Antioxidative and Cytoprotective Efficacy of Ethanolic Extracted Cranberry Pomace against Salmonella Enteritidis Infection in Chicken Liver Cells

Abstract

Salmonella enterica serovar Enteritidis is a globally significant zoonotic foodborne pathogen. Chicken liver is a vital organ that has been recently implicated in several reported human salmonellosis outbreaks in the U.S. One promising strategy for reducing Salmonella in chickens could be through supplementation with natural antimicrobial additives. Ethanolic extracted cranberry pomace (CPOH) is an excellent source of bioactive polyphenolic compounds with antioxidant and antimicrobial activities. However, the protective effect of CPOH against S. Enteritidis-induced chicken hepatic cell damage remains unclear. In this study, we used a chicken hepatoma cell (LMH) infection model to investigate the protective effects and potential mechanisms of CPOH. CPOH increased the viability of S. Enteritidis-infected LMH cells. Furthermore, CPOH reduced the adhesion and invasion of S. Enteritidis to LMH cells. CPOH downregulated the expression of Rho GTPase genes that are essential for Salmonella's entry into LMH cells. Additionally, the expression of antioxidant regulatory genes, such as Nrf2, HO-1, Txn, and Gclc, was increased. Our data show that CPOH effectively protected LMH cells from cell damage through the inhibition of S. Enteritidis adhesion and invasion, as well as the induction of the expression of master antioxidant genes. These findings offer opportunities to develop sustainable, safe, and economic strategies to reduce the colonization and pathogenesis of Salmonella.

Keywords: S. Enteritidis; anti-infection; antioxidant regulatory genes; cranberry pomace extract; cytoprotecting.

Figure 1

Protective effects of different doses of CPOH on LMH cell viability in the absence or presence of Salmonella at different time points: (A) 1 h; (B) 3 h; (C) 6 h; (D) 12 h; (E) 24 h. Control group (Con; LMH cells with growth medium), CPOH1 (LMH cells treated with 1 mg/mL neutral ethanolic extracted cranberry pomace), CPOH2 (LMH cells treated with 2 mg/mL neutral ethanolic extracted cranberry pomace), CPOH4 (LMH cells treated with 4 mg/mL neutral ethanolic extracted cranberry pomace), S (LMH cells infected with S. Enteritidis), S + CPOH1 (LMH cells simultaneously infected with S. Enteritidis and treated with 1 mg/mL neutral ethanolic extracted cranberry pomace), S + CPOH2 (LMH cells simultaneously infected with S. Enteritidis and treated with 2 mg/mL neutral ethanolic extracted cranberry pomace), and S + CPOH4 (LMH cells simultaneously infected with S. Enteritidis and treated with 4 mg/mL neutral ethanolic extracted cranberry pomace). Data are shown as average percentages of live cells, normalized to the Con at different time points. Data are representative of three independent experiments and shown as means ± SD; * p < 0.05 vs. the Con group; ^# p < 0.05 vs. the S group.

Figure 1

Protective effects of different doses of CPOH on LMH cell viability in the absence or presence of Salmonella at different time points: (A) 1 h; (B) 3 h; (C) 6 h; (D) 12 h; (E) 24 h. Control group (Con; LMH cells with growth medium), CPOH1 (LMH cells treated with 1 mg/mL neutral ethanolic extracted cranberry pomace), CPOH2 (LMH cells treated with 2 mg/mL neutral ethanolic extracted cranberry pomace), CPOH4 (LMH cells treated with 4 mg/mL neutral ethanolic extracted cranberry pomace), S (LMH cells infected with S. Enteritidis), S + CPOH1 (LMH cells simultaneously infected with S. Enteritidis and treated with 1 mg/mL neutral ethanolic extracted cranberry pomace), S + CPOH2 (LMH cells simultaneously infected with S. Enteritidis and treated with 2 mg/mL neutral ethanolic extracted cranberry pomace), and S + CPOH4 (LMH cells simultaneously infected with S. Enteritidis and treated with 4 mg/mL neutral ethanolic extracted cranberry pomace). Data are shown as average percentages of live cells, normalized to the Con at different time points. Data are representative of three independent experiments and shown as means ± SD; * p < 0.05 vs. the Con group; ^# p < 0.05 vs. the S group.

Figure 2

Effects of different doses of neutral CPOH on adhesion of S. Enteritidis to LMH cells: S (LMH cells infected with S. Enteritidis (MOI~10)), S + CPOH1 (LMH cells simultaneously infected with S. Enteritidis (MOI~10) and treated with 1 mg/mL neutral ethanolic extracted cranberry pomace), and S + CPOH2 (LMH cells simultaneously infected with S. Enteritidis (MOI~10) and treated with 2 mg/mL neutral ethanolic extracted cranberry pomace). Data are representative of three independent experiments and shown as means ± SD; ^# p < 0.05 vs. S group. CFU, colony-forming unit.

Figure 3

Effects of different doses of neutral CPOH on invasion of S. Enteritidis into LMH cells: S (LMH cells infected with S. Enteritidis (MOI~10)), S + CPOH1 (LMH cells simultaneously infected with S. Enteritidis (MOI~10) and treated with 1 mg/mL neutral ethanolic extracted cranberry pomace), and S + CPOH2 (LMH cells simultaneously infected with S. Enteritidis (MOI~10) and treated with 2 mg/mL neutral ethanolic extracted cranberry pomace). Data are representative of three independent experiments and shown as means ± SD; ^# p < 0.05 vs. the S group. CFU, colony-forming unit.

Figure 4

Effects of different doses of neutral CPOH on expression of invasion-related host cell proteins. The gene expression levels of cdc-42 (A), RhoG (B), and rac-1 (C) were determined in LMH cells treated with various concentrations of neutral CPOH (mg/mL): control group (Con; LMH cells with growth medium), S (LMH cells infected with S. Enteritidis), S + CPOH1 (LMH cells simultaneously infected with S. Enteritidis and treated with 1 mg/mL neutral ethanolic extracted cranberry pomace), and S + CPOH2 (LMH cells simultaneously infected with S. Enteritidis and treated with 2 mg/mL neutral ethanolic extracted cranberry pomace). Gene expression was determined using RT-qPCR and is represented relative to b-actin. Relative gene expression levels were calculated using the 2^−(ΔΔct) method. Data are representative of three independent experiments and shown as means ± SD; * p < 0.05 vs. Con group; ^# p < 0.05 vs. S group.

Figure 5

Effects of different doses of neutral cranberry pomace after 6 h of treatment on expression of 4 antioxidant-related genes, including Nrf2 (A), HO-1 (B), Txn (C), and Gclc (D). Control group (LMH cells with growth medium), CPOH1 (LMH cells treated with 1 mg/mL neutral ethanolic extracted cranberry pomace), CPOH2 (LMH cells treated with 2 mg/mL neutral ethanolic extracted cranberry pomace), S (LMH cells infected with S. Enteritidis), S + CPOH1 (LMH cells simultaneously infected with S. Enteritidis and treated with 1 mg/mL neutral ethanolic extracted cranberry pomace), and S + CPOH2 (LMH cells simultaneously infected with S. Enteritidis and treated with 2 mg/mL neutral ethanolic extracted cranberry pomace). Gene expression was determined using RT-qPCR and is represented relative to b-actin. Relative gene expression levels were calculated using the 2^-(∆∆Ct) method. Data are representative of three independent experiments and shown as means ± SD; * p < 0.05 vs. Con group; ^# p < 0.05 vs. S group.

Figure 6

Effects of different doses of neutral cranberry pomace after 12 h of treatment on the expression of 4 antioxidant-related genes, including Nrf2 (A), HO-1 (B), Txn (C), and Gclc (D). Control group (LMH cells with growth medium), CPOH1 (LMH cells treated with 1 mg/mL neutral ethanolic extracted cranberry pomace), CPOH2 (LMH cells treated with 2 mg/mL neutral ethanolic extracted cranberry pomace), S (LMH cells infected with S. Enteritidis), S + CPOH1 (LMH cells simultaneously infected with S. Enteritidis and treated with 1 mg/mL neutral ethanolic extracted cranberry pomace), and S + CPOH2 (LMH cells simultaneously infected with S. Enteritidis and treated with 2 mg/mL neutral ethanolic extracted cranberry pomace). Gene expression was determined using RT-qPCR and is represented relative to b-actin. Relative gene expression levels were calculated using the 2^-(∆∆Ct) method. Data are representative of three independent experiments and shown as means ± SD; * p < 0.05 vs. Con group; ^# p < 0.05 vs. S group.

Figure 6

Effects of different doses of neutral cranberry pomace after 12 h of treatment on the expression of 4 antioxidant-related genes, including Nrf2 (A), HO-1 (B), Txn (C), and Gclc (D). Control group (LMH cells with growth medium), CPOH1 (LMH cells treated with 1 mg/mL neutral ethanolic extracted cranberry pomace), CPOH2 (LMH cells treated with 2 mg/mL neutral ethanolic extracted cranberry pomace), S (LMH cells infected with S. Enteritidis), S + CPOH1 (LMH cells simultaneously infected with S. Enteritidis and treated with 1 mg/mL neutral ethanolic extracted cranberry pomace), and S + CPOH2 (LMH cells simultaneously infected with S. Enteritidis and treated with 2 mg/mL neutral ethanolic extracted cranberry pomace). Gene expression was determined using RT-qPCR and is represented relative to b-actin. Relative gene expression levels were calculated using the 2^-(∆∆Ct) method. Data are representative of three independent experiments and shown as means ± SD; * p < 0.05 vs. Con group; ^# p < 0.05 vs. S group.