Sodium Phenylbutyrate Ameliorates Inflammatory Response Induced by Staphylococcus aureus Lipoteichoic Acid via Suppressing TLR2/NF-κB/NLRP3 Pathways in MAC-T Cells

Abstract

This study aimed to investigate the anti-inflammatory properties of sodium phenylbutyrate (SPB) against Staphylococcus aureus (S. aureus) lipoteichoic acid (LTA)-stimulated bovine mammary alveolar (MAC-T) cells. Quantitative PCR was performed to examine the effect of SPB on inflammatory cytokines and host defense peptide (HDP) gene expression. Western blot wanalysis was used to detect the effect of SPB on the TLR2/NF-κB/NLRP3 signaling pathway. The results showed that SPB significantly suppressed the expression of TNF-α, IL-1β, IL-6; meanwhile, the markedly decreased expression of LTA-stimulated TLR2, NLRP3, ASC, caspase-1, and IL-1β, and the inhibited IkBα and p65 phosphorylation were also observed. However, increased TAP and Bac5 expression in LTA-stimulated MAC-T cells was further detected. In summary, these results suggest that SPB ameliorates the inflammatory response induced by S. aureus LTA via suppressing the TLR2/NF-κB/NLRP3 signaling pathway, which indicates that SPB may be a potential agent for the treatment of bovine mastitis.

Keywords: NF-κB; NLRP3; TLR2; inflammation; lipoteichoic acid; sodium phenylbutyrate.

Figure 1

The effect of SPB on the cell viability of MAC-T cells. MAC-T cells were cultured in the presence of SPB (0.5, 1, 2, and 4 mM) and LTA (1 mg/mL) for 24 h, and then viability was determined by MTT assay. Each data point shows the mean ± standard error (SE) of triplicates from three independent experiments.

Figure 2

The effect of SPB on LTA-induced TNF-α, IL-1β, IL-6, TAP, and Bac5 production. Gene expression was normalized to the expression of the reference gene 18S rRNA. Each column shows the means ± SE of triplicates of three independent experiments. ^# p < 0.05 vs. control group; ** p < 0.01 vs. LTA group.

Figure 3

The effect of SPB on TLR2 expression. The expression of TLR2 was investigated in MAC-T cells using Western blot analysis. β-actin was used as a reference control. (A) The expression levels of toll-like receptor 2 (TLR2); and (B) The quantification histogram of TLR2 protein expression normalized by β-actin. The values presented are the mean ± SE of three independent experiments. # p < 0.05 vs. control group; ** p < 0.01 vs. LTA group.

Figure 4

The effect of SPB on NF-Κb activation. The NF-κB pathway was explored in MAC-T cells using Western blot analysis. β-actin was used as a reference control. (A) The expression levels of p-IκBα, IκBα, p-p65 and p65; (B) The quantification histogram of p-IκBα protein expression normalized by β-actin; and (C) The quantification histogram of p-p65 protein expression normalized by β-actin. The values presented are the means ± SE of three independent experiments. # p < 0.05 vs. control group; ** p < 0.01 vs. LTA group.

Figure 5

The effect of SPB on NLRP3 inflammasome. The NLRP3 inflammasome pathway was explored in MAC-T cells using Western blot analysis. β-actin was used as a reference control. (A) The expression levels of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC), caspase-1, and interleukin 1 beta (IL-1β) proteins; (B) The quantification histogram of NLRP3 protein expression normalized by β-actin; (C) The quantification histogram of ASC protein expression normalized by β-actin; (D) The quantification histogram of caspase-1 protein expression normalized by β-actin; and (E) The quantification histogram of IL-1β protein expression normalized by β-actin. The values presented are the means ± SE of three independent experiments. # p < 0.05 vs. control group; ** p < 0.01 vs. LTA group.

Figure 6

Schematic representation of protective effects of SPB against Staphylococcus aureus LTA-induced cell inflammatory responses in MAC-T cells.