Protective Effects of Lactobacillus plantarum Lac16 on Clostridium perfringens Infection-Associated Injury in IPEC-J2 Cells

Abstract

Clostridium perfringens (C. perfringens) causes intestinal injury through overgrowth and the secretion of multiple toxins, leading to diarrhea and necrotic enteritis in animals, including pigs, chickens, and sheep. This study aimed to investigate the protective effects of Lactobacillus plantarum (L. plantarum) Lac16 on C. perfringens infection-associated injury in intestinal porcine epithelial cell line (IPEC-J2). The results showed that L. plantarum Lac16 significantly inhibited the growth of C. perfringens, which was accompanied by a decrease in pH levels. In addition, L. plantarum Lac16 significantly elevated the mRNA expression levels of host defense peptides (HDPs) in IPEC-J2 cells, decreased the adhesion of C. perfringens to IPEC-J2 cells, and attenuated C. perfringens-induced cellular cytotoxicity and intestinal barrier damage. Furthermore, L. plantarum Lac16 significantly suppressed C. perfringens-induced gene expressions of proinflammatory cytokines and pattern recognition receptors (PRRs) in IPEC-J2 cells. Moreover, L. plantarum Lac16 preincubation effectively inhibited the phosphorylation of p65 caused by C. perfringens infection. Collectively, probiotic L. plantarum Lac16 exerts protective effects against C. perfringens infection-associated injury in IPEC-J2 cells.

Keywords: Clostridium perfringens; Lactobacillus plantarum; cellular injury; inflammation; intestinal barrier integrity.

Figure 1

Antimicrobial activity of L. plantarum Lac16 on C. perfringens. (A) Agar well diffusion assay—1: de Man–Rogosa–Sharpe (MRS) broth; 2: 100 µg/mL of ampicillin; 3 and 4: LFS. (B) The growth of C. perfringens in different groups was measured at OD600 after 12 h of incubation. (C) Biofilm formation was measured at OD590. (D) C. perfringens levels in the co-culture experiment. (E) pH values of cultures in different groups. Data are presented as the means ± SD for n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 (t test).

Figure 2

Gene expression levels of HDPs in L. plantarum Lac16-treated IPEC-J2 cells. (A) pBD1, (B) pBD2, (C) pBD3, (D) pEP2C. mRNA expression was standardized to β-actin expression. Data are presented as the means ± SD for n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA).

Figure 3

Cytotoxicity and C. perfringens adhesion assays. (A) Concentrations of LDH in the supernatants of IPEC-J2 cells. (B) Adhesions of C. perfringens to IPEC-J2 cells were detected by tryptose sulfite cycloserine (TSC) agar. Adherence ratio of the C. perfringens group was normalized to 100%. (C) Adhesions of C. perfringens (green) to IPEC-J2 cells were detected by fluorescence labeling method. (D) The mean relative fluorescence intensity of fluorescein isothiocyanate (FITC) in panel C. Data are presented as the means ± SD for n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 (t test).

Figure 4

Intestinal barrier functions and tight junction proteins of IPEC-J2 cell monolayers. (A) P_app of IPEC-J2 monolayers based on apical-to-basolateral flux of fluorescein sodium. (B) Mucin production was detected by PAS staining (Mucin, carmine). (C) Relative gene expression level of Mucin 2. mRNA expression was standardized to β-actin expression. (D) Western blot detection of occludin and claudin-1 in IPEC-J2 cells, and the quantitative analysis of the expression levels of bands. β-actin was used as an indicator of protein loading. (E) Immunofluorescence staining of zona occludens-1 (ZO-1, green) in IPEC-J2 cells. Nuclei were counterstained using 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, blue). All images were obtained at 63× magnification in oil. (F) The mean relative fluorescence intensity of ZO-1 in panel E. Data are presented as the means ± SD for n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 (t test).

Figure 5

Relative gene expression levels of inflammatory cytokines during C. perfringens infection in IPEC-J2 cells preincubated with L. plantarum Lac16. (A) IL-1β, (B) IL-6, (C) IL-8, (D)TNF-α. mRNA expression was standardized to β-actin expression. Data are presented as the means ± SD for n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 (t test).

Figure 6

Relative gene expression levels of PRRs during C. perfringens infection in IPEC-J2 cells preincubated with L. plantarum Lac16. (A) TLR1, (B) TLR2, (C) TLR4, (D) NOD1, (E) NOD2. mRNA expression was standardized to β-actin expression. Data are presented as the means ± SD for n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 (t test).

Figure 7

Western blot detection of MAPK and NF-κB pathways in IPEC-J2 cells. (A) Western blot analysis of MAPK and NF-κB pathways. (B) Quantitative analysis of the expression levels of phospho-p38/p38, phospho-JNK/JNK, phospho-ERK/ERK, and phosphor-p65/p65. Data are presented as the means ± SD for n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 (t test).