Ghrelin对奶牛乳蛋白合成的影响及机制研究

Low nutritional quality of cow's milk has increasingly become a severe challenge faced by the healthy development of dairy industry in China. Milk protein is the main substance to determine the nutritional quality of cow's milk. Cow's milk quality could be affected by many factors, where neuroendocrine factor plays an important regulatory role to protein contents in cow's milk. Ghrelin is a neuroendocrine factor with various biological functions. It is reported that ghrelin plays regulatory role to protein synthesis in rats muscle tissue, but the effects and molecular mechanism of ghrelin on protein synthesis of cow's milk have not yet been systematically studied. The project will use western blotting, real-time PCR, ELISA, EMSA and other methods to explore the relativity between the level of ghrelin and the contents of protein in cow's milk, study the influences and mechanism of ghrelin on protein synthesis-related genes expression in mammary epithelial cells, verify the impacts of exogenous ghrelin on protein synthesis of cow's milk so as to identify the effects and molecular mechanism of ghrelin on protein synthesis of cow's milk from molecular, cellular and overall levels. These results will provide scientific basis to further study the regulatory mechanism of protein synthesis of cow's milk and improve the nutritional quality of cow's milk.

乳蛋白是构成牛奶营养品质的主要物质基础，乳蛋白含量是奶业核心竞争力的重要标志。神经内分泌因子对奶牛乳蛋白合成具有重要的调控作用。Ghrelin是一种具有多种生物学功能的神经内分泌因子。已有研究表明， Ghrelin能促进大鼠肌肉组织蛋白合成，而且可以调节反刍动物摄食和能量代谢，提示Ghrelin可能对乳蛋白合成过程中具有潜在调节作用。本项目拟利用Western blot、RT-PCR、ELISA、EMSA等技术，通过分析血液Ghrelin水平与奶牛乳蛋白含量的相关性，检测Ghrelin对乳腺上皮细胞氨基酸摄取能力、乳蛋白合成及相关信号分子的影响，并进行在体验证，从分子、细胞和整体水平上明确Ghrelin对奶牛乳蛋白合成的影响及机制。为深入认识奶牛乳蛋白合成的调控机理提供科学依据，为通过人工干预手段来提高牛奶营养品质奠定理论基础。

本项目以提升乳蛋白含量为目标，系统的研究了Ghrelin对奶牛乳蛋白含量的影响及其机制。首先采取不同泌乳水平的奶牛血清和牛奶，通过分析Ghrelin浓度和乳蛋白含量的相关性，发现Ghrelin浓度和乳蛋白含量呈显著正相关。在奶牛乳腺上皮细胞中，Ghrelin能显著促进β-酪蛋白（CSN2）基因和蛋白的表达，而GHSR1a和Gαs蛋白的特异性抑制剂显著抑制Ghrelin的作用。Ghrelin通过GHSR1a和Gαs蛋白激活奶牛乳腺上皮细胞中ERK1/2、AKT和JNK信号通路，而抑制ERK1/2、AKT和JNK的磷酸化则抑制Ghrelin诱导的CSN2合成。此外，Ghrelin还通过上述通路促进奶牛乳腺上皮细胞的增殖。这些结果说明Ghrelin激活GHSR1a，活化的GHSR1a与Gαs蛋白耦合，通过ERK1/2、AKT和JNK通路增加CSN2和促进细胞增殖，并且这一结果在体外培养的乳腺组织块中得到了进一步的验证。乳房炎是导致乳品质下降的主要原因之一，我们研究发现Ghrelin能通过GHSR1a依赖的ERK1/2和AKT通路抑制LPS诱导的乳房炎细胞模型中炎性因子的产生，增加CSN2的表达；同时Ghrelin还通过p53/Bax/Bcl-2通路抑制LPS诱导的细胞凋亡，从而促进CSN2的表达。以上结果提示Ghrelin能通过多种方式提高乳蛋白含量。这些结果为深入研究乳蛋白合成调控机制及乳房炎的防治提供了科学依据，为通过人工干预手段来提高牛奶营养品质奠定了理论基础。