Astragaloside IV ameliorates necrotizing enterocolitis by attenuating oxidative stress and suppressing inflammation via the vitamin D3-upregulated protein 1/NF-κB signaling pathway

Abstract

Astragaloside IV (AS-IV) is a flavonoid from the plant Astragalus membranaceus (Fisch) Bge that has a wide range of therapeutic effects. The aim of the present study was to examine the effect of AS-IV on rats with necrotizing enterocolitis (NEC) under oxidative stress and inflammation. Newborn Sprague-Dawley rats were induced with NEC by asphyxia and hypothermia applied on 3 consecutive days. The rats were orally administered AS-IV at 25, 50 and 75 mg/kg for 4 days. The results revealed that AS-IV administration prevented NEC-induced decrease in the concentration of malondialdehyde and myeloperoxidase, and increase in the activity of glutathione (GSH) and superoxide dismutase in murine models. AS-IV also inhibited NEC-induced elevation in the levels of interleukin (IL)-6, IL-1β, tumor necrosis factor-α and nuclear factor (NF)-κB. The effects of AS-IV were achieved under inflammation and oxidative stress. Western blotting demonstrated that AS-IV substantially inhibited the phosphorylated (p)-IκBα, NF-κBp65, p-NF-κBp65 protein levels and increased vitamin D3 upregulated protein 1 (VDUP1) and IκBα protein levels. These data indicate that AS-IV may be effective in the protection of NEC-induced ileum degeneration by inhibiting the levels of inflammatory markers and oxidative stress via the regulation of the VDUP1/NF-κB signaling pathway.

Keywords: astragaloside IV; inflammation; necrotizing enterocolitis; oxidative stress; vitamin D3-upregulated protein 1/nuclear factor-κB pathway.

Figure 1.

Representative histopathologic condition of the terminal ileum from each experimental group stained with H&E (original magnification, ×10). Intestinal architecture of a rat from (A) the control group revealed normal histological findings; (B) NEC group and NEC group treated with (C) 25, (D) 50 and (E) 75 mg/kg AS-IV groups. H&E, hematoxylin and eosin; NEC, necrotizing enterocolitis; AS-IV, astragaloside IV.

Figure 2.

(A) GSH, (B) SOD, (C) MDA and (D) MPO concentrations in the distal ileum of rats after 4 days of treatment. Data are presented as the mean ± standard error of the mean in each group (n=10), **P<0.01, ***P<0.001, vs. the control group; ^#P<0.05, ^##P<0.01, vs. the model group. GSH, glutathione; SOD, superoxide dismutase; MDA, malondialdehyde; MPO, myeloperoxidase; NEC, necrotizing enterocolitis; AS-IV, astragaloside IV.

Figure 3.

Effect of AS-IV on inflammatory cytokine production in the serum. The levels of (A) TNF-α, (B) IL-1β and (C) IL-6 were measured by ELISA kits after 4 days of treatment. Data are presented as the means ± standard error of the mean in each group (n=10), **P<0.01, vs. the control group; ^#P<0.05, ^##P<0.01, vs. the model group. AS-IV, astragaloside IV; TNF-α, tumor necrosis factor-α; IL-interleukin; NEC, necrotizing enterocolitis.

Figure 4.

The mRNA expression levels of (A) TNF-α, (B) IL-1β and (C) IL-6 and (D) NF-κB in rat distal ileum after 4 days treatment was detected by reverse-transcription polymerase chain reaction. Data represent the mean ± standard error of the mean in each group (n=5), **P<0.01 and ***P<0.001, vs. the control group; ^#P<0.05, ^##P<0.01 and ^###P<0.001, vs. the model group. TNF-α, tumor necrosis factor-α; NEC, necrotizing enterocolitis; AS-IV, astragaloside IV; IL-interleukin; NF-κB, nuclear factor-κB.

Figure 5.

Protein expression levels in rat distal ileum after 4 days treatment were detected by (A) western blot analysis. The relative expressions of (B) p-NF-κB/NF-κB, (C) p-IκBα/IκBα and (D) VDUP1 were normalized to GAPDH. Data are presented as the means ± standard error of the mean in each group (n=3), **P<0.01, vs. the control group; ^#P<0.05 and ^##P<0.01, vs. the model group. NF-κB, nuclear factor-κB; VDUP1, vitamin D3-upregulated protein 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NEC, necrotizing enterocolitis; AS-IV, astragaloside IV; p, phosphorylated.