本试验旨在通过半体内和体外试验研究灌注脂多糖(lipopolysacchride,LPS)对泌乳奶牛乳中氨基酸组成和蛋白质代谢相关基因表达的影响。半体内试验选用6头经产的荷斯坦奶牛,随机分为对照组和试验组,每组3头。采用交叉试验设计,每期正试期7 d,间隔期14 d;试验组阴外动脉灌注LPS(大肠杆菌型O111: B4,0.01 μg/kg),对照组阴外动脉灌注生理盐水。体外试验以乳腺上皮细胞为模型,基础培养基中LPS添加水平分别为0、0.1和10.0 ng/mL,培养24 h后,采用实时定量-PCR(qRT-PCR)法检测目的基因的相对表达水平。结果表明,阴外动脉灌注LPS后,乳中必需氨基酸、支链氨基酸含量随着灌注时间的增加呈先升高后降低的趋势(P>0.05),灌注后6 h达到最高;乳中非必需氨基酸含量随着灌注时间的增加呈先降低后升高的趋势(P>0.05),灌注后6 h达到最低。LPS提高了乳腺上皮细胞中p70核糖体蛋白S6激酶-1(S6K1)和真核起始因子4E结合蛋白-1(4EBP1)mRNA的表达水平(P>0.05),且具有剂量效应。LPS对雷帕霉素靶点(mTOR)、Janus激酶2(JAK2)mRNA表达水平影响差异不显著(P>0.05),但有提高的趋势。与对照组相比,LPS显著提高了信号转导和转录激活因子5(STAT5)mRNA表达水平(P<0.05)。综上所述,LPS通过干扰mTOR通路和JAK2/STAT5通路影响了乳蛋白及氨基酸组成。
The objective of this experiment was to study the effects of infusion lipopolysacchride (LPS) on the amino acid composition and gene expressions related to protein metabolism in milk of dairy cows by semi in vivo and in vitro tests. Six multipara Holstein lactating cows were selected in the in vivo test, and randomly divided into experimental and control groups with three cows in each group. A cross test design was applied. The test consisted of two experimental periods (7 d each) and an interval period (14 d). Cows in the experimental group were infused LPS (Escherichia coli O111: B4, 0.01μg/kg) into external pudendal artery, as those in the control group were infused physiological salt solution. Mammary epithelial cells were applied in the in vitro test, and the supplemental level of LPS in a basal culture medium was 0, 0.1 and 10.0 ng/mL, respectively. After 24 h, the relative expression level of target genes was determined using qRT-PCR. The results showed that contents of essential amino acids (EAA) and branch-chain amino acids (BCAA) in milk were firstly ascended and then descended (P>0.05) as the infusion time prolonged, and maximum contents appeared at 6 h; the non-essential amino acids (NEAA) content in milk was increased at the beginning and then decreased (P>0.05), and the minimum content appeared at 6 h. LPS increased mRNA expression levels of S6K1 and 4EBP1 in mammary epithelial cells (P>0.05), and the effects showed dosage dependent. LPS did not significantly affect mRNA expression levels of mTOR and JAK2 (P>0.05), but they tended to increased. Compared with the control group, LPS enhanced the mRNA expression of STAT5 significantly (P<0.05). Therefore, LPS affects the amino acid composition in milk by interrupt pathways of mTOR and JAK2/STAT5.
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