Chinese Journal of Animal Nutrition >

2022 , Vol. 34 >Issue 1: 612 - 625

DOI: https://doi.org/10.3969/j.issn.1006-267x.2022.01.056

RESEARCH PAPER

In Vitro Study on Effects of Bamboo Leaf Flavonoids on Antioxidation and Expression of Key Genes in Mammalian Target of Rapamycin Signaling Pathway in Bovine Mammary Epithelial Cells

  • WANG Yutong ,
  • SHEN Yiyuan ,
  • HUO Xiangyu ,
  • NIU Hui ,
  • HAN Siyu ,
  • TONG Jinjin ,
  • XIONG Benhai ,
  • JIANG Linshu
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  • 1. College of Animal Science and Technology, Beijing University of Agriculture, Beijing Key Laboratory of Cow Nutrition, Beijing 102206, China;
    2. Beijing Institute of Animal Husbandry and Veterinary Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received date: 2021-06-23

  Online published: 2022-01-18

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Abstract

The study was conducted to investigate the effects of bamboo leaf flavonoids(BLF)on antioxidant activity and expression of key genes in mammalian target of rapamycin (mTOR) signaling pathway in bovine mammary epithelial cells(BMECs) in vitro. Using a single factor experimental design, BMECs were treated with different concentrations of BLF(0, 0.5, 1.0, 5.0, 10.0, 20.0, 60.0, 100.0 μg/mL)for 3, 6, 12, 24 and 48 h without BLF as control group. The activity of BMECs was detected by CCK-8 assay, and the toxicity of BMECs was detected by lactic dehydrogenase (LDH) method. There were 6 parallel multiple holes in each test, and the test was repeated for 3 times. The effects of BLF on the antioxidant indices of BMECs was detected by flow cytometry and kit method. The changes of mRNA expression levels of apoptotic genes, and the key genes in mTOR signaling pathway were detected by fluorescence quantitative PCR. The results showed as follows:1) the optimum concentrations of BLF on BMECs activity were 1.0, 5.0, 10.0 μg/mL, and the best action time was 12 h. The concentrations of BLF had no toxic effect on BMECs. 2) The results of flow cytometry showed that 5.0 and 10.0 μg/mL BLF extremely significantly decreased the early apoptosis rate and apoptosis/necrosis rate of BMECs(P<0.01), and BLF extremely significantly decreased the total apoptosis rate of BMECs(P<0.01). 3) 5.0 μg/mL BLF extremely significantly decreased the production of intracellular reactive oxygen species (ROS) and the level of mitochondrial membrane potential (MMP)(P<0.01). 4) BLF supplementation extremely significantly increased the activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) (P<0.01), and extremely significantly decreased the content of MDA(P<0.01). 5) The results of fluorescence quantitative PCR showed that 5.0 μg/mL BLF significantly decreased the gene relative expression level of B-cell lymphoma/leukaemia-2-associated X protein (Bax)(P<0.05), BLF extremely significantly increased the gene relative expression level of B-cell lymphoma/leukaemia-2 (Bcl-2)(P<0.01), and extremely significantly decreased the ratio of Bax/Bcl-2 (P<0.01), 5.0 and 10.0 μg/mL BLF significantly decreased the gene relative expression level of cyclooxygenase-2 (COX-2) (P<0.05), and extremely significantly decreased the gene relative expression level of Caspase-3 (P<0.01), and extremely significantly increased the gene relative expression level of eukaryotic translation initiation factor 4E binding protein 1 (EIF-4EBP1)(P<0.01). In addition, BLF extremely significantly increased the gene relative expression level of mTOR, ribosome p70s6 (S6K1) and Cyclin D1 in BMECs (P<0.01), the key regulatory genes of mTOR signaling pathway in BMECs. BLF extremely significantly increased the gene expression levels of β-casein in BMECs(P<0.01). In conclusion, BLF can promote the activity of BMECs, reduce the accumulation of ROS and the level of MMP. BLF can significantly improve the antioxidant capacity of BMECs, reduce the expression of apoptosis-related genes, and affect the synthesis of milk protein by promoting mTOR signaling pathway.

Key words： bamboo leaf flavonoids; anti-oxidative; apoptosis; mTOR signaling pathway

Cite this article

WANG Yutong , SHEN Yiyuan , HUO Xiangyu , NIU Hui , HAN Siyu , TONG Jinjin , XIONG Benhai , JIANG Linshu . In Vitro Study on Effects of Bamboo Leaf Flavonoids on Antioxidation and Expression of Key Genes in Mammalian Target of Rapamycin Signaling Pathway in Bovine Mammary Epithelial Cells[J]. Chinese Journal of Animal Nutrition, 2022 , 34(1) : 612 -625 . DOI: 10.3969/j.issn.1006-267x.2022.01.056

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