基于IGF-1信号通路探讨瘤胃上皮对高精料日粮的分子适应机制

The previous studies showed that the gradually increasing the amount of concentrate could increase the adaptation of rumen epithelium to high concentrate diet in postpartum cows and feedlot and ovine, and this may have a positive effect on reducing the risk of rumen acidosis, however, the mechanism underling the adaption of rumen epithelium to high concentrate diets is still unclear. It is speculated that mechanisms responsible for the adaption of rumen epithelium to high concentrate diets might be related with the stimulation of ruminal epithelium growth by the activation of insulin-like growth factor 1 (IGF-1) signaling pathway, which was caused by high concentrate feeding, and thereby increase the absorption of short chain fatty acid, however, this speculation requires further examination. This project aims to investigate the effect of the different proportion of concentrate on the changes in IGF-1 secretion and its biological effect, and the mRNA and protein expressions of IGF-1 receptor and the key protein involved in activation of IGF-1 receptor signaling pathway; and to investigate the modulation effect of activation of IGF-1 receptor signaling pathway on the changes in the cell cycle and the expression of the cyclin and apoptosis protein and its mechanisms, using an animal experimental model and the culture cells methods of rumen epithelium in vitro. The success of the project will help reveal the molecular adaptation mechanism of ruminal epithelium to high concentrate, and provide some new ideas and theoretical basis for preventing the rumen acidosis.

资料显示，对产后奶牛和育肥期牛羊饲喂精料水平逐步提高的日粮，有利于瘤胃上皮适应高精料日粮，进而降低瘤胃酸中毒的发病风险，但涉及该过程的生物学机制尚不清楚，推测与高营养水平日粮激活胰岛素样生长因子1（IGF-1）信号通路，进而促进瘤胃上皮生长、加快瘤胃内短链脂肪酸吸收有关，但具体机理不明。本项目拟采用动物试验，结合体外细胞培养模型；研究提高日粮精料水平对IGF-1信号通路中关键因子IGF-1内分泌及其生物活性的影响、对瘤胃上皮细胞IGF-1受体和受体后信号转导通路中关键蛋白的影响与机制，探讨IGF-1受体后通路活化对瘤胃上皮细胞周期、细胞周期蛋白与凋亡相关蛋白的表达和功能的调节及其作用机理。研究结果可揭示“日粮→IGF-1→IGF-1受体→IGF-1受体后通路→瘤胃上皮细胞增殖与凋亡→瘤胃上皮生长”通路。项目将从分子水平阐明瘤胃上皮对高精料日粮的适应机制，为预防瘤胃酸中毒提供新的理论依据。

在现代集约化养殖中，为提高反刍动物的生产性能及生产效率，生产者往往采用高精料日粮饲喂模式。与以粗饲料为主的日粮相比，高精料日粮在瘤胃内被瘤胃微生物快速发酵，导致瘤胃内挥发性脂肪酸累积和pH值降低。为适应上述瘤胃内环境的变化，反刍动物瘤胃生理结构会发生相应改变。然而，目前人们对于瘤胃上皮对高精料日粮的适应性机制仍不清楚。因此，本项目以绵羊为反刍家畜代表，从组织、细胞和分子层面，系统研究了绵羊瘤胃上皮对持续性饲喂高精料日粮的适应机制，并探索此适应性过程中瘤胃上皮生长的细胞信号传导机制。项目发现饲喂高精料日粮改变了绵羊瘤胃发酵模式，增加了瘤胃总挥发性脂肪酸产量，发现绵羊瘤胃上皮可通过形态学的改变来适应高精料日粮饲喂模式，发现饲喂高精料日粮可影响瘤胃上皮细胞周期蛋白CyclinE1和细胞周期蛋白激酶CDK2的表达，加快瘤胃上皮细胞周期进程，减弱瘤胃上皮细胞凋亡，进而导致瘤胃上皮乳头发生适应性变化；发现高精料日粮通过提高血液中IGF-1浓度，激活瘤胃上皮细胞“IGF-1→IGF-1R→Ras/Raf/MEK/ERK和PI3K/AKT通路→瘤胃上皮细胞增殖与凋亡→瘤胃上皮生长”这一通路，促进了瘤胃上皮生长，促使瘤胃上皮在形态与功能上适应高精料日粮。在此基础上，项目采用瘤胃上皮细胞原代培养技术，探究了IGF-1促进绵羊瘤胃上皮细胞生长的机理，发现IGF-1可通过缩短瘤胃上皮细胞周期的S期来推进瘤胃上皮细胞细胞周期进程。项目同时发现，丁酸钠可通过缩短瘤胃上皮细胞周期中的S期，推进瘤胃上皮细胞细胞周期进程，发现IGF-1和丁酸共存可影响Ras/Raf/MEK/ERK信号通路、PI3K/AKT信号通路和p38 MAPK信号通路中相关基因和蛋白的表达，进而影响瘤胃上皮细胞增殖和凋亡的生物学进程。项目结果说明，高精料日粮可通过IGF-1激活Ras/Raf/MEK/ERK、PI3K/AKT和p38/MAPK信号通路，进而调控瘤胃上皮细胞周期、细胞凋亡以及细胞增殖等相关蛋白的表达，从而增大瘤胃上皮乳头吸收表面积，促使瘤胃上皮适应高精料日粮的饲喂模式，同时瘤胃内丁酸也有助于瘤胃上皮对高精料日粮的适应。