Effects of Luteolin in an In Vitro Model of Porcine Intestinal Infections

Abstract

Intestinal infections caused by Escherichia coli and Salmonella enterica pose a huge economic burden on the swine industry that is exacerbated by the development of antimicrobial resistance in these pathogens, thus raising the need for alternative prevention and treatment methods. Our aim was to test the beneficial effects of the flavonoid luteolin in an in vitro model of porcine intestinal infections. We infected the porcine intestinal epithelial cell line IPEC-J2 with E. coli and S. enterica subsp. enterica serovar Typhimurium (10⁶ CFU/mL) with or without previous, concurrent, or subsequent treatment with luteolin (25 or 50 µg/mL), and measured the changes in the reactive oxygen species and interleukin-6 and -8 levels of cells. We also tested the ability of luteolin to inhibit the adhesion of bacteria to the cell layer, and to counteract the barrier integrity damage caused by the pathogens. Luteolin was able to alleviate oxidative stress, inflammation, and barrier integrity damage, but it could not inhibit the adhesion of bacteria to IPEC-J2 cells. Luteolin is a promising candidate to be used in intestinal infections of pigs, however, further studies are needed to confirm its efficacy. The use of luteolin in the future could ultimately lead to a reduced need for antibiotics in pig production.

Keywords: AMR; Escherichia coli; IPEC-J2; Salmonella enterica; bacterial infections; flavonoids; intestines; luteolin; pigs; porcine.

Figure 1

Intracellular reactive oxygen species level of IPEC-J2 cells after one-hour treatment with Escherichia coli and luteolin (LUT). Negative control: untreated cells (plain medium only); positive control: cells challenged with 10⁶ CFU/mL E. coli. Group A—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT before challenge with 10⁶ CFU/mL E. coli; Group B—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT concurrently with challenge with 10⁶ CFU/mL E. coli; Group C—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT after challenge with 10⁶ CFU/mL E. coli. Data are shown as means with standard deviation and expressed as relative fluorescence, considering the mean value of control as 100%. N = 6/group. Significant difference: *** p < 0.001, asterisk in gray: compared to the negative (untreated) control; in red: compared to the positive control (E. coli challenge).

Figure 2

Intracellular reactive oxygen species level of IPEC-J2 cells after one-hour treatment with Salmonella Typhimurium and luteolin (LUT). Negative control: untreated cells (plain medium only); positive control: cells challenged with 10⁶ CFU/mL S. Typhimurium. Group A—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT before challenge with 10⁶ CFU/mL S. Typhimurium; Group B—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT concurrently with challenge with 10⁶ CFU/mL S. Typhimurium; Group C—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT after challenge with 10⁶ CFU/mL S. Typhimurium. Data are shown as means with standard deviation and expressed as relative fluorescence, considering the mean value of control as 100%. N = 6/group. Significant difference: * p < 0.05, *** p < 0.001, asterisk in gray: compared to the negative (untreated) control; in red: compared to the positive control (S. Typhimurium challenge).

Figure 3

Interleukin-6 and interleukin-8 levels of IPEC-J2 cells after one-hour treatment with Escherichia coli and luteolin (LUT). Negative control: untreated cells (plain medium only); positive control: cells challenged with 10⁶ CFU/mL E. coli. Group A—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT before challenge with 10⁶ CFU/mL E. coli; Group B—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT concurrently with challenge with 10⁶ CFU/mL E. coli; Group C—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT after challenge with 10⁶ CFU/mL E. coli. Data are shown as means with standard deviation and expressed as relative absorbance, considering the mean value of control as 100%. N = 6/group. Significant difference: * p < 0.5, *** p < 0.001, asterisk in gray: compared to the negative (untreated) control; in red: compared to the positive control (E. coli challenge).

Figure 4

Interleukin-6 and interleukin-8 levels of IPEC-J2 cells after one-hour treatment with Salmonella Typhimurium and luteolin (LUT). Negative control: untreated cells (plain medium only); positive control: cells challenged with 10⁶ CFU/mL S. Typhimurium. Group A—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT before challenge with 10⁶ CFU/mL S. Typhimurium; Group B—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT concurrently with challenge with 10⁶ CFU/mL S. Typhimurium; Group C—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT after challenge with 10⁶ CFU/mL S. Typhimurium. Data are shown as means with standard deviation and expressed as relative absorbance, considering the mean value of control as 100%. N = 6/group. Significant difference: * p < 0.5, ** p < 0.01, *** p < 0.001, asterisk in gray: compared to the negative (untreated) control; in red: compared to the positive control (S. Typhimurium challenge).

Figure 5

Paracellular permeability of IPEC-J2 cells 3 and 24 h after one-hour treatment with Escherichia coli and luteolin (LUT). Negative control: untreated cells (plain medium only); positive control: cells challenged with 10⁶ CFU/mL E. coli. Group A—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT before challenge with 10⁶ CFU/mL E. coli; Group B—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT concurrently with challenge with 10⁶ CFU/mL E. coli; Group C—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT after challenge with 10⁶ CFU/mL E. coli. Data are shown as means with standard deviation and expressed as relative fluorescence, considering the mean value of control as 100%. N = 6/group. Significant difference: * p < 0.5, *** p < 0.001, asterisk in gray: compared to the negative (untreated) control; in red: compared to the positive control (E. coli challenge).

Figure 6

Paracellular permeability of IPEC-J2 cells 3 and 24 h after one-hour treatment with Salmonella Typhimurium and luteolin (LUT). Negative control: untreated cells (plain medium only); positive control: cells challenged with 10⁶ CFU/mL S. Typhimurium. Group A—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT before challenge with 10⁶ CFU/mL S. Typhimurium; Group B—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT concurrently with challenge with 10⁶ CFU/mL S. Typhimurium; Group C—LUT25 or LUT50: cells treated with 25 or 50 μg/mL LUT after challenge with 10⁶ CFU/mL S. Typhimurium. Data are shown as means with standard deviation and expressed as relative fluorescence, considering the mean value of control as 100%. N = 6/group. Significant difference: ** p < 0.01, *** p < 0.001, asterisk in gray: compared to the negative (untreated) control; in red: compared to the positive control (S. Typhimurium challenge).