitis. A: A substantial increase in the messenger ribonucleic acid expression level of LC3B and Beclin-1 was observed in the dextran sulfate sodium (DSS) + resveratrol treatment group compared with the DSS group (P < 0.05); B and C: The Western blotting showed that resveratrol treatment induced significant increases in the LC3-II/I ratio and Beclin-1 level in DSS-induced colitis mice; Mean grey level of Beclin-1 and LC3B protein level was increased in DSS + resveratrol treatment group. ^aP < 0.05. DSS: Dextran sulfate sodium; 5-ASA: 5-aminosalicylic acid; mRNA: Messenger ribonucleic acid; RES: Resveratrol.

Figure 7

Structures of the intestinal epithelial cells and autolysosomes observed by transmission electron microscopy. A: Control group showed normal organelle structure; B: Dextran sulfate sodium induced colitis group showed mitochondrial swelling and ruptures of internal cristae, along with distention of the rough endoplasmic reticulum; C: 5-aminosalicylic acid group revealed that mitochondrial swelling was not obvious, and mitochondrial cristae showed blurred appearance; and D: Resveratrol treatment group showed that organelle structure was basically normal, and autophagosome could be observed. ER: Endoplasmic reticulum, M: Mitochondria.