Levosimendan Inhibits Peroxidation in Hepatocytes by Modulating Apoptosis/Autophagy Interplay

Abstract

BackgroundLevosimendan 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.Methods and FindingsIn 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.ConclusionsLevosimendan 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.

Levosimendan Improves Oxidative Balance in Cardiogenic Shock/Low Cardiac Output Patients

“…Similar beneficial effects against peroxidation, as were observed in the renal model of ischemia/reperfusion, have been observed in anesthetized rats subjected to hepatic ischemia/reperfusion and treated with levosimendan [21]. Finally, in rat hepatocytes, the administration of levosimendan dose-dependently counteracted the injuries caused by oxidative stress, as evidenced by the keeping GSH content and the reduction of TBARS release [24]. In all the above conditions, endothelial and mitochondrial function were found to be ameliorated by levosimendan, which prevented the fall of mitochondrial membrane potential and restored nitric oxide (NO) release.…”

Anti-Vascular Endothelial Growth Factors Protect Retinal Pigment Epithelium Cells Against Oxidation by Modulating Nitric Oxide Release and Autophagy

“…After each treatment, the medium was removed and an MTT solubilization solution was added and mixed in a gyratory shaker until the complete dissolution of formazan crystals. Cell viability was determined by measuring the absorbance through a spectrometer (BS1000 Spectra Count, San Jose, CA, USA) [18-20]. …”

Modulation of Oxidative Stress by 17 β-Estradiol and Genistein in Human Hepatic Cell Lines In Vitro

“…The oxidation of 2,7-dichlorodihydrofluorescein diacetate into 2,7- dichlorodihydrofluorescein was used to assess ROS generation, following the manufacturer’s instructions (Abcam, Cambridge, United Kindom), and as previously performed [14, 15]. Briefly, cells in 96-well plates were stimulated with genistein (10 pM;1 µM) and 17 β estradiol (10 pM; 100 nM), as described for GSH measurement.…”