UDK 619:618.–002:636.2 ESTABLISHMENT OF INFLAMMATORY MODEL OF BOVINE MAMMARY EPITHELIAL CELLS INDUCED BY LIPOTEICHOIC ACID

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.

IL-8, tumor necrosis factor (TNF)-α and other cytokines. This might disturb the proliferation and milk synthesis of bovine mammary epithelial cells (BMECs), resulting in a decrease in milk yield and quality. The amounts and secretory activity of BMECs are related to milk yield (Sun, X.D.al., 2019), and the lipid content of milk, is an important indicator of milk quality (Shen, J. al., 2018). Triglyceride (TG) accounted for more than 98% of milk lipid, (Sheng, R. al., 2015). Fatty acids can be rapidly taken up and converted into lipid droplets by the lactating mammary gland (Viguier, C. al., 2009). Non-esterified fatty acid (NEFA) is a source of fatty acids and can increase milk fat synthesis (Szyda, J. al., 2019). A variety of genes, such as fatty acid synthase (FASN), acetyl coenzyme-A carboxylase 1 (ACACA), and stearoyl-CoA desaturase (SCD), etc., are involved in milk fat synthesis (Nagasawa, Y al., 2018). FASN catalyzes the synthesis of long-chain fatty acids (He, X.J al., 2019), ACACA is the ratelimiting enzyme catalyzing the first reaction step of fatty acid synthesis, and SCD is responsible for catalyzing the synthesis of mono-saturated fatty acids (Qi, L. al., 2014).
With the development of society and the expansion of dairy farming industry, bovine mastitis continues to be one of the most common diseases worldwide and has been seriously impacting on milk yield, milk composition and animal welfare (Seegers H et al., 2003). Mastitis is the persistent inflammatory response of mammary tissue attributed to intra-mammary invasion of pathogens (Rinaldi Met al., 2010). Bovine mammary gland inflammation is mainly caused by changes in metabolism, physiological trauma, and contagious or environmental pathogenic microorganisms (Oviedo-Boyso et al., 2007;Lahouassa H et al., 2007). So for mastitis, it is important to understand the mechanisms controlling the immune response at the molecular level (Wellnitz O and Bruckmaier RM,2012).
Literature Review. The BMECs was first infected when the pathogenic bacteria infect the mammary gland through the milk duct to reach the acinar of the mammary gland，and then inflammatory reactions occur (Park HJ et al., 2016). Bovine mammary epithelial cells (bMECs) are capable of initiating an innate immune response (IIR) to invading bacteria (Zhao and Lacasse, 2008).
Gram-positive bacteria as Staphylococcus aureus (S. aureus) is one of the major contagious pathogens which account for clinical and subclinical bovine mastitis through rapid multiplication and persistent adhesion in mammary gland tissue, and it a chronic and recurrent disease that affectdairy cattle worldwide (Bradley AJ.,2002).S. aureus by the mammary gland is not as well as known therecognition of Escherichia coli, another major pathogen for themammary gland. The counterpart of E. coli outer membrane lipopolysaccharide (LPS), asa proinflammatory bacterial agonist of the mammary glandinnate immune system, has not yet been established for S.aureus.Lipoteichoic acid (LTA) is the main component of S. aureus cell wall and the key cytotoxic factor causing inflammation (Schroder NW et al., 2003;Bougarn S et al., 2010).LTA has beenshown to be an important pattern for immunerecognition of S. aureus (Van Amersfoort E.S. et al., 2003).One of the advantages of LTA as a tool tomodel inflammation, it is a definedbacterial PAMP which targets identifiedpattern recognition receptors (PRR) andincreasingly defined accessory molecules forrecognition and for the signaling cascade. LTA signals through toll like receptor 2(TLR2) in the bovine mammary gland by bMEpC (Schröder N.W.et al., 2003; Henneke P.et al., 2005). In this study, we used a purified commercial preparation of S. aureus LTA to determine whether the bovine mammary gland responds to LTA, to determine the dose-response effects, and to begin to characterize the induced inflammatory response. 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.
Aims. The aims of our work was to establish inflammatory model of bovine mammary epithelial cells induced by lipoteichoic acid Materials and Methods. Bioethics statement The 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. Cultivation and treatment of BMECs 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.
Extraction and purification of total RNA Total RNA was extracted from adherent BMECs using RNAiso Plus (TaKaRa, Dalian, P. R. China, cat. 9109) in accordance with the manufacturer's instructions. The assessment of the quantity and quality of RNA was verified using a NanoDrop 1000 (Thermo Scientific, Co., Ltd., P. R. China). The 260:280 nm optical density value was between 1.8 and 2.0. Then, the first-strand cDNA was synthesized using PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, P. R. China, cat. RR047A).
Cell Enzyme-linked immunosorbent assay (ELISA) BMECs were cultured for 12h, 24 h, and 48h in fresh serum-free medium after treatment with LTA. The medium was collected and centrifuged at 12,000 rpm for 5 min to remove cell debris. The levels of tumor necrosis factor α (TNF-α), and Interleukin-6 (IL-6) in the supernatants of BMECs were detected according to the ELISA kit instructions (Jiangsu Mei Biao Biological Technology Co., Ltd., Jiangsu, P. R. China, cat. MB-4838A\MB-4837A).
RT-qPCR analysis Real-time PCR primers for amplification of mRNA were designed using Primer Premier 5.0 and synthesized by Sangon Biotech (Shanghai, P. R. China, Co., Ltd.). The primers used are in Table 1. Real-time quantitative PCR was performed using TB Green Premix Ex TaqTM II (TaKaRa, Dalian, P. R. China, cat#RR820B) on a 7500 Real-Time PCR system (Applied Biosystems Inc., Foster City, CA). GAPDH was used as a reference gene. The relative gene expression was calculated using the 2-△△Ct method. The results are expressed as means ± SD. Statistical differences were analyzed using a t-test for independent groups. The ANOVA was performed using GraphPad Prism version 6.01 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was declared as *P< 0.05, **P< 0.01, and ***P< 0.001. Each experiment was repeated at least 3 times.
Results CCK-8 and RTCA assay for cell viability CCK-8 assay carried out to examine the viability of cells. The viability of BMECs infected with LTA were lower than that of the normal BMECs. RTAC was used to detect the effect of different concentrations of LTA on the proliferation of BMECs, the results were shown in Figure 2. With the increase of LTA concentration, the proliferation activity of BMEC cells was inhibited. According to the change of cell index value and different proliferation curves, the dynamic detection after LTA treatment of cells was found.

Fig. 2. Effect of LTA on the BMEC proliferation
Secretion of inflammatory cytokines by BMEC It shown that the basal expression of TNF-α and IL-6 protein in the culture supernatant of BMECs in the blank control group was low (Figure 3). BMECs were stimulated by different mass concentrations of LTA for different time, the expression of TNF-α and IL-6 protein Significantly increased. When different mass concentrations of LTA acted on cells for 12h, 24h, and 48h, the expression of TNF-α and IL-6 protein reached its peak when the mass concentration of LTA was 20 ng/μL. At the LTA mass concentration was 40 and 80 ng/μL, the expression of TNF-α and

Fig. 3. Effect of LTA on the BMEC TNF-α and IL6 production. (A) (B)
Expression of inammatory cytokines by BMEC It shown that the TNF-α and IL6 mRNA expression in the culture supernatant of BMECs in the blank control group was low ( Figure 4). As the BMECs were stimulated by different mass concentrations of LTA for different time, the mRNA expression of TNF-α and IL-6 increased significantly. The LTA acted on the cells for 12h and 48h, the mRNA expression of IL-6 did not change significantly with the increase in mass concentration. However, the mRNA expression of TNF-α changes significantly. The mRNA expression of TNF-α and IL6 reached the maximum,when the LTA concentration was 20 ng/μL with different times later. It can be seen that the LTA of 20 ng/μLtreating BMECs for 24 h can induce a significant cellular immune response in BMECs.

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Discussion and Conclusion
Inflammatory response refers to the multi-cytokine involved in the occurrence and development of inflammation by regulating the balance between pro-inflammatory factors and anti-inflammatory systems (Wu T, et al., 2016;Dinarello, C. A. 2009). TNF-α is the earliest and most important inflammatory mediator in the process of inflammation. IL6 can induce B cells to differentiate and produce antibodies, and is a promoter of inflammatory response. LTA is an important component of the cell wall of Staphylococcus aureus and can activate inflammatory cells to cause inflammation. In the process of inflammation, LTA activates macrophages through the TLR2 receptor, which leads to the production of inflammatory cytokines TNF-α and IL6. The secretion of cytokines can induce further activation of inflammatory cells, leading to excessive or uncontrolled inflammatory response, and ultimately causing inflammatory case damage to host tissues and organs (Giovannin A E J, et al., 2017). Due to the immune characteristics of BMECs, specific inflammatory substances can be selected to induce BMECs to produce an immune response. The most used in the experiment is LPS and bacterial culture filtrate. However, there are few reports on the establishment of inflammation models by LTA on BMECs.In this study, LTA was used as a proinflammatory mediator. The two classic acute early cytokines, IL-6 and TNF-α, were selected as the criteria for measuring the success of the model. CCK-8, RTAC, ELISA and qRT-PCR were used to test the method, the results showed that treatment of bovine mammary epithelial cells with 20 ng/μL LTA for 24 h can significantly increase the protein and gene expression levels of TNF-α and IL-6. The establishment of this model could play an important role in screening anti-inflammatory drugs and studying the mechanism of action in the future.
Author's contributions All authors participated in this article design. Ping Xu participated and performed writing and data collection. All authors read and approved the final manuscript. All authors contributed to the draft of the manuscript. All authors gave final approval for publication.