迷迭香酸通过上调PINK1/Parkin介导的线粒体自噬增强巨噬细胞抗菌免疫的机制研究

Rosmarinic acid bolsters antibacterial immunity activity of macrophages by up-regulating PINK1/Parkin-mediated mitophagy

探讨迷迭香酸(rosmarinic acid)通过上调线粒体自噬增强巨噬细胞抗菌免疫的具体分子机制。将巨噬细胞RAW264.7用不同浓度的迷迭香酸预处理后感染金黄色葡萄球菌Staphylococcus aureus,提取细胞总RNA和总蛋白,利用q-PCR和Western blot法分别检测磷酸酶及张力蛋白同源物(PTEN)诱导的激酶1(PTEN induced putative kinase 1,PINK1)转录水平和蛋白表达的变化;使用线粒体分离试剂盒分离巨噬细胞线粒体,结合Western blot法检测E3泛素-蛋白连接酶Parkin在线粒体的募集情况及其磷酸化水平的变化;综合运用免疫共沉淀和激光共聚焦显微技术观察PINK1和Parkin在细胞内的共定位情况并观察迷迭香酸对PINK1/Parkin相互作用的影响;利用线粒体自噬抑制剂Mdivi-1(mitochondrial division inhibitor 1)预处理、小干扰RNA(small interfering RNA,siRNA)敲除技术以及细菌平板计数,检测炎症细胞因子水平和胞内杀菌能力,确证迷迭香酸可以通过上调PINK1/Parkin介导的线粒体自噬增强巨噬细胞抗菌免疫。结果显示,迷迭香酸可显著上调PINK1转录和蛋白水平的表达,促进Parkin由细胞质向线粒体募集并增强其磷酸化水平,促进PINK1和Parkin的相互作用并增强其在巨噬细胞内的共定位;阻断线粒体自噬或敲除PINK1能显著抑制迷迭香酸对巨噬细胞抗菌免疫的促进作用。该研究表明,迷迭香酸通过上调PINK1/Parkin介导的线粒体自噬促进巨噬细胞抗菌固有免疫应答。

This study aims to explore the molecular mechanism through which rosmarinic acid up-regulates mitophagy and enhances antibacterial immunity activity of macrophages. To be specific, RAW264.7 macrophages were treated with rosmarinic acid and then infected with Staphylococcus aureus. The total mRNA and proteins of the cells were then extracted. The mRNA and protein levels of phosphatase and tensin homolog(PTEN)-induced putative kinase 1(PINK1) were detected by q-PCR and Western blot, respectively. Cell mitochondria isolation kit was employed to isolate mitochondria in macrophages. Recruitment of E3 ubiquitin ligase Parkin to mitochondria and the phosphorylation of Parkin were detected by Western blot. Co-immunoprecipitation and laser confocal microscopy were employed to observe the co-localization of PINK1 and Parkin. Mitochondrial division inhibitor 1(Mdivi-1), small interfering RNA(siRNA)-directed gene knockdown, and plate-colony counting were used to detect the levels of inflammatory cytokines and the intracellular antibacterial ability, in an attempt to confirm that rosmarinic acid promotes antibacterial immunity activity of macrophages through strengthening PINK1/Parkin-mediated mitophagy. The results showed that rosmarinic acid up-regulated the mRNA and protein expression of PINK1, promoted the recruitment of Parkin from cytoplasm to mitochondria and the phosphorylation, and enhanced the interaction between PINK1 and Parkin and their co-localization in macrophages. Blocking mitophagy or knocking PINK1 significantly abrogated the promotion of macrophage antibacterial immune response by rosmarinic acid. In summary, rosmarinic acid enhances antibacterial immunity activity of macrophages through up-regulating PINK1/Parkin-mediated mitophagy.