Astragalus polysaccharides and astragaloside IV alleviate inflammation in bovine mammary epithelial cells by regulating Wnt/β-catenin signaling pathway

Abstract

The Wnt/β-catenin signaling regulates cell renewal and repair and is closely associated with inflammation. Astragalus polysaccharides (APS) and astragaloside IV (AS-IV), which are the main active substances extracted from Radix Astragali, protect cells by regulating Wnt signaling in cells, exerting antiinflammatory, antioxidant, and antistress effects. However, the mechanisms by which APS and AS-IV interact with Wnt signaling to achieve their therapeutic effects in bovine mammary epithelial cells (BMECs) are not understood. In this study, we used lipopolysaccharide (LPS)-stimulated BMECs as an in vitro model of inflammation to investigate the effects of APS and AS-IV on Wnt signaling in inflamed BMECs. Drug concentrations were screened using the CCK-8 method, the effect on protein expression was analyzed using immunoblotting, the effect on inflammatory factors using enzyme-linked immunosorbent assay, and the effect on oxidative factors using enzyme labeling and flow cytometry. LPS activated the expression of inflammatory and oxidative factors in cells and inhibited Wnt/β-catenin signaling. APS and AS-IV antagonized the inhibitory effect of LPS, protecting BMECs. They inhibited the expression of the IL-6, IL-8, and TNF-α inflammatory factors, and that of the MDA oxidative factor, and activated Wnt signaling in LPS-stimulated BMECs. Silencing of β-catenin abolished the protective effect of APS and AS-IV against LPS-stimulated BMECs. Thus, APS and AS-IV mediate protective effects in inflammatory BMECs model through activation of the Wnt signaling pathway. Wnt signaling pathway is one of the targets of the inhibitory effects of APS and AS-IV on inflammation.

Fig 1. LPS inhibits the proliferation and expression of β-catenin in BMECs.

BMECs were treated with 0–5 μg/mL LPS for 24 h. (a) The cellular activity of LPS-stimulated BMECs at 24 h was assayed using the CCK-8 method. (b) Effect on the expression of β-catenin in LPS-stimulated BMECs. Western blotting was used to determine the relative levels of β-catenin. β-actin was used as a control. Data are expressed as mean ± SD of 3 independent experiments. *0.01 < P < 0.05, **P < 0.01.

Fig 2. Influence of AS-IV and APS on the biological activity of BMECs.

The viability of BMECs treated with the indicated concentrations of AS-IV and APS was determined using the CCK-8 assay. Data are expressed as mean ± SD of 6 independent experiments. *0.01 < P < 0.05, **P < 0.01.

Fig 3. Effect of siRNA on the expression of β-catenin in BMECs.

C is negative control group; NC is NC-siRNA group; 1225 is 1225-β-catenin -siRNA group; 1990 is 1990-β-catenin-siRNA group. Western blotting was used to determine the relative levels of β-catenin. β-actin was used as a control. Data are expressed as mean ± SD of 3 independent experiments; * represents 0.01 < P < 0.05 and ** represents P < 0.01.

Fig 4. APS and AS-IV regulation of the Wnt/β-catenin signaling pathway in BMECs.

(a) APS and AS-IV activate the Wnt/β-catenin signaling in BMECs. C = Control cells without any processing; APS = cells treated with 1 mg/mL APS; EA = negative control for AS-IV (ethanol concentration was the same as that in AS-IV 100 μg/mL, ethanol concentration 1%); AS-IV50 = cells treated with 50 μg/mL AS-IV; AS-IV75 = cells treated with 75 μg/mL AS-IV; AS-IV100 = cells treated with 100 μg/mL AS-IV. (b) APS and AS-IV activate Wnt/β-catenin signaling in inflammatory state BMECs. A = Control cells without any processing; B = cells treated with LPS (0.5 μg/mL); C = LPS (0.5 μg/mL) + APS (1 mg/mL); D = LPS (0.5 μg/mL) + lincomycin (50 μg /mL); E = LPS (0.5 μg/mL) + penicillin (86U/mL≈50 μg/mL); F = LPS (0.5 μg/mL) + ethanol concentration 1% (vol/vol); G = LPS (0.5 μg/mL) + AS-IV(50 μg/mL); H = LPS (0.5 μg/mL) + AS-IV(75 μg/mL); I = LPS (0.5 μg/mL) + AS-IV(100 μg/mL). (c, d) Silencing of β-catenin abolishes the ability of APS and AS-IV to regulate the Wnt/β-catenin signaling pathway. siNC = cells treated with NC-siRNA alone; siNC + LPS = NC-siRNA+ LPS (0.5 μg/mL); siNC + LPS + APS = NC-siRNA + LPS (0.5 μg/mL) + APS (1 mg/mL); siRNA = cells treated with 1225-siRNA alone; siRNA + LPS = 1225-siRNA + LPS (0.5 μg/mL); siRNA + LPS + APS = 1225-siRNA+LPS (0.5 μg/mL) + APS (1 mg/mL). siNC + EA = NC-siRNA+ ethyl alcohol (1% vol/vol); siNC + LPS + EA = NC-siRNA + LPS (0.5 μg/mL) + ethyl alcohol (1% vol/vol); siNC + LPS + AS-IV = NC-siRNA+LPS (0.5 μg/mL) +100 μg/mL AS-IV (100 μg/mL); siRNA + EA = 1225-siRNA+ ethyl alcohol (1% vol/vol); siRNA + LPS + EA = 1225-siRNA+LPS (0.5 μg/mL) + ethyl alcohol (1% vol/vol); siRNA + LPS + AS-IV = 1225-siRNA + LPS (0.5 μg/mL) +100 μg/mL AS-IV (100 μg/mL). Protein blots were used to determine relative protein expression levels. β-actin was used as a control. Data are expressed as mean ± SD of 3 independent experiments. * represents 0.01 < P < 0.05, ** represents P < 0.01 and letter differences in the graph indicate significant differences between groups, P < 0.05.

Fig 5. APS and AS-IV inhibit the expression of inflammatory factors in BMECs under an inflammatory state.

(a, b) APS and AS-IV suppress the expression of inflammatory factor genes and proteins in LPS-stimulated BMECs. C = Control cells without any processing; LPS = cells treated with LPS (0.5 μg/mL) alone; LPS + APS = LPS (0.5 μg/mL) + APS (1 mg/mL); LPS + EA = LPS (0.5 μg/mL) + ethanol concentration 1% (vol/vol); LPS + AS-IV = LPS (0.5 μg/mL) + AS-IV (100 μg/mL). (c, d) APS and AS-IV fail to inhibit inflammatory factors in BMECs under an inflammatory state after silencing β-catenin. Each experimental group expresses the same meaning as in Fig 4C and 4D. Data are expressed as the mean ± SD of 3 independent experiments. *, # represents 0.01 < P < 0.05, **, ## represents P < 0.01, ns represents P > 0.05, and letter differences in the graphs indicate significant P < 0.05 differences between groups.

Fig 6. APS and AS-IV regulate the accumulation of ROS in BMECs under an inflammatory state.

(a) APS and AS-IV inhibit the LPS-stimulated accumulation of ROS in LPS-stimulated BMECs. (b, c) Effect of APS and AS-IV on the accumulation of ROS in LPS-stimulated BMECs after silencing β-catenin. Each experimental group expresses the same meaning as in Fig 5. Data are expressed as the mean ± SD of 3 independent experiments. *, # represents 0.01 < P < 0.05, **, ## represents P < 0.01, ns represents P > 0.05, and letter differences in the graphs indicate significant P < 0.05 differences between groups.

Fig 7. APS and AS-IV regulate the levels of MDA in BMECs under an inflammatory state.

(a) APS and AS-IV inhibit the LPS-stimulated upregulation of MDA in LPS-stimulated BMECs. (b, c) Effect of APS and AS-IV on the levels of MDA in LPS-stimulated BMECs after silencing β-catenin. Each experimental group expresses the same meaning as in Fig 5. Data are expressed as the mean ± SD of 3 independent experiments. *, # represents 0.01 < P < 0.05, **, ## represents P < 0.01, ns represents P > 0.05, and letter differences in the graphs indicate significant P < 0.05 differences between groups.