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]
维普
万方数据