Gut Liver. 2021;15(1):100-108.

Astragalin Inhibits Nuclear Factor-κB Signaling in Human Colonic Epithelial Cells and Attenuates Experimental Colitis in Mice.

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제1저자 한유민 · 교신저자 김주성

PMID 32390406 PMC7817923 DOI 10.5009/gnl19268 IF 4.1 소화기내과

소속 (4)

Department of Internal Medicine and Healthcare Research Institute, Seoul National University Hospital Healthcare System Gangnam Center, Seoul, Korea.
Department of Internal Medicine and Liver Research Institute, Seoul, Korea.
Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea.
Department of Internal Medicine, CHA Bundang Medical Center, CHA University School of Medicine, Seongnam, Korea.

Abstract

BACKGROUND/AIMS Astragalin (kaempferol-3-O-β-D-glucoside) is a flavonoid isolated from the leaves of persimmon or Rosa agrestis. Astragalin exhibits various anti-inflammatory properties; however, little is known about its therapeutic potential for inflammatory bowel disease (IBD). This study aims to investigate the anti-inflammatory effect of astragalin via blockade of the nuclear factor κB (NF-κB) signaling pathway in human colonic epithelial cells and a murine colitis model.

METHODS HCT-116 and HT-29 human colonic epithelial cells were pretreated with astragalin and stimulated with tumor necrosis factor-α (TNF-α). Cell viability was assessed by the MTS assay. Real-time reverse transcription polymerase chain reaction was used to analyze the messenger RNA expression of the inflammatory cytokines interleukin (IL)-6 and IL-8. The effect of astragalin on the NF-κB pathway was evaluated by Western blot analysis of inhibitor of NF-κB alpha (IκBα) phosphorylation/degradation and by electrophoretic mobility shift assay. Dextran sulfate sodium (DSS)-induced acute murine colitis model was used for in vivo experiments.

RESULTS Astragalin strongly suppressed the expression of proinflammatory cytokines in human colonic epithelial cells in a dose-dependent manner. Western blot analysis showed that astragalin inhibited IκBα phosphorylation/degradation. Additionally, astragalin reduced the DNA binding activity of NF-κB. Astragalin alleviated colon shortening and improved the pathologic scores in DSS-induced acute murine colitis model. Furthermore, astragalin reduced the level of phosphorylated IκBα and decreased the production of the inflammatory cytokines IL-6, IL-8, and TNF-α in the DSS-treated colon mucosa.

CONCLUSIONS Astragalin exerted an anti-inflammatory effect through NF-κB pathway inhibition and attenuated murine colitis. Astragalin is thus a potential therapeutic agent for IBD.

Keywords

AstragalinColitisHuman colonic epithelial cellsInflammatory bowel diseaseNF-kappa B

Figures

**Fig. 1** Effect of astragalin on the growth of colonic epithelial cells. (A) HCT-116 and (B) HT-29 cells were treated with astragalin at different concentrations (0, 20, 40, 60, 80, and 100 μM) for 24 hours, and cell viability was evaluated by an MTS assay.

**Fig. 2** Effect of astragalin on the messenger RNA (mRNA) expression of inflammatory cytokines in colonic epithelial cells. HCT-116 and HT-29 cells were pretreated with astragalin at different concentrations (0, 10, and 50 μM) and stimulated with 10 ng/mL tumor necrosis factor α (TNF-α) for 30 minutes. Reverse transcription polymerase chain reaction was performed to detect the mRNA expression levels of interleukin-6 (IL-6) and IL-8. (A) IL-6 in HCT-116 and (B) HT-29 cells. (C) IL-8 in HCT-116 and (D) HT-29 cells. (E) TNF-α in HCT-116 and (F) HT-29 cells. *p<0.05 compared with TNF-α alone.

**Fig. 3** Effect of astragalin on inhibitor of nuclear factor κB-alpha (IκBα) phosphorylation/degradation in colonic epithelial cells. HCT-116 and HT-29 cells were treated with astragalin at different concentrations (0, 10, and 50 μM) and stimulated with 10 ng/mL tumor necrosis factor α (TNF-α) for 30 minutes. Whole-cell extracts were prepared and analyzed for IκBα and phospho-IκBα expression.

**Fig. 4** Effect of astragalin on the DNA binding activity of nuclear factor κB (NF-κB) in colonic epithelial cells. HCT-116 cells were treated with astragalin at different concentrations (0, 10, and 50 μM) and stimulated with 10 ng/mL tumor necrosis factor α (TNF-α) for 30 minutes. The DNA binding activity was evaluated using electrophoretic mobility shift assay.

**Fig. 5** Effect of astragalin on dextran sulfate sodium (DSS)-induced acute murine colitis. (A) Oral administration of astragalin significantly reduced the degree of body weight loss compared with that of vehicle-treated mice. (B) Colon shortening improved with oral administration of astragalin. (C) Treatment with astragalin significantly improved the histologic scores of mice with colitis compared with those of vehicle-treated mice. (D) Astragalin reduced colitis-induced inhibitor of nuclear factor κB-alpha (IκBα) phosphorylation in the colonic mucosa. (E) Oral administration of astragalin attenuated the destruction of crypts, damage to the epithelium and infiltration of inflammatory cells (H&E). (F) Oral administration of astragalin reduced the level of phosphorylated IκBα in both destroyed epithelial cells and inflammatory cells (immunohistochemical staining). *p<0.05 and ^†p<0.01 compared with vehicle.

**Fig. 6** Effect of astragalin on the messenger RNA (mRNA) expression of inflammatory cytokines in dextran sulfate sodium (DSS)-induced acute murine colitis. Oral administration of astragalin significantly and dose-dependently reduced mRNA expression of (A) IL-6, (B) IL-8, and (C) TNF-α in murine colonic extracts. IL, interleukin; TNF, tumor necrosis factor. *p<0.05 compared with vehicle.

그림 출처: PubMed Central 공개 논문 (PMC7817923)

Conflict of interest statement

CONFLICTS OF INTEREST. No potential conflict of interest relevant to this article was reported.

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