Jinfeng Miao

Taurine attenuates lipopolysaccharide-induced disfunction in mouse mammary epithelial cells

The intent of this study was to evaluate the active defense reaction of mouse mammary epithelial cells and the cytoprotective and anti-inflammatory properties of taurine to lipopolysaccharide (LPS)-induced disfunction in mouse mammary epithelial cells. (1) Primary cultured mouse mammary epithelial cells were stimulated with LPS for 24 h (final concentration=0, 5, 10, 20 μg/mL). Western blotting demonstrated a significant decrease in the secretion of β-casein in the 20 μg/mL LPS treatment group (P<0.05), while nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), lactoferrin (LF) and N-acetyl-β-D-glucosaminidase (NAGase) were all significantly increased following LPS treatment (P<0.01). Furthermore, cell survival was significantly inhibited after treatment with 20 μg/mL LPS; however, neither 5 μg/mL nor 10 μg/mL LPS had any effect on cell survival. Therefore, a level of 10 μg/mL LPS was selected to test the protective effect of taurine on mouse mammary epithelial cells. (2) Primary cultured mouse mammary epithelial cells were treated with 0, 5, 15 or 45 mmol/L taurine for 3 h, followed by 10 μg/mL LPS for 24 h. Taurine significantly attenuated the LPS-induced increase in NAGase activity, NO concentrations and the level of TNF-α, IL-1β, IL-6 and LF. Taurine at 45 mmol/L markedly increased β-casein secretion in response to LPS-induced disfunction. This study demonstrated that the addition of taurine to a culture medium significantly inhibited the LPS-induced release of inflammatory factors and increased β-casein secretion from mammary epithelial cells, thereby providing a possible explanation for the protective effect proposed for taurine in the prevention of LPS-induced disfunction in mammary epithelial cells.

Retinoid protects rats against neutrophil-induced oxidative stress in acute experimental 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-alpha 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-beta-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.

Retinoic acid attenuates lipopolysaccharide-induced inflammatory responses by suppressing TLR4/NF-κB expression in rat mammary tissue

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.

Protective effect of retinoid against endotoxin-induced mastitis in rats

Abstract.Objective:A lipopolysaccharide (LPS) induced mastitis model in rats was used to study the protective effect of retinoid.Materials and methods:Commencing at gestation day 10, retinoid (dissolved in olive oil) or an equal volume of olive oil was administered to rats daily by gavage until parturition. LPS or pyrogen-free physiological saline was inoculated into the mammary gland 72h after parturition and the rats were euthanized at 12h post-infection.Results:Myeloperoxidase (MPO), N-acetyl -β-D- glucosaminidase (NAGase), tumor necrosis factor-alpha (TNF-α), interleukin-8 (IL-8) in mammary tissues and CD4+/CD8+ in peripheral blood were increased and serum MPO and IL-2 in mammary tissues were decreased 12h after LPS infusion. Retinoid decreased MPO, NAGase, and TNF-α in mammary tissue and increased IL-2 in serum. Four thousand and 8000 I.U/kg•d of retinoid significantly decreased the infiltration of PMNs in mammary alveoli and ameliorated the imbalance of CD4+/CD8+ in peripheral blood.Conclusion:These results suggest that retinoid protected against LPS-induced mastitis in a rat model.

The effect of taurine on the toll-like receptors/nuclear factor kappa B (TLRs/NF-κB) signaling pathway in Streptococcus uberis-induced mastitis in rats

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<0.05) were present at 24 h and 48 h PI for TNF-α and at 16 h PI for iNOS. TLR-4 mRNA expression was increased by taurine administration and significant differences were observed at 8h, 16 h and 24 h PI. These results suggest that the in vivo relationship of immunomodulatory reagents with TLRs is complex. Taurine may modulate inflammatory injury induced by S. uberis in mammary glands though TLR-2 and TLR-4. Suppression of inflammation may be related to TLRs/NF-κB and may be one mechanism of taurine action in controlling S. uberis mastitis.

Polysaccharide Nucleic Acid of Bacillus Calmette Guerin Modulates Th1/Th2 Cytokine Gene Expression in Lipopolysaccharide-Induced Mastitis in Rats

The aim of this study was to evaluate, in rats, the changes in the T helper type 1 (Th1)/Th2 radio in mammary glands after an intramammary infusion of lipopolysaccharide (LPS) and to characterize the moderating effects of the polysaccharide nucleic acid of Bacillus Calmette Guerin (BCG-PSN) on the mammary gland. In the control group, the levels of IL-2 and INF-γmRNA expression increased, whereas IL-4 mRNA expression decreased after LPS challenge. As a consequence, the INF-[.gamma]/IL-4 mRNA ratio was significantly higher at 3, 6, and 9 h post-infusion (PI) compared to the control value (0 h; P< 0.01). BCG-PSN increased mRNA expression of both INF-γ and IL-4 before infusion of LPS. LPS challenge significantly the reduced Th1/Th2 cytokine ratio due to Th1 cytokine IFN-γ suppression and Th2 cytokine IL-4 upregulation compared with the control group. A significant reduction of N-acetyl-[.beta]-D-glucosaminidase (NAGase) was observed at 24 h PI in the BCG-PSN treatment group compared to the control group (P < 0.05). Thus, it was demonstrated that level of BCG-PSN might change the Th1/Th2 ratio mainly by enhancing the Th2 immune response. This is the first report of a Th1/Th2 change induced by coliform mastitis and characterization of the effect of BCG-PSN on mammary gland inflammation. This study makes a better understanding of the mechanisms of coliform mastitis and provides a putative novel strategy for the prevention and/or treatment of mastitis.

The role of NADPH oxidase in taurine attenuation of Streptococcus uberis-induced mastitis in rats.

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 (P<0.05) 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 tauriness ability to inhibit the expression of NADPH oxidase.

β-Glucan modulates the lipopolysaccharide-induced innate immune response in rat mammary epithelial cells.

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<0.05), as did the secretion of TNF-α and IL-1β (P<0.05), but IκB and β-casein expression both decreased (P<0.05). Treatment with different concentrations of β-glucan for 12h activated Dectin1/Syk, which subsequently suppressed TLR4, MyD88 and NF-κB expression and TNF-α and IL-1β secretion. However, it restored the IκB and β-casein expression that had been induced by the 40 min incubation with 10 μg/mL LPS. Pretreatment with BAY 11-7082 at 10 µmol/L for 2h partially prevented NF-κB induction by LPS, but the presence of β-glucan prevented this inactivation. BAY 11-7082 could not simultaneously inhibit LPS induction of TLR4, MyD88 and β-glucan activation of Dectin1/Syk in rat mammary epithelial cells. These findings demonstrated that β-glucan activation of Dectin1/Syk attenuated LPS induction of TLR4/MyD88/NF-κB and inhibited the LPS-induced inflammation factors in mammary epithelial cells, thereby providing a possibly protective effect of β-glucan in the prevention of LPS-induced dysfunction in mammary epithelial cells.

Variant innate immune responses of mammary epithelial cells to challenge by Staphylococcus aureus, Escherichia coli and the regulating effect of taurine on these bioprocesses.

Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are important pathogens causing subclinical and clinical bovine mastitis, respectively. Taurine, an organic acid found in animal tissues, has been used for the treatment of various superficial infections and chronic inflammations. We challenged a bovine mammary epithelial cell (MEC) line (MAC-T) or a mouse mammary epithelial cell line (EpH4-Ev) with either E. coli or S. aureus and compared the responses of MECs to these 2 pathogens. We also examined the regulatory effects of taurine on these responses. Receptor analyses showed that both TLR2 and TLR4 are upregulated upon exposure to either E. coli or S. aureus. Taurine pre-treatment dampened upregulation to some extent. E. coli and S. aureus stimulated comparable levels of ROS, which could be inhibited by taurine pre-treatment. E. coli infection elicited a dramatic change in iNOS expression. Taurine significantly decreased iNOS expression in the S. aureus challenged group. Protein microarray demonstrated that 32/40 and 8/40 inflammatory molecules/mediators were increased after E. coli or S. aureus challenge, respectively. The fold changes of most molecules were higher in the E. coli infection group than that in the S. aureus infection group. Taurine negatively regulated the inflammatory profile in both bacterial infections. Pro-inflammatory cytokines (such as TNF-α) connected with TLR activation were down-regulated by taurine pre-treatment. The influence of TAK-242 and OxPAPC on cytokine/molecule expression profiles to E. coli challenge are different than to S. aureus. Some important factors (MyD88, TNF-α, IL-1β, iNOS and IL-6) mediated by TLR activation were suppressed either in protein microarray or special assay (PCR/kits) or both. TAK-242 restrained ROS production and NAGase activity similar to the effect of taurine in E. coli challenge groups. The detection of 3 indices (T-AOC, SOD and MDA) reflecting oxidative stress in vivo, showed that taurine improved the antioxidant ability of cells. We conclude that taurine can regulate the inflammatory response during infection with E. coli and prevent cell damage by affecting the signaling pathways mediated by TLRs and by improving the antioxidant ability of cells. In S. aureus infections, taurines antioxidant ability may be the primary means of resistance to inflammation. This study provides a better understanding of the inflammatory mechanisms of E. coli and S. aureus mastitis, and it provides a putative strategy for the prevention of this disease.

The crosstalk between Dectin1 and TLR4 via NF-κB subunits p65/RelB in mammary epithelial cells.

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.