乙酰乙酸抑制脂多糖诱导的奶牛中性粒细胞炎症信号通路的激活
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摘要
为研究乙酰乙酸(Acetoacetic acid,AcAc)对奶牛中性粒细胞炎症信号通路的影响,以脂多糖(Lipopolysaccharide,LPS)诱导的奶牛中性粒细胞炎症模型为研究对象,利用qRT-PCR、生化和酶联免疫吸附法(ELISA)测定IL-1β、IL-6、TNF-α和IKKβ激酶活性。结果表明:1)qRT-PCR结果显示,与对照组相比较,LPS处理组IL-1β、IL-6和TNF-αmRNA的表达显著增强(P<0.01)。与LPS处理组比较,LPS+AcAc混合处理组,其表达量显著下调(P<0.05);2)生化检测结果显示,与对照组相比较,LPS处理组中性粒细胞IKKβ激酶活性显著增强(P<0.01)。与LPS处理组比较,LPS+AcAc混合处理组IKKβ激酶活性则显著降低(P<0.05);3)ELISA结果显示,与对照组相比较,LPS增加促炎因子TNF-α、IL-6和IL-1β的释放。相对于LPS处理组,LPS+AcAc混合处理组促炎因子的释放则显著降低(P<0.01)。综上,AcAc可以抑制LPS诱导的奶牛中性粒细胞炎症信号通路的激活,具有一定的抗炎功能。
To study the effect of acetoacetic acid(AcAc) on neutrophil inflammatory signaling pathway in dairy cows,a cow neutrophil inflammation model induced by lipopolysaccharide(LPS) was used as the research object.qRT-PCR,biochemistry and enzyme-linked immunosorbent assay(ELISA) methods were adopted in this study.The results showed that:1) Compared with the control group,the qRT-PCR results showed that the mRNA expression levels of IL-1β,IL-6 and TNF-α were significantly increased in LPS treatment group(P<0.01),while their expression levels were significantly decreased in LPS + AcAc treated group than those in LPS treated group(P<0.05);2) Compared with the control group,the biochemical test results showed that the activity of IKKβ kinase was significantly increased in LPS treated group(P<0.01),Its activity was significantly decreased in LPS+AcAc treated group than that in LPS treated group(P<0.05);3) Compared with the control group,the ELISA results showed that LPS increased the release of pro-inflammatory factors TNF-α,IL-6 and IL-1β.These pro-inflammatory factors were significantly decreased in LPS+AcAc treated group than that in LPS treated group(P<0.01).The results suggested that AcAc inhibited the activation of inflammation signaling pathway in lipopolysaccharide-stimulated cow neutrophils and played anti-inflammatory function.
To study the effect of acetoacetic acid(AcAc) on neutrophil inflammatory signaling pathway in dairy cows,a cow neutrophil inflammation model induced by lipopolysaccharide(LPS) was used as the research object.qRT-PCR,biochemistry and enzyme-linked immunosorbent assay(ELISA) methods were adopted in this study.The results showed that:1) Compared with the control group,the qRT-PCR results showed that the mRNA expression levels of IL-1β,IL-6 and TNF-α were significantly increased in LPS treatment group(P<0.01),while their expression levels were significantly decreased in LPS + AcAc treated group than those in LPS treated group(P<0.05);2) Compared with the control group,the biochemical test results showed that the activity of IKKβ kinase was significantly increased in LPS treated group(P<0.01),Its activity was significantly decreased in LPS+AcAc treated group than that in LPS treated group(P<0.05);3) Compared with the control group,the ELISA results showed that LPS increased the release of pro-inflammatory factors TNF-α,IL-6 and IL-1β.These pro-inflammatory factors were significantly decreased in LPS+AcAc treated group than that in LPS treated group(P<0.01).The results suggested that AcAc inhibited the activation of inflammation signaling pathway in lipopolysaccharide-stimulated cow neutrophils and played anti-inflammatory function.
引文
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[19] Sordillo L M,Streicher K L.Mammary gland immunity and mastitis susceptibility[J].Journal of Mammary Gland Biology and Neoplasia,2002,7(2):135-146
[20] Aitken S L,Corl C M,Sordillo L M.Pro-inflammatory and pro-apoptotic responses of TNF-α stimulated bovine mammary endothelial cells[J].Veterinary Immunology and Immunopathology,2011,140(3-4):282-290
[21] Sordillo L M,Pighetti G M,Davis M R.Enhanced production of bovine tumor necrosis factor-α during the periparturient period[J].Veterinary Immunology and Immunopathology,1995,49(3):263-270
[22] Hu Y.Abnormal morphogenesis but intact IKK activation in mice lacking the IKKα subunit of IκB kinase[J].Science,1999,284(5412):316-320
[23] Li Q.Severe liver degeneration in mice lacking the IκB kinase 2 gene[J].Science,1999,284(5412):321-325
[24] Ruocco M G,Maeda S,Park J M,Lawrence T,Hsu L C,Cao Y X,Schett G,Wagner E F,Karin M.IκB kinase (IKK)β,but not IKKα,is a critical mediator of osteoclast survival and is required for inflammation-induced bone loss[J].Journal of Experimental Medicine,2005,201(10):1677-1687
[2] Holtenius P,Holtenius K.New aspects of ketone bodies in energy metabolism of dairy cows:A review[J].Journal of Veterinary Medicine Series A,1996,43(10):579-587
[3] Hammon D S,Evjen I M,Dhiman T R,Goff J P,Walters J L.Neutrophil function and energy status in Holstein cows with uterine health disorders[J].Veterinary Immunology and Immunopathology,2006,113(1-2):21-29
[4] Jain S K,Kannan K,Lim G,Matthews-Greer J,McVie R,Bocchini J A.Elevated blood interleukin-6 levels in hyperketonemic type 1 diabetic patients and secretion by acetoacetate-treated cultured U937 monocytes[J].Diabetes Care,2003,26(7):2139-2143
[5] Loor J J,Everts R E,Bionaz M,Dann H M,Morin D E,Oliveira R,Rodriguez-Zas S L,Drackley J K,Lewin H A.Nutrition-induced ketosis alters metabolic and signaling gene networks in liver of periparturient dairy cows[J].Physiological Genomics,2007,32(1):105-116
[6] Zhou G X,Liu Z.Potential roles of neutrophils in regulating intestinal mucosal inflammation of inflammatory bowel disease[J].Journal of Digestive Diseases,2017,18(9):495-503
[7] Fu S P,Li S N,Wang J F,Li Y,Xie S S,Xue W J,Liu H M,Huang B X,Lv Q K,Lei L C,Liu G W,Wang W,Liu J X.BHBA suppresses LPS-induced inflammation in BV-2 cells by inhibiting NF-κB activation[J].Mediators of Inflammation,2014,2014(2):983401
[8] 李建,王银龙,刘强,木尔扎提,桂光磊,赛务加甫。酮病奶牛乳液与血液中β-羟丁酸浓度的相关性试验[J].中国兽医杂志,2015,51(1):17-19Li J,Wang Y L,Liu Q,Mu E Z T,Gui G L,Sai W J F.The study of relationship of the concentration of β-hydroxybutyric acid in milk with that in serum from ketotic cows[J].Chinese Journal of Veterinary Medicine,2015,51(1):17-19 (in Chinese)
[9] Jain S K,McVie R.Hyperketonemia can increase lipid peroxidation and lower glutathione levels in human erythrocytes in vitro and in type 1 diabetic patients[J].Diabetes,1999,48(9):1850-1855
[10] 张玉明,雷林,蒋兴华,杜希良,高文文,邓清华,王哲,李心慰,刘国文.NEFA对体外培养奶牛中性粒细胞TLR2/4介导的炎症信号通路的影响[J].中国兽医学报,2015,35(4):626-630Zhang Y M,Lei L,Jiang X H,Du X L,Gao W W,Deng Q H,Wang Z,Li X W,Liu G W.Effect of high concentrations of NEFA on TLR2/4-mediated inflammatory signaling pathway in dairy cattle neutrophils cultured in vitro[J].Chinese Journal of Veterinary Science,2015,35(4):626-630 (in Chinese)
[11] Abdelmegeed M A.Acetoacetate activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase in primary cultured rat hepatocytes:Role of oxidative stress[J].Journal of Pharmacology and Experimental Therapeutics,2004,310(2):728-736
[12] Jain S K,McVie R.Hyperketonemia can increase lipid peroxidation and lower glutathione levels in human erythrocytes in vitro and in type 1 diabetic patients[J].Diabetes,1999,48(9):1850-1855
[13] Atabai K,Matthay M A.The pulmonary physician in critical care 5:Acute lung injury and the acute respiratory distress syndrome:Definitions and epidemiology[J].Thorax,2002,57(5):452-458
[14] Shi J J,Zhao Y,Wang Y P,Gao W Q,Ding J J,Li P,Hu L Y,Shao F.Inflammatory caspases are innate immune receptors for intracellular LPS[J].Nature,2014,514(7521):187-192
[15] Ibeagha-Awemu E M,Lee J W,Ibeagha A E,Bannerman D D,Paape M J,Zhao X.Bacterial lipopolysaccharide induces increased expression of toll-like receptor (TLR) 4 and downstream TLR signaling molecules in bovine mammary epithelial cells[J].Veterinary Research,2008,39(2):11
[16] Li Y,Ding H Y,Wang X C,Liu L,Huang D,Zhang R H,Guo L H,Wang Z,Li X B,Liu G W,Wu J J,Li X W.High levels of acetoacetate and glucose increase expression of cytokines in bovine hepatocytes,through activation of the NF-κB signalling pathway[J].Journal of Dairy Research,2016,83(1):51-57
[17] Baeuerle P A,Baichwal V R.NF-κB as a frequent target for immunosuppressive and anti-inflammatory molecules[J].Advances in Immunology,1997,65:111-137
[18] Hoshino K,Takeuchi O,Kawai T;Sanjo H,Ogawa T,Takeda Y,Takeda K,Akira S.Cutting edge:Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide:evidence for TLR4 as the Lps gene product[J].Journal of Immunology,1999,162(7):3749-3752
[19] Sordillo L M,Streicher K L.Mammary gland immunity and mastitis susceptibility[J].Journal of Mammary Gland Biology and Neoplasia,2002,7(2):135-146
[20] Aitken S L,Corl C M,Sordillo L M.Pro-inflammatory and pro-apoptotic responses of TNF-α stimulated bovine mammary endothelial cells[J].Veterinary Immunology and Immunopathology,2011,140(3-4):282-290
[21] Sordillo L M,Pighetti G M,Davis M R.Enhanced production of bovine tumor necrosis factor-α during the periparturient period[J].Veterinary Immunology and Immunopathology,1995,49(3):263-270
[22] Hu Y.Abnormal morphogenesis but intact IKK activation in mice lacking the IKKα subunit of IκB kinase[J].Science,1999,284(5412):316-320
[23] Li Q.Severe liver degeneration in mice lacking the IκB kinase 2 gene[J].Science,1999,284(5412):321-325
[24] Ruocco M G,Maeda S,Park J M,Lawrence T,Hsu L C,Cao Y X,Schett G,Wagner E F,Karin M.IκB kinase (IKK)β,but not IKKα,is a critical mediator of osteoclast survival and is required for inflammation-induced bone loss[J].Journal of Experimental Medicine,2005,201(10):1677-1687
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