Forsythoside A regulates autophagy and apoptosis through the AMPK/mTOR/ULK1 pathway and alleviates inflammatory damage in MAC-T cells

Abstract

Dairy cow mastitis is the most common disease encountered in dairy farming. Lipopolysaccharides (LPS), among the major virulence-related factors produced by Escherichia coli, stimulate mammary gland inflammation and cause its damage, thereby affecting milk yield and quality. Forsythoside A (FTA) is among the main active components of forsythia. Recent pharmacological studies have demonstrated that FTA possesses anti-inflammatory, antiviral, antioxidant, and other biological activities. This study investigated the effects of the FTA-activated AMP-activated protein kinase (AMPK) signaling pathway on LPS-induced autophagy, apoptosis, and inflammatory damage in bovine mammary epithelial (MAC-T) cells. Cell activity was measured using the Cell Counting Kit 8. Moreover, real-time quantitative polymerase chain reaction and western blot analyses were used to detect expression levels of autophagic, apoptotic, and inflammatory factors, as well as those of oxidative stress-related genes and proteins. The annexin-FITC/PI assay and immunofluorescence assay were used to detect the apoptosis rate and LC3B expression, respectively. We found that FTA attenuated LPS-induced inhibition of MAC-T cell proliferation, reduced mRNA expression of related inflammatory factors, relieved oxidative stress, and exerted protective effects on MAC-T cells. Additionally, FTA activated autophagy, attenuated inhibition of autophagy flow, and inhibited apoptosis. Autophagy and apoptosis were mainly regulated through the AMPK/mTOR/ULK1 pathway. The aforementioned FTA-induced effects were inhibited by the administration of Compound C (CC; an AMPK inhibitor). Taken together, these results indicate that FTA can alleviate LPS-induced inflammation and oxidative stress in MAC-T cells, attenuate impairments in autophagy, and inhibit apoptosis. However, these effects were blocked by CC, which suggests that FTA inhibits LPS-induced autophagy, apoptosis, and inflammatory damage in MAC-T cells by activating the AMPK pathway.

Keywords: AMPK/mTOR/ULK1; Apoptosis; Autophagy; FTA; LPS; MAC-T.