Grossini Elena, Bellofatto Kevin, Farruggio Serena, Sigaudo Lorenzo, Marotta Patrizia, Raina Giulia, De Giuli Veronica, Mary David, Pollesello Piero, Minisini Rosalba, Pirisi Mario, Vacca Giovanni
Laboratory of Physiology and Experimental Surgery, Department of Translational Medicine, University Eastern Piedmont "Amedeo Avogadro", Via Solaroli 17, Azienda Ospedaliera Universitaria Maggiore della Carità, corso Mazzini 36, Novara, Italy.
Internal Medicine, Department of Translational Medicine, University Eastern Piedmont "Amedeo Avogadro", Via Solaroli 17, Azienda Ospedaliera Universitaria Maggiore della Carità, corso Mazzini 36, Novara, Italy.
PLoS One. 2015 Apr 16;10(4):e0124742. doi: 10.1371/journal.pone.0124742. eCollection 2015.
Levosimendan protects rat liver against peroxidative injuries through mechanisms related to nitric oxide (NO) production and mitochondrial ATP-dependent K (mitoKATP) channels opening. However, whether levosimendan could modulate the cross-talk between apoptosis and autophagy in the liver is still a matter of debate. Thus, the aim of this study was to examine the role of levosimendan as a modulator of the apoptosis/autophagy interplay in liver cells subjected to peroxidation and the related involvement of NO and mitoKATP.
In primary rat hepatocytes that have been subjected to oxidative stress, Western blot was performed to examine endothelial and inducible NO synthase isoforms (eNOS, iNOS) activation, apoptosis/autophagy and survival signalling detection in response to levosimendan. In addition, NO release, cell viability, mitochondrial membrane potential and mitochondrial permeability transition pore opening (MPTP) were examined through specific dyes. Some of those evaluations were also performed in human hepatic stellate cells (HSC). Pre-treatment of hepatocytes with levosimendan dose-dependently counteracted the injuries caused by oxidative stress and reduced NO release by modulating eNOS/iNOS activation. In hepatocytes, while the autophagic inhibition reduced the effects of levosimendan, after the pan-caspases inhibition, cell survival and autophagy in response to levosimendan were increased. Finally, all protective effects were prevented by both mitoKATP channels inhibition and NOS blocking. In HSC, levosimendan was able to modulate the oxidative balance and inhibit autophagy without improving cell viability and apoptosis.
Levosimendan protects hepatocytes against oxidative injuries by autophagic-dependent inhibition of apoptosis and the activation of survival signalling. Such effects would involve mitoKATP channels opening and the modulation of NO release by the different NOS isoforms. In HSC, levosimendan would also play a role in cell activation and possible evolution toward fibrosis. These findings highlight the potential of levosimendan as a therapeutic agent for the treatment or prevention of liver ischemia/reperfusion injuries.
左西孟旦通过与一氧化氮（NO）生成及线粒体ATP依赖性钾通道（mitoKATP）开放相关的机制保护大鼠肝脏免受过氧化损伤。然而，左西孟旦是否能调节肝脏中凋亡与自噬之间的相互作用仍存在争议。因此，本研究的目的是探讨左西孟旦作为过氧化肝细胞中凋亡/自噬相互作用调节剂的作用以及NO和mitoKATP的相关参与情况。
在遭受氧化应激的原代大鼠肝细胞中，进行蛋白质印迹法以检测内皮型和诱导型NO合酶同工型（eNOS、iNOS）的激活、凋亡/自噬以及对左西孟旦反应的生存信号检测。此外，通过特定染料检测NO释放、细胞活力、线粒体膜电位和线粒体通透性转换孔开放（MPTP）。其中一些评估也在人肝星状细胞（HSC）中进行。用左西孟旦对肝细胞进行预处理可剂量依赖性地抵消氧化应激造成的损伤，并通过调节eNOS/iNOS激活来减少NO释放。在肝细胞中，虽然自噬抑制降低了左西孟旦的作用，但在泛半胱天冬酶抑制后，对左西孟旦的细胞存活和自噬增加。最后，mitoKATP通道抑制和NOS阻断均阻止了所有保护作用。在HSC中，左西孟旦能够调节氧化平衡并抑制自噬，但未改善细胞活力和凋亡。
左西孟旦通过自噬依赖性抑制凋亡和激活生存信号来保护肝细胞免受氧化损伤。这些作用将涉及mitoKATP通道开放以及不同NOS同工型对NO释放的调节。在HSC中，左西孟旦也将在细胞激活以及可能向纤维化发展中发挥作用。这些发现突出了左西孟旦作为治疗或预防肝脏缺血/再灌注损伤治疗药物的潜力。
