FOXA2对SARA引起的反刍动物肝脏炎症的调节作用与机制研究

High-yielding dairy cows are often fed with high ratio concentrate diet, and usually suffer from subacute ruminal acidosis (SARA) in veterinary clinic. Under SARA status, lipopolysaccharide (LPS) produced in the rumen is absorbed and enter the liver from portal vein, thus cause liver inflammatory response. Our initial study showed that transcription factor FOXA2 could reduce inflammatory cytokine secretion in the liver of dairy cows. However, the exact mechanism involved in it is still unclear. In this project, dairy goats will be used as model. SARA will be induced by feeding high ratio concentrate diet. Portal and hepatic catheters will be inserted by surgical operation. Punch biopsy of liver tissue, signal pathway analysis, immunofluorescent observation, chromatin compaction assay, DNA methylation assay, chromatin immunoprecipitation assay techniques will be used in this project. The study of the project includes: transcription control of LPS on gene expression of FOXA2 in liver tissues; effects of FOXA2 on TLR4-NF-κB signal pathway of liver tissues; regulation mechanism of FOXA2 on inflammatory response in liver will be further revealed by LPS infusion experiment from portal vein; molecular mechanism of FOXA2 on inflammatory response of primary hepatocytes in vitro; by up-regulating FOXA2 expression to interfere inflammatory response of liver caused by SARA. The results of this project will provide new target point and theoretical basis for veterinary clinical control of liver inflammation caused by SARA and restoration of liver function in dairy cows.

高产奶牛因饲喂高比例精料日粮，临床上常发生亚急性瘤胃酸中毒(SARA)。SARA状态下瘤胃中产生的脂多糖（LPS）被吸收后由门静脉进入肝脏引起炎症反应。前期研究发现，转录因子FOXA2可以减轻肝脏炎性因子的分泌，但其确切机制尚不清楚。本项目以奶山羊为模型动物，人工诱发SARA，手术安装门静脉和肝静脉血管瘘，应用肝脏组织活体取样技术、信号通路分析、免疫荧光观察、染色质重塑分析、DNA甲基化分析、染色质免疫共沉淀等方法研究： LPS对肝脏组织中FOXA2基因表达的转录调控；FOXA2对肝脏组织中TLR4-NF-κB信号通路的影响；通过门静脉LPS灌注试验进一步研究FOXA2对肝脏炎症反应的调节机制；体外研究FOXA2对肝细胞炎症反应的分子机制；通过上调FOXA2表达，干预SARA引起的肝脏炎症反应。研究结果可为兽医临床上SARA引起的奶牛肝脏炎症的防治和肝脏功能的修复提供新的靶点和理论依据。

饲喂高精料日粮引起的亚急性瘤胃酸中毒（SARA）是临床上高产泌乳奶牛常见的多发病。SARA状态下瘤胃中产生的脂多糖（LPS）被吸收后由门静脉进入肝脏而引发肝脏稳态的失衡，严重危害奶牛的健康。前期研究发现，转录因子FOXA2可抑制肝脏炎性因子的分泌，对肝脏稳态的维持起到重要的调控作用。本项目通过饲喂高精料日粮成功诱发了奶牛SARA，瘤胃中产生的LPS进入肝脏后激活了TLR4/NF-κB和MAPK信号通路，肝细胞的自噬和氧化应激水平升高。饲喂高精料日粮可通过启动子区域的DNA甲基化和染色质压缩而抑制肝脏组织中FOXA2的表达。FOXA2启动子区域的甲基化水平升高与去甲基化酶TET1的表达量降低有关，染色质压缩程度上升与去乙酰化酶HDAC3表达的升高相关。因此，高精料日粮诱导的奶牛SARA通过表观遗传修饰降低了肝脏组织中FOXA2的表达，进而激活NF-κB和MAPK信号通路，肝细胞自噬和氧化应激水平升高，从而引起肝脏稳态的失衡与肝脏功能受损。体外研究结果发现，LPS处理奶牛肝细胞可激活TLR4/NF-κB炎性信号通路，内质网应激、自噬和凋亡水平升高，同时降低FOXA2的表达。抑制TLR4可缓解LPS引起的炎症反应、内质网应激、自噬和凋亡，FOXA2的表达水平升高。LPS刺激下，过表达FOXA2可降低LPS引起的NF-κB介导的炎症反应、内质网应激、自噬和凋亡水平。而干扰FOXA2表达可升高奶牛肝细胞的炎症反应、自噬和凋亡水平。门静脉LPS灌注试验进一步证明了以上结果。体外研究结果表明，FOXA2处于TLR4下游和NF-κB上游，FOXA2对肝细胞的炎症反应、内质网应激、自噬和凋亡起到重要的调控作用，在维持肝细胞的稳态中发挥重要功能。对饲喂高精料日粮的泌乳湖羊添加延胡索酸二钠能有效防止SARA的发生，并可通过表观遗传修饰升高肝脏组织中FOXA2的表达水平，降低炎症反应、坏死性凋亡、焦亡、内质网应激和自噬水平，从而减轻高精料日粮对肝脏的损伤作用。本项目揭示了LPS 对肝脏组织中FOXA2 基因表达的转录调控机理，明确了FOXA2 对LPS引起的反刍动物肝脏稳态失衡的调节作用与分子机制，证实了调控瘤胃代谢对肝脏FOXA2表达与肝功能的修复具有重要的作用。研究结果可为兽医临床上SARA 引起的肝脏炎症的综合防治和肝脏功能的修复提供新的靶点和理论依据。