Kittur Farooqahmed S, Lin Yuan, Arthur Elena, Hung Chiu-Yueh, Li P Andy, Sane David C, Xie Jiahua
Department of Pharmaceutical Sciences, Biomanufacturing Research Institute & Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA.
School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, China.
Biochem Biophys Rep. 2019 Jan 9;17:157-168. doi: 10.1016/j.bbrep.2019.01.004. eCollection 2019 Mar.
Recombinant human erythropoietin (rhuEPO) and asialoerythropoietin (asialo-rhuEPO) are cardioprotective. However, the protective effects of rhuEPO could not be translated into clinical practice because of its hematopoiesis-associated side effects while non-erythropoietic asialo-rhuEPO is unavailable in large quantities for clinical studies. This study was designed to investigate the cardiomyocyte protective potential of plant-produced asialo-rhuEPO (asialo-rhuEPO) against staurosporine (STS)-induced injury in HL-1 murine cardiomyocytes and identify cellular pathway(s) responsible for its cardioprotection.
HL-1 cardiomyocytes were simultaneously treated with STS and asialo-rhuEPO. Cellular injury, apoptosis, and cell viabilities were measured by LDH assay, Hoechst staining and trypan blue exclusion method, respectively while western blotting was used to study its effects on apoptosis and autophagy hallmarks.
Our results showed that 20 IU/ml asialo-rhuEPO provided 39% protection to cardiomyocytes compared to STS-treated cells, which is 2-fold better than that of mammalian cell-produce rhuEPO (rhuEPO). Asialo-rhuEPO was found to suppress activation of proapoptotic kinase Mst1 (mammalian Sterile-20-like kinase 1) and FOXO3, leading to inhibition of apoptotic pathway and restoration of autophagy as indicated by the reduction of fragmented/condensed nuclei, altered ratios of Bax/Bcl2, p-Bad/Bad, cytosol/mitochondrial cyt and caspase-3 activation, and the restored levels of autophagy markers Beclin1, p62 and LC3B-II. Additionally, Akt was found to be activated and FOXO3 was phosphorylated on Ser253, suggesting inhibition of FOXO3 transcriptional function.
Asialo-rhuEPO-mediated cardioprotection occurs through activation of PI3K/Akt pathway leading to suppression of Mst1 activation and promoting cardiomyocyte survival.
Asialo-rhuEPO could be used to modulate Mst1 activity elevated under numerous pathological states.
重组人促红细胞生成素（rhuEPO）和去唾液酸促红细胞生成素（去唾液酸 - rhuEPO）具有心脏保护作用。然而，由于rhuEPO存在与造血相关的副作用，其保护作用无法转化为临床应用，而用于临床研究的非促红细胞生成的去唾液酸 - rhuEPO无法大量获得。本研究旨在探讨植物产生的去唾液酸 - rhuEPO对星形孢菌素（STS）诱导的HL - 1小鼠心肌细胞损伤的心肌保护潜力，并确定其心脏保护作用的细胞途径。
HL - 1心肌细胞同时用STS和去唾液酸 - rhuEPO处理。分别通过乳酸脱氢酶（LDH）测定、Hoechst染色和台盼蓝排斥法测量细胞损伤、凋亡和细胞活力，同时使用蛋白质印迹法研究其对凋亡和自噬标志的影响。
我们的结果表明，与STS处理的细胞相比，20 IU/ml的去唾液酸 - rhuEPO为心肌细胞提供了39%的保护，比哺乳动物细胞产生的rhuEPO（rhuEPO）好2倍。发现去唾液酸 - rhuEPO可抑制促凋亡激酶Mst1（哺乳动物无活性20样激酶1）和FOXO3的激活，导致凋亡途径的抑制和自噬的恢复，表现为碎片化/浓缩核减少、Bax/Bcl2、p - Bad/Bad、胞质/线粒体细胞色素c和半胱天冬酶 - 3激活的比率改变，以及自噬标志物Beclin1、p62和LC3B - II水平的恢复。此外，发现Akt被激活且FOXO3在Ser253处磷酸化，表明FOXO3转录功能受到抑制。
去唾液酸 - rhuEPO介导的心脏保护作用通过激活PI3K/Akt途径发生，导致Mst1激活的抑制并促进心肌细胞存活。
去唾液酸 - rhuEPO可用于调节在多种病理状态下升高的Mst1活性。
