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. 2022 Oct 27;44(11):5234-5246.
doi: 10.3390/cimb44110356.

Quercetin Alleviates Lipopolysaccharide-Induced Cell Damage and Inflammation via Regulation of the TLR4/NF-κB Pathway in Bovine Intestinal Epithelial Cells

Xiaoxiao Gong  1 , Yinghao Huang  1 , Qianbo Ma  1 , Maocheng Jiang  1 , Kang Zhan  1   2   3 , Guoqi Zhao  1   2   3
Affiliations

Quercetin Alleviates Lipopolysaccharide-Induced Cell Damage and Inflammation via Regulation of the TLR4/NF-κB Pathway in Bovine Intestinal Epithelial Cells

Xiaoxiao Gong et al. Curr Issues Mol Biol. .

Abstract

Acute diarrhoea and intestinal inflammation represent one of the most prevalent clinical disorders of milk production, resulting in enormous annual financial damage for the dairy sector. In the context of an unsatisfactory therapeutic effect of antibiotics, the natural products of plants have been the focus of research. Quercetin is an important flavonoid found in a variety of plants, including fruits and vegetables, and has strong anti-inflammatory effects, so it has received extensive attention as a potential anti-inflammatory antioxidant. However, the underlying basis of quercetin on inflammatory reactions and oxidative tension generated by lipopolysaccharide (LPS) in bovine intestinal epithelial cells (BIECs) is currently unexplained. This research aimed to determine the influence of quercetin on LPS-induced inflammatory reactions, oxidative tension, and the barrier role of BIECs. Our findings demonstrated that BIEC viability was significantly improved in LPS-treated BIEC with 80 μg/mL quercetin compared with the control group. Indicators of oxidative overload and genes involved in barrier role revealed that 80 μg/mL quercetin efficiently rescued BIECs from oxidative and barrier impairment triggered by 5 μg/mL LPS. In addition, the mRNA expression of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, as well as chemokines CXCL2, CXCL5, CCL5, and CXCL8, was diminished in LPS-treated BIECs with 80 μg/mL quercetin compared with LPS alone. Furthermore, the mRNA expression of toll-like receptor 4 (TLR4), CD14, myeloid differential protein-2 (MD2), and myeloid differentiation primary response protein (MyD88) genes associated with the TLR4 signal mechanism was markedly reduced by the addition of quercetin to LPS-modulated BIECs, indicating that quercetin can suppress the TLR4 signal mechanism. We performed Western blotting on the NF-κB signalling mechanism and compared it with immunofluorescence to further corroborate this conclusion. The LPS treatment enhanced the proportions of p-IκBα/GAPDH and p-p65/GAPDH. Compared with the LPS-treated group, quercetin administration decreased the proportions of p-IκBα/GAPDH and p-p65/GAPDH. In addition, immunofluorescence demonstrated that quercetin greatly reduced the LPS-induced nuclear translocation of NF-κB p65 in BIECs. The benefits of quercetin on inflammatory reactions in LPS-induced BIECs may be a result of its capacity to inhibit the TLR4-mediated NF-κB signalling mechanism. These findings suggest that quercetin can be used as an anti-inflammatory reagent to treat intestinal inflammation induced by LPS release.

Keywords: anti-inflammatory; antioxidant; barrier function; fruit and vegetable extracts; quercetin.

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Conflict of interest statement

Regarding the manuscript’s topic, we can confirm that there are no possible conflicts of interest with any financial companies.

Figures

Figure 1
Figure 1
Impacts of quercetin on viable cells in LPS-induced BIECs. (A) Outcomes of quercetin on viable cells in BIECs. BIECs were treated with 40, 80, 120, 160, 200, or 240 µg/mL quercetin for 12 h. (B) Impacts of quercetin on cell vitality in LPS-induced BIECs. BIECs were given 5 µg/mL LPS and various densities of quercetin (40, 80, or 120 µg/mL) for 6 h. Records from the control group were employed to normalise the findings of each experimental group. The results are mean ± SEM (n = 6). Various lowercase letters in the line graph reveal significant differences (p < 0.05).
Figure 2
Figure 2
Impacts of quercetin on oxidative features in LPS-induced BIECs. BIECs were given 5 µg/mL LPS and 80 µg/mL quercetin. After 6 h of culture, the cells were gathered. The results are mean ± SEM (n = 6). Various lowercase letters in the line graph show significant changes (p < 0.05).
Figure 3
Figure 3
Impact of quercetin on barrier function in LPS-induced BIECs. BIECs were treated with 80 μg/mL quercetin in the environment of 5 μg/mL LPS for 6 h; The mRNA representation of zo-1, occluding, claudin 1, and claudin 4 in BIECs was examined by qRT-PCR. The results are mean ± SEM (n = 6). Various lowercase letters in the line graph reveal significant changes (p < 0.05).
Figure 4
Figure 4
Impact of quercetin on the mRNA expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in LPS-induced BIECs. BIECs were treated with 80 μg/mL quercetin in the involvement of 5 μg/mL LPS for 6 h. The mRNA expressions of IL-1β, IL-6, and TNF-α in BIECs were examined by qRT-PCR. The results are mean ± SEM (n = 6). Various lowercase letters in the line graph demonstrate significant changes (p < 0.05).
Figure 5
Figure 5
Quercetin’s effect on the mRNA expression of chemokines in LPS-stimulated BIECs. BIECs were treated with 80 μg/mL quercetin in an environment of 5 μg/mL LPS for 6 h. The mRNA expressions of CCL2, CCL5, CXCL2, CXCL5, and CXCL8 in BIECs were measured by qRT-PCR. The results are mean ± SEM (n = 6). Various lowercase letters in the line graph reveal significant changes (p < 0.05).
Figure 6
Figure 6
Influence of quercetin on the TLR4 signalling system in LPS-induced BIECs. BIECs were treated with 80 μg/mL quercetin in an environment of 5 μg/mL LPS for 6 h. The mRNA expressions of TLR4, TLR2, MD2, CD14, MyD88, IRF3 and NF-κB in BIECs were tested by qRT-PCR. The results are mean ± SEM (n = 6). Various lowercase letters in the line graph demonstrate significant changes (p < 0.05).
Figure 7
Figure 7
Impacts of quercetin on the NF-κB signalling system. BIECs were treated with 80 μg/mL quercetin in an environment of 5 μg/mL LPS for 6 h. (A) Western blot evaluation of p-IκBα and p-p65. (B) Relative protein expression levels of p-IκBα to GAPDH. (C) Relative protein expression levels of p-p65 to GAPDH. The results are mean ± SEM (n = 3). Various lowercase letters in line graphs reveal significant changes (p < 0.05).
Figure 8
Figure 8
Impact of quercetin on the NF-κB (p65) signalling mechanism in LPS-induced BIECs. BIECs were treated with 80 μg/mL quercetin in an environment of 5 μg/mL LPS for 6 h. The immunofluorescence for phosphorylation-NF-κB (p-p65) (red) was conducted and the nuclear dye 4′, 6-diamidino-2-phenylindole (DAPI; blue) was included. Scale bar = 20 µm.
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