Zhang Mingming, Niu Xiaolin, Hu Jianqiang, Yuan Yuan, Sun Shuhong, Wang Jiaxing, Yu Wenjun, Wang Chen, Sun Dongdong, Wang Haichang
Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, China.
Department of Cardiology, Tangdu Hospital, Fourth Military Medical University, Xi'an, China.
PLoS One. 2014 Oct 14;9(10):e110580. doi: 10.1371/journal.pone.0110580. eCollection 2014.
The aim of the present study was to investigate the role of Lin28a in protecting against hypoxia/reoxygenation (H/R)-induced cardiomyocytes apoptosis under high glucose/high fat (HG/HF) conditions.
Primary cardiomyocytes which were isolated from neonatal mouse were randomized to be treated with lentivirus carrying Lin28a siRNA, Lin28acDNA 72 h before H/R (9 h/2 h). Cardiomyocytes biomarkers release (LDH and CK), cardiomyocytes apoptosis, mitochondria biogenesis and morphology, intracellular reactive oxygen species (ROS) production, ATP content and inflammatory cytokines levels after H/R injury in high glucose/high fat conditions were compared between groups. The target proteins of Lin28a were examined by western blot analysis.
Our results revealed that Lin28a cDNA transfection (overexpression) significantly inhibited cardiomyocyte apoptotic index, improved mitochondria biogenesis, increased ATP production and reduced ROS production as compared with the H/R group in HG/HF conditions. Lin28a siRNA transfection (knockdown) rendered the cardiomyocytes more susceptible to H/R injury as evidenced by increased apoptotic index, impaired mitochondrial biogenesis, decreased ATP production and increased ROS level. Interestingly, these effects of Lin28a were blocked by pretreatment with the PI3K inhibitor wortmannin. Lin28a overexpression increased, while Lin28a knockdown inhibited IGF1R, Nrf-1, Tfam, p-IRS-1, p-Akt, p-mTOR, p-p70s6k, p-AMPK expression levels after H/R injury in HG/HF conditions. Moreover, pretreatment with wortmannin abolished the effects of Lin28a on the expression levels of p-AKT, p-mTOR, p-p70s6k, p-AMPK.
The present results suggest that Lin28a inhibits cardiomyocytes apoptosis by enhancing mitochondrial biogenesis and function under high glucose/high fat conditions. The mechanism responsible for the effects of Lin28a is associated with the PI3K/Akt dependent pathway.
本研究旨在探讨Lin28a在高糖/高脂（HG/HF）条件下对缺氧/复氧（H/R）诱导的心肌细胞凋亡的保护作用。
将从新生小鼠分离的原代心肌细胞随机分组，在H/R（9小时/2小时）前72小时用携带Lin28a小干扰RNA（siRNA）、Lin28a互补DNA（cDNA）的慢病毒处理。比较各组在HG/HF条件下H/R损伤后心肌细胞生物标志物释放（乳酸脱氢酶和肌酸激酶）、心肌细胞凋亡、线粒体生物发生和形态、细胞内活性氧（ROS）产生、三磷酸腺苷（ATP）含量及炎性细胞因子水平。通过蛋白质免疫印迹分析检测Lin28a的靶蛋白。
我们的结果显示，与HG/HF条件下的H/R组相比，Lin28a cDNA转染（过表达）显著抑制心肌细胞凋亡指数，改善线粒体生物发生，增加ATP产生并减少ROS产生。Lin28a siRNA转染（敲低）使心肌细胞对H/R损伤更敏感，表现为凋亡指数增加、线粒体生物发生受损、ATP产生减少及ROS水平升高。有趣的是，Lin28a的这些作用被磷脂酰肌醇-3-激酶（PI3K）抑制剂渥曼青霉素预处理所阻断。在HG/HF条件下H/R损伤后，Lin28a过表达增加，而Lin28a敲低抑制胰岛素样生长因子1受体（IGF1R）、核呼吸因子-1（Nrf-1）、线粒体转录因子A（Tfam）、磷酸化胰岛素受体底物-1（p-IRS-1）、磷酸化蛋白激酶B（p-Akt）、磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）、磷酸化核糖体蛋白S6激酶（p-p70s6k）、磷酸化腺苷酸活化蛋白激酶（p-AMPK）的表达水平。此外，渥曼青霉素预处理消除了Lin28a对p-AKT、p-mTOR、p-p70s6k、p-AMPK表达水平的影响。
目前的结果表明，在高糖/高脂条件下，Lin28a通过增强线粒体生物发生和功能来抑制心肌细胞凋亡。Lin28a发挥作用的机制与PI3K/Akt依赖性途径有关。
