Mechanism of Bamboo Leaf Flavonoids Regulating Antioxidant Pathway and Autophagy to Alleviate Oxidative Stress in Dairy Cow Mammary Epithelial Cells
The purpose of this experiment was to explore the effect of bamboo leaf flavonoids(BLF)on the autophagy and antioxidant pathway of dairy cow mammary epithelial cells under oxidative stress,and to ex-plore the correlation between autophagy and Kelch-like ECH-associated protein 1(Keap1)-nuclear factor ery-throid-2-related factor 2(Nrf2)signaling pathway by adding autophagy inhibitor Mdivi-1.The MAC-T cell line of dairy cow mammary epithelial cells was used as the research object.The MAC-T treated in the mainte-nance medium was set as the control group.The MAC-T treated with the maintenance medium containing(80 μg/mL)BLF was set as the BLF group.The MAC-T treated with the maintenance medium containing(800 μmol/L)hydrogen peroxide(H2O2)was set as the H2O2 group(induced oxidative stress).The MAC-T treated with the maintenance medium containing(80 μg/mL)BLF and the maintenance medium containing(800 µmol/L)H2O2 were set as the BLF+H2O2 group.The MAC-T treated with the maintenance medium containing(80 μg/mL)BLF,(1 μmol/L)Mdivi-1 and(800 μmol/L)H2O2 was set as BLF+H2O2+Midiv group.The MAC-T treated with the maintenance medium containing(1 µmol/L)Mdivi-1 was set as Midiv group.After the culturing,real-time fluorescence quantitative PCR was used to detect the expression of autoph-agy and Keap1-Nrf2 signaling pathway-related genes,Western blot was used to detect the expression of autoph-agy-related proteins,Keap1-Nrf2 and extracellular signal-regulated kinase(MAPK)signaling pathway-related proteins,and immunofluorescence technique was used to detect transcription factor EB(TFEB)protein expres-sion,Nrf2 protein expression and nuclear translocation.The results showed as follows:1)BLF pretreatment could significantly increase the expression levels of autophagy markers Beclin1 and Parkin proteins in oxidative damaged cells(P＜0.05),significantly increase the expression levels of lysosomal function-related proteins ca-thepsin D(CTSD)and lysosomal associated membrane protein 1(LAMP1)(P＜0.05),significantly increase the phosphorylation degree of adenosine monophosphate activated protein kinase(AMPK)protein and the ex-pression level of TFEB protein(P＜0.05),promote TFEB nuclear translocation,and significantly increase the relative expression levels of lysosomal-related genes such as ATPase H+transporting V1 subunit H(ATP6V1H),cathepsin B(CTSB)and tripeptidyl peptidase 1(TPP1)and autophagy-related gene microtu-bule-associated protein 1 light chain 3(LC3)(P＜0.05).2)BLF pretreatment significantly reduced the ex-pression level of Keap1 protein in oxidative damaged cells(P＜0.05),resulting in the stabilization of Nrf2 pro-tein in the cytoplasm and the transfer of Nrf2 protein to the nucleus,which activated the transcription and trans-lation of downstream Nrf2 target genes heme oxygenase-1(HO-1)and NAD(P)H:quinone oxidoreductase 1(NQO1).However,inhibition of autophagy by Mdivi-1 inhibited the promotion of BLF on the Keapl-Nrf2 signaling pathway.In summary,this study found that BLF alleviates the oxidative stress of dairy cow mammary epithelial cells by promoting autophagy and lysosomal function and activating Keap1-Nrf2 signaling pathway,and there is a crosstalk between autophagy and antioxidant signaling pathways.[Chinese Journal of Animal Nutrition,2024,36(7):4677-4692]
万方数据
维普
