A20 Alleviates the Inflammatory Response in Bovine Endometrial Epithelial Cells by Promoting Autophagy

Abstract

Endometritis represents a prevalent condition in perinatal dairy cows. Bovine endometrial epithelial cells (BEECs), as the primary interface between cavity and the external environment, are particularly vulnerable to infection by pathogenic bacteria following parturition. A20 is essential for regulating inflammation and modulating immune responses. Nevertheless, the exact role of A20 in the BEECs in response to inflammatory response is not fully understood. An endometritis model infected by Escherichia coli (E. coli) in vivo and a BEECs inflammation model induced with lipopolysaccharide (LPS) in vitro were built to investigate the function and governing mechanisms of A20 in endometritis. The results showed that infection with E. coli resulted in endometrial damage, inflammatory cell infiltration, and upregulation of inflammatory factors in dairy cows. Furthermore, A20 expression was upregulated in the endometrium of cows with endometritis and in BEECs following LPS stimulation. A20 overexpression attenuated the level of proinflammatory cytokines in LPS-stimulated BEECs; conversely, A20 knockdown lead to an exacerbated response to LPS stimulation. The overexpression of A20 was shown to activate autophagy and suppress the NF-κB signaling pathway in LPS-stimulated BEECs. However, blocking autophagy with chloroquine notably attenuated the anti-inflammatory effect of A20, leading to the activation of the NF-κB signaling pathway. In summary, the study demonstrated that A20's suppression of inflammation in LPS-stimulated BEECs is associated with the activation of autophagy. Therefore, the A20 protein showed potential as a novel treatment focus for managing endometritis in dairy cows.

Keywords: A20; LPS; autophagy; bovine endometrial epithelial cells; inflammatory response.

Figure 1

Endometrial inflammatory damage of dairy cow caused by E. coli. (A) Histopathological changes in endometrium by HE staining. (B) The mRNA expression levels of inflammatory factors in bovine endometrium by qRT-PCR. The data represented the mean ± SEM from three separate experiments. ** p < 0.01 vs. the control group.

Figure 2

The expression level of A20 in bovine endometrium infected with E. coli and LPS-stimulated BEECs. (A) The protein expression level of A20 in bovine endometrium by immunohistochemical staining. (B) Quantitative analysis of A20 protein expression in bovine endometrium. (C) The mRNA expression level of A20 in bovine endometrium by qRT-PCR. (D,F) The protein and mRNA expression level of A20 following stimulation with 1 μg/mL LPS in BEECs by Western blot and qRT-PCR. (E) Quantitative analysis of A20 protein expression in BEECs. The data represent the mean ± SEM from three separate experiments. * p < 0.05, ** p < 0.01 vs. the control group.

Figure 3

Silencing ATG5 enhanced LPS-induced inflammatory response in BEECs. (A) The mRNA expression levels of proinflammatory cytokines in BEECs by qRT-PCR. (B) The protein expression levels of ATG5, LC3 II, p62, p-IκBα, and p-p65 in BEECs by Western blot. (C) Quantitative analysis of ATG5, LC3 II, p62, p-IκBα, and p-p65. The data represent the mean ± SEM from three separate experiments. * p < 0.05, ** p < 0.01 vs. the negative control (NC) group, and # p < 0.05, ## p < 0.01 vs. the NC + LPS group.

Figure 4

A20 inhibited the inflammatory responses induced by LPS in BEECs. (A) The mRNA expression levels of proinflammatory cytokines upon A20 knockdown in BEECs by qRT-PCR. (B) The mRNA expression levels of proinflammatory cytokines upon A20 overexpression in BEECs by qRT-PCR. The data represent the mean ± SEM from three separate experiments. ** p < 0.01 vs. the NC group, and ## p < 0.01 vs. the NC + LPS group.

Figure 5

CQ-induced autophagy inhibition attenuated A20-mediated suppression of NF-κB signaling. (A) The protein expression levels of A20, p-IκBα, p-p65, and LC3 II by Western blot. (B) Quantitative analysis of A20, p-IκBα, p-p65, and LC3 II. (C) p65 protein nuclear translocation with immunofluorescence. (D) Quantification analysis of nuclear translocation of p65. The data represent the mean ± SEM from three separate experiments. * p < 0.05, ** p < 0.01.