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.