FoxO1 regulates TLR4/MyD88/MD2-NF-κB inflammatory signalling in mucosal barrier injury of inflammatory bowel disease

Abstract

In this study, FoxO1 transgenic mice (transgenic, FoxO1-Tg) and C57BL/6 wild-type (wild-type, FoxO1-WT) mice were used to establish chronic colitis by drinking water containing dextran sulphate sodium (DSS). Afterwards, we observed the life changes in mice and assessed the pathological changes by H&E tissue staining. In addition, the TLR4/MyD88/MD2-NF-κB inflammatory signals were detected. As a result, under DSS treatment, the activation level of TLR4/MyD88/MD2-NF-κB inflammatory signal was higher in FoxO1-Tg mice than that in FoxO1-WT mice. Meanwhile, the intestinal mucosal tissue damage was more severe, the down-regulation of tight junction protein level was more significant and the life quality was decreased to a higher degree in FoxO1-Tg mice compared with those in FoxO1-WT mice. Caco-2 cells were used to mimic the intestinal mucosal barrier model for in vitro assays. In addition, lentiviral packaging FoxO1 overexpressing plasmid was transfected into Caco-2 cells for FoxO1 overexpression. TNF-α intervention was performed for intestinal mucosal inflammatory response model. Consequently, the down-regulation of FoxO1 inhibited the activation of TLR4/MyD88/MD2-NF-κB inflammatory signal, decreased the mucosal barrier permeability and up-regulated the expression of tight junction protein. By contrast, the overexpression of FoxO1 increased the mucosal barrier permeability and down-regulated the level of tight junction protein.

Keywords: inflammatory bowel disease; inflammatory signal; tight junction; transcription factor FoxO1.

Figure 1

Expression levels of FoxO1 in intestinal tissues of IBD patients (Control: healthy population; IBD: inflammatory bowel disease; UC: ulcerative colitis; and CD: Crohn's disease). A, IHC was used to detect the expression of FoxO1 in intestinal tissues of IBD patients (brown for positive FoxO1 protein staining). As a result, the protein level of FoxO1 was higher in UC and CD patients than Control. B, ELISA was used to determine the protein level of FoxO1 in intestinal tissue of IBD patients (UC+CD). Comparison between groups, P<.05

Figure 2

Effects of FoxO1 by evaluating bodyweight, DAI score and pathological condition of mice with chronic colitis. A, The intestinal histopathological scores in mice showed that the pathological scores were 0 in FoxO1‐WT‐Con and FoxO1‐Tg‐Con groups. However, after DSS intervention, the pathological scores were significantly higher in FoxO1‐Tg‐DSS group than those of FoxO1‐WT‐DSS group. Comparison between groups, ^* P<.05. B, The change in intestinal tissue length in mice: The length of intestinal tissue was significantly lower in FoxO1‐Tg‐DSS group than that in FoxO1‐WT‐DSS group. Comparison between groups, ^* P<.05. C, DAI scores in mice showed no significant changes in FoxO1‐WT‐Con and FoxO1‐Tg‐Con mice during the experiment, whereas DAI scores were significantly changed in FoxO1‐Tg‐DSS and FoxO1‐WT‐DSS groups. Comparison with FoxO1‐WT‐DSS group at the same time‐point, ^* P<.05. D, Bodyweight changes of mice: The bodyweight was gradually increased in FoxO1‐WT‐Con and FoxO1‐Tg‐Con mice under normal feeding, without significant difference. However, the bodyweight was decreased in FoxO1‐Tg‐DSS and FoxO1‐WT‐DSS groups. Comparison with the FoxO1‐WT‐DSS group at the same time‐point, ^* P<.05. E, HE staining showed that the intestinal mucosa epithelium was intact, and the intestinal gland composed of lamina propria and mucosa muscle layer was arranged regularly in FoxO1‐WT‐Con group under light microscope. However, the colonic mucosa was defective, with decreased goblet cells and destructive or even disappeared gland, and large number of lymphocytes were infiltrated in the submucosa and even the muscle layer in FoxO1‐WT‐DSS group, which was more severe in FoxO1‐Tg‐DSS group than FoxO1‐WT‐DSS group. F,G, The expression of FoxO1 in mouse intestinal tissue: The expression of FoxO1 was increased in FoxO1‐WT‐DSS and FoxO1‐Tg‐DSS groups after DSS intervention, and the expression of FoxO1 was significantly higher in FoxO1‐Tg‐DSS group than that in FoxO1‐WT‐DSS group. Comparison between groups, ^* P < .05

Figure 3

Effects of FoxO1 on the expression of tight junction protein in mice with chronic colitis. A, Expression of ZO‐1 protein in mouse intestinal tissue by IHC: ZO‐1 protein was strongly expressed in intestinal tissues of FoxO1‐WT‐Con and FoxO1‐Tg‐Con groups, which was significantly down‐regulated in FoxO1‐WT‐DSS and FoxO1‐Tg‐DSS groups. Meanwhile, the expression of ZO‐1 was significantly lower in FoxO1‐Tg‐DSS group than that of FoxO1‐WT‐DSS group. B, Expression of occludin protein in mouse intestinal tissue by IHC: Occludin protein was strongly expressed in intestinal tissues of FoxO1‐WT‐Con and FoxO1‐Tg‐Con groups, which was significantly down‐regulated in FoxO1‐WT‐DSS and FoxO1‐Tg‐DSS groups. Meanwhile, the expression of occludin was significantly lower in FoxO1‐Tg‐DSS group than that of FoxO1‐WT‐DSS group. C, Expression of FoxO1 protein in mouse intestinal tissue by IHC: The expression levels of FoxO1 were significantly increased in FoxO1‐WT‐DSS and FoxO1‐Tg‐DSS groups after DSS intervention, whereas the expression levels of FoxO1 were significantly higher in FoxO1‐Tg‐DSS group than those in FoxO1‐WT‐DSS group. D,E, The protein expression of ZO‐1 and occludin in intestinal tissue by Western blot. After DSS intervention, the expression levels of ZO‐1 and occludin were down‐regulated, whereas the expression levels of ZO‐1 and occludin were significantly lower in FoxO1‐Tg‐DSS than those in FoxO1‐WT‐DSS group. Comparison between groups, ^* P<.05

Figure 4

Effects of FoxO1 on intestinal mucosal villus structure, permeability and expression of inflammatory signals in mice with chronic colitis. A, Changes in the intestinal mucosal microvillus structure of mice. The intestinal mucosal microvillus was neatly arranged in FoxO1‐WT‐Con and FoxO1‐Tg‐Con mice, with clear structure. The intestinal mucosa microvillus structure was significantly changed in FoxO1‐WT‐DSS and FoxO1‐Tg‐DSS groups, with obvious loss of microvillus structure. In addition, the lesion degree was significantly more severe in FoxO1‐Tg‐DSS group than that in FoxO1‐WT‐DSS group. B, FITC‐D permeability assay revealed that the permeability was significantly higher in FoxO1‐WT‐DSS and FoxO1‐Tg‐DSS groups than that in Con group. Comparison between groups ^* P<.05. C,D, Western blot assay for TIL4/MyD88/MD2‐NF‐kB signalling. The activation levels of TIL4/MyD88/MD2‐NF‐kB signalling were lower in FoxO1‐WT‐Con and FoxO1‐Tg‐Con groups. After DSS intervention, the levels of key proteins, TLR4, MyD88 and MD2 were up‐regulated. Comparison between groups, ^* P<.05

Figure 5

Effects of FoxO1 silencing by siRNA on Caco‐2 cell permeability. A‐C, The protein and mRNA expression of FoxO1 after FoxO1 silencing. After FoxO1 silencing by siRNA, both protein and mRNA levels of FoxO1 were significantly down‐regulated. Comparison between groups, ^* P<.05. D,E, After TNF‐α intervention, the apoptotic level was not significantly different between the Control group and the siRNA‐Negative group, whereas the apoptotic rate was significantly decreased in the siRNA‐FoxO1 group. Comparison between groups, ^* P<.05. F, Cell resistance assay showed no significant difference between the Control group and the siRNA‐Negative group, whereas cell resistance was significantly up‐regulated in the siRNA‐FoxO1 group. Comparison between groups, ^* P<.05. G, The permeability change of FITC‐D was not significantly different between the Control group and the siRNA‐Negative group, whereas the permeability was significantly down‐regulated in the siRNA‐FoxO1 group. Comparison between groups, ^* P<.05

Figure 6

Effects of FoxO1 overexpression on Caco‐2 cell permeability. A‐C, The protein and mRNA expression of FoxO1 after overexpression of FoxO1. After overexpression of FoxO1, both protein and mRNA levels of FoxO1 were significantly up‐regulated. Comparison between groups, ^* P<.05. D,E, After TNF‐α intervention, the apoptotic level was not significantly different between the Control group and the siRNA‐Negative group, whereas the apoptotic rate was significantly increased in the siRNA‐FoxO1 group. Comparison between groups, ^* P<.05. F, Cell resistance assay showed no significant difference between the Control group and the siRNA‐Negative group, whereas cell resistance was significantly down‐regulated in the siRNA‐FoxO1 group. Comparison between groups, ^* P<.05. G, The permeability change of FITC‐D was not significantly different between the Control group and the siRNA‐Negative group, whereas the permeability was significantly up‐regulated in the siRNA‐FoxO1 group. Comparison between groups, ^* P<.05

Figure 7

Effects of down‐regulation/overexpression of FoxO1 on inflammatory signalling and tight junction protein expression in inflammatory model. A,B, Expression of TIL4/MyD88/MD2‐NF‐kB signal. The down‐regulation of FoxO1 led to the suppressed TIL4/MyD88/MD2‐NF‐kB signalling pathway, whereas the levels of key proteins TLR4, MyD88 and MD2 were down‐regulated. After FoxO1 overexpression, the levels of key proteins TLR4, MyD88 and MD2 were increased. Comparison between groups, ^* P<.05. C,D, The expression of tight junction proteins ZO‐1 and occludin. After TNF‐α intervention, the expression levels of tight junction protein were down‐regulated. The down‐regulation of FoxO1 inhibited the down‐regulation of ZO‐1 and occludin proteins, whereas after overexpression of FoxO1, the down‐regulation of ZO‐1 and occludin proteins was more obvious. Comparison between groups, ^* P<.05