Anti-Inflammatory and Antimicrobial Effects of Estradiol in Bovine Mammary Epithelial Cells during Staphylococcus aureus Internalization

Figure 5

Role of MAPKs p38, JNK, and ERK1/2 in E2-treated bMECs during S. aureus internalization. (a) Participation of MAPKs in S. aureus internalization determined by CFU units. bMECs were incubated (30 min, 1 or 2 h) with pharmacological inhibitors of p38 (5 μM, SB203580), JNK (20 μM, SP600125), or ERK1/2 (2.5 μM, U0126) prior to infection with S. aureus (2 h). Values were determined considering the control (bMECs cultured with the vehicle 1% ethanol) as 100% internalization. (b) The same procedure was used to determine S. aureus internalization by flow cytometry using the LIVE/DEAD BacLight Bacterial viability kit. Fluorescence intensity was estimated from 10,000 events. (c) Phosphorylation was measured in bMECs that were treated with 50 pg/mL E2 and/or challenged with S. aureus by flow cytometry. The phosphorylated MAPK concentrations (U/mL) are represented: (a) pp38, (b) pJNK, and (c) pERK1/2. SB203580: p38 inhibitor. SP600125: JNK1/2 inhibitor. U0126: ERK1/2 inhibitor. According to manufacturer’s protocol 300 events were obtained. In (a) and (b) different letters indicate significant changes among treatments (), and in (c) different letters above the bars indicate significant changes among the four treatments within the same MAPK evaluated (). Vehicle: 1% ethanol.

(a)

(b)

(c)