Metabolomic Profiles of Bovine Mammary Epithelial Cells Stimulated by Lipopolysaccharide

Yixin Huang,Jing Jiang,Qipin Xu,Zhengzhong Luo,Suizhong Cao,Qiao Luo,Xueping Yao,Yanchun Hu,Zhihua Ren,Shumin Yu,Liuhong Shen,Yongxin Yang

doi:10.1038/s41598-019-55556-2

Abstract

Bovine mammary epithelial cells (bMECs) are the main cells of the dairy cow mammary gland. In addition to their role in milk production, they are effector cells of mammary immunity. However, there is little information about changes in metabolites of bMECs when stimulated by lipopolysaccharide (LPS). This study describes a metabolomics analysis of the LPS-stimulated bMECs to provide a basis for the identification of potential diagnostic screening biomarkers and possible treatments for bovine mammary gland inflammation. In the present study, bMECs were challenged with 500 ng/mL LPS and samples were taken at 0 h, 12 h and 24 h post stimulation. Metabolic changes were investigated using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF MS) with univariate and multivariate statistical analyses. Clustering and metabolic pathway changes were established by MetaboAnalyst. Sixty-three differential metabolites were identified, including glycerophosphocholine, glycerol-3-phosphate, L-carnitine, L-aspartate, glutathione, prostaglandin G2, α-linolenic acid and linoleic acid. They were mainly involved in eight pathways, including D-glutamine and D-glutamic acid metabolism; linoleic acid metabolism; α-linolenic metabolism; and phospholipid metabolism. The results suggest that bMECs are able to regulate pro-inflammatory, anti-inflammatory, antioxidation and energy-producing related metabolites through lipid, antioxidation and energy metabolism in response to inflammatory stimuli.

Highlights

Bovine mastitis is common and is economically one of the most important diseases that seriously affects the health and welfare of dairy cows
Expressed proteins that were associated with inflammatory model of Bovine mammary epithelial cells (bMECs) induced by LPS were screened by isobaric tag for relative and absolute quantification, combined with 2-dimensional liquid chromatography- tandem mass spectrometry (2D-LC-MS/MS), indicating that LPS treatment could affect the synthesis of protein and fat in bMECs6
We found that the mRNA expression of chemokine (c-c motif) ligand (CCL)-2, IL-6 and tumor necrosis factor-α (TNF-α) in LPS12h increased significantly (p < 0.01) compared to Control

Summary

Introduction

Bovine mastitis is common and is economically one of the most important diseases that seriously affects the health and welfare of dairy cows It is an inflammatory condition of the mammary gland, reducing milk quality and production, impairing fertility, causing mortality[1,2], and is a food-borne disease that increases human public health risks[3]. Studies have shown that simulation of bMECs with LPS can generate strong immune responses, upregulating the expression of pathogen-associated molecular patterns (PAMPs), activating nuclear factor-κB (NF-κB) signaling pathway, and increasing the secretion of inflammatory cytokines[3,5]. In a study of bMECs4, the mRNA expression levels of Toll-like receptor 4 (TLR4) and TLR2 were significantly increased by stimulation with 0.01 μg/mL, 1 μg/mL, 5 μg/mL and 10 μg/mL LPS for 24 h when compared to controls (without LPS). We used HPLC-Q-TOF MS technique, with the aim of identifying differentially produced metabolites and the relevant pathways of the inflammatory response in bMECs after LPS stimulation

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