黄精调控PINK1介导的线粒体自噬延缓大鼠内皮祖细胞复制性衰老

Rhizoma polygonati attenuate the senescence of endothelial progenitor cells of rats via PINK1 mediated mitophagyFeng Jing-yue1,Ye Li-bing2,Shi Yong-fang1, Liang Bing1*,Qin Zhen1*(1.School of basic medical science,Guizhou Medical University,Guiyang 550025,China 2. The Fifth Hospital of Guangzhou Medical University,Guangdong 510799,China)

关键词:  黄精  内皮祖细胞  复制性衰老  细胞功能  线粒体功能  线粒体自噬

Key words:  Rhizoma Polygonati  endothelial progenitor cells  replicative senescence  fuctional activities  mitochondrial function  mitophagy

摘要:

目的 动态观察黄精含药血清对大鼠骨髓内皮祖细胞(endothelial progenitor cells,EPCs)复制性衰老进程中线粒体功能和线粒体自噬水平的影响,探讨黄精是否通过调节磷酸酶和张力蛋白同源物诱导激酶1(PTEN-induced putative kinase 1, PINK1)介导的线粒体噬自延缓EPCs衰老。方法 分离培养大鼠骨髓 EPCs 并鉴定,将培养至第4、6、8代的 EPCs各分为7组：对照组(10 %空白血清),黄精低、中、高剂量组(10%黄精低、中、高剂量含药血清),黄精低、中、高剂量组+3-甲基腺嘌呤(3-methyl adenine,3MA),各组干预 48 h后,采用β-半乳糖苷酶染色法检测细胞衰老程度；CCK-8 法检测细胞增殖功能；Transwell 小室检测迁移功能；体外成血管试剂盒检测成血管功能；JC-1 法检测细胞线粒体膜电位；化学发光法检测 ATP 含量；流式细胞仪检测胞内ROS水平；Western blot检测线粒体自噬相关蛋白PINK1、Pink1/ E3泛素连接酶(E3 ubiquitin ligases,Parkin)、微管相关蛋白轻链3(microtubule-associated protein lightchain 3,LC3)、p62的表达。结果 体外培养EPCs 至第4、6、8代,其衰老程度逐渐加重,细胞增殖、迁移和成小管功能损伤逐渐加重；胞内ROS水平逐渐升高,线粒体膜电位和ATP 含量逐渐降低；PINK1、Parkin、LC3蛋白表达下调,p62蛋白上调(P<0.05)。经黄精干预后,EPCs 衰老程度减轻,细胞增殖、迁移和成小管功能有所改善；胞内ROS水平和线粒体膜电位降低、 ATP 含量升高；PINK1、Parkin、LC3蛋白表达上调,p62蛋白下调(P<0.05)；3-MA可显著抑制黄精对EPCs的上述作用。结论 黄精可通过促进PINK介导的线粒体自噬水平,维护线粒体功能来延缓大鼠EPCs复制性衰老。

Abstract:

Abstract：Aim To observe dynamically the effect of Rhizoma Polygonati（RP） on the mitochondrial function and mitophagy in the replicative senescence process of endothelial progenitor cells (EPCs) derived from rat bone marrow, investigating the attenuate effect of RP on the senescence of EPCs is related with Pink1 mediated mitophagy. Methods EPCs derived from rat bone marrow were cultured and characterized. The 4th, 6th, and 8th passages of EPCs were divided into seven groups：a control group (treated with 10% blank serum)，RP-low group, RP-mid group，and RP-high group (treated with 10% RP low，medium，and high dose drug-containing serum，respectively), RP-low，RP-mid and RP-high groups with 3-methyl adenine（3-MA）. Following a 48-hour intervention，the positive rate of cell senescence was assessed using beta-galactosidase. Cellular fuction of proliferation，migration，and tubule formation were evaluated using CCK-8，transwell chamber，and in vivo angiogenesis kit，respectively. Additionally，the levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP)，and ATP content were measured using flow cytometry, JC-1, and chemiluminescence method.The expression of mitophagy related proteins PINK1, E3 ubiquitin ligases(Parkin)、3 microtubule-associated protein lightchain 3（LC3）and p62 was detected by Western-blot. Results EPCs were subcultured to the 4th，6th and 8th passage，the positive rate of cell senescence increased gradually，accompanied by a significant decrease in proliferation，migration and tubule formation of EPCs.The level of ROS increased but the MMP and ATP content both decreased.The expression of PINK1, Parkin, and LC3 was down-regulated while p62 protein was up-regulated (P<0.05). However，RP treatment could reduce the positive rate of cell senescence ，enhance the functional activities of EPCs，decrease the ROS level and increase the MMP and ATP content (P<0.05). The expression of PINK1, Parkin, and LC3 was up-regulated while p62 protein was down-regulated after RP treatment. 3-MA significantly inhibited the above effects of RP on EPCs. Conclusion RP could attenuate the replicative senescence of EPCs in rats by maintaining the mitochondrial function through PINK-mediated mitophagy.