Si-Wu Water Extracts Protect against Colonic Mucus Barrier Damage by Regulating Muc2 Mucin Expression in Mice Fed a High-Fat Diet

Abstract

A high-fat diet (HFD) could cause gut barrier damage. The herbs in si-wu (SW) include dang gui (Angelica sinensis (Oliv.) Diels), shu di huang (the processed root of Rehmannia glutinosa Libosch.), chuan xiong (rhizome of Ligusticum chuanxiong Hort.), and bai shao (the root of Paeonia lactiflora f. pilosella (Nakai) Kitag.). Si-wu water extracts (SWE) have been used to treat blood deficiency. Components of one herb from SW have been reported to have anti-inflammatory and anti-obesity activities. However, there have been no reports about the effects of SWE on gut barrier damage. Therefore, the aim of the study was to explore the effect of SWE on gut barrier damage. In this study, we found that SWE effectively controlled body weight, liver weight, and feed efficiency, as well as decreased the serum TC level in HFD-fed mice. Moreover, SWE and rosiglitazone (Ros, positive control) increased the colonic alkaline phosphatase (ALP) level, down-regulated serum pro-inflammatory cytokine levels, and reduced intestinal permeability. In addition, SWE increased goblet cell numbers and mucus layer thickness to strengthen the mucus barrier. After supplementation with SWE and rosiglitazone, the protein expression of CHOP and GRP78 displayed a decrease, which improved the endoplasmic reticulum (ER) stress condition. Meanwhile, the increase in Cosmc and C1GALT1 improved the O-glycosylation process for correct protein folding. These results collectively demonstrated that SWE improved the mucus barrier, focusing on Muc2 mucin expression, in a prolonged high-fat diet, and provides evidence for the potential of SWE in the treatment of intestinal disease-associated mucus barrier damage.

Keywords: O-glycosylation; endoplasmic reticulum stress; high-fat diet; mucus barrier; si-wu water extract.

Figure 1

Effects of SWE on growth performance in the HFD−fed mice. (A) schema showing the animal groups and treatments, (B) body weight change, (C) food intake, (D) feed efficiency (body weight gain/food intake), (E) epididymis fat weight, (F) liver weight, (G) serum TC level, (H) serum TG level. Values are expressed as mean ± S.E.M. (* p < 0.05, ** p < 0.001, vs. SWE). Means marked with superscript letters are significantly different to the others (p < 0.05). n = 6−8 per group.

Figure 2

Effects of SWE on colonic mucosal morphology, intestinal permeability, and inflammation. (A) representative photographs of the distal colon sections with H&E staining (400×). Scale bar: 50 μm; red arrow indicates inflammatory infiltration. The a, b, c, d in subgraph A represent the order of the pictures for easy elaboration, (B) Histological score of the colon tissues (n = 5–7), (C) ALP level in colon, (D) LPS level, (E) D-La level, (F) DAO level, (G) serum TNF-α level. (H) serum IL-6 level, (I) serum IL-1β level. Values are expressed as mean ± S.E.M. Data with different superscript letters are significantly different (p < 0.05). n = 4–8 per group.

Figure 3

Effects of SWE on colonic mucus thickness and number of goblet cells in HFD-fed mice. (A) representative AB/PAS-stained distal colon sections showing mucus layer thickness and goblet cells within the colon tissue, scale bar: 50 μm, (B) manually measured colonic mucus layer thickness, (C) number of goblet cells per crypt (5–8 sections per animal). Values are expressed as mean ± S.E.M. Data with different superscript letters are significantly different (p < 0.05). n = 4–6 per group.

Figure 4

Effects of SWE on the content and distribution of colonic mucin. (A) representative immunofluorescent staining in the distal colon using an antibody against Muc2 (red) with DAPI (blue). Scale bar: 100 μm, magnification 200×, (B) fluorescent images showing O-glycans by using UEA1 staining. Scale bar: 100 μm, magnification 200×, (C) average optical density (AOD) of Muc2, (D) average optical density of UEA1, (E) Muc2 protein band diagrams, (F) Muc2 protein expression. Values are expressed as mean ± S.E.M. Means marked with superscript letters are significantly different to the others (p < 0.05). n = 4–8 per group.

Figure 5

Effects of SWE on colonic ER stress markers and mucin O-glycosylation in the colon. Western blot analysis: (A) protein abundance of GRP78 and CHOP, (B) GRP78 protein expression, (C) CHOP protein expression, (D) protein abundance of C1GALT1 and Cosmc, (E) C1GALT1 protein expression. (F) Cosmc protein expression. ELISA analysis: (G) C1GALT1 level in colon tissue, (H) Cosmc level in colon tissue. Values are expressed as mean ± S.E.M. Different letters indicate significant differences (p < 0.05). n = 4–8 per group.