Impact of Escherichia coli and Lipopolysaccharide on the MAPK Signaling Pathway, MMPs, TIMPs, and the uPA System in Bovine Mammary Epithelial Cells

Bovine mastitis is a condition typically induced by various pathogens, with Escherichia coli (<i>E. coli</i>) being a common causative agent known for its propensity to cause persistent infections. In experimental models of bovine mastitis, lipopolysaccharide (LPS), a key component of the <i>E. coli</i> cell wall, is frequently employed as an inducer. The extracellular matrix (ECM) is regulated by MMPs, TIMPs, and the uPA system. They collectively participate in ECM degradation and remodeling and have been identified as promising targets for mastitis treatment. However, investigations into the precise mechanisms underlying <i>E. coli</i> and LPS-induced mastitis, as well as the relationship between bovine mastitis and the MAPK signaling pathway, remain limited. In this study, bovine mammary epithelial cells (BMECs) were treated in vitro with 10⁶ CFU/mL heat-inactivated <i>E. coli</i>, 7.5 µg/mL LPS, or a combination of both. The treatments resulted in varying degrees of activation of the MAPK signaling pathway, specifically ERK1/2, JNK, and P38. BMECs were exposed to MAPK inhibitors (the JNK inhibitor SP600125, the ERK inhibitor PD98059, and the P38 inhibitor SB203580) after treatments with heat-inactivated <i>E. coli</i> (10⁶ CFU/mL), LPS (7.5 µg/mL), or a combination of the two for 6, 12, 24, and 48 h. The mRNA and protein levels of <i>MMP-1</i>, <i>MMP-2</i>, <i>MMP-3</i>, <i>MMP-9</i>, <i>MMP-13</i>, <i>TIMP-1</i>, <i>TIMP-2</i>, <i>uPA</i>, <i>uPAR</i>, and <i>PAI-1</i> were assessed using RT-qPCR and Western blot analysis. The findings indicated that heat-inactivated <i>E. coli</i> and LPS stimulated the expression of MAPK mRNAs <i>(ERK1/2</i>, <i>P38</i>, <i>and JNK</i>) in BMECs, along with corresponding increases in the phosphorylated proteins. Furthermore, MAPK inhibitors substantially upregulated the expression of TIMP-1, TIMP-2, and PAI-1. However, no significant changes were observed in the mRNA and protein levels of <i>MMP-1</i>, <i>MMP-2</i>, <i>MMP-3</i>, <i>MMP-9</i>, <i>MMP-13</i>, <i>uPA</i>, or <i>uPAR</i>. In conclusion, heat-inactivated <i>E. coli</i> and LPS can activate the MAPK signaling pathway in BMECs. Inhibiting this signaling pathway can modulate the expression of <i>TIMP-1</i>, <i>TIMP -2</i>, and <i>PAI-1</i> at both mRNA and protein levels.