title: Punicalagin reduces H2O2-induced cytotoxicity and apoptosis in PC12 cells by modulating the levels of reactive oxygen species.
abstract: BACKGROUND
  Oxidative stress has long been linked to neuronal cell death in many neurodegenerative diseases. Antioxidant conventional supplements are poorly effective in preventing neuronal damage caused by oxidative stress due to their inability to cross the blood brain barrier. Hence the use of molecules extracted from plants and fruits such as phenolics, flavonoids, and terpenoids compounds constitute a new wave of antioxidant therapies to defend against free radicals.
  
  
  OBJECTIVE
  In this study we examined the effects of punicalagin, a ellagitannin isolated from the pomegranate juice, on a rat adrenal pheochromocytoma cell line, treated with hydrogen peroxide, evaluating the viability, oxidation potential, mitochondrial function, and eventual apoptosis.
  
  
  METHODS
  This study was performed on PC12 cells pretreated with punicalagin (0.5, 1, 5, 10 e 20 µM) 24 hours before of the damage by hydrogen peroxide (H2O2). H2O2concentration (300 µM) used in our study was determined by preliminary experiments of time course. The cell viability and ROS production were evaluated by MTS assay and cytofluorometry assays, respectively. Subsequently, the number of apoptotic-positive cells and mitochondrial transmembrane potential, were measured by flow cytometry, in the same experimental paradigm. Finally, the expression of Bax and enzymatic activity of Caspase 3, some of the principle actors of programmed cell death, were investigated by semiquantitative PCR and utilizing a colorimetric assay kit, respectively.
  
  
  RESULTS
  We found that pretreatment with punicalagin protected the cells from H2O2-induced damage. In particular, the protective effect seemed to be correlated with a control both in radical oxygen species production and in mitochondrial functions. In fact the cells treated with H2O2showed an altered mitochondrial membrane integrity while the pretreatment with punicalagin retained both the cellular viability and the mitochondrial membrane potential similar to the control. Furthermore, the punicalagin, modulated the apoptotic cascade triggered reducing Bax gene expression and Caspase 3 activity.
  
  
  CONCLUSIONS
  Results of the present study demonstrated a neuroprotective effect of punicalagin on H2O2-induced PC12 cell death, including mitochondria damage and expression of apoptotic gene Bax; therefore we hypothesize a possible prevent role for this molecule in neurodegenerative diseases related to oxidative stress.
authors.affiliation: a CNR-ICRM Istituto di"Chimica del Riconoscimento Molecolare", c/o Istituto di Biochimica e Biochimica Clinica , Università Cattolica del Sacro Cuore , Rome , Italy.
authors.forename: Maria Elisabetta
authors.identifier: http://orcid.org/0000-0002-4615-7826
authors.initials: ME
authors.lastname: Clementi
authors.affiliation: b Istituto di Patologia Generale , Università Cattolica del Sacro Cuore , Rome , Italy.
authors.forename: Giovambattista
authors.initials: G
authors.lastname: Pani
authors.affiliation: a CNR-ICRM Istituto di"Chimica del Riconoscimento Molecolare", c/o Istituto di Biochimica e Biochimica Clinica , Università Cattolica del Sacro Cuore , Rome , Italy.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Sampaolese
authors.affiliation: c Istituto di Farmacologia , Università Cattolica del Sacro Cuore , Rome , Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Tringali
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D005786:Gene Expression Regulation; D006861:Hydrogen Peroxide / Q000633:toxicity*; D047348:Hydrolyzable Tannins / Q000494:pharmacology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects; D009474:Neurons / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D016716:PC12 Cells; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D000975:Antioxidants; C494828:Bax protein, rat; D047348:Hydrolyzable Tannins; D018696:Neuroprotective Agents; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; C013515:ellagitannin; C115642:punicalagin; D006861:Hydrogen Peroxide; C505410:Casp3 protein, rat; D053148:Caspase 3
keywords: Apoptosis; Mitochondrial dysfunction; Neurodegeneration; Oxidative stress; Punicalagin