Lactobacillus casei Zhang Counteracts Blood-Milk Barrier Disruption and Moderates the Inflammatory Response in Escherichia coli-Induced Mastitis

Abstract

Escherichia coli is a common mastitis-causing pathogen that can disrupt the blood-milk barrier of mammals. Although Lactobacillus casei Zhang (LCZ) can alleviate mice mastitis, whether it has a prophylactic effect on E. coli-induced mastitis through intramammary infusion, as well as its underlying mechanism, remains unclear. In this study, E. coli-induced injury models of bovine mammary epithelial cells (BMECs) and mice in lactation were used to fill this research gap. In vitro tests of BMECs revealed that LCZ significantly inhibited the E. coli adhesion (p < 0.01); reduced the cell desmosome damage; increased the expression of the tight junction proteins claudin-1, claudin-4, occludin, and zonula occludens-1 (ZO-1; p < 0.01); and decreased the expression of the inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 (p < 0.01), thereby increasing trans-epithelial electric resistance (p < 0.01) and attenuating the lactate dehydrogenase release induced by E. coli (p < 0.01). In vivo tests indicated that LCZ significantly reduced the injury and histological score of mice mammary tissues in E. coli-induced mastitis (p < 0.01) by significantly promoting the expression of the tight junction proteins claudin-3, occludin, and ZO-1 (p < 0.01), which ameliorated blood-milk barrier disruption, and decreasing the expression of the inflammatory cytokines (TNF-α, IL-1β, and IL-6) in mice mammary tissue (p < 0.01). Our study suggested that LCZ counteracted the disrupted blood-milk barrier and moderated the inflammatory response in E. coli-induced injury models, indicating that LCZ can ameliorate the injury of mammary tissue in mastitis.

Nisin Z attenuates lipopolysaccharide-induced mastitis by inhibiting the ERK1/2 and p38 mitogen-activated protein kinase signaling pathways

“…Studies have demonstrated that E. coli (Zheng et al, 2021) or LPS (Kan et al, 2019) could induce lesions in the mouse mammary gland tissue, increasing the levels of TNF-α, IL-6, and IL-1β. These inflammatory indices were significantly downregulated after intervention with anti-inflammatory substances, alleviating LPSinduced inflammation.…”

N6-Methyladenosine Modification Profile in Bovine Mammary Epithelial Cells Treated with Heat-Inactivated Staphylococcus aureus

“…Staphylococcus aureus is one of the most common pathogens that often causes subclinical mastitis [ 34 ]. Previous studies have shown that bovine mammary epithelial cells are the first line of defense against the invasion of mammary glands by microorganisms such as S. aureus [ 1 , 35 ], which leads to the release of various chemokines and cytokines [ 36 ]. Although there is currently much research into mastitis, the molecular mechanism of mastitis caused by S. aureus is still unclear.…”

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

“…Meanwhile, the desmosome structure of monolayer cells was detected by TEM in this study, which also confirmed the successful construction of the monolayer dense bovine mammary epithelial cell model. Predecessor's researches have shown that human and bovine mammary epithelial cells polarize when cultured on permeable transwell supports to form an epithelial barrier, that is, monolayer cells are in a state of high transmembrane resistance (TEER > 600 W•cm 2 ) (25,48). In addition, our study showed that the TEER of the cells was significantly lower than CON group after 1 h of heat stress but then recovered, and the difference was extremely significant compared with the CON group.…”

“…In detail, cells were seeded in upper chamber of transwell (membrane: 0.33 cm 2 ), 200 mL medium was added in upper chamber and 600 mL medium in lower chamber. The TEER value is subtracted from the blank (with no cells) to obtain the actual measured value and expressed as W•cm 2 .When it reached 400 W•cm 2 , four treatments were performed (25). Besides, cell counting kit-8 kits (Beyotime) were used to detect cell viability.…”

“…Mammary tissue samples were collected for hematoxylineosin staining, immunofluorescence detection, transmission electron microscopy analysis, relative mRNA expression levels detection, inflammatory cytokines expression and antioxidant index detection. Specifically, hematoxylin-eosin staining of pathological sections of mice mammary gland samples was performed as follows: Fixed mammary tissues in 4% cell tissue fixative (Solarbio, Beijing, China) immediately after sampling, used hematoxylin-eosin to stain, and used a microscope to observe; Immunofluorescence detection of tight junction proteins (ZO-1, claudin 1, and occludin) and transmission electron microscopy analysis of tight junction structure as Zheng et al (25); Real-time quantitative PCR was performed as following: total RNA was extracted from mammary gland tissues with Trizol reagent (TaKaRa Bio, Kusatsu, Japan), and RNA concentration and purity were determined with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA) referred to Zhao et al (31). The primers used in our study lists in Table 2, and the relative mRNA expression levels of each target gene were detected using the method of described before (32); Two milliliter of RIPA tissue lysate (Solarbio, Beijing, China) was added to each sample, centrifuged at 10000-14000 × g for 4 min, and removed the supernatant for inflammatory cytokines expression measurement using enzyme-linked immunosorbent assay (ELISA) with mouse ELISA kits (Thermo Fisher); The glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activity, T-AOC, and MDA content were determined using commercial detection kits (A042, A005, A001-3, and A015; Nanjing JianCheng Bioengineering Institute, Nanjing, China) according to the method provided by the manufacturer.…”