基于3D培养探讨PINK1-Parkin/线粒体自噬缓解猪乳腺细胞氧化应激机理及营养调控

Mammary gland damage caused by oxidative stress could affect the lactation performance of sows and reduce the growth performance of piglets. Mitophagy is very important for alleviating oxidative stress damage, and antioxidant might alleviate oxidative stress and improve lactation performance through mitophagy. Nowadays, monolayer cell culture, which is difficult to simulate in vivo environment, is used as the research model. Therefore, this project is conducted to study the effects of oxidative stress on the mitophagy function of pig mammary epithelial cells (PMECs) by using a 3D culture model of PMECs challenged with hydrogen peroxide, to investigate the role of mitophagy mediated by PINK1-Parkin pathway on the regulation of oxidative stress damage in PMECs. Meanwhile, isoflavones were used as antioxidants to study the effects of antioxidants on the regulation mechanism in mitochondrial function and PINK1-Parkin pathway of PMECs in order to explore the mechanism of isoflavones in improving cellular oxidative stress. The aim of this project is to clarify the core mechanism of mitophagy mediated by PINK1-Parkin pathway in the regulation of oxidative stress in PMECs, and to alleviate oxidative damage of mammary gland cells through nutritional regulation, so as to provide a theoretical and technical basis for improving lactation performance of sow.

氧化应激造成乳腺功能损伤影响母猪泌乳性能，降低仔猪生长性能。线粒体自噬对缓解氧化应激损伤非常重要，抗氧化剂可能通过线粒体自噬有效缓解氧化应激改善泌乳性能。目前多采用难模拟体内环境的单层细胞培养作为研究模型。因此，本项目利用猪乳腺上皮细胞3D培养模型，以过氧化氢诱导应激损伤，研究氧化应激对猪乳腺上皮细胞的线粒体自噬功能的影响；过表达及沉默PINK1基因，探讨PINK1-Parkin介导线粒体自噬在调控猪乳腺上皮细胞氧化应激损伤中的作用机制；通过添加异黄酮，研究抗氧化剂对猪乳腺上皮细胞线粒体功能和PINK1-Parkin通路的调控作用，探讨异黄酮改善细胞氧化应激的作用机制。以期阐明PINK1-Parkin介导线粒体自噬调控猪乳腺上皮细胞氧化应激中的核心作用机制，通过营养调控缓解乳腺细胞氧化损伤，为提高母猪泌乳性能提供理论和技术基础。

氧化应激造成乳腺功能损伤影响母猪泌乳性能，降低仔猪生长性能。线粒体自噬对缓解氧化应激损伤非常重要，抗氧化剂可能通过线粒体自噬有效缓解氧化应激改善泌乳性能。目前多采用难模拟体内环境的单层细胞培养作为研究模型。项目通过构建猪乳腺上皮细胞3D培养和组织培养模型，以过氧化氢、4-HNE等氧化诱导剂诱导应激损伤，发现氧化应激降低猪乳腺上皮细胞的增殖和线粒体功能，抑制线粒体自噬；添加抗氧化剂白藜芦醇及异黄酮后缓解了氧化应激对猪乳腺上皮细胞的损伤，且均通过PINK1-Parkin介导线粒体自噬实现。RNA-seq结果表明白藜芦醇通过TNF信号通路，NF-RB通路，MAPK通路等信号通路调控线粒体自噬通路。结果阐明PINK1-Parkin介导线粒体自噬调控猪乳腺上皮细胞氧化应激中的核心作用机制，通过抗氧化剂白藜芦醇和异黄酮在体内外缓解了猪乳腺细胞氧化损伤，为提高母猪泌乳性能提供理论和技术基础。