Effects of Chinese Propolis in Protecting Bovine Mammary Epithelial Cells against Mastitis Pathogens-Induced Cell Damage

Abstract

Chinese propolis (CP), an important hive product, can alleviate inflammatory responses. However, little is known regarding the potential of propolis treatment for mastitis control. To investigate the anti-inflammatory effects of CP on bovine mammary epithelial cells (MAC-T), we used a range of pathogens to induce cellular inflammatory damage. Cell viability was determined and expressions of inflammatory/antioxidant genes were measured. Using a cell-based reporter assay system, we evaluated CP and its primary constituents on the NF-κB and Nrf2-ARE transcription activation. MAC-T cells treated with bacterial endotoxin (lipopolysaccharide, LPS), heat-inactivated Escherichia coli, and Staphylococcus aureus exhibited significant decreases in cell viability while TNF-α and lipoteichoic acid (LTA) did not. Pretreatment with CP prevented losses in cell viability associated with the addition of killed bacteria or bacterial endotoxins. There were also corresponding decreases in expressions of proinflammatory IL-6 and TNF-α mRNA. Compared with the mastitis challenged cells, enhanced expressions of antioxidant genes HO-1, Txnrd-1, and GCLM were observed in CP-treated cells. CP and its polyphenolic active components (primarily caffeic acid phenethyl ester and quercetin) had strong inhibitive effects against NF-κB activation and increased the transcriptional activity of Nrf2-ARE. These findings suggest that propolis may be valuable in the control of bovine mastitis.

Figure 1

Effects of propolis on mastitis pathogen-induced cell viability decreases and cell apoptosis in MAC-T cells. (a) MAC-T cells were treated with propolis and/or various mastitis pathogens, including proinflammatory cytokine (TNF-α 25 ng/mL), bacterial cell wall components (LPS, 1 μg/mL; LTA, 10 μg/mL), and heat-killed mastitis strains (Escherichia coli and Staphylococcus aureus, 107 particles/mL) with indicated concentrations of propolis for 24 h. The CCK-8 assay was performed to determine cell viability. ^## P < 0.01 and ^### P < 0.001 significantly different from untreated cells. ^∗ P < 0.05 and ^∗∗ P < 0.01 significantly different from mastitis pathogens-treated cells. (b) After being pretreated with or without Chinese propolis (15 μg/mL) for 1 h, MAC-T cells were challenged with various mastitis pathogens for 24 h. Cell apoptosis was analyzed by flow cytometry analysis using annexin V-FITC and PI staining. The data are expressed as the mean ± SD (n = 3). ^∗ P < 0.05 and ^∗∗ P < 0.01.

Figure 2

Effects of CP treatment on proinflammatory responses following mastitis pathogen challenges in MAC-T cells. Quantitative PCR analysis of inflammatory cytokine genes, IL-6 (a), IL-8 (b), and TNF-α (c), showing gene expressions after 6 h incubation of MAC-T cells with each different mastitis pathogen, including proinflammatory cytokine (TNF-α 25 ng/mL), bacterial cell wall components (LPS, 1 μg/mL; LTA, 10 μg/mL), and heat-killed mastitis strains (E. coli and S. aureus, 10⁷ particles/mL). Ct values of target genes were normalized to the value of β-actin and relative gene expressions in TNF-α control group were arbitrarily set to one. The data are shown as mean ± SD from three independent experiments and were analyzed by one-way ANOVA with the Student-Newman-Keuls method. The means with different superscripts are significantly different (P < 0.05).

Figure 3

Effects of CP treatment on cellular antioxidant defense gene expressions following mastitis pathogen challenges in MAC-T cells. Quantitative PCR analysis of cellular antioxidant defense genes, HO-1 (a), Txnrd-1 (b), and GCLM (c), showing gene expressions after 6 h incubation of MAC-T cells with each different mastitis pathogen, including proinflammatory cytokine (TNF-α 25 ng/mL), bacterial cell wall components (LPS, 1 μg/mL; LTA, 10 μg/mL), and heat-killed mastitis strains (E. coli and S. aureus, 10⁷ particles/mL). Ct values of target genes were normalized to the value of β-actin and relative gene expressions in TNF-α control group were arbitrarily set to one. The data are shown as mean ± SD from three independent experiments and were analyzed by one-way ANOVA with the Student-Newman-Keuls method. The means with different superscripts are significantly different (P < 0.05).

Figure 4

Time and dose effects of CP treatment on inflammatory cytokine and cellular antioxidant defense gene expressions in TNF-α stimulated MAC-T cells. MAC-T cells were preincubated with different concentrations of propolis for 1 h and then stimulated with 25 ng/mL TNF-α for various time periods (time effects, right) or preincubated with various concentrations of propolis (dose effect, left) and then stimulated with 25 ng/mL TNF-α for 6 h. IL-6 (a), IL-8 (b), TNF-α (c), HO-1 (d), Txnrd-1 (e), and GCLM (f) mRNA expressions were quantified by real-time PCR. Ct values of target genes were normalized to the value of β-actin. Relative gene expression in TNF-α stimulation for 6 h was arbitrarily set to one. The data are shown as mean ± SD from three independent experiments. ^# P < 0.05 compared to the normal control and ^∗significantly different from the TNF-α treated control (P < 0.05). ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001.

Figure 5

Chinese propolis and its major bioactive compounds suppressed mastitis pathogen-induced NF-κB activation and induces ARE transcriptional activity. (a) Effects of CP on mastitis pathogen-induced NF-κB promoter activation in HEK-293T cells. Cells were pretreated with 20 μg/mL CP and identified isolated active compounds from propolis (caffeic acid, 50 μM; CAPE, 5 μM; chrysin, 50 μM; ferulic acid, 50 μM; galangin, 25 μM; kaempferol, 50 μM; pinocembrin, 50 μM; and quercetin, 50 μM) for 1 h and then stimulated with TNF-α (25 ng/mL) for another 12 h. ^### P < 0.001 compared to the vehicle control; ^∗ P < 0.05 and ^∗∗∗ P < 0.001 compared to TNF-α control. (b) Effects of propolis on mastitis pathogens-induced ARE promoter activation in HEK-293T cells. Cells were treated with CP or identified isolated active compounds. tBHQ (15 μM) treatment for 7 h was used as ARE positive control. ^∗ P < 0.05 and ^∗∗∗ P < 0.001 compared to the vehicle control. The data represent the mean ± SD of four independent experiments.