Caffeic Acid Prevented LPS-Induced Injury of Primary Bovine Mammary Epithelial Cells through Inhibiting NF-κB and MAPK Activation

Figure 2

CA decreased cell apoptosis through inhibiting the mitochondria apoptosis pathway caused by LPS. The changes in apoptosis, mitochondrial membrane potential, and apoptosis-related protein levels of bMEC after treatment with LPS or CA. Cells were pretreated with indicated concentrations (10, 25, and 50 μg/mL) of CA or serum-free media for 3 h before stimulation with LPS (50 μg/mL) for 12 h. (a) The cell apoptosis was measured by flow cytometry following annexin V/propidium iodide (PI) staining. Cells that were positive for annexin V, but not PI, which are present in the lower right quadrant, were early apoptotic cells. Cells that were both annexin V and PI positive, which are present in the upper right quadrant, were late apoptotic cells. (b) Bivariate JC-1 analysis of mitochondrial membrane potential (ΔΨm) in cells by flow cytometry. Fluorescence of cells stained with JC-1 for the indicating treatments. In nondamaged cells, JC-1 forms red-emitting aggregates in the mitochondrial matrix, which are present in the upper right quadrant (high ΔΨm). A loss of red fluorescence and an increase in cytoplasmic green-emitting monomers signal the disruption of ΔΨm, which are present in the lower right quadrant (low ΔΨm). (c) The rate of red to green fluorescence intensity in different groups. The values represent the magnitude of ΔΨm; the data represent the means of 3 independent experiments. vs. the control group, vs. the group treated with LPS only, and vs. the group treated with LPS only. (d) The total cellular proteins were collected; then, the protein levels of Bcl-2, Bax, and casepase-3 were detected by western blotting. Upper panel: western blot was performed to related protein levels; one out of three independent experiments is shown. Lower panel: quantification of related proteins normalized to actin levels. Data are shown as the (). vs. the control group; vs. the group treated with LPS only.

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