Protective Mechanism of Bamboo Leaf Flavonoids on Hydrogen Peroxide-Induced Pyroptosis of Bovine Mammary Epithelial Cells Was Discussed Based on Silent Information Regulator 1/p65 Pathway
This study aimed to explore the mechanism by which bamboo leaf flavonoids(BLF)pretreatment alleviates pyroptosis in bovine mammary epithelial cells(bMECs)induced by hydrogen peroxide(H2O2).bMECs were used as the research subjects,and control group,BLF group,H2O2 group,BLF+H2O2 group,BAY 11-7082+H2O2 group,BLF+BAY 11-7082+H2O2 co-treatment group,EX 527+H2O2 group and BLF+EX 527+H2O2 co-treatment group were set up in this experiment.BLF was administered at a concentration of 80 µg/mL,and H2O2 was used at 800 µmol/L.BAY 11-7082 and EX 527 were used to inhibit the expression of p65 and silent information regulator 1(SIRT1),respectively.Gene and protein expression levels in the cells were detected using PCR and Western Blot.The number of pyroptotic cells was determined by flow cytometry.The fluorescence intensity of apoptosis-associated speck-like protein(ASC)spots was detected by immunofluo-rescence to observe the role of the SIRTl/p65 pathway in BLF-mediated alleviation of H2O2-induced bMECs pyroptosis.The results showed as follows:1)p65 mRNA and protein expression levels were significantly in-creased in H2O2-induced bMECs pyroptosis(P＜0.05),and significantly decreased following BLF treatment(P＜0.05).BLF and BAY 11-7082 significantly inhibited p65 expression and nuclear translocation(P＜0.05),effectively alleviating bMECs pyroptosis.The expression levels of pyroptosis-related genes and proteins in bMECs were significantly reduced by BLF and BAY 11-7082(P＜0.05),as well as the fluorescence intensity of ASC spots was reduced.2)SIRT1 mRNA and protein expression levels were significantly decreased in H2O2-induced bMEC pyroptosis(P＜0.05),and significantly increased following BLF treatment(P＜0.05).EX 527 significantly inhibited SIRT1 expression,while promoted p65 expression and nuclear translocation(P＜0.05).EX 527 also significantly increased the expression levels of pyroptosis-related genes and proteins in bMECs(P＜0.05),and enhanced ASC spots fluorescence intensity.In summary,80 µg/mL BLF alleviates H2O2-induced bMECs pyroptosis and NLRP3 inflammasome activation by inhibiting p65 expression and nuclear translocation.SIRT1 expression is downregulated in H2O2-induced bMEC pyroptosis,and BLF alleviates bMECs pyroptosis by upregulating SIRT1 expression,thereby inhibiting p65 expression and exerting a protec-tive effect on the cells.[Chinese Journal of Animal Nutrition,2024,36(9):5949-5969]
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