JAK/STAT3通路调控蹄组织中MMP-2/9活性在SARA引发的蹄叶炎发病中的作用与营养干预研究

Laminitis is one of the most important disease issues of high producing dairy cows in China. Previous studies showed that subacute rumen acidosis(SARA) can improve the matrix metalloproteinase 2 (MMP-2) and matix metallopoteinase 9 (MMP-9) activity in lamina tissue, this lead to the lamina dysfunction of the barrier function and connection, and laminitis may occur. However, there is little research on the exploration of the exact mechanism of the increase in MMP activity caused by SARA, and the future preventative measures are not yet clear. Based on these previous researches, three research objectives have been identified for this research project, with the hope that the outcome will reveal the mechanism of the laminitis occurrence induced by the SARA at the cellular and molecular level, and bring the dairy industry closer to finding an effective prevention strategy. The first goal of this project is to examine the effect of inflammation induced by SARA on the activation of JAK/STAT3 signalling pathwary. The second goal was to determine the role of JAK/STAT3 signalling pathway in the regulation of the activity of MMP-2 and MMP-9 in lamina tissue at the levels of transcriptional regulation, zymogen activation and enzyme inhibition in a laminitis model. The third goal is to test the effects of quercetin as a feed additive in preventing the increase in the activity of MMP-2 and MMP-9 in lamina tissue, and to explore the signalling pathway involved in the reduction of MMP-2 and MMP-9 activity induced by quercetin addition during SARA.

蹄叶炎是严重危害我国奶牛健康的一类疾病。我们及他人研究发现，亚急性瘤胃酸中毒（SARA）致奶牛蹄组织中基质金属蛋白酶MMP-2与MMP-9的酶活性升高，蹄组织结构受损，奶牛发生蹄叶炎，但其中的确切机制与干预措施尚不清楚。本课题拟在此基础上，通过SARA诱导蹄叶炎试验及体外建立蹄角细胞受损模型，应用Real-time PCR、Western blot和免疫组化等方法：1、解析SARA引发的炎症反应对蹄组织中JAK/STAT3信号通路活化的影响；2、从转录、酶原活化和酶活抑制三个层面，阐明蹄组织中JAK/STAT3通路对MMP-2/9酶活性的调控作用与机制；3、查明槲皮素干预SARA下蹄组织中MMP-2/9酶活性升高的效应及其信号转导机制。课题将在组织、细胞和分子水平上阐明SARA诱发蹄叶炎的机制，并为蹄叶炎的防治提供思路和理论依据。

前期研究表明，瘤胃酸中毒可诱发反刍动物蹄叶炎，其机制与酸中毒损伤瘤胃上皮屏障功能，致消化道内源性LPS移位进入宿主体内，诱发炎性反应有关。然而，酸中毒诱发蹄叶炎的具体机制不清楚，推测与炎性反应激活MMP-2/9有关，且JAK/STAT3通路参与了该过程的调节作用。为厘清相关机制，本项目从组织、细胞及分子层面，通过体内外手段，系统并深入的探索了瘤胃酸中毒诱发蹄叶炎的生物学机制。通过两个动物饲养实验，结果阐明了瘤胃酸中毒可诱发蹄叶炎，降低瘤胃pH值，提高瘤胃总挥发性脂肪酸、血液乳酸和游离脂多糖含量，导致蹄小叶形态结构严重受损，酸中毒同时提高蹄小叶组织中致炎因子和金属蛋白酶-9的相对表达量，并显著提高JAK2和STAT3的mRNA相对表达量；在此基础上，本研究通过两个动物实验，研究了高精料日粮下通过瘤胃及真胃灌注槲皮素对蹄组织的保护作用，发现槲皮素可降低蹄部炎症和金属蛋白酶的表达量，显示出较好的预防蹄叶炎效应。体外培养实验表明，LPS可剌激蹄组织发生炎性反应，而在此基础上应用槲皮素则可显著降低蹄组织炎性反应，但对JAK/STAT3通路相关基因表达无显著影响。结果说明，酸中毒可活化蹄小叶内JAK/STAT3通路，促进金属蛋白酶的表达，致蹄小叶结构完整性被破坏，诱发蹄叶炎；而添加槲皮素可有效缓解蹄叶炎症状，但其作用机制不涉及JAK/STAT3通路。项目培养研究生5名，发表10篇论文，其中SCI论文5篇。