Anti-inflammation and Anti-Pyroptosis of Epicatechin Gallate in LPS-Stimulated MAC-T Cells and Mouse Mammary Glands

Plant-active ingredients serve as an alternative treatment for mastitis, one of the most costly diseases in the dairy industry, to decrease drug-resistant bacteria and antibiotic residues in the food chain because of antibiotic abuse. Epigallocatechin gallate (ECG) has been proven to have the efficacy of anti-inflammation, anti-tumor, and anti-apoptosis. However, its potential for the anti-inflammation and anti-pyroptosis effects in cow mastitis remains to be elucidated. In this research, bovine mammary alveolar cells (MAC-T) and the mouse mammary gland were treated with lipopolysaccharide (LPS) in combination with ECG. The MTT assay showed no effect of ECG on MAC-T viability at 1, 15, and 30 μg/mL, while Hematoxylin-Eosin staining indicated that ECG administration at 10, 20, and 40 mg/kg alleviated the inflammatory changes and inflammatory cell infiltration in the mammary gland induced by LPS. ELISA assay showed that ECG significantly and dose-dependently decreased LPS-induced levels of proinflammatory factors (IL-1β, IL-6, and TNF-α) both <i>in vivo</i> and<i> in vitro</i>. Furthermore, TUNEL analysis demonstrated that ECG dose-dependently lowered LPS-induced membrane rupture of MAC-T cells. Importantly, ELISA assay and immunofluorescence analyses revealed that ECG significantly suppressed the protein levels of NOD-like receptor protein 3 (NLRP3) inflammasome and the N-terminus of gasdermin D (GSDMD-N), as well as the phosphorylation of nuclear factor kappa (NF-κB) p65 and IκB (inhibitor of NF-κB). In conclusion, ECG inhibits inflammation and pyroptosis in MAC-T cells and mouse mammary glands, as well as the NF-κB pathway and NLRP3 inflammasome.