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). H₂O₂ concentration (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 H₂O₂ showed 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.

Discussion: 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.

Keywords:

• Oxidative stress
• Punicalagin
• Neurodegeneration
• Apoptosis
• Mitochondrial dysfunction

Acknowledgements

The authors of the present study would like to thank Dr Lucia Lisi for her contribution to the experimental work described and for her expert technical assistance.

ORCID

Maria Elisabetta Clementi http://orcid.org/0000-0002-4615-7826

Additional information

Funding

The present study was financed by the Italian National Research Council (CNR) funding.