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. 2022 May 11;14(5):337.
doi: 10.3390/toxins14050337.

Phloretin Protects Bovine Rumen Epithelial Cells from LPS-Induced Injury

Kexin Wang  1 , Qian Lei  1 , Huimin Ma  1 , Maocheng Jiang  1 , Tianyu Yang  1 , Qianbo Ma  1 , Osmond Datsomor  1 , Kang Zhan  1 , Guoqi Zhao  1
Affiliations

Phloretin Protects Bovine Rumen Epithelial Cells from LPS-Induced Injury

Kexin Wang et al. Toxins (Basel). .

Abstract

Lipopolysaccharide (LPS) is an endotoxin that induces immune and inflammatory responses in the rumen epithelium of dairy cows. It is well-known that flavonoid phloretin (PT) exhibits anti-oxidative, anti-inflammatory and antibacterial activity. The aim of this research was to explore whether PT could decrease LPS-induced damage to bovine rumen epithelial cells (BRECs) and its molecular mechanisms of potential protective efficacy. BRECs were pretreated with PT for 2 h and then stimulated with LPS for the assessment of various response indicators. The results showed that 100 µM PT had no significant effect on the viability of 10 µg/mL LPS-induced BRECs, and this dose was used in follow-up studies. The results showed that PT pre-relieved the decline in LPS-induced antioxidant indicators (T-AOC and GSH-PX). PT pretreatment resulted in decreased interleukin-1β (IL-1β), IL-6, IL-8, tumor necrosis factor-α (TNF-α) and chemokines (CCL2, CCL5, CCL20) expression. The underlying mechanisms explored reveal that PT may contribute to inflammatory responses by regulating Toll-like receptor 4 (TLR4), nuclear transcription factor-κB p65 (NF-κB p65), and ERK1/2 (p42/44) signaling pathways. Moreover, further studies found that LPS-induced BRECs showed decreased expression of claudin-related genes (ZO-1, Occludin); these were attenuated by pretreatment with PT. These results suggest that PT enhances the antioxidant properties of BRECs during inflammation, reduces gene expression of pro-inflammatory cytokines and chemokines, and enhances barrier function. Overall, the results suggest that PT (at least in vitro) offers some protective effect against LPS-induced ruminal epithelial inflammation. Further in vivo studies should be conducted to identify strategies for the prevention and amelioration of short acute rumen acidosis (SARA) in dairy cows using PT.

Keywords: bovine rumen epithelial cells; inflammation; lipopolysaccharide; oxidation resistance; phloretin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Dose–Effect of Phloretin and LPS on the viability of BRECs. (A) Dose–effect of different concentrations of Phloretin (0, 1, 10, 20, 40, 60, 80, 100 and 1000 µM) for 12 h on the viability of BRECs. Results are measured relative to 0 µM Phloretin (100%). (B) The apoptosis rate of cells treated with 100 μM PT for 12 h was measured by flow cytometry. Apoptotic cells were expressed as percentage of the total cells. (C) Cell viability induced by LPS at different concentrations (0, 1, 10, 20 and 40 μg/mL) and time points (3, 6 h). Results are measured relative to 0 μg/mL LPS (100%). PT, Phloretin; LPS, lipopolysaccharide. The apoptosis rate of cells was evaluated using an independent samples t-test. Other data were determined by ANOVA followed by Duncan’s multiple range test. All data were presented as mean ± SEM. Different lowercase letters (a–c) on the bar chart indicate significant differences (n = 3, p < 0.05).
Figure 2
Figure 2
Effect of Phloretin on LPS-induced cell viability. BRECs were pretreated with Phloretin (100 μM) for 2 h and then induced by LPS (10 μg/mL) for 6 h. PT, Phloretin; LPS, lipopolysaccharide. Data were presented as a percentage of the CON group. Data as determined by ANOVA followed by Duncan’s multiple range test were presented as mean ± SEM.
Figure 3
Figure 3
Effect of Phloretin on LPS-induced oxidative properties of BRECs. BRECs were pretreated with Phloretin (100 μM) for 2 h and then induced by LPS (10 μg/mL) for 6 h. (A) Total antioxidant capacity (T-AOC) activity in BRECs. (B) Superoxide dismutase (SOD) activity in BRECs. (C) Glutathione peroxidase (GSH-PX) activity in BRECs. (D) Catalase (CAT) activity in BRECs. PT, Phloretin; LPS, lipopolysaccharide. Data as determined by ANOVA followed by Duncan’s multiple range test were presented as mean ± SEM. Different lowercase letters (a–d) on the bar chart indicate significant differences (n = 3, p < 0.05).
Figure 4
Figure 4
Effect of PT on LPS-induced the mRNA expression of inflammatory cytokine gene in BRECs. BRECs were pretreated with Phloretin (100 μM) for 2 h and then induced by LPS (10 μg/mL) for 6 h. (A) Interleukin-1β (IL-1β) mRNA level in BRECs. (B) IL-6 mRNA level in BRECs. (C) IL-8 mRNA level in BRECs. (D) Tumor necrosis factor-α (TNF-α) mRNA level in BRECs. (E) TLR2 mRNA level in BRECs. (F) TLR4 mRNA level in BRECs. PT, Phloretin; LPS, lipopolysaccharide. Data as determined by ANOVA followed by Duncan’s multiple range test were presented as mean ± SEM. Different lowercase letters (a–c) on the bar chart indicate significant differences (n = 3, p < 0.05).
Figure 5
Figure 5
Effect of Phloretin on LPS-induced the mRNA expression of chemokine gene in BRECs. BRECs were pretreated with Phloretin (100 μM) for 2 h and then induced by LPS (10 μg/mL) for 6 h. (A) CXCL8 mRNA level in BRECs. (B) CCL2 mRNA level in BRECs. (C) CCL5 mRNA level in BRECs. (D) CCL20 mRNA level in BRECs. PT, Phloretin; LPS, lipopolysaccharide. Data as determined by ANOVA followed by Duncan’s multiple range test were presented as mean ± SEM. Different lowercase letters (a–c) on the bar chart indicate significant differences (n = 3, p < 0.05).
Figure 6
Figure 6
Effect of Phloretin on LPS-induced the expression of p-p65 and p-p42/44 in BRECs. BRECs were pretreated with Phloretin (100 μM) for 2 h and then induced by LPS (10 μg/mL) for 6 h. (A) The expression of p-p65 and p-p42/44 was determined by Western blot analysis. Immunofluorescence analysis for (B) p-p65 and (C) p-p42/44. Scale bar = 50 μm. Quantification of Fluorescence intensity of p-p65 and p-p42/44 was counted by ImageJ. PT, Phloretin; LPS, lipopolysaccharide. Data as determined by ANOVA followed by Duncan’s multiple range test were presented as mean ± SEM. Different lowercase letters (a–c) on the bar chart indicate significant differences (n = 3, p < 0.05).
Figure 7
Figure 7
Effects of PT on LPS-induced expression of tight junction proteins in BRECs. BRECs were pretreated with Phloretin (100 μM) for 2 h and then induced by LPS (10 μg/mL) for 6 h. (A) ZO-1, Claudin-1 and Occludin mRNA levels in BRECs. (B) The expression of tight junction proteins was determined by Western blot analysis. PT, Phloretin; LPS, lipopolysaccharide. Data as determined by ANOVA followed by Duncan’s multiple range test were presented as mean ± SEM. Different lowercase letters (a–c) on the bar chart indicate significant differences (n = 3, p < 0.05).
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This study was supported by the National Natural Science Foundation of China (No. 31972589) and China Agriculture Research System of MOF and MARA.

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