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Streptococcus uberis (S. uberis) is an important causative agent of mastitis, leading to significant economic losses to dairy industry. This research used a mouse mastitis model to investigate the protective effects of taurine on mammary inflammatory response and blood-milk barrier integrity in S. uberis challenge. The results showed that taurine attenuated S. uberis-induced mammary histopathological changes, especially neutrophil infiltration. The S. uberis-induced expression of pro-inflammatory mediators were decreased significantly by taurine. Further, we demonstrated that taurine limited the S. uberis-induced inflammatory responses via inhibiting the activation of NF-κB and MAPK signaling pathways. Inflammation usually disrupts the mammary barrier system. The recovery of claudin-3 and occludin expressions indicated that attenuation of inflammatory response by taurine can protect the integrity of blood-milk barrier in S. uberis infection. Taken together, our results reveal that the development of taurine as an effective prevention and control strategy for S. uberis-induced mastitis.

Due to its antioxidant properties, resveratrol may relieve the cellular oxidative injury induced by Streptococcus uberis (S. uberis) infection. However, the underlying molecular mechanisms remain unknown. Herein, we used S. uberis to challenge C57BL/6 mice or a mouse mammary epithelial cell line (EpH4-Ev), and the regulatory molecular mechanism of resveratrol on hosts' oxidative injury were investigated. The results showed that gavage of resveratrol alleviate the inflammatory responses and oxidative injury of mammary gland tissues induced by S. uberis infection via activating Nrf2 signaling pathways. To further understand the molecular mechanism, inhibitor of Nrf2 (ML385) and siRNA targeting p62 were used in mammary epithelial cells. The findings indicated that resveratrol mediates Keap1 degradation by activating p62, induces the expression of Nrf2 and its downstream antioxidant signaling pathways, and ameliorates oxidative damage during S. uberis infection. Collectively, these outcomes suggested that resveratrol can function as an activator of the p62-Keap1/Nrf2 signaling pathway to improve oxidative injury caused by S. uberis in mammary glands as well as in EpH4-Ev cells. Therefore, resveratrol may be useful to prevent and control S. uberis-induced bovine mastitis by relieving oxidative stress.

Mammary epithelial cells (MECs) play an important role against Streptococcus uberis infection which is one of the main causes of bovine mastitis and a potential threat to human health. Toll-like receptors (TLRs) and their mediated signaling pathways are critical in both innate and infection responses, yet their roles in anti-S. uberis infection in MECs remains poorly defined. In this work we investigated the regulatory mechanisms of TLR2 in inflammatory responses, where WT and TLR2−/− mice were euthanized at 15–18 days gestation, and mammary gland tissues were collected aseptically. The mouse MECs (MMECs) were isolated by combined digestion with type I collagenase, hyaluronidase and trypsin. We challenged MMECs with S. uberis and quantified antioxidant capacity as well as reactive oxygen species (ROS), proinflammatory cytokines and cell damage at different times. The loss of TLR2 function in MMECs results in more serious cell damage, increased cell adhesion, and significantly decreased ROS and mitochondrial ROS (mROS) with bactericidal function in response to S. uberis infection. Moreover, it was observed that the antioxidant capacity declined, and the production of TLR2-mediated cytokines (except CXC ligand 15) also were reduced. We demonstrated that TLR2 can mediate cellular anti-infective processes in MMECs by regulating the production of ROS and mROS and the secretion of cytokines. The results suggest an unpredicted role of TLR2 in MMECs in response to S. uberis infection.

Mammary epithelial cells (MECs), as part of the functional unit of the udder, are not only responsible for the synthesis of many components in milk that provide necessary nutritional and immunological support to the offspring, but also playing essential roles in the reaction to mastitis pathogens and the initiation of the immune signaling pathway. There are contributions of MECs to the signaling and production of pathogen associated molecular patterns (PAMPs) such as LPS, lipoteichoic acid (LTA), and β-glucans, but the crosstalk of different PAMPs induces signalings and productions in rat MEC that need further study. In the present study, we have demonstrated that β-glucan up-regulates Dectin1 and LPS up-regulates TLR4 directly, as confirmed by generation of siDectin1 and siTLR4 in rat MECs. Then our results have described that either β-glucan or LPS can activate RelB and/or p65 in rat MECs. Furthermore, the association of p65 and RelB has been analyzed that collaboration of β-glucan and LPS promotes p65/RelB heterodimers, producing inflammatory responses in rat MECs. In conclusion, summary of our present results suggests that β-glucan can be considered as a potential immuno-modulator, which s with TLR4 via NF-κB subunits to initiate and regulate the innate immunity in rat MECs.

In order to evaluate the role of taurine on the oxidative stress mediated by NADPH oxidase in Streptococcus uberis-induced (S. uberis) mastitis, rats were administered daily (per os) 100mg/kg of taurine (group TS) or an equal volume of physiological saline (group CS) from gestation day 14 until parturition. Seventy-two hours after parturition, approximately 100cfu of S. uberis was infused into each of 2 mammary glands. Pretreatment with taurine significantly decreased mRNA and protein expression of p47phox and p22phox in mammary tissues. The total anti-oxidation capability (T-AOC) levels and superoxide dismutase (SOD) activities decreased, while malondialdehyde (MDA) levels increased both in mammary tissues and serum of rats with intramammary S. uberis infusion. Gavage administration of taurine moderated this change. Concentrations of interleukin-1β (IL-1β) and IL-6 in mammary glands decreased as a result of taurine administration. Significant differences (P0.05) were present at 48 and 72 h post S. uberis-infusion (PI) for IL-1β and 72 h PI for IL-6. Our data indicate that, in S. uberis-induced mastitis, taurine has the ability of regulating redox conditions which leads to the suppression of oxidative stress and secretion of proinflammatory cytokines. This phenomenon may be ascribed to taurines's ability to inhibit the expression of NADPH oxidase.

Mastitis, caused by mammary pathogenic bacteria which are frequent implications of Escherichia coli, is an important disease affecting women and dairy animals worldwide. The β-glucan binding of dectin-1 can induce its own intracellular signaling and can mediate a variety of cellular responses. This work was to investigate the effect of β-glucan on the lipopolysaccharide (LPS)-induced inflammatory response and related innate immune signaling in primary rat mammary epithelial cells. Cells were treated with serum-free medium added with a DMSO solution containing β-glucans at concentrations of 0, 1, 5, 25 μmol/L for 12h, and then exposed to 10 μg/mL LPS for 40 min. Moreover, cells were pretreated with BAY 11-7082 to inhibit NF-κB and then successively exposed to 5 μmol/L β-glucan, 10 μg/mL LPS, 5 μmol/L β-glucan and 10 μg/mL LPS, according to the specific experimental design. Normal control cultures contained an equal volume of DMSO, which was collected at the same time. After incubating rat mammary epithelial cells for 40 min with 10 μg/mL LPS, TLR4, MyD88 and NF-κB expression all increased (P

To investigate whether taurine ameliorates mammary damage in a rat model of S. uberis mastitis by suppressing inflammation related to the toll-like receptors/nuclear factor kappa B (TLRs/NF-κB) signaling pathway. Starting on gestation day 14 and continuing until parturition, 100 mg/kg of taurine (group TS) or an equal volume of physiological saline (group CS) was administered daily to rats. Seventy-two hours after parturition, rats were infused with 100 cfu of S. uberis into each of 2 mammary glands. The resultant inflammation, evidenced by swelling, degeneration of secretory epithelium, increased tissue loss and neutrophil (PMN) infiltration was observed. Pretreatment with taurine attenuated inflammatory changes and significantly decreased mRNA expression of TLR-2 (8 h post S. uberis-injection, PI), NF-κB p65 (16 h and 24 h PI), and NF-κB DNA binding activity (16 h PI). Tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) levels were also decreased. Significant differences (P

The retinoids, a group of natural or synthetic derivatives of vitamin A, exert various anti-neoplastic and immunomodulatory actions. Recent studies have demonstrated that retinoic acid protects rats against lipopolysaccharide (LPS)-induced mastitis, but the mechanism of action is unclear. In the present study, an LPS-induced rat mastitis model and primary cultures of rat mammary epithelial cells were used to investigate the effect of retinoic acid on the TLR4/NF-kappaB signaling pathway. The data indicated that toll-like receptor 4 (TLR4) gene expression reached its peak value earlier in retinoic acid-treated rats than in the control group, and that retinoic acid significantly decreased NF-kappaB DNA binding activity and the level of IL-1beta in the mammary gland. The animal study result was confirmed by an in vitro cell culture system trial. TLR4 protein expression and NF-kappaB DNA binding activity were significantly decreased in primary rat mammary epithelial cells pretreated with 1mumol/l retinoic acid at 1h post-LPS stimulation. IL-1beta gene expression was also significantly decreased at 2, 4 and 8h post-LPS stimulation. These findings demonstrate that direct action by retinoic acid leads to attenuation of the LPS-induced inflammatory response by suppression of the TLR4/NF-kappaB signalling system, thereby providing a novel explanation for the underlying effect proposed for retinoic acid in the protection of mammary tissue during LPS-induced acute mastitis.

Activated polymorphonuclear neutrophilic leukocytes (PMN) are able to produce large quantities of bactericidal molecules such as reactive oxygen species (ROS) that are associated with tissue damage in models of inflammatory mastitis. In this study, the putative protective effect of retinoid was evaluated in a lipopolysaccharide (LPS) induced mastitis model in rats. Commencing at 10 d of gestation, retinoid (dissolved in olive oil) or an equal volume of olive oil were administered daily by gavage to pregnant rats until parturition. LPS or pyrogen-free physiological saline were infused into the mammary gland 72 h after parturition. At pre-infusion (defined as 0 h) and at 2, 4, 8, 16 and 24 h post-infusion, six rats from each group were euthanized. Retinoid administration decreased PMN accumulation in mammary alveoli, significantly decreased the level of TNF-α in mammary tissues and IL-8 in serum at the different time points. ROS release was significantly increased after LPS infusion and was reduced by retinoid at 16 h PI. Retinoid reduced N -acetyl- β - d -glucosaminidase (NAGase) activity in both serum and mammary tissue at 8 h PI. Intercellular adhesion molecule 1 (ICAM-1) mRNA expression reached its peak value earlier in retinoid treated rats than in the control group. Overall, the results suggest that activated PMN play an important role in the pathogenesis of acute mastitis and retinoid administration may be an effective tool for protecting mammary tissue against PMN-induced oxidative stress during LPS-induced acute mastitis.