EGCG Alleviates H2O2-Induced Inflammatory Injury and Apoptosis in Bovine Mammary Epithelial Cells Through Nrf2 Pathway Activation and p38MAPK Pathway Inhibition

Abstract

Epigallocatechin-3-gallate (EGCG) is a potential antioxidant that protects cells from oxidative damage. However, EGCG is less studied in oxidative stress-induced inflammation in bovine mammary epithelial cells (BMECs). Therefore, the present study sought to investigate the protective effects of EGCG on hydrogen peroxide (H₂O₂)-induced oxidative stress, inflammation, and apoptosis, and related mechanisms involved in BMECs using the H₂O₂-induced BMECs as an in vitro cell model of oxidative stress and inflammation response. The BMECs were treated with H₂O₂ (600 μM) and EGCG (5 μM), respectively, while the cells without any treatment were regarded as the controls. The protective effects of EGCG were investigated by quantitative real-time fluorescence PCR, western blot, ELISA, CCK-8, and so forth. The results showed that the treatment of BMECs with H₂O₂ significantly decreased the anti-oxidation ability of the cells, increased the expression of inflammation-related factors, and induced apoptosis. Furthermore, the functional recovery test showed that EGCG significantly improved the resistance to oxidative stress, inflammation, and apoptosis in H₂O₂-induced BMECs. The study of the protective mechanisms of EGCG in BMECs showed that EGCG could enter the nucleus by activating nuclear factor erythroid 2-related factor 2 (Nrf2) and exert the effects of anti-oxidation and anti-inflammation upon treatment with BMECs alone. The Nrf2 knockdown assay (siNrf2) showed that siNrf2 upregulated the mRNA expression of inflammatory factors and apoptosis-related genes in BMECs, increased reactive oxygen species (ROS) accumulation and mitochondrial damage, and downregulated mRNA expression of antioxidant genes. Similarly, EGCG reduced ROS production in BMECs by inhibiting p38 mitogen-activated protein kinase (p38MAPK) phosphorylation, thereby reducing the mRNA expression of related genes in the NF-κB/caspase-3 pathway when p38MAPK was inhibited with the p38MAPK inhibitor SB203580. Overall, the experimental results showed that EGCG could improve the antioxidant function of BMECs by activating the Nrf2 and inhibiting the p38MAPK pathways, reducing inflammation and mitochondrial damage. This study provides a theoretical basis for further study of exogenous EGCG to prevent mastitis in dairy cows.