苜蓿黄酮对脂多糖刺激下乳成分合成相关基因表达的影响
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摘要
为探索苜蓿黄酮对脂多糖(LPS)刺激下乳成分合成相关基因表达的影响,采用体外细胞培养技术,将奶牛乳腺上皮细胞分成4个组:1)基础培养基(Con);2)基础培养基中加入1μg/mL的LPS(L);3)基础培养基中加入1μg/mL的LPS和75μg/mL苜蓿黄酮(L+F);4)基础培养基中加入75μg/mL苜蓿黄酮(F)。细胞在37℃,5%CO2的培养箱中培养12h后,对其乳成分合成相关基因的表达进行测定。结果表明:1)相对于对照组,L组和L+F组的酪氨酸激酶(JAK2)表达显著降低(P<0.01),信号转导子和转录激活子(STAT5)和真核起始因子4E(eIF4E)表达显著升高(P<0.05)。L+F组真核细胞始动因子4E结合蛋白1(4EBP1)的表达显著高于F组(P<0.01),核糖体S6蛋白激酶1(S6K1)的表达显著高于L组和对照组(P<0.05)。2)F组胆固醇调节元件结合蛋白1(SREBP1)的表达显著低于L组(P<0.05),过氧化物酶体增殖物激活受体γ(PPAR-γ)的表达则相反;F组脂肪酸转运蛋白1(FATP1)和脂肪酸转运蛋白4(FATP4)的表达显著低于L和L+F组(P<0.01),脂肪酸结合蛋白3(FABP3)和乙酰辅酶A羧化酶(ACACA)的表达则相反;对照组硬脂酰辅酶A去饱和酶1(SCD1)的表达显著高于其他各组(P<0.01)。3)L+F组葡萄糖转运蛋白1(Glut1)(P<0.05)、葡萄糖转运蛋白4(Glut4)(P<0.05)和葡萄糖转运蛋白8(Glut8)(P<0.01)的表达显著高于对照组,F组己糖激酶2(HK2)的表达显著低于L组和L+F组(P<0.05),而β-1,4-半乳糖基转移酶(β-1,4-Gal T)的表达最低。因此,在无LPS刺激下,添加苜蓿黄酮可能会抑制乳脂合成;在LPS刺激下,添加苜蓿黄酮可能会促进乳蛋白和乳糖的合成。
The aim of this study was to examine the effect of alfalfa flavonoids(AF)on the gene expression of milk component synthesis in bovine mammary epithelial cells(BMECs)induced by lipopolysaccharide(LPS).BMECs were cultured on media without LPS+AF(Con),and with 1μg/mL LPS(L),1μg/mL LPS+75μg/mL AF(L+F),75μg/mL AF(F)at 37℃ with 5% CO2 for 12 h.The expression of gene related to milk component synthesis was evaluated.The results showed:1)Compared with the control group,the L and L+F treatment groups showed significant decrease in JAK2 expression(P<0.01),but significant increase in the expressions of STAT5 and eIF4 E(P<0.01).The relative expressions of 4 EBP1 and S6 K1 were significantly higher in the L+F treatment group than in the F treatment group(P<0.01),L treatment and control groups(P<0.05),respectively.2)The relative expression of SREBP1 was significantly lower in the F treatment group than that of the L treatment group(P<0.05),whereas the PPAR-γshowed the opposite result.The relative expressions of FATP1 and FATP4 were significantly lower in the F treatment group than in the L and L+F treatment groups(P<0.01),whereas the FABP3 and ACACAhad the opposite result.The relative expression of SCD1 was significantly higher in the control group than in other groups(P<0.01).3)Compared with the control group,the L+F treatment group showed significantly higher expression levels of Glut1(P<0.05),Glut8(P<0.01)and Glut4(P<0.05).The relative expression of HK2 was significantly lower in the F treatment group than in the L+F and L treatment groups(P<0.05),and the relative expression ofβ-1,4-Gal T was the lowest in the F treatment group.This study showed that alfalfa flavonoids might inhibit milk fat synthesis of BMECs cultured without LPS.Alfalfa flavonoids might promote lactose and protein synthesis of BMECs induced by LPS.
The aim of this study was to examine the effect of alfalfa flavonoids(AF)on the gene expression of milk component synthesis in bovine mammary epithelial cells(BMECs)induced by lipopolysaccharide(LPS).BMECs were cultured on media without LPS+AF(Con),and with 1μg/mL LPS(L),1μg/mL LPS+75μg/mL AF(L+F),75μg/mL AF(F)at 37℃ with 5% CO2 for 12 h.The expression of gene related to milk component synthesis was evaluated.The results showed:1)Compared with the control group,the L and L+F treatment groups showed significant decrease in JAK2 expression(P<0.01),but significant increase in the expressions of STAT5 and eIF4 E(P<0.01).The relative expressions of 4 EBP1 and S6 K1 were significantly higher in the L+F treatment group than in the F treatment group(P<0.01),L treatment and control groups(P<0.05),respectively.2)The relative expression of SREBP1 was significantly lower in the F treatment group than that of the L treatment group(P<0.05),whereas the PPAR-γshowed the opposite result.The relative expressions of FATP1 and FATP4 were significantly lower in the F treatment group than in the L and L+F treatment groups(P<0.01),whereas the FABP3 and ACACAhad the opposite result.The relative expression of SCD1 was significantly higher in the control group than in other groups(P<0.01).3)Compared with the control group,the L+F treatment group showed significantly higher expression levels of Glut1(P<0.05),Glut8(P<0.01)and Glut4(P<0.05).The relative expression of HK2 was significantly lower in the F treatment group than in the L+F and L treatment groups(P<0.05),and the relative expression ofβ-1,4-Gal T was the lowest in the F treatment group.This study showed that alfalfa flavonoids might inhibit milk fat synthesis of BMECs cultured without LPS.Alfalfa flavonoids might promote lactose and protein synthesis of BMECs induced by LPS.
引文
[1]占今舜,詹康,刘明美,霍永久,林淼,赵国琦,杨富裕.苜蓿草颗粒饲料对鹅屠宰性能、器官和血液生化指标的影响[J].草业学报,2015,24(8):181-187Zhan J S,Zhan K,Liu M M,Huo Y J,Lin M,Zhao G Q,Yang F Y.Effect of alfalfa pellet feed on slaughter performance,organ weights and blood biochemical indices of geese[J].Acta Prataculturae Sinica,2015,24(8):181-187(in Chinese)
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[3]Gohlke A,Ingelmann C J,Nürnberg G,Weitzel J M,Hammon H M,G9rs S,Starke A,Wolffram S,Metges C C.Influence of4-week intraduodenal supplementation of quercetin on performance,glucose metabolism,and mRNA abundance of genes related to glucose metabolism and antioxidative status in dairy cows[J].Journal of Dairy Science,2013,96(11):6986-7000
[4]Gessner D K,Koch C,Romberg F J,Winkler A,Dusel G,Herzog E,Most E,Eder K.The effect of grape seed and grape marc meal extract on milk performance and the expression of genes of endoplasmic reticulum stress and inflammation in the liver of dairy cows in early lactation[J].Journal of Dairy Science,2015,98(12):8856-8868
[5]Cui K,Guo X D,Tu Y,Zhang N F,Ma T,Diao Q Y.Effect of dietary supplementation of rutin on lactation performance,ruminal fermentation and metabolism in dairy cows[J].Journal of Animal Physiology and Animal Nutrition,2015,99:1065-1073
[6]Piotrowska-Tomala K K,Siemieniuch M J,Szóstek A Z,Korzekwaa A J,Woclawek-Potocka I,Galvaoa A M,Okuda K,Skarzynski D J.Lipopolysaccharides,cytokines,and nitric oxide affect secretion of prostaglandins and leukotrienes by bovine mammary gland epithelial cells[J].Domestic Animal Endocrinology,2012,43(4):278-288
[7]张养东.脂多糖对泌乳奶牛乳脂肪和乳蛋白影响及其机理研究[D].哈尔滨:东北农业大学,2011Zhang Y D.Effects of lipolysacchride on the milk fat and protein synthesis in dairy cow[D].Harbin:Northeast Agricultural University,2011(in Chinese)
[8]臧长江,张养东,王加启,胡涛,卜登攀,金迪,周凌云,李发弟.脂多糖对泌乳奶牛乳中氨基酸组成及蛋白质代谢相关基因表达的影响[J].动物营养学报,2012,24(9):1770-1777Zang C J,Zhang Y D,Wang J Q,Hu T,Bu D P,Jin D,Zhou L Y,Li F D.Effects of lipolysacchride on amino acid composition and gene expression related to protein metabolism in milk of dairy cows[J].Chinese Journal of Animal Nutrition,2012,24(9):1770-1777(in Chinese)
[9]詹康,贡笑笑,左晓昕,陈银银,占今舜,赵国琦.奶牛乳腺上皮细胞系的培养与鉴定[J].动物营养学报,2015,27(8):2544-2550Zhan K,Gong X X,Zuo X X,Chen Y Y,Zhan J S,Zhao G Q.Culture and identification of the bovine mammary epithelial cell line[J].Chinese Journal of Animal Nutrition,2015,27(8):2544-2550(in Chinese)
[10]占今舜,刘明美,詹康,赵国琦.苜蓿黄酮对奶牛乳腺上皮细胞乳蛋白、乳脂和乳糖合成的影响[J].中国畜牧杂志,2017,53(1):91-95Zhan J S,Liu M M,Zhan K,Zhao G Q.Effects of alfalfa flavonoids on lactoprotein,milk fat and lactose synthesis in bovine mammary epithelial cells[J].Chinese Journal of Animal Science,2017,53(1):91-95(in Chinese)
[11]Liu X,Robinson G W,Wagner K U,Garrett L,Wynshaw-Boris A,Hennighausen L.Stat5ais mandatory for adult mammary gland development and lactogenesis[J].Genes&Development,1997,11(2):179-186
[12]Shillingford J M,Miyoshi K,Robinson G W,Grimm S L,Rosen J M,Neubauer H,Pfeffer K,Hennghausen L.Jak2is an essential tyrosine kinase involved in pregnancy-mediated development of mammary secretory epithelium[J].Molecular Endocrinology,2002,16(3):563-570
[13]王立娜.氨基酸与STAT5A基因互作对奶牛乳腺上皮细胞泌乳的调节作用及机理[D].哈尔滨:东北农业大学,2014Wang L N.Effect of interaction between amino acids and STAT5A on lactation of dairy cow mammary epithelial cells and its mechanism[D].Harbin:Northeast Agricultural University,2014(in Chinese)
[14]Inoki K,Ouyang H,Li Y,Guan K L.Signaling by target of rapamycin proteins in cell growth control[J].Microbiology&Molecular Biology Reviews,2005,69(1):79-100
[15]刘立新,林叶,张莉,李庆章.脂多糖对奶牛乳腺上皮细胞毒性作用及乳蛋白合成的影响[J].东北农业大学学报,2015,46(6):61-66Liu L X,Lin Y,Zhang L,Li Q Z.Cytotoxicity of LPS and effects on milk protein synthesis in dairy cow mammary epithelialcells[J].Journal of Northeast Agricultural University,2015,46(6):61-66(in Chinese)
[16]张蕊,张宜辉,邵丹,王来娣,龚道清.硬脂酰辅酶A去饱和酶基因的功能与调控[J].生命科学,2013,25(4):378-382Zhang R,Zhang Y H,Shao D,Wang L D,Gong D Q.The function and regulation of stearoyl-CoA desaturase gene[J].Chinese Bulletin of Life Sciences,2013,25(4):378-382(in Chinese)
[17]Matsumata M,Inada H,Osumi N.Fatty acid binding proteins and the nervous system:Their impact on mental conditions[J].Neuroscience Research,2014,102:47-55
[18]李楠.SREBP1在奶牛乳腺上皮细胞乳脂合成中的功能研究[D].哈尔滨:东北农业大学,2014Li N.Function ofSREBPl in the milk fat synthesis of dairy cow mammary gland epithelial cells[D].Harbin:Northeast Agricultural University,2014(in Chinese)
[19]Desvergne B,Michalik L,Wahli W.Transcriptional regulation of metabolism[J].Physiological Reviews,2006,86(2):465-514
[20]Kadegowda A K G,Bionaz M,Piperova L S,Erdman R A,Loor J J.Peroxisome proliferator-activated receptor-γactivation and long-chain fatty acids alter lipogenic gene networks in bovine mammary epithelial cells to various extents[J].Journal of Dairy Science,2009,92(9):4276-4289
[21]李文清,王加启,南雪梅,孙鹏.奶牛乳糖合成及泌乳相关基因和细胞信号通路的研究进展[J].中国畜牧兽医,2012,39(11):104-110Li W Q,Wang J Q,Nan X M,Sun P.Research progress on genes and signal pathways associated with lactose synthesis and lactation in dairycows[J].China Animal Husbandry&Veterinary Medicine,2012,39(11):104-110(in Chinese)
[22]孙晓旭.葡萄糖对奶牛乳腺上皮细胞乳糖合成的影响[D].哈尔滨:东北农业大学,2013Sun XX.Effect of glucose on lactose synthesis in bovine mammary epithelial cells[D].Harbin:Northeast Agricultural University,2013(in Chinese)
[2]占今舜,刘明美,赵国琦.黄酮的作用及其在反刍动物上的应用[J].中国饲料,2014,23:13-15Zhan J S,Liu M M,Zhao G Q.Effects of flavonoids and its application in ruminant[J].China Feed,2014,23:13-15(in Chinese)
[3]Gohlke A,Ingelmann C J,Nürnberg G,Weitzel J M,Hammon H M,G9rs S,Starke A,Wolffram S,Metges C C.Influence of4-week intraduodenal supplementation of quercetin on performance,glucose metabolism,and mRNA abundance of genes related to glucose metabolism and antioxidative status in dairy cows[J].Journal of Dairy Science,2013,96(11):6986-7000
[4]Gessner D K,Koch C,Romberg F J,Winkler A,Dusel G,Herzog E,Most E,Eder K.The effect of grape seed and grape marc meal extract on milk performance and the expression of genes of endoplasmic reticulum stress and inflammation in the liver of dairy cows in early lactation[J].Journal of Dairy Science,2015,98(12):8856-8868
[5]Cui K,Guo X D,Tu Y,Zhang N F,Ma T,Diao Q Y.Effect of dietary supplementation of rutin on lactation performance,ruminal fermentation and metabolism in dairy cows[J].Journal of Animal Physiology and Animal Nutrition,2015,99:1065-1073
[6]Piotrowska-Tomala K K,Siemieniuch M J,Szóstek A Z,Korzekwaa A J,Woclawek-Potocka I,Galvaoa A M,Okuda K,Skarzynski D J.Lipopolysaccharides,cytokines,and nitric oxide affect secretion of prostaglandins and leukotrienes by bovine mammary gland epithelial cells[J].Domestic Animal Endocrinology,2012,43(4):278-288
[7]张养东.脂多糖对泌乳奶牛乳脂肪和乳蛋白影响及其机理研究[D].哈尔滨:东北农业大学,2011Zhang Y D.Effects of lipolysacchride on the milk fat and protein synthesis in dairy cow[D].Harbin:Northeast Agricultural University,2011(in Chinese)
[8]臧长江,张养东,王加启,胡涛,卜登攀,金迪,周凌云,李发弟.脂多糖对泌乳奶牛乳中氨基酸组成及蛋白质代谢相关基因表达的影响[J].动物营养学报,2012,24(9):1770-1777Zang C J,Zhang Y D,Wang J Q,Hu T,Bu D P,Jin D,Zhou L Y,Li F D.Effects of lipolysacchride on amino acid composition and gene expression related to protein metabolism in milk of dairy cows[J].Chinese Journal of Animal Nutrition,2012,24(9):1770-1777(in Chinese)
[9]詹康,贡笑笑,左晓昕,陈银银,占今舜,赵国琦.奶牛乳腺上皮细胞系的培养与鉴定[J].动物营养学报,2015,27(8):2544-2550Zhan K,Gong X X,Zuo X X,Chen Y Y,Zhan J S,Zhao G Q.Culture and identification of the bovine mammary epithelial cell line[J].Chinese Journal of Animal Nutrition,2015,27(8):2544-2550(in Chinese)
[10]占今舜,刘明美,詹康,赵国琦.苜蓿黄酮对奶牛乳腺上皮细胞乳蛋白、乳脂和乳糖合成的影响[J].中国畜牧杂志,2017,53(1):91-95Zhan J S,Liu M M,Zhan K,Zhao G Q.Effects of alfalfa flavonoids on lactoprotein,milk fat and lactose synthesis in bovine mammary epithelial cells[J].Chinese Journal of Animal Science,2017,53(1):91-95(in Chinese)
[11]Liu X,Robinson G W,Wagner K U,Garrett L,Wynshaw-Boris A,Hennighausen L.Stat5ais mandatory for adult mammary gland development and lactogenesis[J].Genes&Development,1997,11(2):179-186
[12]Shillingford J M,Miyoshi K,Robinson G W,Grimm S L,Rosen J M,Neubauer H,Pfeffer K,Hennghausen L.Jak2is an essential tyrosine kinase involved in pregnancy-mediated development of mammary secretory epithelium[J].Molecular Endocrinology,2002,16(3):563-570
[13]王立娜.氨基酸与STAT5A基因互作对奶牛乳腺上皮细胞泌乳的调节作用及机理[D].哈尔滨:东北农业大学,2014Wang L N.Effect of interaction between amino acids and STAT5A on lactation of dairy cow mammary epithelial cells and its mechanism[D].Harbin:Northeast Agricultural University,2014(in Chinese)
[14]Inoki K,Ouyang H,Li Y,Guan K L.Signaling by target of rapamycin proteins in cell growth control[J].Microbiology&Molecular Biology Reviews,2005,69(1):79-100
[15]刘立新,林叶,张莉,李庆章.脂多糖对奶牛乳腺上皮细胞毒性作用及乳蛋白合成的影响[J].东北农业大学学报,2015,46(6):61-66Liu L X,Lin Y,Zhang L,Li Q Z.Cytotoxicity of LPS and effects on milk protein synthesis in dairy cow mammary epithelialcells[J].Journal of Northeast Agricultural University,2015,46(6):61-66(in Chinese)
[16]张蕊,张宜辉,邵丹,王来娣,龚道清.硬脂酰辅酶A去饱和酶基因的功能与调控[J].生命科学,2013,25(4):378-382Zhang R,Zhang Y H,Shao D,Wang L D,Gong D Q.The function and regulation of stearoyl-CoA desaturase gene[J].Chinese Bulletin of Life Sciences,2013,25(4):378-382(in Chinese)
[17]Matsumata M,Inada H,Osumi N.Fatty acid binding proteins and the nervous system:Their impact on mental conditions[J].Neuroscience Research,2014,102:47-55
[18]李楠.SREBP1在奶牛乳腺上皮细胞乳脂合成中的功能研究[D].哈尔滨:东北农业大学,2014Li N.Function ofSREBPl in the milk fat synthesis of dairy cow mammary gland epithelial cells[D].Harbin:Northeast Agricultural University,2014(in Chinese)
[19]Desvergne B,Michalik L,Wahli W.Transcriptional regulation of metabolism[J].Physiological Reviews,2006,86(2):465-514
[20]Kadegowda A K G,Bionaz M,Piperova L S,Erdman R A,Loor J J.Peroxisome proliferator-activated receptor-γactivation and long-chain fatty acids alter lipogenic gene networks in bovine mammary epithelial cells to various extents[J].Journal of Dairy Science,2009,92(9):4276-4289
[21]李文清,王加启,南雪梅,孙鹏.奶牛乳糖合成及泌乳相关基因和细胞信号通路的研究进展[J].中国畜牧兽医,2012,39(11):104-110Li W Q,Wang J Q,Nan X M,Sun P.Research progress on genes and signal pathways associated with lactose synthesis and lactation in dairycows[J].China Animal Husbandry&Veterinary Medicine,2012,39(11):104-110(in Chinese)
[22]孙晓旭.葡萄糖对奶牛乳腺上皮细胞乳糖合成的影响[D].哈尔滨:东北农业大学,2013Sun XX.Effect of glucose on lactose synthesis in bovine mammary epithelial cells[D].Harbin:Northeast Agricultural University,2013(in Chinese)