Effect Of Retinoid On LPS Induced Inflammatory Responses In Rat Mammary Tissue

In this study, effects of retinoid on mammary defense and the protective mechanism of retinoid on mammary gland were first evaluated on experimental mastitis induced by LPS in rats. Optimized primary culture of rat mammary epithelial cells and its inflammatory model was also established to discuss the protective mechanism of retinoid.1 Protective effect of retinoid against LPS-induced mastitis in ratsA LPS induced mastitis model in rats was used to study the protective effect of retinoid. Commencing at gestation day 10, retinoid (dissolved in soy oil) or an equal volume of soy oil was administered to rats daily by gavage until parturition. LPS or pyrogen-free physiological saline was inoculated into the mammary gland 72 h after parturition and the rats were euthanized at 12 h post-infection. 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, superoxide dismutase (SOD), total anti-oxidizing capability (T-AOC) and interleukin-2 (IL-2) in mammary tissues were decreased 12 h after LPS infusion. Retinoid decreased MPO, NAGase, TNF-αin mammary tissue and increased IL-2 in serum. Four thousand and 8000 I.U/kg·d of retinoid significantly decreased the infiltration of PMN in mammary alveoli, ameliorated the imbalance of CD4+/CD8+ in peripheral blood and increased SOD and T-AOC in serum. These results suggest that retinoid protects against LPS-induced mastitis in a rat model.2 Effect of retinoid on inflammatory cytokines secretion in rats acute experimental mastitisIn this study, the putative protective effect of retinoid on inflammatory cytokines secretion was evaluated in a LPS-induced mastitis model in rats. Commencing at 10 d of gestation, retinoid (dissolved in soy oil) or an equal volume of soy 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. The results showed that retinoid administration decreased toll-like receptor-4 (TLR-4) and nuclear factor-κB (NF-κB) p65 mRNA in mammary tissues, significantly decreased NF-κB and DNA binding activity, decreased the secretion of TNF-αand interleukin-1β(IL-1β) in mammary gland. Overall, the results suggest that retinoid protects the mammary gland through decreasion the activity of TLR-4 and NF-κB and inhibition the inflammtory factors secreting.3 Effect of retinoid on the activity of neutrophils in rats acute experimental mastitisActivated 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 LPS induced mastitis model in rats. Commencing at 10 d of gestation, retinoid (dissolved in soy oil) or an equal volume of soy 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-a 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 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 can adjust PMN activity, may be an effective tool for protecting mammary tissue against PMN-induced oxidative stress during LPS-induced acute mastitis.4 Optimized culture and inflammatory model establishment of rat mammary epithelial cellsIn this study, rat mammary epithelial cells were cultured and the inflammatory model was established. Primary culture of rat mammary epithelial cells (rMEC) were separated through digestion by collagenase and hyaluronidase, cells grown well in DMEM/F12 when supplemented with insulin, transferrin, epidermal growth factor (EGF), hydrocortisone and other nutritional factors. Cells were identified by cytokeratin andβ-casein, the results shown that the cell purity was up to 95%, and had perfect biological function. Stimulation of rMEC with LPS for 24 h elicited a marked increase in mRNA expression for TNF-α, IL-1βand inducible nitric oxide synthase (iNOS), the expression showed a dose-dependent manner from 1 to 10μg of LPS per ml,20μg LPS stimulation showed a slightly decrease in those genes expression due to the cell toxicity effect. Secretion of TNF-α, IL-1βand nitric oxide (NO) was also increased when induced by LPS, and with a maximal level when stimulated with 10μg of LPS per ml. In this study, we successfully established the inflammatory models of primary culture rat mammary epithelial cells which would be important in further investigation.5 Modulation and mechanism of retinoid in LPS-induced inflammation in primary rat mammary epithelial cellsIn order to study the protective mechanism of retinoid on mammary gland, LPS induced inflammatory model in rat mammary epithelial cells was used in this study. Primary culture of rat mammary epithelial cells were separated through digestion by collagenase and hyaluronidase, cells were pretreated with 1μmol/L retinoid before stimulation with 10μg/mL LPS. The result showed that TLR-4 protein expression and NF-κB DNA binding activity was significantly decreased in primary rMEC pretreated with 1μmol/L retinoid at 1 h post LPS stimulation. LPS stimulation significantly increased the TNF-α, IL-1β, cytokine-induced neutrophil chemoattractant (CINC)-1, iNOS and ICAM-1 gene expression, and retinoid treatment could down-regulate TNF-αgene expression at 2,4, 8,12 h, IL-1βat 2,4,8 h and 8,12 h iNOS, ICAM-1 gene expression. These findings demonstrate that direct action by retinoid leads to attenuate LPS-induced inflammatory responses by suppressing TLR-4/NF-κB signaling system, thereby providing a novel explanation for some of underlying effect proposed for retinoid in the protection of mammary tissue during LPS-induced acute mastitis.In conclusion, retinoid may protect the mammary gland by down-regulating TLR-4/NF-κB signaling pathway and inhibiting the over activation of PMN. The results suggest that retinoid can be used as a potential candidate to prevent mastitis.