Mesenchymal stem cell transplantation improves chronic colitis-associated complications through inhibiting the activity of toll-like receptor-4 in mice

Abstract

Background: A variety of extra-intestinal manifestations (EIMs), including hepatobiliary complications, are associated with inflammatory bowel disease (IBD). Mesenchymal stem cells (MSCs) have been shown to play a potential role in the therapy of IBD. This study was designed to investigate the effect and mechanism of MSCs on chronic colitis-associated hepatobiliary complications using mouse chronic colitis models induced by dextran sulfate sodium (DSS).

Methods: DSS-induced mouse chronic colitis models were established and treated with MSCs. Severity of colitis was evaluated by disease activity index (DAI), body weight (BW), colon length and histopathology. Serum lipopolysaccharide (LPS) levels were detected by limulus amebocyte lysate test (LAL-test). Histology and liver function of the mice were checked correspondingly. Serum LPS levels and bacterial translocation of mesenteric lymph nodes (MLN) were detected. Pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1β (IL-1β), interleukin-17A (IL-17A), Toll receptor 4 (TLR4), TNF receptor-associated factor 6 (TRAF6) and nuclear factor kappa B (NF-κB) were detected by immunohistochemical staining, western blot analysis and real-time PCR, respectively.

Results: The DSS-induced chronic colitis model was characterized by reduced BW, high DAI, worsened histologic inflammation, and high levels of LPS and E. coli. Liver histopathological lesions, impaired liver function, enhanced proteins and mRNA levels of TNF-α, IFN-γ, IL-1β, IL-17A, TLR4, TRAF6 and NF-κB were observed after DSS administration. MSCs transplantation markedly ameliorated the pathology of colon and liver by reduction of LPS levels and proteins and mRNA expressions of TNF-α, IFN-γ, IL-1β, IL-17A, TLR4, TRAF6 and NF-κB.

Conclusions: MSCs can improve chronic colitis-associated hepatobiliary complications, probably by inhibition of enterogenous endotoxemia and hepatic inflammation through LPS/TLR4 pathway. MSCs may represent a novel therapeutic approach for chronic colitis-associated hepatobiliary complications.

Keywords: Chronic colitis; Hepatobiliary complications; Lipopolysaccharide; Mesenchymal stem cells; Toll receptor 4.

Fig. 1

Characterization of UC-MSCs. a Flow cytometric analysis of cell surface markers on UC-MSCs. b adipogenesis differentiation of UCMSCs was analyzed by Oil-Red-O staining. c Osteogenesis differentiation was analyzed by Alizarin Red-S staining. US-MSC, umbilical cordderived mesenchymal stem cell

Fig. 2

The chronic DSS-induced colitis model was established. a, c Body weight (BW) was assessed daily and expressed as percentage of baseline BW. b, d Disease activity index (DAI), consisting of BW loss, stool consistency and OB, was measured daily. The DSS + vehicle group (b) showed more severe inflammation in the colon compared to the control group a, while the inflammation in the colon in the DSS + MSCs group was ameliorated. e Samples of colon. f Colon length. g Morphology score. h H&E staining (× 200). i Histological score (^aP < 0.05 vs. the DSS + vehicle group; ^bP < 0.05 vs. the control group). Data were expressed as mean ± SD

Fig. 3

MSCs alleviated chronic colitis-associated hepatobiliary disorders. The DSS + vehicle group showed more severe hepatobiliary disorders compared to that of the control group, while MSCs ameliorated hepatobiliary disorders associated with chronic colitis. a Representative sections samples of liver (H&E and MT staining; original magnifications, × 200). b Histological score (^aP < 0.05 vs. the DSS + vehicle group; ^bP < 0.05 vs. the control group). c, d Liver function, ALT and AST levels in the DSS + vehicle group were increased and ALB level were lowered compared to that of the control group. ALT and AST levels in the DSS + MSCs group were significantly lower and ALB levels were increased (^aP < 0.05 vs. the DSS + vehicle group; ^bP < 0.05 vs. the control group). TBIL and DBIL levels were not changed (^aP > 0.05 vs. the DSS + vehicle group; ^bP > 0.05 vs. the control group). Data were expressed as mean ± SD

Fig. 4

Effect of MSCs on intestinal permeability. (A) Bacteria cultured from MLN. Viable bacteria are mainly Escherichia coli in the control group (a), DSS + vehicle group (b), and DSS + MSCs group (c). (B) Differences in incidence of bacterial translocation to MLN were displayed, Bacterial translocation was increased in the DSS + vehicle group (90%) compared with that of the control group (0%), while bacterial translocation to mesenteric lymph nodes decreased in the DSS + MSCs group (15%). (C) The changes of LPS levels. LPS level in the DSS + vehicle group were increased obviously compared with the control group. In the DSS + MSCs group, the LPS levels were lowered compared with that of the DSS + vehicle group. Data were expressed as mean ± SD. ^aP < 0.05 vs. the DSS + vehicle group; ^bP < 0.05 vs. the control group

Fig. 5

MSCs suppressed the expressions of TNF-α, IFN-γ, IL-1β and IL-17A mRNA and proteins in the liver tissues of chronic DSS-induced colitis. a MSCs changed the expressions of TNF-α, IFN-γ, IL-1β and IL-17A mRNA in liver tissues of chronic colitis-associated hepatobiliary disorders. Data were expressed as mean ± SD. ^aP < 0.05 vs. the DSS + vehicle group; ^bP < 0.05 vs. the control group. b Representative western blots showing TNF-α, IFN-γ, IL-1β and IL-17A proteins expressions. c Expressions of TNF-α, IFN-γ, IL-1β and IL-17A staining in the DSS + vehicle group were higher than that of the control group, while in the DSS + MSCs group, these expressions decreased. d The expressions of the TNF-α, IFN-γ, IL-1β, and IL-17A protein in liver tissues were significantly increased in the DSS + vehicle group compared with that of the control group, while they were decreased in the DSS + MSCs group. e Quantification of cytokines by immunohistochemistry (original magnifications, × 400)

Fig. 6

MSCs suppressed the expressions of TLR4, TRAF6 and NF-κB mRNA and proteins in the liver tissues of chronic DSS-induced colitis. a MSCs changed the expressions of TLR4, TRAF6 and NF-κB mRNA in liver tissues of chronic colitis-associated hepatobiliary disorders. Data were expressed as mean ± SD. ^aP < 0.05 vs. the DSS + vehicle group; ^bP < 0.05 vs. the control group. b Representative western blots showing TLR4, TRAF6 and NF-κB protein expressions. c Expressions of TLR4, TRAF6 and NF-κB staining in the DSS + vehicle group were higher than that of the control group, while in the DSS + MSCs group, these expressions decreased. d The expressions of the TNF-α, IFN-γ, IL-1β and IL-17A proteins in liver tissues were increased in the DSS + vehicle group compared with that of the control group, while they were decreased in the DSS + MSCs group. e Quantification of cytokines by image analysis of immunohistochemistry (original magnifications, × 400)

Fig. 7

Serum LPS levels depicted positive correlation with TLR4 mRNA expression in liver tissues and HAI-Knodell score. Pearson’s correlation analysis between LPS and TLR4Mrna a, LPS and histological score (b) were done