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

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 ac...

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Autores principales: Liu, Songqi, Guo, Wenjin, Jia, Yuxi, Ye, Bojian, Liu, Shu, Fu, Shoupeng, Liu, Juxiong, Hu, Guiqiu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8727745/
https://www.ncbi.nlm.nih.gov/pubmed/35003099
http://dx.doi.org/10.3389/fimmu.2021.782989
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author Liu, Songqi
Guo, Wenjin
Jia, Yuxi
Ye, Bojian
Liu, Shu
Fu, Shoupeng
Liu, Juxiong
Hu, Guiqiu
author_facet Liu, Songqi
Guo, Wenjin
Jia, Yuxi
Ye, Bojian
Liu, Shu
Fu, Shoupeng
Liu, Juxiong
Hu, Guiqiu
author_sort Liu, Songqi
collection PubMed
description 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 inflammatory response in primary bovine mammary gland epithelial cells (BMECs) and its anti-inflammatory mechanism. First, BMECs were isolated and amplified from the udders of Holstein 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), interleukin-6 (IL-6), and interleukin-1β (IL-1β), while menthol was able to inhibit this effect. The inhibitory effect of menthol on proinflammatory factors was significantly reduced when autophagy was blocked using 3-Methyladenine (5μg/ml), an inhibitor of autophagy. Furthermore, lipopolysaccharide treatment reduced the expression levels of milk lipids and milk proteins, which were inhibited 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 significantly enhance autophagic flux. Interestingly, treatment of BMECs with menthol (200 μM) promoted 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, autophagy was significantly inhibited. Autophagy levels were significantly decreased after blocking 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 AMPK/ULK1/Nrf-2/autophagy pathway.
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spelling pubmed-87277452022-01-06 Menthol Targeting AMPK Alleviates the Inflammatory Response of Bovine Mammary Epithelial Cells and Restores the Synthesis of Milk Fat and Milk Protein Liu, Songqi Guo, Wenjin Jia, Yuxi Ye, Bojian Liu, Shu Fu, Shoupeng Liu, Juxiong Hu, Guiqiu Front Immunol Immunology 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 inflammatory response in primary bovine mammary gland epithelial cells (BMECs) and its anti-inflammatory mechanism. First, BMECs were isolated and amplified from the udders of Holstein 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), interleukin-6 (IL-6), and interleukin-1β (IL-1β), while menthol was able to inhibit this effect. The inhibitory effect of menthol on proinflammatory factors was significantly reduced when autophagy was blocked using 3-Methyladenine (5μg/ml), an inhibitor of autophagy. Furthermore, lipopolysaccharide treatment reduced the expression levels of milk lipids and milk proteins, which were inhibited 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 significantly enhance autophagic flux. Interestingly, treatment of BMECs with menthol (200 μM) promoted 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, autophagy was significantly inhibited. Autophagy levels were significantly decreased after blocking 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 AMPK/ULK1/Nrf-2/autophagy pathway. Frontiers Media S.A. 2021-12-22 /pmc/articles/PMC8727745/ /pubmed/35003099 http://dx.doi.org/10.3389/fimmu.2021.782989 Text en Copyright © 2021 Liu, Guo, Jia, Ye, Liu, Fu, Liu and Hu https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Liu, Songqi
Guo, Wenjin
Jia, Yuxi
Ye, Bojian
Liu, Shu
Fu, Shoupeng
Liu, Juxiong
Hu, Guiqiu
Menthol Targeting AMPK Alleviates the Inflammatory Response of Bovine Mammary Epithelial Cells and Restores the Synthesis of Milk Fat and Milk Protein
title Menthol Targeting AMPK Alleviates the Inflammatory Response of Bovine Mammary Epithelial Cells and Restores the Synthesis of Milk Fat and Milk Protein
title_full Menthol Targeting AMPK Alleviates the Inflammatory Response of Bovine Mammary Epithelial Cells and Restores the Synthesis of Milk Fat and Milk Protein
title_fullStr Menthol Targeting AMPK Alleviates the Inflammatory Response of Bovine Mammary Epithelial Cells and Restores the Synthesis of Milk Fat and Milk Protein
title_full_unstemmed Menthol Targeting AMPK Alleviates the Inflammatory Response of Bovine Mammary Epithelial Cells and Restores the Synthesis of Milk Fat and Milk Protein
title_short Menthol Targeting AMPK Alleviates the Inflammatory Response of Bovine Mammary Epithelial Cells and Restores the Synthesis of Milk Fat and Milk Protein
title_sort menthol targeting ampk alleviates the inflammatory response of bovine mammary epithelial cells and restores the synthesis of milk fat and milk protein
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8727745/
https://www.ncbi.nlm.nih.gov/pubmed/35003099
http://dx.doi.org/10.3389/fimmu.2021.782989
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