| In this study, effects of β-glucan on mammary defense and the protective mechanism of β-glucan 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 β-glucan. The results were as follows:1Dectin1activation of β-glucan produces an anti-mastitis effect in the ratIn order to study the protective effect of β-glucan on rat experimental acute mastitis, on gestation day10, β-glucan was administered to rats daily by gavage at either0(control),0.1mg/kg·d,1mg/kg·d and10mg/kg·d until parturition. lipopolysaccharide (LPS) or pyrogen-free physiological saline was inoculated into the mammary gland72h after parturition and the rats were euthanized at12h post-infection. In comparison with negative control, LPS significantly increased dectinl (P<0.05) and toll-like receptor4(TLR4)(P<0.05) mRNA and protein expression in mammary tissues. Tumor necrosis factor (TNF)-α (P <0.05), interleukin (IL)-1β(P<0.05), N-acetyl-β-D-glucose-minidase (NAGase)(P<0.05), inducible nitric oxide synthase (iNOs)(P<0.05) in mammary tissues and serum, and myeloperoxidase (MPO)(P<0.05) in mammary tissues were significantly increased12h after LPS infusion. By comparison with positive control, P-glucan administration could enhance Dectin1(P<0.05) mRNA and protein expression while down regulate the expression of TLR4(P<0.05). Level of TNF-α (P<0.05), IL-1β (P<0.05) in mammary tissues and serum, MPO (P<0.05), NAGase (P<0.05) and iNOs (P<0.05) activity in mammary tissues was decreased, but the level of IL-2(P<0.05) in serum was increased by β-glucan. Histology of mammary gland showed β-glucan reduced the count of PMN and recovered the secretion of milk. Overall, the results indicate that β-glucan may protect mammary tissue against LPS-induced rat acute mastitis.2Auto-defense and protective effects of β-glucan in rat mammary epithelial cells on lipopolysaccharide inductionThe protective effects of β-glucan on lipopolysaccharide (LPS) induced inflammatory responses in mammary epithelial cells were quantifed using primary cultured rat mammary epithelial cells. Rat mammary epithelial cells were treated with β-glucan (final concentration0,1,5,25μg/mL) dissolved in DMSO for12h, followed by1μg/mL LPS for40min. Negative control were treated with DMSO at all times. In comparison with negative control, LPS significantly enhanced CD14(P<0.05), MD2(P<0.05) mRNA expression, increased TLR4(P<0.05) mRNA and protein expression, increased MyD88(P <0.05) and dectinl (P<0.05) protein expression, decreased IκBα (P<0.05) and β-casein (P<0.05) protein expression, increased NF-κB binding DNA activity (P<0.05), increased tumor necrosis factor-a (TNF-a)(P<0.05), interleukin-1p (IL-1β)(P<0.05) secretion after LPS infusion for40min. By comparison with positive control, pretreated with all doses group of β-glucan could decrease CD14(P<0.05), MD2(P<0.05) mRNA expression, β-glucan (PL.PH) suppressed the protein expression of TLR4(P<0.05) and MyD88(P<0.05), all doses group of P-glucan enhanced the mRNA and protein expression of Dectinl(P <0.05), and increased Syk(P<0.05), β-casein(P<0.05) protein expression, β-glucan (PL,PH) increased IκBα(P<0.05) protein expression but suppressed NF-κB binding DNA activity (P<0.05). All doses group of β-glucan could decrease TNF-α (P<0.05), IL-1β (P <0.05) secretion. While LPS up-regulated the signal pathway of TLR4/MyD88/IκBα(-)/NF-KB, β-casein could protect the P-casein secretion and decrease the level of TNF-a, IL-1β from rat mammary epithelial cells with dose-dependant, via up-regulating the signal pathway of Dectinl/Syk/1κBα(-) and down-regulating the signal pathway of TLR4/MyD88/IκBα(-) for suppressed NF-κB binding DNA activity.3Effects of β-glucan regulating on associated proteins in auto-defensive signal pathway with time-course in rat mammary epithelial cellsThe signal pathways of β-glucan on lipopolysaccharide (LPS) induced inflammatory responses in mammary epithelial cells were examined using primary cultured rat mammary epithelial cells with time-dependant. Rat mammary epithelial cells were treated with P-glucan at final concentration5μg/mL dissolved in DMSO while control group were treated with DMSO for12h, then all cells were infused with10μg/mL LPS for Oh,0.5h and1h. In comparison with Oh control group, LPS significantly enhanced the protein expression of TLR4, MyD88and Syk at0.5h (P<0.05),1h (P<0.05), decreased the protein expression of IκBα at0.5h (P<0.01),1h (P<0.01), enhanced NF-κB binding DNA activity at0.5h (P<0.01),1h (P<0.01), increased the level of TNF-α at0.5h (P <0.05),1h (P<0.05) in rat mammary epithelial cells. By comparison with corresponding time control group, pretreated with β-glucan could decreased the protein expression of MyD88at0.5h (P<0.05),1h (P<0.05) increased the protein expression of Dectin1at0.5h (P<0.05), increased the protein expression of Syk,IκBα at0.5h (P<0.05),1h (P <0.05), suppressed NF-κB binding DNA activity at0.5h (P<0.01),1h (P<0.01) decreased the level of TNF-α at0.5h (P<0.05),1h (P<0.05) in rat mammary epithelial cells. Additionally, LPS enhanced the immunofluorescence of TLR-4at0.5h compared to Oh control group, and pretreated with β-glucan increased the immunofluorescence of Dectinl at0.5h compared with corresponding time control group in rat mammary epithelial cells. While LPS up-regulated the signal pathway of TLR4/MyD88/IκBα(-)/NF-κB, P-casein could decrease the level of TNF-a via up-regulating the signal pathway of Dectinl/Syk/IκBα(-) and antagonizing the signal protein MyD88for suppressing NF-κB binding DNA activity in rat mammary epithelial cells.4Mechanism of β-glucan regulating on TLR4and Dectinl signal pathway in rat mammary epithelial cellsInhibition pretreatment with TLR4, Syk and NF-κB in rat mammary epithelial cells respectively, the effects and signal pathway of infusing P-glucan and LPS with cells were examined. Pretreatment with5μmol/L TLR4inhibitor,150nmol/L Syk inhibitor and10μmol/L NF-κB inhibitor in rat mammary epithelial cells for2h respectively. All cells were treated with DMSO(control group),5μg/mL β-glucan(treatment group1), DMSO (treatment group2) and5μg/mL β-glucan (treatment group3) for12h, then treated with DMSO(control group), DMSO (treatment group1),10μg/mL LPS(treatment group2) and10μg/mL LPS(treatment group3) for Oh,0.5h,1h and2h.In rat mammary epithelial cells of inhibition TLR4, by comparison with Oh control group, treatment group1increased the protein expression of Dectinlat Oh (P<0.05),0.5h (P<0.05),1h (P<0.05) and Syk (P<0.05) at all times, and these two protein expressions peaked at1h together. Treatment group2increased the protein expression of TLR4(P<0.05) and MyD88(P<0.05) at all times, and TLR4peaked at0.5h but MyD88peaked at1h. Treatment group2also decreased IκBα at Oh (P<0.05) and0.5h (P<0.05), enhanced NF-κB binding DNA activity (P<0.01) at all times, increased the level of TNF-α 0.5h (P<0.05),1h (P<0.05) and2h (P<0.05), enhanced NF-κB binding DNA activity (P<0.05) at all times. In comparison with treatment group2at the same time, treatment group1increased the protein expression of Dectin1at Oh (P<0.05),0.5h (P<0.05),1h (P<0.05) and Syk (P<0.05) at all times, decreased the protein expression of TLR4(P <0.05) and MyD88(P<0.05) at all times, increased IκBα at Oh (P<0.05) and0.5h (P <0.05), attenuated NF-κB binding DNA activity (P<0.01)at all times, decreased the level of TNF-α at0.5h (P<0.05),1h (P<0.05),2h (P<0.05) and IL-1βat1h (P<0.05) Treatment group3increased the protein expression of Syk at Oh (P<0.05),1h (P<0.05),2h (P<0.05) and Dectin1(P<0.05) at all times, attenuated NF-κB binding DNA activity at Oh (P<0.05),2h (P<0.05), others were the same as treatment group1.In rat mammary epithelial cells of inhibition Syk, by comparison with Oh control group, treatment group1increased the protein expression of Syk at1h (P<0.05),2h (P<0.05) and Dectin1(P<0.05) at all times. Treatment group2increased the protein expression of TLR4(P<0.05) at all times, and MyD88at0.5h (P<0.05), also decreased IκBα at2h (P<0.05), enhanced NF-κB binding DNA activity at0.5h (P<0.01),1h (P <0.01),2h (P<0.01) increased the level of TNF-α at0.5h (P<0.05),1h (P<0.05) and IL-1β at0.5h (P<0.05). Treatment group3increased the protein expression of Dectin1(P<0.05) at all times, increased Syk at1h (P<0.05)and2h (P<0.05), enhanced NF-κB binding DNA activity at0.5h (P<0.01),1h (P<0.01),2h (P<0.01).In comparison with treatment group2at the same time, treatment group1increased the protein expression of Syk at1h (P<0.05),2h (P<0.05) and Dectinl (P<0.05) at all times, decreased the protein expression of TLR4(P<0.05) at all times and MyD88at0.5h (P<0.05), increased IκBα at2h (P<0.05), attenuated NF-κB binding DNA activity at0.5h (P<0.01),1h (P<0.01),2h (P<0.01), decreased the level of TNF-a at0.5h (P<0.05),1h (P<0.05) and IL-1β at0.5h (P<0.05). Treatment group3decreased the protein expression of MyD88at0.5h(P>0.05), attenuated NF-κB binding DNA activity at0.5h (P>0.05),1h (P>0.05),2h (P>0.05), others were the same as treatment group1.In rat mammary epithelial cells of inhibition NF-κB, by comparison with Oh control group, treatment group1increased the protein expression of Dectinl and Syk at Oh (P<0.05),0.5h (P<0.05),1h (P<0.01), and these two protein expressions peaked at1h together. Treatment group2increased the protein expression of TLR4(P<0.05) and MyD88(P<0.05) at all times, and TLR4peaked at0.5h but MyD88peaked at1h. Treatment group2also decreased IκBα at1h(P<0.05)and2h(P<0.05), enhanced NF-κB binding DNA activity at0.5h (P<0.05),1h (P<0.01).increased the level of TNF-α at0.5h (P<0.05),1h (P<0.05),2h (P<0.05) and IL-1βat0.5h (P<0.05),1h (P<0.05). Treatment group3increased the protein expression of Syk (P<0.05) at all times, increased Dectin1at Oh (P<0.05),0.5h (P<0.05) and1h (P<0.05), enhanced NF-κB binding DNA activity at0.5h (P<0.05),1h (P<0.05) and2h (P<0.05). In comparison with treatment group2at the same time, treatment group1increased the protein expression of Dectinlat Oh (P<0.05),0.5h (P<0.05),1h (P<0.05) and Syk at1h (P<0.05), decreased the protein expression ofTLR4at Oh (P<0.05),0.5h (P<0.05) and2h (P<0.05) and MyD88(P<0.05) at all times, increased IκBα at0.5h (P<0.05),1h (P<0.05) and2h (P<0.05), attenuated NF-κB binding DNA activity at0.5h (P<0.05),1h (P<0.05), decreased the level of TNF-a at0.5h (P<0.05),1h (P<0.05),2h (P<0.05) and IL-1β at0.5h (P<0.05),1h (P<0.05). Treatment group3increased the protein expression of Syk at2h (P<0.05), decreased the protein expression of TLR4and MyD88at0.5h (P<0.05),1h (P<0.05) and2h (P<0.05), increased IκBα at0.5h (P<0.05),2h (P<0.05), activated NF-κB binding DNA activity at1h (P<0.05),2h (P<0.05), decreased the level of TNF-α at0.5h (P<0.05),1h (P<0.05),2h (P<0.05) and IL-1β at0.5h (P<0.05),1h (P<0.05)The Dectinl/Syk signal way and partial activity of NF-κB were not inhibited using TLR4inhibitor in rat mammary epithelial cells. The activity of NF-κB and TLR4/MyD88signal way were not inhibited using Syk inhibitor in rat mammary epithelial cells. The TLR4/MyD88and Dectinl/Syk signal way were not inhibited using NF-κB inhibitor in rat mammary epithelial cells. In conclusion, β-glucan alone could activate the Dectinl/Syk signal way, LPS alone could activate the TLR4/MyD88signal way or both P-glucan and LPS could activate the Dectinl/Syk via antaganizing TLR4/MyD88while these corresponding three inhibitors was pretreated with rat mammary epithelial cells. |
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