BACKGROUND

Clostridium butyricum is a probiotic widely used in animal husbandry, and there is evidence to suggest that it can alleviate intestinal inflammation in pigs and may be related to its lipoteichoic acid (LTA), but the mechanism is still unclear.

OBJECTIVE

This study aimed to determine the regulatory effect and potential mechanism of C. butyricum LTA on LPS-stimulated inflammation in intestinal porcine epithelial line-J2 (IPEC-J2).

METHODS

IPEC-J2 cells were treated with LPS and different concentrations of LTA (0.05, 0.1 and 0.15 mM). After treatment of 0.5, 1.5 and 4.5 h, the cell culture media were collected for the measurement of TNF-α and IL-10 by using ELISA kits, and the cells were collected for RT-qPCR and Western blotting detections. Further elucidating the pathway of LTA regulating IL-10 and TNF-α gene expression by inhibiting key proteins in the toll-like receptor pathway with antagonists C34, PDTC, SB230580 and U0126.

RESULTS

High-dose LTA significantly promoted the secretion of the anti-inflammatory factor IL-10 in IPEC-J2 cells, and inhibited the expression and secretion of pro-inflammatory TNF-α in the short term. LTA inhibited the gene expression of TLR4 in LPS-stimulated cells and reduced the protein phosphorylation levels of p38, ERK1/2 and p65. The inhibition of TLR4, p38, ERK1/2 and p65 reduced the TNF-α gene expression caused by LPS; LTA increased TLR2 gene expression, inhibition of p38, ERK and p65 rather than TLR4 reduced the IL-10 gene expression.

CONCLUSION

Our study found that C. butyricum LTA was an important component of C. butyricum regulating the inflammatory response of IPEC-J2 cells. LTA mainly reduced the expression of TNF-α by inhibiting TLR4, while stimulating TLR2 increased the expression of IL-10. Downstream p65, p38 and ERK1/2 were involved in regulating both TNF-α and IL-10. However, TLR4 was only related to the increase in TNF-α caused by LPS and not to the increase in IL-10 caused by LTA. Our work supplemented the probiotic mechanism of C. butyricum and provided a theoretical basis for the application of C. butyricum LTA.