Punicalagin protects bovine endometrial epithelial cells against lipopolysaccharide-induced inflammatory injury

Abstract

Objective: Bovine endometritis is one of the most common reproductive disorders in cattle. The aim of this study was to investigate the anti-inflammation potential of punicalagin in lipopolysaccharide (LPS)-induced bovine endometrial epithelial cells (bEECs) and to uncover the underlying mechanisms.

Methods: bEECs were stimulated with different concentrations (1, 10, 30, 50, and 100 μg/ml) of LPS for 3, 6, 9, 12, and 18 h. MTT assay was used to assess cell viability and to identify the conditions for inflammatory injury and effective concentrations of punicalagin. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess gene expression of pro-inflammatory cytokines. Western blotting was used to assess levels of inflammation-related proteins.

Results: Treatment of bEECs with 30 µg/ml LPS for 12 h induced cell injury and reduced cell viability. Punicalagin (5, 10, or 20 µg/ml) pretreatment significantly decreased LPS-induced productions of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α) in bEECs. Molecular research showed that punicalagin inhibited the activation of the upstream mediator nuclear factor-κB (NF-κB) by suppressing the production of inhibitor κBα (IκBα) and phosphorylation of p65. Results also indicated that punicalagin can suppress the phosphorylation of mitogen-activated protein kinases (MAPKs) including p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK).

Conclusions: Punicalagin may attenuate LPS-induced inflammatory injury and provide a potential option for the treatment of dairy cows with Escherichia coli endometritis.

Keywords: Bovine endometrial epithelial cell; Cytokine; Inflammatory injury; Punicalagin.

Fig. 1

Punicalagin cytotoxicity in bEECs Cells were treated with different concentrations (5, 10, 20, 25, 30, and 50 µg/ml) of punicalagin for 24 h. Cell viability was evaluated by MTT assay. Data represent the mean±SEM of three independent experiments. ^* P<0.05 vs. the control group

Fig. 2

Cell viability of bEECs induced by LPS Cells were incubated with different concentrations (1, 10, 30, 50, and 100 µg/ml) of LPS for 3, 6, 9, 12, and 18 h. Cell viability was significantly reduced by 30 µg/ml of LPS at 12 h (^* P<0.05). Data represent the mean±SEM of three independent experiments. ^* P<0.05, ^** P<0.01 vs. the control (Con) group

Fig. 3

Morphology of bEECs induced by LPS Cells were incubated with or without 30 µg/ml LPS for 12 h and cell morphology was observed using an inverted microscope. (a–c) Control group: the distribution of bEECs was compact and regular. (d–f) Model group: bEECs exposed to 30 µg/ml LPS for 12 h became enlarged, lost their cuboidal shape, and showed disrupted cell-cell contacts (arrowheads). (a, d) 4× microscope; (b, e) 10× microscope; (c, f) 20× microscope

Fig. 4

Effect of punicalagin on LPS-induced pro-inflammatory cytokine mRNA expression Cells were pretreated with punicalagin (5, 10, and 20 µg/ml) for 2 h and exposed to 30 µg/ml LPS for 3, 6, 9, and 12 h. The levels of IL-1β (a), IL-6 (b), IL-8 (c), and TNF-α (d) mRNAs were quantified using RT-PCR analysis. Data represent the mean±SEM of three independent experiments. ^## P<0.01, ^### P<0.001 vs. the control (Con) group. ^* P<0.05, ^** P<0.01, ^*** P<0.001 vs. the model (Mod) group. Con: control; Mod: treated with LPS (30 µg/ml) only; Low: punicalagin (5 µg/ml)+LPS (30 µg/ml); Mid: punicalagin (10 µg/ml)+LPS (30 µg/ml); High: punicalagin (20 µg/ml)+LPS (30 µg/ml)

Fig. 5

Effect of punicalagin on LPS-induced NF-κB activation Cells were pretreated with punicalagin (5, 10, and 20 µg/ml) for 2 h, exposed to 30 µg/ml LPS for 15, 45, and 90 min, and analyzed by Western blotting. IκBα (a) and phosphorylated p65 (b) were analyzed using anti-IκBα and phosphor-specific anti-p65 antibodies. Data represent the mean±SEM of three independent experiments. ^# P<0.05, ^### P<0.001 vs. the control (Con) group. ^* P<0.05, ^** P<0.01, ^*** P<0.001 vs. the model (Mod) group. Con: control; Mod: treated with LPS (30 µg/ml) only; Low: punicalagin (5 µg/ml)+LPS (30 µg/ml); Mid: punicalagin (10 µg/ml)+LPS (30 µg/ml); High: punicalagin (20 µg/ml)+LPS (30 µg/ml)

Fig. 6

Effect of punicalagin on LPS-induced MAPK activation Cells were pretreated with punicalagin (5, 10, and 20 µg/ml) for 2 h, exposed to 30 µg/ml LPS for 15, 45, and 90 min, and analyzed by Western blotting. Phosphorylation levels of p38 (a), JNK (b), and ERK (c) were analyzed using phospho-specific anti-p38, phospho-specific anti-JNK, and phospho-specific anti-ERK antibodies. Data represent the mean±SEM of three independent experiments. ^### P<0.001 vs. the control (Con) group. ^* P<0.05, ^*** P<0.001 vs. the model (Mod) group. Con: control; Mod: treated with LPS (30 µg/ml) only; Low: punicalagin (5 µg/ml)+LPS (30 µg/ml); Mid: punicalagin (10 µg/ml)+LPS (30 µg/ml); High: punicalagin (20 µg/ml)+LPS (30 µg/ml)