Astragaloside II alleviates the symptoms of experimental ulcerative colitis in vitro and in vivo

Abstract

Background: Ulcerative colitis (UC) is a chronic inflammatory intestinal disease, and its morbidity is rising worldwide. Previous study indicated that astragaloside II (AS II), a monomeric compound, was used to treat bowel disease. However, the effects of AS II on UC remains unclear. Thus, this study aimed to investigate the therapeutic effects of AS II on experimental UC in vitro and in vivo.

Methods: CCD-18Co cells were stimulated by 1 μg/mL LPS to mimic UC in vitro. In addition, dextran sulfate sodium (DSS)-induced UC mouse model was established in vivo. CCK-8 assay was used to detect cell proliferation in vitro. Moreover, the concentrations of inflammatory factors interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), nitric oxide (NO), superoxide dismutase (SOD) and malondialdehyde (MDA) in CCD-18Co cells and colon tissues were determined by ELISA, respectively. Meanwhile, the expressions of hypoxia-inducible factor 1α (HIF-α), phospho-inhibitor of NF-κB (p-IκB) and phospho-NF-κB p65 (p-p65) were detected by western blotting in vitro and in vivo, respectively.

Results: In this study, the levels of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 were significantly increased in lipopolysaccharide (LPS)-stimulated CCD-18Co cells. However, LPS-induced inflammatory response was markedly alleviated by AS II. In addition, LPS-induced HIF-α, p-IκB and p-p65 proteins increases were markedly ameliorated by AS II treatment. Moreover, AS II reduced disease activity index (DAI) scores and increased the colon lengths in DSS-treated mice. Meanwhile, AS II decreased the levels of IL-6, TNF-α, IL-1β, NO, MPO and MDA, and increased the level of SOD in colon of DSS-treated mice. Furthermore, AS II downregulated the expressions of HIF-α, p-IκB and p-p65 in DSS-induced UC in mice.

Conclusion: Our findings indicated that AS II could alleviate inflammatory response in LPS-induced CCD-18Co cells and in DSS-induced UC in mice. In conclusion, AS II may serve as a potential agent for the treatment of UC.

Keywords: Astragaloside II; dextran sulfate sodium; lipopolysaccharide; ulcerative colitis.

Figure 1

AS II attenuated oxidative stress damage in LPS-stimulated CCD-18Co cells via decreasing the production of inflammatory factors. A. The chemical structure of AS II. B. CCD-18Co cells were treated with AS II (0, 0.1, 0.33, 1, 3 μM) for 48 h. CCK-8 assay was used to detect the viability of CCD-18Co cells. C. CCD-18Co cells were treated with LPS (1 μg/mL) for 0, 12, 24 and 48 h. In addition, CCD-18Co cells were treated with 1 μM AS II and 1 μg/mL LPS for 48 h. The level of IL-6 in the culture media was measured with ELISA. D. The level of TNF-α in the culture media was measured with ELISA. E. The level of IL-β in the culture media was measured with ELISA. F. CCD-18Co cells were treated with 1 μg/mL LPS or/and 1 μM AS II for 48 h. The level of NO in cells was measured with ELISA. G. The level of SOD in cells was measured with ELISA. H. The level of MDA in cells was measured with ELISA. *P<0.05, **P<0.01 compared with 0 h group; ##P<0.01 compared with 48 h group.

Figure 2

AS II inhibited the levels of HIF-α, p-p65 and p-IκB in LPS-treated CCD-18Co cells in vitro. CCD-18Co cells were treated with 1 μg/mL LPS or/and 1 μM AS II for 48 h. A. Relative expression of HIF-α in CCD-18Co cells was detected by qRT-PCR. B. Expression levels of HIF-α, p-IκB and p-p65 in CCD-18Co cells were detected with western blotting. β-actin was used as an internal control. C. The relative expression of HIF-α was quantified via normalization to β-actin. D. The relative expression of p-IκB was quantified via normalization to β-actin. E. The relative expression of p-p65 was quantified via normalization to β-actin. **P<0.01 compared with control group; ##P<0.01 compared with LPS_1 μg/ml group.

Figure 3

AS II attenuated DSS-induced UC in mice. A. Drug administration in a mouse model of DSS-induced UC. B. Mice were treated with AS II (0, 10, 30, 50 or 80 mg/kg) for 0, 2, 4, 6, 8 and 10 day and the body weight of mice were monitored. C. The mice were treated with DSS, DSS plus AS II (30 or 50 mg/kg) for 0, 2, 4, 6, 8 and 10 day. HE staining was performed by photomicrography (magnification at 400×). D. Body weights of mice in each group were monitored. E. Disease activity index (DAI) of mice in each group was measured. F. The colon lengths of mice in each group were evaluated. **P<0.01 compared with control group; #P<0.05, ##P<0.01 compared with DSS group.

Figure 4

AS II attenuated oxidative stress damage in DSS-induced UC mice via decreasing the levels of inflammatory factors. The mice were treated with DSS, DSS and 30 mg/kg AS II or DSS plus 50 mg/kg AS II for 0, 2, 4, 6, 8 and 10 day. A-C. The levels of IL-6, TNF-α or IL-β in the colon tissues were measured with ELISA. D-G. The levels of NO, MPO, SOD and MDA in colon tissues were measured with ELISA. **P<0.01 compared with control group; #P<0.05, ##P<0.01 compared with DSS group.

Figure 5

AS II inhibited the expression of inflammatory proteins in DSS-induced UC mice. The mice were treated with DSS, DSS and 30 mg/kg AS II or DSS and 50 mg/kg AS II for 0, 2, 4, 6, 8 and 10 day. A. The level of p-p65 in colon tissues was detected by immunohistochemical staining assay (magnification at 400×). B. Expression levels of HIF-α, p-IκB and p-p65 in colon tissues were detected with western blotting. β-actin was used as an internal control. C-E. The relative expressions of HIF-α, p-IκB and p-p65 were quantified via normalization to β-actin. **P<0.01 compared with control group; #P<0.05, ##P<0.01 compared with DSS group.