Abstract
Nuclear factor E2-related factor 2 (NRF2) plays an anti-inflammatory role in several pathological processes, but its function in lipopolysaccharide- (LPS-) induced goat endometrial epithelial cells (gEECs) is still unknown. We designed a study to investigate the function of NRF2 in LPS-induced gEECs. LPS was found to increase the NRF2 expression and the nuclear abundance of NRF2 in gEECs in a dose-dependent manner. NRF2 knockout (KO) not only increased the expression of LPS-induced proinflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8) but also increased the expression of TLR4, p-IκBα/IκBα, and p-p65/p65 proteins. Immunoprecipitation experiments showed that NRF2 directly binds to p65 in the nucleus and inhibits the binding of p65 to downstream target genes (TNF-α, IL-1β, IL-6, and IL-8). Even though a NF-κB/p65 inhibitor (PDTC) reduced the LPS-induced NRF2 expression and nuclear abundance of NRF2, overexpressing TNF-α reversed the inhibitory effects of PDTC on the NRF2 expression and on its abundance in the nucleus. Similarly, knockdown of the proinflammatory cytokines (TNF-α, IL-1β, IL-6, or IL-8) significantly decreased the LPS-induced NRF2 expression and NRF2 in the nucleus. In conclusion, our data suggest that proinflammatory cytokines induced by LPS through the TLR4/NF-κB pathway promote the NRF2 expression and its translocation into the nucleus. Our work also suggests that NRF2 inhibits the expression of proinflammatory cytokines by directly binding to p65.
Highlights
After giving birth, animals often suffer from endometritis as a result of infection, making it difficult for them to have healthy pregnancies in the future as well as produce milk
Immunoblotting was used to detect the expression of Nuclear factor E2-related factor 2 (NRF2); lamin B was loaded as the nucleus control, and βactin was loaded as the cytoplasm control (Figure 1(a))
The analysis of the gray value of protein bands showed that the LPS -increased expression of NRF2 in goat endometrial epithelial cells (gEECs) in a dosedependent manner (Figure 1(b))
Summary
Animals often suffer from endometritis as a result of infection, making it difficult for them to have healthy pregnancies in the future as well as produce milk. Animals that have issues with pregnancies or milk production contribute to economic loss on farms [1]. There are many species of bacteria that cause endometritis, such as Escherichia coli, Arcanobacterium pyogenes, Prevotella spp., Fusobacterium necrophorum, and Fusobacterium nucleatum, most of which are gram-negative [4, 5]. Lipopolysaccharide (LPS) is a substance composed of lipids and polysaccharides that composes the outer cell wall of gram-negative bacteria and reported as the main toxic factor of gram-negative bacteria causing an immune response [6, 7]. In the study presented here, LPS was used to simulate infection by gram-negative bacteria
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