Protective effect of low-dose lactulose in dextran sulfate sodium induced ulcerative colitis model of rats

Abstract

Although low-dose lactulose has shown a good theoretical foundation for the treatment of ulcerative colitis (UC) in previous studies, the exact effects and mechanism remain unclear. The rats were randomly distributed into 5 groups, i.e., normal drinking water was provided for an initial 14 days in blank control group, 4% dextran sulfate sodium was used for modeling in the remaining 4 groups. During the 15-24th day, rats in the blank control group were administered with 0.9% saline (0.5 ml/d) by gavage. In the rest 4 groups, rats were administered 0.9% saline (0.5 ml/d, UC model), mesalazine (400 mg/kg/d), lactulose (1000 mg/kg/d), and lactulose + mesalazine (two-drug combination) by gavage. In addition to symptoms and pathological changes, serum IL-6, TNF-α, and High-sensitivity C-reactive protein(Hs-CRP) by ELISA analysis, mRNA and protein expression levels of TLR-2, TLR-4, Nuclear factor-κB(NF-κB), IL-6, and TNF-α in colon tissues by RT-qPCR and WB analyses respectively. Meanwhile, short-chain fatty acid(SCFAs) and intestinal flora were analyzed. Low-dose lactulose improved symptoms (diarrhea, blood in stool, weight loss) and pathological inflammation. In addition to serum IL-6, TNF-α, and Hs-CRP, the mRNA and protein expression levels of TLR-2, TLR-4, NF-κB, IL-6 and TNF-α in the colon were down-regulated with the intervention of lactulose.Meanwhile, lactulose decreased the ileocecal PH, increased SCFAs and altered the intestinal flora. Low-dose lactulose may be beneficial to UC by regulating TLRs/NF-κB pathway, reducing ileocecal PH, increasing SCFAs, regulating intestinal flora and improving the intestinal mucosal barrier. Meanwhile, low-dose lactulose and mesalazine may have additive effects upon combination.

Keywords: Intestinal flora; Lactulose; Short-chain fatty acids; Ulcerative colitis.

Fig. 1

Symptoms of rats in 5 different treatment groups. (A) The data show changes in the body weight during 24 days in 5 treatment groups. (B) The image shows the DAI score during 24 days of treatment in 5 treatment groups of rats.

Fig. 2

Colonic histopathological observations in treatment 5 groups of rats. (a) The images show the length of the colon. (b) The data show the comparison of 5 groups concerning the length of the colon. (c) The data present the colon mucosa damage index in 5 different treatment groups. (d) The data show histologic scores in 5 different treatment groups. Data are presented as mean ± SD, and *indicates P < 0.05.

Fig. 3

Representative images show HE-stained colon tissues in 5 different treatment groups (Low magnification: × 50, Medium magnification: × 100).

Fig. 4

The data present the expression levels of serum inflammatory factors of (a) IL-6, (b) TNF-α, and (c) Hs-CRP. Data are presented as mean ± SD, and * indicates p < 0.05.

Fig. 5

RT-qPCR analysis determines the gene expression levels. The images show mRNA levels of (a) IL-6, (b) TNF-α, (c) NF-κB, (d) TLR-2, and (e) TLR-4. * indicates P < 0.05.

Fig. 6

(a) Representative images show the WB bands of different proteins, including IL-6, TNF-α,NF-κB, TLR-2, and TLR-4, as well as β-actin as the internal control. Western blot analysis showed the protein expression comparison of (b) IL-6, (c) TNF-α, (d) NF-κB, (e) TLR-2 and (f) TLR-4.

Fig. 7

The TEM observations show the ultrastructure of colonic mucosa in 5 different groups, regarding (a) microvilli (× 5000), (b) tight junction, intermediate junction, and bridging grains (× 10,000), and (c) morphology of mitochondria (× 20,000).

Fig. 8

The pH levels in the ileocecal region, transverse colon, and sigmoid colon of 5 treatment groups. Data are presented as mean ± SD; * indicates P < 0.05; “ns “ represents no significant difference.

Fig. 9

Alpha diversity and beta diversity analyses of gut microbiota in 5 different treatment groups. The data show (a) the abundance of microbiota (ACE index) and (b) the diversity of microbiota (Shannon index). (c) The data present the PcoA approach of microbiota in 5 different treatment groups of rats.

Fig. 10

The microbiota composition in 5 different treatment groups. The data show microbiota composition and comparison of microbiota in rats on the phylum (a), class (b), order (c), family (d), genus (e), and species levels (f).

Fig. 11

Characteristics of intestinal microbiota composition by LEfSe analysis in 5 groups. (a) Distribution histogram of LDA value, LDA > 4. (b) The cladogram based on LEfSe.