Cheng Cheng, Sha Zhou, Chen Xiang, Zhang Wei, Shi Li-Yun
Nanjing University of Chinese Medicine Nanjing 210023, China.
Zhongguo Zhong Yao Za Zhi. 2022 Dec;47(23):6450-6456. doi: 10.19540/j.cnki.cjcmm.20220726.401.
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.
本研究旨在探讨迷迭香酸上调巨噬细胞线粒体自噬并增强其抗菌免疫活性的分子机制。具体而言，用迷迭香酸处理RAW264.7巨噬细胞，然后用金黄色葡萄球菌感染。随后提取细胞的总mRNA和蛋白质。分别通过q-PCR和蛋白质免疫印迹法检测磷酸酶和张力蛋白同源物（PTEN）诱导的假定激酶1（PINK1）的mRNA和蛋白质水平。使用细胞线粒体分离试剂盒分离巨噬细胞中的线粒体。通过蛋白质免疫印迹法检测E3泛素连接酶帕金蛋白向线粒体的募集以及帕金蛋白的磷酸化。采用免疫共沉淀和激光共聚焦显微镜观察PINK1和帕金蛋白的共定位。使用线粒体分裂抑制剂1（Mdivi-1）、小干扰RNA（siRNA）介导的基因敲低以及平板菌落计数来检测炎性细胞因子水平和细胞内抗菌能力，以试图证实迷迭香酸通过增强PINK1/帕金蛋白介导的线粒体自噬来促进巨噬细胞的抗菌免疫活性。结果表明，迷迭香酸上调了PINK1的mRNA和蛋白质表达，促进了帕金蛋白从细胞质向线粒体的募集及其磷酸化，并增强了巨噬细胞中PINK1与帕金蛋白之间的相互作用及其共定位。阻断线粒体自噬或敲除PINK1可显著消除迷迭香酸对巨噬细胞抗菌免疫反应的促进作用。综上所述，迷迭香酸通过上调PINK1/帕金蛋白介导的线粒体自噬来增强巨噬细胞的抗菌免疫活性。
