NOX2/ROS通路调控猪胎盘血管生成的作用机制研究

The birth weight of piglets significantly affects survival and postnatal growth of newborn piglets. Placental angiogenesis abnormality is the major cause of low birth weight (LBW) of piglets. However, the mechanism of the differences in placental angiogenesis among the litter remains unclear. Our previous study demonstrated that the density of placental vessels was diminished in LBW piglets, and the expression levels of placental angiogenic markers (CD31 and VEGF) were noticeably decreased, as well as NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS) pathway was activated in the LBW piglets. Thus, we propose that NOX2/ROS is a negative regulation pathway of placental angiogenesis. On the basis of previous study, firstly, we would investigate the role and mechanism of NOX2 in placental angiogenesis on pig iliac endothelium cell lines by agonists, inhibitors and gene editing method. Secondly, we would examine the effect of inhibition NOX2-derived ROS on placental angiogenesis and birth weight of piglets through dietary supplementation NOX2 inhibitor and ROS scavenger in the sows during gestation. This study could not only reveal the mechanism of NADPH oxidase 2-derived ROS in the regulation of angiogenesis in porcine placenta, but also provide theoretical basis in the nutritional strategy to improve placental angiogenesis and birth weight of piglets.

仔猪初生重直接影响其生长发育和成活率，而胎盘血管生成异常是导致仔猪初生重低的重要原因。迄今对同窝胎猪不同胎盘间血管生成差异的调节机制尚不清楚。申请人前期研究发现，初生重低的仔猪其胎盘血管密度显著降低、血管生成标记物（CD31和VEGF）表达量显著下调；同时发现NADPH氧化酶亚型2（NOX2）/活性氧（ROS）通路被激活。据此申请人提出如下科学假设：NOX2/ROS是胎盘血管生成的负调控通路。本项目拟在前期基础上，以猪血管内皮细胞为模型，采用NOX2表达调控和药物阻断策略，研究NOX2在血管生成中的作用及机制；然后以不同营养状况的母猪为对象，采用NOX2天然抑制剂和ROS清除剂，研究NOX2/ROS通路对胎盘血管生成和仔猪初生重的影响。研究结果不仅可揭示NOX2/ROS通路调控猪胎盘血管生成的作用机制，同时也为研发促进胎盘血管发育、提高仔猪初生重的营养策略提供理论依据。

宫内发育迟缓易导致仔猪初生重低，影响新生仔猪的存活和后期的生长发育。胎盘血管生成异常是导致仔猪初生重低的重要原因。活性氧（ROS）主要由细胞内源氧化酶途径和线粒体呼吸链所产生，在心血管系统中，烟酰胺腺嘌呤二核苷酸磷酸氧化酶（NADPH）是其主要来源之一。本实验室前期数据提示：NOX2/ROS通路可能是胎盘血管生成的重要抑制因素。为此，项目首先分析了不同初生重仔猪的胎盘血管生成、氧化应激及NADPH氧化酶表达的差异规律；随后，研究了NOX2/ROS通路在猪体外血管生成中的作用及其机制；最后，以妊娠母猪为对象，验证了不同营养水平对胎盘NOX2表达、胎盘氧化应激、血管生成和仔猪初生重的影响。主要结果如下：（1）低初生重仔猪的胎盘氧化应激水平增加，线粒体功能和胎盘血管生成下降。尽管NOX2和NOX4在胎盘中均可表达，但NOX2表达量是NOX4的5.7倍，且NOX2在低初生重仔猪胎盘的内皮细胞中高表达。（2）NOX2是抑制猪血管内皮细胞血管生成的关键靶点，其分子机制是：NOX2生成线粒体ROS，降低细胞核内STAT3的磷酸化水平，进而下调VEGF-A的转录活性，最终抑制猪内皮细胞血管生成。（3）母猪妊娠中期饲喂高能量水平饲粮（消化能为13.43 MJ/kg）上调胎盘血管中NOX2的表达，提高胎盘氧化应激水平，降低胎盘线粒体功能和血管密度并最终降低仔猪初生重。当妊娠母猪饲粮消化能为12.41 MJ/kg时，不仅可降低饲料成本，更有利于母猪繁殖性能的发挥，主要体现在仔猪初生重和低体重仔猪比例的改善。本研究结果不仅揭示了NOX2/ROS通路调控猪胎盘血管生成的作用机制，可作为鉴定低初生重仔猪胎盘血管生成异常的新靶点，而且为研制促进胎盘血管发育、提高仔猪初生重的营养策略提供了科学依据。