内毒素引起奶牛乳腺组织表观遗传变化及其机制研究

In practical dairy production, endotoxin can gain an entry into the body in a large amount due to high-concentrate diet feeding, poor hygienic environment, heat stress as well as diseases such as mastitis. Our previous study showed that endotoxin in the mammary arterial blood could result in epigenetic alterations in the mammary tissue of dairy cows. However, the extent of epigenetic alterations caused by endotoxin and the mechanisms by which endotoxin causes epigenetic alterations in the mammary tissue of dairy cows remain unclear. Therefore, the in vitro technique of mammary epithelial cell culture and the in vivo endotoxin infusion approach are employed in this project to investigate the effects of endotoxin on the acetylation of histone H3 and H4 and the methylation of specific genes associated with milk fat and milk protein synthesis in the mammary gland of dairy cows. Furthermore, the activity of histone H3 and H4 acetylase and deacetylase as well as DNA methyltransferase is assayed to reveal the mechanisms by which endotoxin causes epigenetic alterations in the mammary gland of dairy cows. Meanwhile, the histone deacetylase and DNA methyltransferase inhibitors are used to investigate the efficacy of blocking endotoxin-induced epigenetic alterations in the mammry gland of dairy cows. The objectives of this project are to clarify the epigenetic alterations in the mammary tissue of dairy cows caused by endotoxin and to unravel the mechanisms of the endotoxin-induced epigenetic alterations in the mammary tissue of dairy cows, so as to provide a theoretical foundation for more effective control of the adverse effects of endotoxin on lactation in dairy cows by using epigenetic manipulatory means.

在奶牛生产中，因饲喂高精料日粮、圈舍环境卫生不良、夏季热应激以及发生乳房炎等，内毒素较多地进入奶牛体内和乳腺中。我们前期研究表明，乳腺动脉血中内毒素可引起乳腺组织发生表观遗传变化。但是，关于内毒素引起奶牛乳腺组织表观遗传变化的程度及机制，目前尚不清楚。本项目拟应用乳腺上皮细胞培养技术和奶牛活体灌注法，研究内毒素对乳腺组蛋白H3和H4乙酰化及乳脂肪和乳蛋白合成相关基因甲基化的影响，探讨内毒素是否通过改变组蛋白H3和H4乙酰转移酶和去乙酰化酶及DNA甲基转移酶的活性来造成乳腺组织表观遗传的变化，并探索采用组蛋白去乙酰化酶活性抑制剂和DNA甲基转移酶活性抑制剂来阻断内毒素对乳腺表观遗传不利作用的效果。本项目旨在阐明内毒素引起奶牛乳腺组织表观遗传变化的规律，揭示内毒素影响奶牛乳腺乳脂肪和乳蛋白合成的表观遗传学机制，为今后应用表观遗传调控手段来更为有效地消除内毒素对奶牛泌乳的负面影响提供理论依据。

我国奶牛生产中由于优质牧草严重不足，饲养者不得不通过增加日粮中精料比例来满足奶牛的营养需要。高精料日粮容易造成亚急性瘤胃酸中毒，使瘤胃pH下降和内毒素（LPS）释放增加。LPS进入体内和乳腺后导致泌乳性能下降。本项目旨在阐明LPS引起奶牛乳腺组织表观遗传变化的规律，揭示LPS影响奶牛乳腺乳脂肪和乳蛋白合成的表观遗传学机制，为今后从表观遗传调控角度更有效地消除LPS对奶牛泌乳的负面影响提供理论依据。为此，本项目开展了如下研究：（1）应用细胞培养技术研究LPS对奶牛乳腺上皮细胞表观遗传的影响；（2）应用奶牛乳腺上皮细胞培养技术研究LPS对组蛋白乙酰转移酶（HAT）和组蛋白去乙酰化酶（HDAC）活性及DNA甲基转移酶（DNMT）活性的影响；（3）应用奶牛乳腺上皮细胞培养技术研究HDAC和DNMT活性抑制剂阻断LPS引起的表观遗传变化的效果；（4）采用颈静脉灌注法研究LPS对奶牛乳腺组织表观遗传变化和产奶性能的影响。结果显示：（1）随着LPS剂量（0、1、10、100、1000 EU/mL; 1 EU = 0.1 ng）增加，乳腺上皮细胞组蛋白H3的乙酰化水平降低，而组蛋白H4的乙酰化水平不受LPS的显著影响。基因组甲基化水平、基因组发生甲基化的基因数量以及泌乳基因如乙酰辅酶A羧化酶1 （ACACA）、短链脂酰辅酶A合成酶2（ACSS2）和核糖体S6激酶1（S6K1）启动子甲基化水平，均随着LPS剂量从0升高到10 EU/mL而增加；泌乳基因mRNA表达量与甲基化水平呈负相关。（2）HDAC活性随着LPS剂量增加而升高，HAT和DNMT活性则不受LPS的影响。（3）HDAC的抑制剂丁酸钠能够显著提高组蛋白H3乙酰化水平。添加丁酸钠可有效消除LPS对泌乳相关基因如ACACA、长链脂酰辅酶A合成酶2（ACSL1）、ACSS2、S6K1、信号转导与转录激活因子5A（STAT5A）及3种酪蛋白（CSN1S1、CSN2、CSN3）基因表达量的不利影响。（4）对泌乳奶牛从颈静脉连续灌注低剂量LPS（100 EU/kg体重）使干物质采食量和产奶量降低；灌注LPS对乳腺中大部分基因甲基化程度影响不显著，但使硬酯酰辅酶A去饱和酶（SCD）基因甲基化水平升高，使STAT5A基因的甲基化水平降低。