Establishment of inflammatory model of bovine mammary epithelial cells induced by lipoteichoic acid

Abstract

The mammary gland of the cow is particularly susceptible to infections of a wide range of pathogenic bacteria, including both Gram-positive and Gram-negative bacteria. The endotoxins of these pathogenic bacteria include peptidoglycan (PGN), lipoteichoic acid (LTA) and lipopolysaccharide (LPS), and they are the pathogen-associated molecular patterns (PAMPs) to induce mastitis. Cow mastitis is a detrimental factor in dairy farming industry. Lipoteichoic acid (LTA) is the main component of Staphylococcus aureus cell wall and the key cytotoxic factor causing inflammation. The aims of our work was to establish inflammatory model of study procedures were approved by the Animal Care and Use Committee of the Sumy National Agricultural University, Sumy, Ukraine, and the Henan Institute of Science and Technology, Xinxiang, China, and performed in accordance with the animal welfare and ethics guidelines.
 The BMECs harvested from mid-lactation dairy cow milk were isolated by our laboratory. Briefly, the base medium for this cell is DMEM/F-12 (Gibco, USA, cat.12400-024). The complete growth medium included 10% fetal bovine serum (Biological Industries, Israel, cat.04-011-1A/B), DMEM/F-12, and 10 ng/mL epidermal growth factor (Sigma, USA, cat. E4127). Cells were maintained at 37℃in an incubator containing 5% CO2. When cells grew to 80% confluency, the cells were rinsed twice with PBS, and then the primary mammary epithelial cells were trypsinized with 0.25% trypsin plus 0.02% EDTA and passaged. In this study, one inflammatory bovine mammary epithelial cell (BMEC) model was established by infecting the cells with LTA. The BMEC viability induced by LTA were evaluated. The expressions of pro-inflammatory cytokines (TNF-α and IL-6) were measured by ELISA and RT- qPCR. The results showed that the treatment of BMECs with LTA at 20 ng/μL for 24 h obviously improved TNF-α and IL-6 protein and gene expression levels. The establishment of the model will play an important role in the screening of anti-inflammatory drugs and the study of the mechanism of action in the future.