Figure Legends
Figure 1: Effect of LCZ on cell growth and the blood-milk barrier. (A) The effect of LCZ on cell viability was determined by the LDH method. (B) The effect of LCZ on the change of TEER was determined using a Millicell Electrical Resistance System-2. Change of TEER represents TEER of monolayer cells after treatment - TEER of monolayer cells before treatment. (C) Transmission electron microscopy (TEM) was used to detect the structure of desmosomes. Scale bars: 1 μm. Data shown are means ± SEM (n = 9). Means with different superscripts indicate significant differences (P < 0.05), whereas means with the same superscripts indicate treatments that are not significantly different (P > 0.05).
Figure 2: Effect of LCZ on the adhesion rate of E. coli to BMECs. The data shown are means ± SEM (n = 9). Means with different superscripts indicate significant differences (P < 0.05), whereas means with the same superscripts indicate treatments that are not significantly different (P > 0.05).
Figure 3: Effect of LCZ on the expression of tight junction proteins was determined by western blotting. Quantification of tight junction proteins was determined by densitometry and was normalized to β-Actin. Data shown are means ± SEM (n = 9). Means with different superscripts indicate significant differences (P < 0.05), whereas means with the same superscripts indicate treatments that are not significantly different (P > 0.05).
Figure 4: Effect of LCZ on the transcriptional levels of inflammatory cytokines of BMECs. Levels of TNF-α, IL-1β, and IL-6 were measured by ELISA. Data shown are means ± SEM (n = 9). Means with different superscripts indicate significant differences (P < 0.05), whereas means with the same superscripts indicate treatments that are not significantly different (P > 0.05).
Figure 5: Effect of LCZ on E. coli -induced histopathological impairment of mammary tissue in mice. (A) Injury score of mice mammary tissue from the Control (a), E. coli (b), LCZ + E. coli(c), and LCZ (d) groups. The injury score ranged from 1–5, where 1 indicated no damage, 2 indicated slight redness, 3 indicated redness and slight bleeding, 4 indicated redness and bleeding, and 5 indicated redness and obvious bleeding. (B) Histological score of hematoxylin-eosin stained pathological sections from the Control (a),E. coli (b), LCZ + E. coli (c), and LCZ (d) groups. Hematoxylin-eosin staining of formalin-fixed mammary gland. Histopathological sections of mammary gland. Scale bars: 100 μm. The histological score ranged from 1–5, where 1 indicated the absence of histological features (e.g., necrosis, neutrophils, and lymphocytes), 2 indicated minimal histological features (i.e., individual neutrophils), 3 indicated mild histological features (i.e., a small amount of neutrophils), 4 indicated moderate histological characteristics (i.e., many neutrophils and slight damage to glandular structure), and 5 indicated severe histological characteristics (i.e., a large number of neutrophils and severe damage to glandular structure). Data shown are means ± SEM (n = 10). Means with different superscripts indicate significant differences (P < 0.05), and means with the same superscripts indicate treatments that are not significantly different (P > 0.05).
Figure 6: Effect of LCZ on thee tight junction structure of mice mammary tissue. Transmission electron microscopy (TEM) was used to characterize the structure of the tight junction. Scale bars: 50 μm (A) and 10 μm (B).
Figure 7: Effect of LCZ on the expression of tight junction proteins of mice mammary tissue. (A-C) Effect of LCZ on the localization of claudin-3, occludin, and ZO-1. Blue shows nuclei (DAPI), red shows claudin-3 and occludin, and green shows ZO-1. Scale bars: 100 μm. (D) Prophylactic effect of LCZ on the expression levels of the tight junction proteins claudin-3, occludin, and ZO-1 was determined by western blotting. Quantification of tight junction proteins was determined by densitometry and was normalized to β-Actin. Data shown are means ± SEM (n = 10). Means with different superscripts indicate significant differences (P < 0.05), whereas means with the same superscripts indicate treatments that are not significantly different (P > 0.05).
Figure 8: Effect of LCZ on the transcriptional levels of inflammatory cytokines of mice mammary tissue. Levels of TNF-α, IL-1β, and IL-6 were measured by ELISA. Data shown are means ± SEM (n = 10). Means with different superscripts indicate significant differences (P < 0.05), whereas means with the same superscripts indicate treatments that are not significantly different (P > 0.05).