Novel organic selenium source hydroxy-selenomethionine counteracts the blood-milk barrier disruption and inflammatory response of mice under heat stress

Abstract

Heat stress (HS) in summer has caused huge economic losses to animal husbandry production recently. When mammary gland is exposed to high temperatures, it will cause blood-milk barrier damage. Hydroxy-selenomethionine (HMSeBA) is a new selenium source with better guarantee of animals' production performance under stress, but whether it has protective effect on heat stress-induced blood-milk damage is still unclear. We established mammary epithelial cells and mice heat stress injury models to fill this research gap, and hope to provide theoretical basis for using HMSeBA to alleviate heat stress damage mammary gland. The results showed that (1) Heat stress significantly decreases in vitro transepithelial electrical resistance (TEER) and cell viability (P < 0.01), and significantly decreases clinical score, histological score, and total alveoli area of mice mammary gland tissue (P < 0.01). (2) HMSeBA significantly increases TEER and fluorescein sodium leakage of HS-induced monolayer BMECs (P < 0.01), significantly improves the milk production and total area of alveoli (P < 0.01), and reduces clinical score, histological score, mRNA expression of heat stress-related proteins, and inflammatory cytokines release of heat-stressed mice (P < 0.01). (3) HMSeBA significantly improves tight junction structure damage, and significantly up-regulated the expression of tight junction proteins (ZO-1, claudin 1, and occludin) as well as signal molecules PI3K, AKT, and mTOR (P < 0.01) in heat-stressed mammary tissue. (4) HMSeBA significantly increases glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), and superoxide dismutase release (SOD) (P < 0.01) and significantly reduce malondialdehyde (MDA) expression (P < 0.01) in heat-stressed mammary tissue. In conclusion, this study implemented heat-stressed cell and mice model and showed that HMSeBA significantly regulate antioxidant capacity, inhibited inflammation, and regulate tight junction proteins expression in blood-milk barrier via PI3K/AKT/mTOR signaling pathway, so as to alleviate mammary gland damage and ensure its structure and function integrity.

Keywords: antioxidant activity; blood-milk barrier; heat stress; hydroxy-selenomethionine; inflammatory response.

Figure 1

Establishment of the monolayer BMEC model (n = 9). (A) Morphology observations of monolayer BMECs using optical microscope; 4× and 10× indicate the magnification of the microscope objective. (B) Effect of culture time on the TEER of monolayer BMECs. ^**indicates extremely significant differences between the BMECs and CON group (P < 0.01), and no marker indicates a non-significant difference between the BMECs and CON group (P > 0.05). (C) TEM image of the desmosome structure of monolayer BMECs. TEER means transepithelial electrical resistance. The red arrow and the circled part indicate the desmosome structure. BMECs indicates bovine mammary epithelial cells.

Figure 2

Establishment of the heat-stressed monolayer BMEC model (n = 9). (A) Effects of HS on cell viability of BMECs. (B) Effects of HS on TEER of BMECs. ^**Indicates that the BMEC group had a very significant difference compared with CON group (P < 0.01). ^*Indicates that the BMEC group had a significant difference compared with CON group (0.01 < P < 0.05), and no marker indicates that the BMEC group had no significant difference compared with CON group (P > 0.05). BMECs means bovine mammary epithelial cells. HS means heat stress. TEER means transepithelial electrical resistance.

Figure 3

Effects of HS on anus temperature, dehydration degree, and clinical score of mice (n = 12). (A) Anus temperature. (B) Dehydration degree. Dehydration degree was described by changes in the body weight of the mice. (C) Clinical score. ^a-b Different lowercase letters indicate extremely significant differences between two groups (P < 0.01), while the same lowercase letters indicate no significant difference between two groups (P > 0.05).

Figure 4

Effects of HS on mammary gland tissue of mice (n = 12). (A) Mammary gland tissue surface. (B) Clinical score. (C) hematoxylin-eosin-stained pathological sections. (D) Histological score. (E) Number of alveoli. (F) Total area of alveoli. ^a-c Different lowercase letters indicate extremely significant differences between two groups (P < 0.01), while the same lowercase letters indicate no significant difference between two groups (P > 0.05).

Figure 5

Effects of HMSeBA on permeability of monolayer BMECs under HS (n = 9). (A) TEER. (B) Fluorescein sodium leakage (30 min). (C) Fluorescein sodium leakage (2 h). ^a-d Different lowercase letters indicate extremely significant differences between two groups (P < 0.01), while the same lowercase letters indicate no significant difference between two groups (P > 0.05). BMECs means bovine mammary epithelial cells. TEER means transepithelial electrical resistance.

Figure 6

Effects of HMSeBA on milk production of mice under HS (n = 12). (A) Early lactation (0-4 day). (B) Transition (5-8 day). (C) Mid-lactation (9-12 day). ^a-d Different lowercase letters indicate extremely significant differences between two groups (P < 0.01), while the same lowercase letters indicate no significant difference between two groups (P > 0.05).

Figure 7

Effects of HMSeBA on mammary gland tissue of mice under HS (n = 12). (A) Clinical score. (B) Clinical score. (C) hematoxylin-eosin-stained pathological sections. (D) Histological score. (E) Number of alveoli. (F) Total area of alveoli. ^a-d Different lowercase letters indicate extremely significant differences between two groups (P < 0.01), while the same lowercase letters indicate no significant difference between two groups (P > 0.05). ns, no significant difference.

Figure 8

Effects of HMSeBA on the mRNA expression of heat stress related protein in mammary gland of mice under HS (n = 12). The relative mRNA expression levels of HSF1 (A) and HSP70 (B) were measured using real-time quantitative PCR. ^a-c Different lowercase letters indicate extremely significant differences between two groups (P < 0.01), while the same lowercase letters indicate no significant difference between two groups (P > 0.05).

Figure 9

Effects of HMSeBA on the concentration of inflammatory cytokines in mammary gland of mice under HS (n = 12). The interleukin (IL)-1β (A), tumor necrosis factor (TNF)-a (B), and IL-6 (C) expression levels were measured with an enzyme-linked immunosorbent assay. ^a-d Different lowercase letters indicate extremely significant differences between two groups (P < 0.01), while the same lowercase letters indicate no significant difference between two groups (P > 0.01).

Figure 10

Effects of HMSeBA on the tight junction of mammary gland tissues of mice under HS (n = 12). (A) Tight junction structure under transmission electron microscopy. The relative mRNA expression levels of ZO-1 (B), claudin 1 (C), and occludin (D) were measured using real-time quantitative PCR. ^a-c Different lowercase letters indicate extremely significant differences between two groups (P < 0.01), while the same lowercase letters indicate no significant difference between two groups (P > 0.05).

Figure 11

Effects of HMSeBA on the immunofluorescence of tight junction proteins in the mammary gland of mice under HS (20 ×) (n = 12). Effects of HMSeBA on immunofluorescence of tight junction protein ZO-1 (A), claudin 1 (B), and occludin (C) were determined using immunofluorescence. DAPI marks the nucleus of neutrophils, and the green label marks the tight junction proteins.

Figure 12

Effects of HMSeBA on the mRNA expression of PI3K/AKT/mTOR in the mammary gland of mice under HS (n = 12). The relative mRNA expression levels of PI3K (A), AKT (B), and, mTOR (C) were measured using real-time quantitative PCR. ^a-d Different lowercase letters indicate extremely significant differences between two groups (P < 0.01), while the same lowercase letters indicate no significant difference between two groups (P > 0.05).

Figure 13

Effects of HMSeBA on the levels of antioxidant indexes in mammary gland of mice under HS (n = 12). The levels of glutathione peroxidase (GSH-Px) (A), total antioxidant capacity (T-AOC) (B), superoxide dismutase (SOD) (C), and malondialdehyde (MDA) (D) were measured with an enzyme-linked immunosorbent assay. ^a-d Different lowercase letters indicate extremely significant differences between two groups (P < 0.01), while the same lowercase letters indicate no significant difference between two groups (P > 0.05).