Effects of Astragaloside Ⅳ on Expression of Milk Protein and Milk Fat Synthesis-Related Factors in Lipopolysaccharide-Stimulated Milk Bovine Mammary Epithelial Cells

FAN Jiaqi; ZHOU Xuezhang

doi:10.3969/j.issn.1006-267x.2022.10.063

2022 , Vol. 34 >Issue 10: 6702 - 6713

DOI: https://doi.org/10.3969/j.issn.1006-267x.2022.10.063

RESEARCH PAPER

Effects of Astragaloside Ⅳ on Expression of Milk Protein and Milk Fat Synthesis-Related Factors in Lipopolysaccharide-Stimulated Milk Bovine Mammary Epithelial Cells

FAN Jiaqi ,
ZHOU Xuezhang

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College of Life Sciences, Ningxia University, Yinchuan 750000, China

Received date: 2022-04-15

Online published: 2022-10-17

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Abstract

The purpose of this experiment was to study the effects of astragaloside Ⅳ (AS-Ⅳ) on the expression of milk fat and milk protein synthesis-related genes and the content of lipid peroxides in lipopolysaccharide (LPS)-stimulated milk bovine mammary epithelial cells (BMECs). BMECs were stimulated with 0.5 μg/mL LPS for 24 h to induce inflammation, and the concentration and time of AS-Ⅳ were screened by CCK-8 assay. The experiment was divided into 8 groups (3 repetitions in each group), the control group, LPS stimulation group (LPS group, stimulated with 0.5 μg/mL LPS for 24 h) and 6 AS-Ⅳ intervention groups, that is, after 0.5 μg/mL LPS stimulation for 24 h, different concentrations of AS-Ⅳ were added and continued to culture, divided into: 100 μg/mL AS-Ⅳ stimulation group for 1 h (LPS+100 AS-Ⅳ 1 h group), 100 μg/mL AS-Ⅳ stimulation group for 3 h (LPS+100 AS-Ⅳ 3 h group), 100 μg/mL AS-Ⅳ stimulation group for 6 h (LPS+100 AS-Ⅳ 6 h group), 25 μg/mL AS-Ⅳ stimulation group for 6 h (LPS+25 AS-Ⅳ 6 h group), 50 μg/mL AS-Ⅳ stimulation group for 6 h (LPS+50 AS-Ⅳ 6 h group), 100 μg/mL AS-Ⅳ stimulation group for 6 h (LPS+100 AS-Ⅳ 6 h group). The expression of milk protein and milk fat-related genes in cells was detected by real-time quantitative PCR; the contents of lipid peroxide (LPO) and malondialdehyde (MDA) in cell supernatant were detected by kits; the lipid droplets in cells were observed by fluorescence microscope The expression of mammalian target protein of rapamycin (mTOR) in cells was detected by Western blot; astragaloside Ⅳ-5(6)-carboxydiacetate fluorescein succinimidyl ester complex (ASIV-CDFA) was used to simulate the expression of BMECs uptake of AS-Ⅳ. The results showed that compared with the control group, LPS could induce the deposition of lipid peroxides in BMECs, and the contents of LPO and MDA were significantly increased (P < 0.05); milk proteins synthesized αs1-casein (CSN1S1) and β-casein (CSN2) genes relative expression levels decreased significantly (P < 0.05); milk fat synthesis fatty acid binding protein 3 (FABP3), fatty acid synthase (FASN), sterol regulatory element binding protein 1 (SREBP1) genes relative expression levels decreased significantly (P < 0.05). Compared with the LPS group, after AS-Ⅳ stimulated BMECs, the relative expression level of CSN2 gene was significantly increased (P < 0.05), but the relative expression level of CSN1S1 gene was not activated; AS-Ⅳ could significantly inhibit the content of MDA in inflammatory cells, 25, 50, 100 μg/mL AS-Ⅳ stimulated cells for 6 h could significantly inhibit the content of LPO in inflammatory cells; 100 μg/mL AS-Ⅳ stimulation for 1 h significantly increased the relative expression levels of FASN, acetyl-CoA carboxylase (ACC), lipoprotein lipase (LPL) genes (P < 0.05), 25 μg/mL AS-Ⅳ stimulation for 6 h significantly increased the relative expression level of peroxisome proliferator-activated receptorγ (PPARγ) gene (P < 0.05), 50 μg/mL AS-Ⅳ stimulated cells for 6 h significantly increased the relative expression level of LPL gene (P < 0.05), and significantly increased the content of lipid droplets in inflammatory cells (P < 0.05). The expression of phosphorylated mammalian target of rapamycin (p-mTOR) protein in inflammatory cells was significantly activated by AS-Ⅳ stimulated cells for 6 h (P < 0.05). ASIV-CDFA was used to observe the absorption of AS-Ⅳ by inflammatory cells. The cellular absorption of ASIV-CDFA increased with the increase of AS-Ⅳ concentration, but did not increase with the prolongation of time. In conclusion, AS-Ⅳ can regulate the expression of milk fat and milk protein synthesis-related genes in LPS-stimulated BMECs. AS-Ⅳ has a slowing effect on the inhibition of lipid droplet expression in LPS-induced BMECs, and has an inhibitory effect on activates lipid peroxides.

Key words： astragaloside Ⅳ; bovine mammary epithelial cells; milk fat; milk protein

Cite this article

FAN Jiaqi , ZHOU Xuezhang . Effects of Astragaloside Ⅳ on Expression of Milk Protein and Milk Fat Synthesis-Related Factors in Lipopolysaccharide-Stimulated Milk Bovine Mammary Epithelial Cells[J]. Chinese Journal of Animal Nutrition, 2022 , 34(10) : 6702 -6713 . DOI: 10.3969/j.issn.1006-267x.2022.10.063

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