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  

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

作　　者：樊嘉琦 周学章[1] FAN Jiaqi;ZHOU Xuezhang(College of Life Sciences,Ningxia University,Yinchuan 750000,China)

机构地区：[1]宁夏大学生命科学学院,银川750000

出　　处：《动物营养学报》2022年第10期6702-6713,共12页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：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)。

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0.5μg/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,0.5μg/mL LPS刺激24 h)以及6个AS-Ⅳ干预组,即在0.5μg/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100μg/mL AS-Ⅳ刺激1 h组(LPS+100 AS-Ⅳ1 h组)、100μg/mL AS-Ⅳ刺激3 h组(LPS+100 AS-Ⅳ3 h组)、100μg/mL AS-Ⅳ刺激6 h组(LPS+100 AS-Ⅳ6 h组)、25μg/mL AS-Ⅳ刺激6 h组(LPS+25 AS-Ⅳ6 h组)、50μg/mL AS-Ⅳ刺激6 h组(LPS+50 AS-Ⅳ6 h组)、100μg/mL AS-Ⅳ刺激6 h组(LPS+100 AS-Ⅳ6 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、100μg/mL的AS-Ⅳ刺激细胞6 h可显著抑制炎症细胞中LPO的含量;100μg/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),25μg/mL AS-Ⅳ刺激6 h显著升高过氧化酶体增殖物激�The purpose of this experiment was to study the effects of astragalosideⅣ(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 0.5μg/mL LPS for 24 h to induce inflammation,and the concentration and time of AS-Ⅳwere 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 0.5μg/mL LPS for 24 h)and 6 AS-Ⅳintervention groups,that is,after 0.5μg/mL LPS stimulation for 24 h,different concentrations of AS-Ⅳwere added and continued to culture,divided into:100μg/mL AS-Ⅳstimulation group for 1 h(LPS+100 AS-Ⅳ1 h group),100μg/mL AS-Ⅳstimulation group for 3 h(LPS+100 AS-Ⅳ3 h group),100μg/mL AS-Ⅳstimulation group for 6 h(LPS+100 AS-Ⅳ6 h group),25μg/mL AS-Ⅳstimulation group for 6 h(LPS+25 AS-Ⅳ6 h group),50μg/mL AS-Ⅳstimulation group for 6 h(LPS+50 AS-Ⅳ6 h group),100μg/mL AS-Ⅳstimulation group for 6 h(LPS+100 AS-Ⅳ6 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;astragalosideⅣ-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 synthesizedαs1-casein(CSN1S1)andβ-casein(CSN2)genes relative expression levels decreased significantly(P<0.05);milk fat synthesis fatty acid binding protein 3(FABP3),fatty acid synthas

关 键 词：黄芪甲苷 奶牛乳腺上皮细胞 乳脂 乳蛋白 

分 类 号：S823[农业科学—畜牧学]