动物营养学报 >

2022 , Vol. 34 >Issue 10: 6702 - 6713

DOI: https://doi.org/10.3969/j.issn.1006-267x.2022.10.063

研究论文 RESEARCH PAPER

黄芪甲苷对脂多糖刺激的奶牛乳腺上皮细胞乳蛋白及乳脂合成相关因子表达的影响

  • 樊嘉琦 ,
  • 周学章
展开
  • 宁夏大学生命科学学院, 银川 750000
樊嘉琦(1997-),女,河北唐山人,博士研究生,从事动物病原生物学研究。E-mail:2090883224@qq.com

收稿日期: 2022-04-15

  网络出版日期: 2022-10-17

基金资助

宁夏回族自治区重点研发计划(2021BEF02021);宁夏回族自治区科技创新领军人才培育项目(2021GKLRLX10)

收起

Effects of Astragaloside Ⅳ on Expression of Milk Protein and Milk Fat Synthesis-Related Factors in Lipopolysaccharide-Stimulated Milk Bovine Mammary Epithelial Cells

  • FAN Jiaqi ,
  • ZHOU Xuezhang
Expand
  • College of Life Sciences, Ningxia University, Yinchuan 750000, China

Received date: 2022-04-15

  Online published: 2022-10-17

Fold

摘要

本试验旨在研究黄芪甲苷(AS-Ⅳ)对脂多糖(LPS)刺激的奶牛乳腺上皮细胞(BMECs)乳脂及乳蛋白合成相关基因表达及脂质过氧化物含量的影响。使用0.5 μg/mL的LPS刺激BMECs 24 h诱导炎症,采用CCK-8试验筛选AS-Ⅳ浓度及时间。试验分为8组(每组3个重复),分别为对照组(C组)、LPS刺激组(LPS组, 0.5 μg/mL LPS刺激24 h)以及6个AS-Ⅳ干预组,即在0.5 μg/mL LPS刺激24 h后,加入不同浓度的AS-Ⅳ继续培养, 6个AS-Ⅳ干预组分别为100 μg/mL AS-Ⅳ刺激1 h组(LPS+100 AS-Ⅳ 1 h组)、100 μg/mL AS-Ⅳ刺激3 h组(LPS+100 AS-Ⅳ 3 h组)、100 μg/mL AS-Ⅳ刺激6 h组(LPS+100 AS-Ⅳ 6 h组)、25 μg/mL AS-Ⅳ刺激6 h组(LPS+25 AS-Ⅳ 6 h组)、50 μg/mL AS-Ⅳ刺激6 h组(LPS+50 AS-Ⅳ 6 h组)、100 μg/mL AS-Ⅳ刺激6 h组(LPS+100 AS-Ⅳ 6 h组)。通过实时荧光定量PCR法检测细胞中乳蛋白和乳脂相关基因的表达;试剂盒检测细胞上清液中脂质过氧化物(LPO)及丙二醛(MDA)含量;荧光显微镜观察细胞中脂滴的分泌;蛋白印迹法检测细胞中哺乳动物雷帕霉素靶蛋白(mTOR)表达;使用黄芪甲苷-5(6)-羧基二乙酸荧光素琥珀酰亚胺酯复合物(ASIV-CDFA)模拟BMECs对AS-Ⅳ的摄取。结果显示:与对照组相比, LPS会诱导BMECs的脂质过氧化物的沉积, LPO、MDA含量显著升高(P＜0.05);乳蛋白合成αS1-酪蛋白(CSN1S1)、β-酪蛋白(CSN2)基因相对表达量显著降低(P＜0.05);乳脂合成脂肪酸结合蛋白3(FABP3)、脂肪酸合成酶(FASN)、固醇调节元件结合蛋白1(SREBP1)基因相对表达量显著降低(P＜0.05)。与LPS组相比, AS-Ⅳ在刺激BMECs后, CSN2基因相对表达量显著升高(P＜0.05),但不激活CSN1S1基因相对表达量; AS-Ⅳ可显著抑制炎症细胞中MDA的含量, 25、50、100 μg/mL的AS-Ⅳ刺激细胞6 h可显著抑制炎症细胞中LPO的含量; 100 μg/mL AS-Ⅳ刺激1 h显著升高FASN、乙酰辅酶A羧化酶(ACC)、脂蛋白酯酶(LPL)基因相对表达量(P＜0.05), 25 μg/mL AS-Ⅳ刺激6 h显著升高过氧化酶体增殖物激活受体γ(PPARγ)基因相对表达量(P＜0.05), 50 μg/mL AS-Ⅳ刺激细胞6 h显著升高LPL基因相对表达量(P＜0.05),并且显著增加炎症细胞中脂滴的含量(P＜0.05), 25、100 μg/mL的AS-Ⅳ刺激细胞6 h可显著激活炎症细胞中磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)的表达(P＜0.05)。使用ASIV-CDFA观察炎症细胞对AS-Ⅳ的吸收情况, ASIV-CDFA的细胞吸收量随AS-Ⅳ浓度升高而增多,但不随时间的延长而增多。综上所述, AS-Ⅳ可以调控LPS刺激的BMECs中乳脂及乳蛋白合成相关基因的表达, AS-Ⅳ对LPS诱导的BMECs中抑制脂滴表达具有减缓效果,对脂质过氧化物的激活具有抑制效果。

关键词： 黄芪甲苷; 奶牛乳腺上皮细胞; 乳脂; 乳蛋白

本文引用格式

樊嘉琦 , 周学章 . 黄芪甲苷对脂多糖刺激的奶牛乳腺上皮细胞乳蛋白及乳脂合成相关因子表达的影响[J]. 动物营养学报, 2022 , 34(10) : 6702 -6713 . DOI: 10.3969/j.issn.1006-267x.2022.10.063

Abstract

The purpose of this experiment was to study the effects of astragaloside Ⅳ (AS-Ⅳ) on the expression of milk fat and milk protein synthesis-related genes and the content of lipid peroxides in lipopolysaccharide (LPS)-stimulated milk bovine mammary epithelial cells (BMECs). BMECs were stimulated with 0.5 μg/mL LPS for 24 h to induce inflammation, and the concentration and time of AS-Ⅳ were screened by CCK-8 assay. The experiment was divided into 8 groups (3 repetitions in each group), the control group, LPS stimulation group (LPS group, stimulated with 0.5 μg/mL LPS for 24 h) and 6 AS-Ⅳ intervention groups, that is, after 0.5 μg/mL LPS stimulation for 24 h, different concentrations of AS-Ⅳ were added and continued to culture, divided into: 100 μg/mL AS-Ⅳ stimulation group for 1 h (LPS+100 AS-Ⅳ 1 h group), 100 μg/mL AS-Ⅳ stimulation group for 3 h (LPS+100 AS-Ⅳ 3 h group), 100 μg/mL AS-Ⅳ stimulation group for 6 h (LPS+100 AS-Ⅳ 6 h group), 25 μg/mL AS-Ⅳ stimulation group for 6 h (LPS+25 AS-Ⅳ 6 h group), 50 μg/mL AS-Ⅳ stimulation group for 6 h (LPS+50 AS-Ⅳ 6 h group), 100 μg/mL AS-Ⅳ stimulation group for 6 h (LPS+100 AS-Ⅳ 6 h group). The expression of milk protein and milk fat-related genes in cells was detected by real-time quantitative PCR; the contents of lipid peroxide (LPO) and malondialdehyde (MDA) in cell supernatant were detected by kits; the lipid droplets in cells were observed by fluorescence microscope The expression of mammalian target protein of rapamycin (mTOR) in cells was detected by Western blot; astragaloside Ⅳ-5(6)-carboxydiacetate fluorescein succinimidyl ester complex (ASIV-CDFA) was used to simulate the expression of BMECs uptake of AS-Ⅳ. The results showed that compared with the control group, LPS could induce the deposition of lipid peroxides in BMECs, and the contents of LPO and MDA were significantly increased (P < 0.05); milk proteins synthesized αs1-casein (CSN1S1) and β-casein (CSN2) genes relative expression levels decreased significantly (P < 0.05); milk fat synthesis fatty acid binding protein 3 (FABP3), fatty acid synthase (FASN), sterol regulatory element binding protein 1 (SREBP1) genes relative expression levels decreased significantly (P < 0.05). Compared with the LPS group, after AS-Ⅳ stimulated BMECs, the relative expression level of CSN2 gene was significantly increased (P < 0.05), but the relative expression level of CSN1S1 gene was not activated; AS-Ⅳ could significantly inhibit the content of MDA in inflammatory cells, 25, 50, 100 μg/mL AS-Ⅳ stimulated cells for 6 h could significantly inhibit the content of LPO in inflammatory cells; 100 μg/mL AS-Ⅳ stimulation for 1 h significantly increased the relative expression levels of FASN, acetyl-CoA carboxylase (ACC), lipoprotein lipase (LPL) genes (P < 0.05), 25 μg/mL AS-Ⅳ stimulation for 6 h significantly increased the relative expression level of peroxisome proliferator-activated receptorγ (PPARγ) gene (P < 0.05), 50 μg/mL AS-Ⅳ stimulated cells for 6 h significantly increased the relative expression level of LPL gene (P < 0.05), and significantly increased the content of lipid droplets in inflammatory cells (P < 0.05). The expression of phosphorylated mammalian target of rapamycin (p-mTOR) protein in inflammatory cells was significantly activated by AS-Ⅳ stimulated cells for 6 h (P < 0.05). ASIV-CDFA was used to observe the absorption of AS-Ⅳ by inflammatory cells. The cellular absorption of ASIV-CDFA increased with the increase of AS-Ⅳ concentration, but did not increase with the prolongation of time. In conclusion, AS-Ⅳ can regulate the expression of milk fat and milk protein synthesis-related genes in LPS-stimulated BMECs. AS-Ⅳ has a slowing effect on the inhibition of lipid droplet expression in LPS-induced BMECs, and has an inhibitory effect on activates lipid peroxides.

Key words： astragaloside Ⅳ; bovine mammary epithelial cells; milk fat; milk protein

参考文献

[1] 侯昆,李欣,沈义媛,等.青蒿素对奶牛乳腺上皮细胞乳脂合成相关基因表达的影响[J].动物营养学报,2021,33(11):6407-6419. HOU K,LI X,SHEN Y Y,et al.Effects of artemisinin on expression of milk fat synthesis related genes in mammary epithelial cells of dairy cows[J].Chinese Journal of Animal Nutrition,2021,33(11):6407-6419.(in Chinese)
[2] BAUMAN D E,MATHER I H,WALL R J,et al.Major advances associated with the biosynthesis of milk[J].Journal of Dairy Science,2006,89(4):1235-1243.  
[3] SHENNAN D B,PEAKER M.Transport of milk constituents by the mammary gland[J].Physiological Reviews,2000,80(3):925-951.  
[4] VYAS D,UWIZEYE A,MOHAMMED R,et al.The effects of active dried and killed dried yeast on subacute ruminal acidosis,ruminal fermentation,and nutrient digestibility in beef heifers[J].Journal of Animal Science,2014,92(2):724-732.  
[5] 冯雪.金黄色葡萄球菌入侵牛乳腺上皮细胞对乳脂合成的影响[D].硕士学位论文.呼和浩特:内蒙古大学,2018. FENG X.Effects of staphylococcus aureus invasion on milk fat synthesis in bovine mammary epithelial cells[D].Master's Thesis.Hohhot:Inner Mongolia University,2018.(in Chinese)
[6] 杨晓茹.大肠杆菌及其LPS对奶牛乳腺上皮细胞摄取硬脂酸和乳脂合成的影响[D].硕士学位论文.呼和浩特:内蒙古大学,2021. YANG X R.Effects of Escherichia coli and ITS LPS on stearic acid uptake and milk fat synthesis in mammary epithelial cells of dairy cows[D].Master's Thesis.Hohhot:Inner Mongolia University,2021.(in Chinese)
[7] 单旭菲.PRL抵抗在LPS导致BMEC乳蛋白合成功能降低中的作用[D].硕士学位论文.大庆:黑龙江八一农垦大学,2019. DAN X F.The effect of prolactin resistant on lactoprotein decrease induce by LPS in BMEC[D].Master's Thesis.Daqing:Heilongjiang Bayi Agricultural University,2019.(in Chinese)
[8] ZHANG W Y,LI X Z,XU T,et al.Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis[J].Experimental Cell Research,2016,349(1):45-52.  
[9] 李楠,高学军,关力.中药王不留行对奶牛乳腺细胞增殖及泌乳的影响[J].中国畜牧兽医,2011,38(6):45-48. LI N,GAO X J,GUAN L.Effects of semen vaccariae on the proliferation and lactation fuction of bovine mammary gland epithelial cell[J].China Animal Husbandry&Veterinary Medicine,2011,38(6):45-48.(in Chinese)
[10] 郑国军,罗天瑶,王刚,等.发酵黄芪对奶牛泌乳及体细胞的影响[J].现代畜牧科技,20205):5-7. ZHENG G J,LUO T Y,WANG G,et al.Effects of fermented astragalus membranaceus on lactation and somatic cells of dairy cows[J].Modern Animal Husbandry Science&Technology,2020(5):5-7.(in Chinese)
[11] 王凤鸽.黄芪甲苷对氨诱导的奶牛乳腺上皮细胞氧化应激和凋亡的缓解作用及其机制研究[D].博士学位论文.长春:吉林大学,2019. WANG F G.Protective effect and mechanism of astragaloside Ⅳ against oxidative stress and apoptosis induced by ammonia in bovine mammary epithelial cells[D].Ph.D.Thesis.Changchun:Jilin University,2019.(in Chinese)
[12] 樊嘉琦.黄芪多糖及黄芪甲苷对奶牛乳腺上皮细胞Wnt/β-catenin信号影响机制的研究[D].硕士学位论文.银川:宁夏大学,2021. FAN J Q.Study of the molecular mechanism of APS and AS-Ⅳ regulating Wnt/β-catenin signaling in BMECs[D].Master's Thesis.Yinchuan:NingXia University,2021.(in Chinese)
[13] 郝颖,赵艳丽,闫素梅,等.维生素A对过氧化氢诱导的奶牛乳腺上皮细胞内乳脂合成的影响[J].动物营养学报,2021,33(11):6398-6406. HAO Y,ZHAO Y L,YAN S M,et al.Effects of vitamin A on milk fat synthesis in mammary epithelial cells of dairy cows induced by hydrogen peroxide[J].Chinese Journal of Animal Nutrition,2021,33(11):6398-6406.(in Chinese)
[14] 李思妍.BLV对MAC-T乳成分合成功能的影响[D].硕士学位论文.大庆:黑龙江八一农垦大学,2021. LI S Y.Effects of BLV on milk composition synthesis function in MAC-T milk[D].Master's Thesis.Daqing:Heilongjiang Bayi Agricultural University,2021.(in Chinese)
[15] 王小龙.奶牛乳腺炎应激条件下乳中脂肪酸变化及其调控[D].博士学位论文.扬州:扬州大学,2014. WANG X L.Regulation of synthesis and metabolism of fatty acids under the stress of mastitis in dairy cow[D].Ph.D.Thesis.Yangzhou:Yangzhou University,2014.(in Chinese)
[16] 杨德英,曹随忠,余树民,等.奶牛隐性乳房炎对牛奶品质的影响[J].湖北农业科学,2009,48(1):132-134. YANG D Y,CAO S Z,YU S M,et al.Effects of subclinical mastitis on milk quality of cows[J].Hubei Agricultural Sciences,2009,48(1):132-134.(in Chinese)
[17] WU Y J,SUN Y W,DONG X W,et al.The synergism of PGN,LTA and LPS in inducing transcriptome changes,inflammatory responses and a decrease in lactation as well as the associated epigenetic mechanisms in bovine mammary epithelial cells[J].Toxins,2020,12(6):387.
[18] 姜丽丽.HPLC-UV法测定黄芪干燥根中黄芪甲苷的含量[J].安徽农业科学,2014,42(22):7381-7383. JIANG L L.Determination of astragaloside Ⅳ in dried roots of Astragalus membranaceus by HPLC-UV[J].Journal of Anhui Agricultural Sciences,2014,42(22):7381-7383.(in Chinese)
[19] XU F,CUI W Q,WEI Y,et al.Astragaloside Ⅳ inhibits lung cancer progression and metastasis by modulating macrophage polarization through AMPK signaling[J].Journal of Experimental&Clinical Cancer Research CR,2018,37(1):207.
[20] 田青,SPITZER A J,赵凤启.脂多糖对大鼠乳腺上皮细胞酪蛋白基因表达的影响[J].动物营养学报,2020,32(7):3365-3373. TIAN Q,SPITZER A J,ZHAO F Q.Effects of lipopolysaccharide on casein gene expression in rat mammary epithelial cells[J].Chinese Journal of Animal Nutrition,2020,32(7):3365-3373.(in Chinese)
[21] KOBAYASHI K,OYAMA S,UEJYO T,et al.Underlying mechanisms involved in the decrease of milk secretion during Escherichia coli endotoxin induced mastitis in lactating mice[J].Veterinary Research,2013,44(1):119.
[22] 周鹏,张玉梅,刘彪,等.乳类食物中β-酪蛋白的结构及营养功能[J].中国食物与营养,2020,26(4):52-56. ZHOU P,ZHANG Y M,LIU B,et al.β-casein of milk:structure and nutritional function[J].Food and Nutrition in China,2020,26(4):52-56.(in Chinese)
[23] 胡耀,宋洁,王丽芳,等.黄花蒿乙醇提取物对脂多糖诱导损伤奶牛乳腺细胞中乳蛋白合成的影响[J].动物营养学报,2021,33(9):5236-5245. HU Y,SONG J,WANG L F,et al.Effects of ethanol extract of Artemisia annua on milk protein synthesis in lipopolysaccharide-induced injury mammary cells of dairy cows[J].Chinese Journal of Animal Nutrition,2021,33(9):5236-5245.(in Chinese)
[24] 王宇彤,沈义媛,霍相羽,等.体外法研究竹叶黄酮对奶牛乳腺上皮细胞抗氧化及哺乳动物雷帕霉素靶蛋白信号通路中关键基因表达的影响[J].动物营养学报,2022,34(1):612-625. WANG Y T,SHEN Y Y,HUO X Y,et al.In vitro study on effects of bamboo leaf flavonoids on antioxidation and expression of key genes in mammalian target of rapamycin signaling pathway in bovine mammary epithelial cells[J].Chinese Journal of Animal Nutrition,2022,34(1):612-625.(in Chinese)
[25] 汪文婧,程储记,陈旭昕,等.BMSC对内毒素诱导的肺泡上皮细胞氧化应激的影响[J].转化医学杂志,2021,10(4):206-210,255. WANG W J,CHENG C J,CHEN X X,et al.Effects of bone marrow mesenchymal stem cells on lipopolysacharride induced alveolar epithelial cells in oxidative stress[J].Translational Medicine Journal,2021,10(4):206-210,255.(in Chinese)
[26] WANG J F,ZHANG X,HE X J,et al.LPS-induced reduction of triglyceride synthesis and secretion in dairy cow mammary epithelial cells via decreased SREBP1 expression and activity[J].Journal of Dairy Research,2018,85(4):439-444.  
[27] WANG J F,LIAN S,SONG J,et al.Liver X receptor α participates in LPS-induced reduction of triglyceride synthesis in bovine mammary epithelial cells[J].The Journal of Dairy Research,2020,87(4):456-462.  
[28] ZHAO Y L,GUO X Y,YAN S M,et al.Acetate regulates milk fat synthesis through the mammalian target of rapamycin/eukaryotic initiation factor 4E signaling pathway in bovine mammary epithelial cells[J].Journal of Dairy Science,2021,104(1):337-345.  
[29] DUAN Y H,LI F N,TAN K R,et al.Key mediators of intracellular amino acids signaling to mTORC1 activation[J].Amino Acids,2015,47(5):857-867.  
Options
文章导航

/

〈 〉

    联系我们

  • 地址：北京市海淀区圆明园西路2号
    　　　中国农业大学动科动医大楼153室
  • 邮编：100193
  • 电话：010－62817823
  • 网址：http://www.chinajan.com
  • E-mail：cjan@chinajan.com
京公网安备 11010802041926号    京ICP备17050989号-2