DISCUSSION
LCZ has a promising prophylactic effect against several inflammatory diseases (Zhang et al., 2017; Wang et al., 2013; Wang et al., 2016), and recent research has shown that the oral administration of LCZ can have a prophylactic effect against E. coli mastitis in mice (Ma et al., 2018). Here, we performed both in vitro and in vivo tests to characterize the prophylactic effect of LCZ on E. coli -stimulated BMECs and mice mammary injury as well as elucidate its underlying mechanism. Our study showed that LCZ pretreatment could significantly ameliorate the injury of BMECs induced by E. coli , which is consistent with the findings of previous studies (Dinić et al., 2017; Wang et al., 2018; Chen et al., 2017). We also explored the prophylactic effect of LCZ on E. coli- induced mastitis in mice by intramammary injection. We found that pre-injection of LCZ could reduce the number of neutrophils and relieve damage to mammary tissue, which was also consistent with the results of our in vitro test. Recent studies have shown that intramammary injection of Lactococcusdoes not cause inflammatory reactions. After intramammary injection ofLactococcus , immune proteins, such as the acute phase proteins haptoglobin and milk amyloid A, in mammary glands of healthy Holstein cows were significantly expressed (Crispie et al., 2008; Pyorala 2003; Eckersall et al., 2006), and no major bovine mastitis pathogen was detected (Pellegrino et al., 2017). Therefore, our findings indicate that LCZ shows high potential for preventing bovine mastitis, which might be related to its ability to promote the production of lactic acid, antibacterial peptides, and other beneficial substances (Lebeer et al., 2018).
Several mammary pathogenic bacteria, including E. coli , cause mastitis. During mastitis, the blood-milk barrier becomes leaky, and molecules can cross the barrier into milk (Lehmann et al., 2013; Nguyen & Neville, 1998). TEER reflects the integrity of the monolayer cells, the ion conductance of the pathway adjacent to monolayer epithelial cells, as well as the pore size of tight junctions (Zucco et al., 2006). Our study showed that LCZ pretreatment could significantly mitigate the reduction in TEER caused by E. coli . This result corroborated the findings of several previous studies showing that Lactobacillus could significantly promote the TEER of Caco-2 (Horibe et al., 1997) and NCM460 cells (Qiu et al., 2017; Liu et al., 2010). Accordingly, we propose that LCZ has a positive effect on enhancing the densification of monolayer BMECs. The same conclusion was obtained based on TEM observations of the desmosome structure between cells.
In exploring the underlying mechanism of the prophylactic effect of LCZ against E. coli- induced cell and blood-milk barrier damage, we found that LCZ pretreatment could significantly reduce the adhesion rate of E. coli to BMECs. These results are consistent with recent studies showing that Lactobacillus could significantly inhibit the adhesion of E. coli toin vitro Caco-2 (Behbahani et al., 2019) and HT-29 cells (Dhanani & Bagchi, 2013). Therefore, the prophylactic effect of LCZ on BMECs could thus be mediated by inhibiting the adhesion of E. coli .
The blood-milk barrier is formed by mammary epithelial cells through special connecting structures, such as tight junctions. Tight junction proteins, such as claudin, occludin, and ZO-1, are known to maintain the structure and functional integrity of tight junctions (Guo et al., 2019; Shen et al., 2006). In this study, we found that LCZ pretreatment could significantly up-regulate the expression levels of the tight junction proteins of BMECs, which is consistent with the results of the TEER. These results are consistent with those of Karczewski et al. (2010) and Wang et al. (2018), who found that Lactobacillus plantarum could effectively promote the expression of occludin and ZO-1 in human intestinal cells as well as claudin-1, occludin, and ZO-1 in IPEC-J2 cells. Moreover, Johnson et al. (2008) found that Lactobacillus rhamnosus could effectively promote the protein expression of claudin-1 and ZO-1 in T84 epithelial cells. The results of the western blot, immunofluorescence, and TEM in vivo test also showed that pre-injection of LCZ could enhance the tight junction structure and up-regulate the expression of tight junction proteins of mammary alveolar lumen in mice. This result is also consistent with our in vitro test as well as previous studies, including Patel et al. (2012), Mennigen et al. (2009), and Karczewski et al. (2010), showing that Lactobacillus can significantly promote the tightness of the intestinal epithelial barrier both in mice and humans by increasing the expression levels of tight junction proteins. Consequently, our study indicates that LCZ could promote the integrity of the epithelial barrier by up-regulating the expression of tight junction proteins in BMECs; meanwhile, intramammary injection of LCZ could effectively prevent E. coli- induced mastitis of mice from significantly down-regulating the expression of tight junction proteins in the blood-milk barrier, thereby alleviating damage to mammary tissue.
Several studies have indicated that pro-inflammatory cytokines act as important regulators in the disruption of tight junctions induced by inflammation (Schulzke et al., 2009; Zhang et al., 2018). The integrity of the blood-milk barrier can help mammary epithelial cells resist infection by external bacteria, thereby relieving the inflammatory reaction. Inflammatory cytokines, such as TNF-α and IL-1β, disrupt tight junctions, and IL-6 is related to host defense against inflammatory disease (Cheng et al., 2018; Rochfort et al., 2014). Our study showed that LCZ pretreatment can significantly down-regulate the expression of TNF-α, IL-6, and IL-1β induced by E. coli . This result is consistent with Wu et al. (2016), who found that Lactobacilluscan effectively reduce the significant up-regulation of TNF-α, IL-1β, IL-18, and IL-8 mRNA expression caused by E. coli . Furthermore, previous studies have shown that the inflammatory response can cause a decrease in the expression of acinar tight junction proteins (Oguro et al., 2011; Hartwig et al., 2003) and thereby affect barrier function. Our in vitro test showed that LCZ could significantly inhibit the expression of inflammatory cytokines; consequently, we also measured the expression of inflammatory cytokines in mammary tissue in mice. We found that pre-injection of LCZ could significantly reduce the expression levels of inflammatory cytokines. Cytokines are known to play an important role in regulating the inflammatory response of the mammary gland and have important physiological and pathological effects on the blood-milk barrier. They can damage tight junction structure, thereby increasing the permeability of the epithelial barrier and causing pathogens in the acinus to enter the mammary tissue (Bruewer et al., 2006; Shen & Turner, 2006). Therefore, our study indicates that the prophylactic effect of LCZ was achieved by inhibiting the expression of inflammatory cytokines in both BMECs and mice mammary tissue.
In conclusion, our study demonstrates that LCZ has a prophylactic effect on E. coli- induced BMEC damage and mastitis of mice by ameliorating the disruption in the blood-milk barrier disruption and suppressing inflammatory responses. The mechanism involved included the promotion of the expression of tight junction proteins by LCZ and the inhibition of the expression of important inflammatory cytokines in both BMECs and mice mammary tissue. This study provides new insights into the protective effect of LCZ, which may serve as a effective prophylactic agent to preserve the blood-milk barrier function during mastitis.