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Levosimendan inhibits peroxidation in hepatocytes by modulating apoptosis/autophagy interplay.

Grossini E, Bellofatto K, Farruggio S, Sigaudo L, Marotta P, Raina G, De Giuli V, Mary D, Pollesello P, Minisini R, Pirisi M, Vacca G - PLoS ONE (2015)

Bottom Line: 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.Such effects would involve mitoKATP channels opening and the modulation of NO release by the different NOS isoforms.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT

Background: 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.

Methods and findings: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus

Effects of levosimendan on autophagy caused by peroxidation in hepatocytes.In A and B, effects of levosimendan on Beclin 1 and LC3I/II, respectively. Densitometric analysis of Beclin 1 and of LC3II and an example of lanes taken in one of the 5 different experiments performed for each experimental protocol are reported. Abbreviations are as in Figs 1–6. In A, b, c, d, e, f, g P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e P <0.05 vs c; d, f, g P <0.05 vs e. In B, b, c, d, e P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e P <0.05 vs c; d, f, g P <0.05 vs e. The results of densitometric analysis are expressed as means of 5 independent experiments (%) ± SD (indicated by the bars).
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pone.0124742.g007: Effects of levosimendan on autophagy caused by peroxidation in hepatocytes.In A and B, effects of levosimendan on Beclin 1 and LC3I/II, respectively. Densitometric analysis of Beclin 1 and of LC3II and an example of lanes taken in one of the 5 different experiments performed for each experimental protocol are reported. Abbreviations are as in Figs 1–6. In A, b, c, d, e, f, g P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e P <0.05 vs c; d, f, g P <0.05 vs e. In B, b, c, d, e P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e P <0.05 vs c; d, f, g P <0.05 vs e. The results of densitometric analysis are expressed as means of 5 independent experiments (%) ± SD (indicated by the bars).

Mentions: In A, B and C, effects of levosimendan on p-Bax, LC3I/II and p-Akt, respectively. Densitometric analysis of p-Bax, LC3II and p-Akt and an example of lanes taken in one of the 3 different experiments performed for each experimental protocol are reported. Abbreviations are as in Figs 1–7. In A, b, c, d, e P <0.05 vs a; e P <0.05 vs b, c, d. In B, b, c, d, e P <0.05 vs a; c, d, e P <0.05 vs b; d, e P <0.05 vs c; e P <0.05 vs d. In C, b, c, d, e P <0.05 vs a; c, d, e P <0.05 vs b; d, e P <0.05 vs c; e P <0.05 vs d. The results of densitometric analysis are expressed as means of 3 independent experiments (%) ± SD (indicated by the bars).


Levosimendan inhibits peroxidation in hepatocytes by modulating apoptosis/autophagy interplay.

Grossini E, Bellofatto K, Farruggio S, Sigaudo L, Marotta P, Raina G, De Giuli V, Mary D, Pollesello P, Minisini R, Pirisi M, Vacca G - PLoS ONE (2015)

Effects of levosimendan on autophagy caused by peroxidation in hepatocytes.In A and B, effects of levosimendan on Beclin 1 and LC3I/II, respectively. Densitometric analysis of Beclin 1 and of LC3II and an example of lanes taken in one of the 5 different experiments performed for each experimental protocol are reported. Abbreviations are as in Figs 1–6. In A, b, c, d, e, f, g P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e P <0.05 vs c; d, f, g P <0.05 vs e. In B, b, c, d, e P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e P <0.05 vs c; d, f, g P <0.05 vs e. The results of densitometric analysis are expressed as means of 5 independent experiments (%) ± SD (indicated by the bars).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4400069&req=5

pone.0124742.g007: Effects of levosimendan on autophagy caused by peroxidation in hepatocytes.In A and B, effects of levosimendan on Beclin 1 and LC3I/II, respectively. Densitometric analysis of Beclin 1 and of LC3II and an example of lanes taken in one of the 5 different experiments performed for each experimental protocol are reported. Abbreviations are as in Figs 1–6. In A, b, c, d, e, f, g P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e P <0.05 vs c; d, f, g P <0.05 vs e. In B, b, c, d, e P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e P <0.05 vs c; d, f, g P <0.05 vs e. The results of densitometric analysis are expressed as means of 5 independent experiments (%) ± SD (indicated by the bars).
Mentions: In A, B and C, effects of levosimendan on p-Bax, LC3I/II and p-Akt, respectively. Densitometric analysis of p-Bax, LC3II and p-Akt and an example of lanes taken in one of the 3 different experiments performed for each experimental protocol are reported. Abbreviations are as in Figs 1–7. In A, b, c, d, e P <0.05 vs a; e P <0.05 vs b, c, d. In B, b, c, d, e P <0.05 vs a; c, d, e P <0.05 vs b; d, e P <0.05 vs c; e P <0.05 vs d. In C, b, c, d, e P <0.05 vs a; c, d, e P <0.05 vs b; d, e P <0.05 vs c; e P <0.05 vs d. The results of densitometric analysis are expressed as means of 3 independent experiments (%) ± SD (indicated by the bars).

Bottom Line: 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.Such effects would involve mitoKATP channels opening and the modulation of NO release by the different NOS isoforms.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT

Background: 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.

Methods and findings: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus