AUTHOR=Liu Songqi , Guo Wenjin , Jia Yuxi , Ye Bojian , Liu Shu , Fu Shoupeng , Liu Juxiong , Hu Guiqiu 

TITLE=Menthol Targeting AMPK Alleviates the Inflammatory Response of Bovine Mammary Epithelial Cells and Restores the Synthesis of Milk Fat and Milk Protein

JOURNAL=Frontiers in Immunology

VOLUME=Volume 12 - 2021

YEAR=2021

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2021.782989

DOI=10.3389/fimmu.2021.782989

ISSN=1664-3224

ABSTRACT=Mastitis is one of the most serious diseases that causes losses in the dairy industry, seriously impairing milk production and milk quality, and even affecting human health. Menthol is a cyclic monoterpene compound obtained from the stem and leaves of peppermint, which has a variety of biological activities, including anti-inflammatory and antioxidant activity. The purpose of this study was to investigate the preventive effect of menthol on the lipopolysaccharide-induced in-flammatory response in primary bovine mammary gland epithelial cells (BMECs) and its an-ti-inflammatory mechanism. First, BMECs were isolated and amplified from the udders of Hol-stein cows by enzymatic hydrolysis. BMECs were treated with menthol (10, 50, 100, 200 μM) for 1h, followed by lipopolysaccharide (5μg/ml) for 12 h.. Lipopolysaccharide treatment upregulated the protein levels of cyclooxygenase-2 (COX-2) and inducible nitric
oxide synthase (INOS) and the mRNA abundance of tumor necrosis factor α (TNF-α), interleu-kin-6 (IL-6), and interleukin-1β (IL-1β), while menthol was able to inhibit this effect. The inhibi-tory effect of menthol on proinflammatory factors was significantly reduced when autophagy was blocked using 3-Methyladenine (5μg/ml), an inhibitor of autophagy. Furthermore, lipopolysac-charide treatment reduced the expression levels of milk lipids and milk proteins, which were in-hibited by menthol. In addition, menthol (200 μM) treatment was able to significantly upregulate the expression level of autophagy-related protein LC3B, downregulate the expression level of P62, promote the expression abundance of autophagy-related gene mRNA, and enhance signifi-cantly enhance autophagic flow. Interestingly, treatment of BMECs with menthol (200 μM) pro-moted the phosphorylation of AMP-activated protein kinase (AMPK) and unc-51 like kinase 1 (ULK1) and increased the nuclear localization of nuclear factor-E2 associated factor 2 (Nrf-2). When the AMPK pathway was blocked using compound C (10μg/ml), an inhibitor of AMPK, au-tophagy was significantly inhibited. Autophagy levels were significantly decreased after block-ing the Nrf-2 pathway using ML385 (5μg/ml), an inhibitor of Nrf-2. Overall, the data suggest that menthol activates the AMPK-ULK1 pathway to initiate the onset of autophagy and maintains the level of autophagy through the AMPK-Nrf-2 pathway. In conclusion, the findings suggest that menthol may alleviate the inflammatory response in BMECs via the AMPN/ULK1/Nrf-2/autophagy pathway.