Premorbid Steatohepatitis Increases the Seriousness of Dextran Sulfate Sodium-induced Ulcerative Colitis in Mice

Abstract

Background and aims: The concurrence of nonalcoholic steatohepatitis (NASH) and ulcerative colitis (UC) is increasingly seen in clinical practice, but the underlying mechanisms remain unclear. This study aimed to develop a mouse model of the phenomenon by combining high-fat high-cholesterol diet (HFHCD)-induced NASH and dextran sulfate sodium (DSS)-induced UC, that would support mechanistic studies.

Methods: Male C57BL/6 mice were randomly assigned to two groups receiving either a chow diet or HFHCD for 12 weeks of NASH modeling. The mice were the divided into four subgroups for UC modeling: (1) A control group given a chow diet with normal drinking water; (2) A colitis group given chow diet with 2% DSS in drinking water; (3) A steatohepatitis group given HFHCD with normal drinking water; and (4) A steatohepatitis + colitis group given HFHCD with 2% DSS in drinking water.

Results: NASH plus UC had high mortality (58.3%). Neither NASH nor UC alone were fatal. Although DSS-induced colitis did not exacerbate histological liver injury in HFHCD-fed mice, premorbid NASH significantly increased UC-related gut injury compared with UC alone. It was characterized by a significantly shorter colon, more colonic congestion, and a higher histopathological score (p<0.05). Inflammatory (tumor necrosis factor-alpha, interleukin 1 beta, C-C motif chemokine ligand 2, and nuclear factor kappa B) and apoptotic (Bcl2, Bad, Bim, and Bax) signaling pathways were significantly altered in distal colon tissues collected from mice with steatohepatitis + colitis compared with the other experimental groups.

Conclusions: Premorbid steatohepatitis significantly aggravated DSS-induced colitis and brought about a lethal phenotype. Potential links between NASH and UC pathogeneses can be investigated using this model.

Keywords: Inflammatory bowel disease; Mortality.; Mouse model; Nonalcoholic fatty liver disease; Ulcerative colitis.

Fig. 1. Overview of the hybrid model

Male C57bl/6 mice were fed a chow diet or HFHCD for 12 weeks of NASH modeling and were then divided into four subgroups (control, colitis, steatohepatitis, and steatohepatitis + colitis) before being challenged with 2% DSS in drinking water. Normal drinking water was the control). DSS, dextran sulfate sodium; HFHCD, high-fat high-cholesterol diet.

Fig. 2. The combination of NASH and UC led to significant weight loss and high mortality in mice.

(A) Body weight and (B, C) histological features of 12 weeks of NASH modeling. (D) DSS treatment significantly reduced mouse body weight in both control and NASH mice. (E) The combination of NASH and UC led to high mortality. (F) Images of freshly harvested mouse livers. *p<0.05, **p<0.01. NASH, nonalcoholic steatohepatitis; UC, ulcerative colitis.

Fig. 3. NASH-related phenotypes.

Mice received 20 weeks of combined NASH and UC modeling. (A) body weight and liver index. (B) Relative fat body weight and lean body weight. (C) Liver injury indicated by ALT and AST. (D, E) Plasma and hepatic triglyceride and cholesterol levels. *p<0.05, **p<0.01, ***p<0.001. ALT, aminotransferase; AST, aspartate aminotransferase; NASH: nonalcoholic steatohepatitis; UC: ulcerative colitis.

Fig. 4. Liver injury and inflammation.

Liver samples were harvested from each group of mice for further analysis. (A) Representative HE staining of each group. (B) Corresponding histopathological scores. (C) Representative Sirius Red staining of each group. (D) Hepatic LPS level of each group. (E) Relative mRNA expression of key inflammatory markers. *p<0.05, **p<0.01, ***p<0.001. HE, hematoxylin-eosin; LPS, lipopolysaccharide

Fig. 5. UC-related phenotypes of the combined model.

(A) Colitis severity indicated by DAI score. (B) Colon length. (C) Relative colon weight. (D) Representative HE staining and corresponding histopathological score. *p<0.05, **p<0.01, ***p<0.001. DAI, disease activity index; HE, hematoxylin-eosin.

Fig. 6. Premorbid NASH exacerbated colon injury and inflammation of UC.

(A) Relative mRNA expression of key inflammatory markers in colon tissue. (B) Representative IHC staining and quantification of the positive staining area. IHC, immunohistochemical staining; NASH, nonalcoholic steatohepatitis; UC, ulcerative colitis.

Fig. 7. Premorbid NASH disturbed colon inflammatory and apoptotic pathways in UC.

(A) Western blots of the relative expression of NF-κB and BAX in colon tissue. (B) Relative mRNA expression of apoptosis signaling. NASH, nonalcoholic steatohepatitis; NF-κB, nuclear factor kappa B; UC, ulcerative colitis.