The CORM ALF-186 Mediates Anti-Apoptotic Signaling via an Activation of the p38 MAPK after Ischemia and Reperfusion Injury in Retinal Ganglion Cells

PLoS One. 2016 Oct 20;11(10):e0165182. doi: 10.1371/journal.pone.0165182. eCollection 2016.

Abstract

Purpose: Ischemia and reperfusion injury may induce apoptosis and lead to sustained tissue damage and loss of function, especially in neuronal organs. While carbon monoxide is known to exert protective effects after various harmful events, the mechanism of carbon monoxide releasing molecules in neuronal tissue has not been investigated yet. We hypothesize that the carbon monoxide releasing molecule (CORM) ALF-186, administered after neuronal ischemia-reperfusion injury (IRI), counteracts retinal apoptosis and its involved signaling pathways and consecutively reduces neuronal tissue damage.

Methods: IRI was performed in rat´s retinae for 1 hour. The water-soluble CORM ALF-186 (10 mg/kg) was administered intravenously via a tail vein after reperfusion. After 24 and 48 hours, retinal tissue was harvested to analyze mRNA and protein expression of Bcl-2, Bax, Caspase-3, ERK1/2, p38 and JNK. Densities of fluorogold pre-labeled retinal ganglion cells (RGC) were analyzed 7 days after IRI. Immunohistochemistry was performed on retinal cross sections.

Results: ALF-186 significantly reduced IRI mediated loss of RGC. ALF-186 treatment differentially affected mitogen-activated protein kinases (MAPK) phosphorylation: ALF-186 activated p38 and suppressed ERK1/2 phosphorylation, while JNK remained unchanged. Furthermore, ALF-186 treatment affected mitochondrial apoptosis, decreasing pro-apoptotic Bax and Caspase-3-cleavage, but increasing anti-apoptotic Bcl-2. Inhibition of p38-MAPK using SB203580 reduced ALF-186 mediated anti-apoptotic effects.

Conclusion: In this study, ALF-186 mediated substantial neuroprotection, affecting intracellular apoptotic signaling, mainly via MAPK p38. CORMs may thus represent a promising therapeutic alternative treating neuronal IRI.

MeSH terms

  • Animals
  • Apoptosis / drug effects*
  • Caspase 3 / genetics
  • Caspase 3 / metabolism
  • Cells, Cultured
  • Coordination Complexes / chemistry
  • Coordination Complexes / pharmacology*
  • Disease Models, Animal
  • Female
  • Imidazoles / pharmacology
  • Male
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • Neuroprotective Agents / pharmacology*
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Pyridines / pharmacology
  • RNA, Messenger / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Reperfusion Injury / metabolism
  • Reperfusion Injury / pathology*
  • Retinal Ganglion Cells / cytology
  • Retinal Ganglion Cells / metabolism
  • Retinal Ganglion Cells / pathology
  • Signal Transduction / drug effects*
  • bcl-2-Associated X Protein / genetics
  • bcl-2-Associated X Protein / metabolism
  • p38 Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • p38 Mitogen-Activated Protein Kinases / metabolism*

Substances

  • Coordination Complexes
  • Imidazoles
  • Neuroprotective Agents
  • Proto-Oncogene Proteins c-bcl-2
  • Pyridines
  • RNA, Messenger
  • bcl-2-Associated X Protein
  • triscarbonyl(histidinato)molybdate(III)
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • p38 Mitogen-Activated Protein Kinases
  • Caspase 3
  • SB 203580

Grant support

This work was conducted at the Department of Anesthesiology and Intensive Care, Medical Center – University of Freiburg, Faculty of Medicine, Freiburg, Germany and granted by The German Research Foundation (DFG, Grants: Go 2158/3-1 and Bi 1567/2-1). The funder had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Alfama Ltd. provided support in the form of supplying Carlos C. Romão with the ALF-186 compound - which is not commercially available - but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the 'author contributions' section.