甘氨酸对奶山羊乳腺炎性应答的调节及其作用机制研究
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摘要
本试验在体内条件下研究了甘氨酸对奶山羊乳腺炎性应答的调节及其信号通路的变化。选用15只泌乳期经产健康萨能奶山羊,分为对照组、脂多糖(LPS)模型组和3个甘氨酸干预组,每组3只,饲养管理程序一致。对照组奶山羊不注射LPS和甘氨酸,以1 mL生理盐水取代; LPS模型组奶山羊于左右乳区通过乳头管分别注射1 mL LPS溶液(4μg/乳区); 3个甘氨酸干预组奶山羊分别按照1、5、25 g/(只·d)的剂量,于左右乳区通过乳头管分别注射0.5 mL甘氨酸,连续注射5 d,第6天在左右乳区通过乳头管注射1 mL LPS(4μg/乳区)。各组奶山羊在注射LPS或生理盐水24 h后屠宰取乳腺组织样。制作乳腺组织切片,苏木精-伊红(HE)染色后用于观察病理变化;采用实时定量PCR法检测乳腺组织中白细胞介素(IL)-1β、IL-6、IL-8、肿瘤坏死因子-α(TNF-α)、环氧合酶-2(COX-2)、Toll样受体(TLR) 2、TLR4、TLR9、髓样分化因子88(MyD88)、β-干扰素TIR结构域衔接蛋白(TRIF)基因的表达水平;采用Western blotting法检测乳腺组织中核转录因子-κB(NF-κB) p65蛋白的表达水平。结果显示:注射LPS 24 h后,3个甘氨酸干预组试验羊体温降至39.5℃以下。与LPS模型组相比,3个甘氨酸干预组炎性细胞侵润明显减少,乳腺细胞坏死程度明显减轻;高、中、低剂量甘氨酸干预均显著下调了IL-1β、IL-8和COX-2基因的表达水平(P<0.05),只有中剂量甘氨酸干预显著下调了IL-6、TNF-α基因的表达水平(P <0.05);高、中、低剂量甘氨酸干预均显著下调了TLR4基因的表达水平(P <0.05),只有中剂量甘氨酸干预显著下调了TLR2、TLR9基因的表达水平(P <0.05);高、中、低剂量甘氨酸干预均显著下调了MyD88基因的表达水平(P<0.05),同时低、高剂量甘氨酸干预还显著下调了TRIF基因的表达水平(P<0.05);高、中、低剂量甘氨酸干预均显著下调了核转录因子NF-κB p65蛋白的表达水平(P<0.05)。综上可知:1)甘氨酸通过下调TLR4及其下游关键信号分子MyD88和TRIF基因的表达,抑制转录因子NF-κB p65磷酸化,从而减少炎性因子IL-1β、IL-6、IL-8、TNF-α、COX-2的分泌。2)本研究初步阐明了甘氨酸通过抑制TLR-NF-κB信号通路从而抑制炎性细胞的激活或活性,阻止其炎症介质的释放,进而阻碍炎症反应过程实现。
This experiment was conducted to investigate the regulation of glycine on mammary inflammatory response in dairy goats in vivo and its signaling pathway changes. Fifteen healthy lactating Saanen dairy goats with similar body weight,lactation period and milk yield were selected and the goats were randomly divided into 5 groups with 3 goats in each group. Dairy goats in lipopolysaccharide( LPS) model group were infused1 mL LPS solution( 4 μg/mL) via each udder; dairy goats in glycine intervention groups were infused three doses [1,5 and 25 g/( goat · d) ] of glycine separately which lasting 5 days,and 1 mL LPS solution( 4 μg/mL) via each udder were infused on the sixth day; dairy goats in control group were infused without glycine and LPS,but were infused 1 mL normal saline. Dairy goats in each group were slaughtered and sampling 24 h after LPS or normal saline infusion. Real time quantitative PCR method was used to assay the gene expression levels of interleukin( IL)-1β,IL-6,IL-8,tumor necrosis factor-α( TNF-α),cyclooxygenase-2( COX-2),Toll like receptor( TLR) 2,TLR4,TLR9,myeloid differentiation factor 88( MyD88) and TIR-domain-containing adapter-inducing interferon-β( TRIF) in mammary tissue,the Western blotting method was used to detect the protein expression level of nuclear transcription factor-κB( NF-κB) p65 in mammary tissue,and the mammary tissue section and hematoxylin-eosin( HE) staining was used to observe the changes of histopathology. The results showed that the goats' body temperature of the glycine intervention groups decreasedbelow39.5 ℃ at 24 h after LPS infusion. Compared with LPS model group,the inflammatory cell invasion and necrosis of mammary gland cells were obviously reduced in the 3 glycine intervention groups; the gene expression levels of IL-1β,IL-8 and COX-2 were significantly decreased in the low,middle and high dose glycine intervention groups( P<0.05),and the gene expression levels of IL-6 and TNF-α were significantly decreased in middle dose glycine intervention group( P < 0.05); the gene expression level of TLR4 was significantly decreased in the low,middle and high dose glycine intervention groups( P<0.05),and the gene expression levels of TLR2 and TLR9 were significantly decreased in middle dose glycine intervention group( P <0.05); the gene expression level of MyD88 was significantly decreased in the low,middle and high dose glycine intervention groups( P<0.05),and the gene expression level of TRIF was significantly decreased in lowand high dose glycine intervention groups( P<0.05); the protein expression level of NF-κB p65 was significantly decreased in the low,middle and high dose glycine intervention groups( P<0.05). The results demonstrate that glycine reduces the secretion of inflammatory factors IL-1β,IL-6,IL-8,TNF-α and COX-2 by down-regulating the gene expression of TLR4 and its downstream key signaling molecules MyD88 and TRIF,and inhibiting the phosphorylation of nuclear transcription factor NF-κB p65. By the results,we preliminarily illuminate that glycine inhibits the activation or activity of inflammatory cells by inhibiting TLR-NF-κB signaling pathway,and prevents the potential inflammatory mediators releasing,such as toxic cytokines and prostaglandins,thus hinders the realization of inflammatory response.[Chinese Journal of Animal Nutrition,2019,31(7):3110-3122]
This experiment was conducted to investigate the regulation of glycine on mammary inflammatory response in dairy goats in vivo and its signaling pathway changes. Fifteen healthy lactating Saanen dairy goats with similar body weight,lactation period and milk yield were selected and the goats were randomly divided into 5 groups with 3 goats in each group. Dairy goats in lipopolysaccharide( LPS) model group were infused1 mL LPS solution( 4 μg/mL) via each udder; dairy goats in glycine intervention groups were infused three doses [1,5 and 25 g/( goat · d) ] of glycine separately which lasting 5 days,and 1 mL LPS solution( 4 μg/mL) via each udder were infused on the sixth day; dairy goats in control group were infused without glycine and LPS,but were infused 1 mL normal saline. Dairy goats in each group were slaughtered and sampling 24 h after LPS or normal saline infusion. Real time quantitative PCR method was used to assay the gene expression levels of interleukin( IL)-1β,IL-6,IL-8,tumor necrosis factor-α( TNF-α),cyclooxygenase-2( COX-2),Toll like receptor( TLR) 2,TLR4,TLR9,myeloid differentiation factor 88( MyD88) and TIR-domain-containing adapter-inducing interferon-β( TRIF) in mammary tissue,the Western blotting method was used to detect the protein expression level of nuclear transcription factor-κB( NF-κB) p65 in mammary tissue,and the mammary tissue section and hematoxylin-eosin( HE) staining was used to observe the changes of histopathology. The results showed that the goats' body temperature of the glycine intervention groups decreasedbelow39.5 ℃ at 24 h after LPS infusion. Compared with LPS model group,the inflammatory cell invasion and necrosis of mammary gland cells were obviously reduced in the 3 glycine intervention groups; the gene expression levels of IL-1β,IL-8 and COX-2 were significantly decreased in the low,middle and high dose glycine intervention groups( P<0.05),and the gene expression levels of IL-6 and TNF-α were significantly decreased in middle dose glycine intervention group( P < 0.05); the gene expression level of TLR4 was significantly decreased in the low,middle and high dose glycine intervention groups( P<0.05),and the gene expression levels of TLR2 and TLR9 were significantly decreased in middle dose glycine intervention group( P <0.05); the gene expression level of MyD88 was significantly decreased in the low,middle and high dose glycine intervention groups( P<0.05),and the gene expression level of TRIF was significantly decreased in lowand high dose glycine intervention groups( P<0.05); the protein expression level of NF-κB p65 was significantly decreased in the low,middle and high dose glycine intervention groups( P<0.05). The results demonstrate that glycine reduces the secretion of inflammatory factors IL-1β,IL-6,IL-8,TNF-α and COX-2 by down-regulating the gene expression of TLR4 and its downstream key signaling molecules MyD88 and TRIF,and inhibiting the phosphorylation of nuclear transcription factor NF-κB p65. By the results,we preliminarily illuminate that glycine inhibits the activation or activity of inflammatory cells by inhibiting TLR-NF-κB signaling pathway,and prevents the potential inflammatory mediators releasing,such as toxic cytokines and prostaglandins,thus hinders the realization of inflammatory response.[Chinese Journal of Animal Nutrition,2019,31(7):3110-3122]
引文
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[3]SENTHILKUMAR R,SENGOTTUVELAN M,NALINI N.Protective effect of glycine supplementation on the levels of lipid peroxidation and antioxidant enzymes in the erythrocyte of rats with alcohol-induced liver injury[J].Cell Biochemistry and Function,2004,22(2):123-128.
[4]SREEKUMAR A,POISSON L M,RAJENDIRAN TM,et al.Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression[J].Nature,2009,457(7231):910-914.
[5]WANG J,ALEXANDER P,WU L,et al.Dependence of mouse embryonic stem cells on threonine catabolism[J].Science,2009,325(5939):435-439.
[6]HOWARD A,TAHIR I,JAVED S,et al.Glycine transporter GLYT1 is essential for glycine-mediated protection of human intestinal epithelial cells against oxidative damage[J].The Journal of Physiology,2010,588(6):995-1009.
[7]LOCASALE J W,GRASSIAN A R,MELMAN T,et al.Phosphoglycerate dehydrogenase diverts glycolytic flux and contributes to oncogenesis[J].Nature Genetics,2011,43(9):869-874.
[8]ZHANG W C,SHYH-CHANG N,YANG H,et al.Glycine decarboxylase activity drives non-small cell lung cancer tumor-initiating cells and tumorigenesis[J].Cell,2012,148(1/2):259-272.
[9]WHEELER MD,THURMAN R G.Production of superoxide and TNF-αfrom alveolar macrophages is blunted by glycine[J].The American Journal of Physiology:Lung Cellular and Molecular Physiology,1999,277(5):L952-L959.
[10]WU H W,YUN K M,HAN D W,et al.Effects of glycine on phagocytosis and secretion by Kupffer cells in vitro[J].World Journal of Gastroenterology,2012,18(20):2576-2581.
[11]FROH M,THURMAN R G,WHEELER MD.Molecular evidence for a glycine-gated chloride channel in macrophages and leukocytes[J].American Journal of Physiology:Gastrointestinal and Liver Physiology,2002,283(4):G856-G863.
[12]QI R B,ZHANG J Y,LU D X,et al.Glycine receptors contribute to cytoprotection of glycine in myocardial cells[J].Chinese Medical Journal,2007,120(10):915-921.
[13]张兴夫.不同日粮模式对泌乳奶牛乳腺乳蛋白合成影响的研究[D].博士学位论文.呼和浩特:内蒙古农业大学,2013.
[14]NRC.Nutrient requirements of goat:angora,dairy,and meat goats in temperate and tropical countries[S].Washington,D.C.:National Academy Press,1981
[15]金公亮.奶山羊饲养标准[J].畜牧兽医杂志,1989(2):7-12.
[16]威廉·C雷布汉.奶牛疾病学[M].赵德明,沈建忠,译.北京:中国农业大学出版社,1999
[17]BRADLEY A J.Bovine mastitis:an evolving disease[J].The Veterinary Journal,2002,164(2):116-128.
[18]BLUMS,SELA S,HAMMER-MUNTZ O,et al.Identification of adaptive traits of bovine mastitis Escherichia coli strains[J].Veterinary Immunology and Immunopathology,2009,128(1/2/3):262.
[19]BURTON J L,ERSKINE R J.Immunity and mastitis.Some newideas for an old disease[J].The Veterinary Clinics of North America:Food Animal Practice,2003,19(1):1-45.
[20]LORD P C,WILMOTH L M,MIZEL S B,et al.Expression of interleukin-1 alpha and beta genes by human blood polymorphonuclear leukocytes[J].The Journal of Clinical Investigation,1991,87(4):1312-1321.
[21]HEINRICH P C,CASTELL J V,ANDUS T.Interleukin-6 and the acute phase response[J].Biochemical Journal,1990,265(3):621-636.
[22]PALTER S F,MULAYIMN,SENTURK L,et al.Interleukin-8 in the human fallopian tube[J].The Journal of Clinical Endocrinology&Metabolism,2001,86(6):2660-2667.
[23]LAHOUASSA H,MOUSSAY E,RAINARD P,et al.Differential cytokine and chemokine responses of bovine mammary epithelial cells to Staphylococcus aureus and Escherichia coli[J].Cytokine,2007,38(1):12-21.
[24]HATA A N,BREYER R M.Pharmacology and signaling of prostaglandin receptors:multiple roles in inflammation and immune modulation[J].Pharmacology&Therapeutics,2004,103(2):147-166.
[25]TSUJIMOTO T,KAWARATANI H,KITAZAWA T,et al.Decreased phagocytic activity of Kupffer cells in a rat nonalcoholic steatohepatitis model[J].World Journal of Gastroenterology,2008,14(39):6036-6043.
[26]ALARCON-AGUILAR F J,ALMANZA-PEREZ J,BLANCAS G,et al.Glycine regulates the production of pro-inflammatory cytokines in lean and monosodium glutamate-obese mice[J].European Journal of Pharmacology,2008,599(1/2/3):152-158.
[27]WANG G,WANG Y,GUAN F L,et al.The effect of combination of glycine and methylprednisolone on Kupffer cells of liver after hemorrhagic shock in rats[J].Chinese Journal of Surgery,2006,44(5):349-352.
[28]GARCIA-MACEDO R,SANCHEZ-MUOZ F,AL-MANZA-PEREZ J C,et al.Glycine increases mRNAadiponectin and diminishes pro-inflammatory adipokines expression in 3T3-L1 cells[J].European Journal of Pharmacology,2008,587(1/2/3):317-321.
[29]SPITTLER A,REISSNER C M,OEHLER R,et al.Immunomodulatory effects of glycine on LPS-treated monocytes:reduced TNF-αproduction and accelerated IL-10 expression[J].The FASEB Journal,1999,13(3):563-571.
[30]ALVARADO-VSQUEZ N,ZAMUDIO P,CERNE,et al.Effect of glycine in streptozotocin-induced diabetic rats[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology,2003,134(4):521-527.
[31]WHEELER MD,ROSE ML,YAMASHIMA S,et al.Dietary glycine blunts lung inflammatory cell influx following acute endotoxin[J].American Journal of Physiology:Lung Cellular and Molecular Physiology,2000,279(2):L390-L398.
[32]IKEJIMA K,IIMURO Y,FORMAN D T,et al.A diet containing glycine improves survival in endotoxin shock in the rat[J].American Journal of PhysiologyGastrointestinal and Liver Physiology,1996,271(1):G97-G103.
[33]TSUNE I,IKEJIMA K,HIROSE M,et al.Dietary glycine prevents chemical-induced experimental colitis in the rat[J].Gastroenterology,2003,125(3):775-785.
[34]KONASHI S,TAKAHASH K,AKIBA Y.Effects of dietary essential amino acid deficiencies on immunological variables in broiler chickens[J].British Journal of Nutrition,2000,83(4):449-456.
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