Clostridium perfringens beta2 toxin induced in vitro oxidative damage and its toxic assessment in porcine small intestinal epithelial cell lines.

Abstract

Clostridium perfringens beta2 (CPB2), a key virulence factor, is produced by C. perfringens type C that is the main pathogenic microorganism causing diarrhea in piglets. However, little is known concerning the toxic damage effect of CPB2 on intestinal cells of piglets. In present study, CPB2 toxin obtained by genetic recombination technology was evaluated for its cytotoxicity property using the intestinal porcine epithelial (IPEC-J2) cells, which aims to attempt to understand and explain its mechanism of action in porcine small intestinal epithelial cells. IPEC-J2 cells were treated with different concentrations of CPB2 toxin (5, 10, 20, 30, 40, and 50μg/mL), and MTT assay results showed that the cell viability of CPB2-treated IPEC-J2 cells decreased in a dose-dependent manner. Lactate dehydrogenase (LDH) assay results revealed that CPB2 significantly increased the LDH release, relative to the control. The expression of tumor necrosis factor α (TNF-α) and interleukin 8 (IL-8) gradually increased, while the expression of interleukin 10 (IL-10) gradually decreased in IPEC-J2 cells with increasing concentration of CPB2 (10-30μg/mL), as analyzed by quantitative real-time PCR (RT-qPCR). Also, CPB2 increased the content of intracellular reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) of IPEC-J2 cells. Western blot and immunofluorescence results demonstrate that CPB2 decreased the expression of zonula occludens (ZO-1), claudin12 (CLDN12) and occludin (OCLN) in IPEC-J2 cells. In addition, CPB2 increased Bax expression, and inhibited Bcl-2 and Bcl-xL expression, as measured by Western blot. Considering all of these findings, it was concluded that CPB2 toxin shows significant cytotoxicity, cell growth inhibition and increase in cell permeability in IPEC-J2 cells in a concentration-dependent manner, thus leading to abnormal cell apoptosis and functions in porcine small intestinal epithelial cells.