Nrf2-ARE Signaling Partially Attenuates Lipopolysaccharide-Induced Mammary Lesions via Regulation of Oxidative and Organelle Stresses but Not Inflammatory Response in Mice

Abstract

The incidence of mastitis is high during the postpartum stage, which causes severe pain and hinders breast feeding in humans and reduces milk production in dairy cows. Studies suggested that inflammation in multiple organs is associated with oxidative stress and nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element pathway is one of the most important antioxidant pathways, but the effects of Nrf2 on antioxidation in the mammary gland during mastitis are still unclear. In this study, intramammary lipopolysaccharide (LPS) challenge was carried out in wild-type (WT) and Nrf2 knockout mice. Results showed that the expression of Nrf2 affected the expression of milk protein genes (Csn2 and Csn3). Importantly, LPS treatment increased the expression of Nrf2 and HO-1 and the content of glutathione in the mammary gland of WT mice, but not in Nrf2(-/-) mice. The expression levels of glutathione synthesis genes (GCLC, GCLM, and xCT) were lower in Nrf2(-/-) mice than in WT mice. Moreover, mitochondrial-dependent apoptotic and endoplasmic reticulum stress were significantly relieved in WT mice compared with that in Nrf2(-/-) mice. In summary, the expression of Nrf2 may play an important role in prevention of oxidative and organelle stresses during endotoxin-induced mastitis in mouse mammary gland.

Figure 1

Effects of Nrf2 genotype on lactation performance and expression of milk protein genes in mice. Effects of Nrf2 genotype on (a) milk production on the 6th day to 11th day of lactation (n = 5), the concentrations of (b) milk triglyceride, (c) lactose, and (d) milk protein on day 12 in lactation (n = 5), and relative mRNA abundance of (e) Csn1s1, (f) Csn1s2, (g) Csn2, and (h) Csn3 in the mammary glands of WT, Nrf2(+/-), and Nrf2(-/-) mice treated with either LPS or PBS (n = 6). In all panels, t test was used to determine the differences between LPS and PBS treatments. ANOVA followed by Tukey's multiple comparison was used to determine the differences among three genotypes of mice. Data represent mean ± SEM. All data marked with ^∗ represent a significant difference (P < 0.05).

Figure 2

Effects of LPS and Nrf2 on morphological changes and cell infiltration of the mammary gland in WT, Nrf2(+/-), and Nrf2(-/-) mice. (a) Morphological change, (b) H&E staining, and (c) statistical result of H&E staining. In (c), t test was used to determine the differences between LPS and PBS treatments. Data represent mean ± SEM (n = 3), and data marked with ^∗ represent a significant difference (P < 0.05).

Figure 3

Effects of LPS and Nrf2 genotype on inflammatory cytokines. Effects of LPS and Nrf2 genotype (+/+, +/-, and -/-) on relative mRNA abundance of (a) TNF-α, (b) IL-1β, (c) CXCR2, and (d) CCL3 in the mammary glands. In all panels, t test was used to determine the differences between LPS and PBS treatments. Data represent mean ± SEM (n = 6). All data marked with ^∗ represent a significant difference (0.01 < P < 0.05), and marked with ^∗∗ represent a highly significant difference (P < 0.01).

Figure 4

Protein-protein interaction (PPI) network analysis in WT and Nrf2(-/-) mice, respectively (n = 4). (a) Summary of PPI subnets in WT mice between LPS and PBS treatments. (b) Summary of PPI subnets in Nrf2(-/-) mice between LPS and PBS treatments.

Figure 5

Effects of LPS and Nrf2 phenotype (+/+, +/-, and -/-) on oxidative stress and antioxidation in the mammary gland. (a) mRNA expression changes in genes of antioxidation in the LPS treatment groups between WT and Nrf2 (-/-) mice. Data represents the log₂(foldchange) (n = 4). Data marked with ^∗∗ represent 0.001 < P_adj < 0.01, and marked with ^∗∗∗ represent P_adj < 0.001. (b–k): (b) T-AOC, the protein abundance of (g) GSH, (h) GSH/GSSG ratio, mRNA abundance of (c) NQO1, (d) CAT, (e) Nrf2, (f) HO-1, (i) GCLC, (j) GCLM, and (k) xCT in mice. T test was used to determine the differences between LPS and PBS treatments. ANOVA followed by Tukey's multiple comparison was used to determine the differences among three genotypes of mice. Data represent mean ± SEM (n = 6). The data marked with ^∗ represent a significant difference (0.01 < P < 0.05), and marked with ^∗∗ or ^## represent a highly significant difference (P < 0.01).

Figure 6

Effects of LPS and Nrf2 phenotype on mitochondrial-dependent apoptotic and endoplasmic reticulum stress. Effects of LPS and Nrf2 phenotype (+/+, +/-, and -/-) on relative mRNA abundance of (a) BAX, (b) Bcl-xl, (c) BAX/Bcl-xl, (d) GPR78, and (e) CHOP in the mammary gland of mice. In all panels, t test was used to determine the differences between LPS and PBS treatments. Data represent the mean ± SEM (n = 6). All data marked with ^∗ or ^# represent a significant difference (0.01 < P < 0.05), and marked with ^∗∗ or ^## represent a highly significant difference (P < 0.01).