Metformin Ameliorates Inflammatory Bowel Disease by Suppression of the STAT3 Signaling Pathway and Regulation of the between Th17/Treg Balance

Abstract

Objective: Metformin is used to treat type 2 diabetes. We sought to determine whether metformin reduces inflammation, by regulating p-signal transducer and activator of transcription 3 (STAT3) expression and T-helper 17 (Th17) cell proliferation, in a mouse model of inflammatory bowel disease (IBD).

Methods: IBD mice were administered metformin for 16 days and their tissues were analyzed. AMP-activated protein kinase (AMPK), the mammalian target of rapamycin (mTOR), p-STAT3 and p-STAT5 in the spleen and lymph nodes were detected using immunohistochemistry and confocal microscopy. Gene expression was determined using quantitative PCR assays, and protein expression levels were measured using western blotting and enzyme-linked immunosorbent assays. Human HT-29 cell proliferation was evaluated using MTT assays.

Results: Metformin reduced disease activity index scores and inhibited weight loss. Metformin also decreased the colonic histological score and inflammatory mediators and increased colon lengths increased. Treatment with metformin inhibited the expression of interleukin (IL)-17, p-STAT3, and p-mTOR. In contrast, metformin treatment increased expression levels of p-AMPK and Foxp3. In addition, expression of inflammatory cytokines decreased in a dose-dependent manner in inflamed human HT-29 cells cultured with metformin at various concentrations.

Conclusions: Metformin attenuates IBD severity and reduces inflammation through the inhibition of p-STAT3 and IL-17 expression. Our results have increased our understanding of this chronic inflammatory disease, and support the strategy of using p-STAT3 inhibitors to treat IBD.

Fig 1. Metformin prevents induction of inflammatory bowel disease (IBD).

Metformin (1 mg/mouse) was administered daily to mice with IBD for 16 days. (A) Weight of mice with IBD. (B and C) disease activity index (DAI) score and colon length. (D) Histopathlogical analysis showed tissue degradation in mice with IBD, and metformin-treated IBD mice. All tissue sections were counterstained with hematoxylin (100× magnification). The scale bar indicates 50 μm. (E) mRNA levels of tumor necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β) in the colon tissues of IBD mice and metformin-treated IBD mice were determined by qPCR. Data are presented as the mean ± SD from three independent experiments (*P < 0.05, **P < 0.03, ***P < 0.001).

Fig 2. Metformin treatment suppresses inflammation and p-signal transducer and activator of transcription 3 (STAT3) expression, but enhances the expression of AMP-activated protein kinase (AMPK) in a mouse model of inflammatory bowel disease (IBD).

(A) Immunohistochemical detection of interleukin (IL)-1β, IL-6, IL-8, TNF-α, and vascular endothelial growth factor (VEGF) in the colon tissues of IBD mice and metformin-treated IBD mice. All tissue sections were counterstained with hematoxylin (400× magnification). The scale bar indicates 50 μm. (B) Expression of p-STAT3, p-mTOR, and p-AMPK proteins were determined by western blotting. Data are presented as the mean ± SD from three independent experiments (*P < 0.05, **P < 0.03).

Fig 3. Metformin controls mRNA expression levels of inflammatory cytokines and the differentiation of Th17 and Treg cells through the regulation of signal transducer and activator of transcription 3 (STAT3) and STAT5 phosphorylation in lymph nodes.

(A) mRNA levels of proinflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, IL-17, and interferon (IFN)-γ] decreased in the lymph nodes of metformin-treated inflammatory bowel disease (IBD) mice. (B) Lymph node tissue sections from IBD and metformin-treated IBD mice were subjected to immunostaining to determine the presence of CD4⁺IL-17⁺ or CD4⁺CD25⁺Foxp3⁺ cells. Data are presented as the mean ± SD from three independent experiments (*P < 0.05, **P < 0.03).

Fig 4. Metformin controls the mRNA expression levels of inflammatory cytokines and the differentiation of Th17 and Treg cells through the regulation of signal transducer and activator of transcription 3 (STAT3) and STAT5 phosphorylation in the spleen.

(A) mRNA levels of proinflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, IL-17, and interferon (IFN)-γ] decreased in the spleens of metformin-treated inflammatory bowel disease (IBD) mice. (B) Spleen tissue sections of IBD and metformin-treated IBD mice were subjected to immunostaining to determine the presence of CD4⁺IL-17⁺ or CD4⁺CD25⁺Foxp3⁺ cells. Data are presented as the mean ± SD from three independent experiments (*P < 0.05, **P < 0.03).

Fig 5. Metformin treatment reduces the mRNA levels of proinflammatory cytokines in human HT-29 cells.

(A) Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-8 mRNA expression levels decreased in human HT-29 cells stimulated with LPS and then treated with metformin. (B) The production of vascular endothelial growth factor (VEGF) decreased in human HT-29 cells stimulated with lipopolysaccharide (LPS) and then treated with metformin. (C) Cell viability was not altered by various concentrations of metformin. Data are presented as the mean ± SD from three independent experiments (*P < 0.05, **P < 0.03, ***P < 0.001).