Reboredo-Rodriguez 2017 MiPschool Obergurgl

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Reboredo-Rodriguez Patricia
Protective effect of a polyphenol-rich strawberry extract on oxidative and endoplasmic reticulum stresses in HepG2 cell line.

Link: MitoEAGLE

Reboredo-Rodriguez P, Giampieri F, Forbes-Hernandez TY, Gasparrini M, Afrin S, Cianciosi D, Varela-Lopez A, Battino M (2017)

Event: MiPschool Obergurgl 2017

COST Action MitoEAGLE

The endoplasmic reticulum (ER) is a specialized organelle for folding and trafficking proteins. It is highly sensitive to change in cellular homeostasis and extracellular stimuli, such as production of reactive oxygen species (ROS) and oxidative stress. Several chronic and degenerative diseases have been associated with ER and oxidative stresses [1]. The use of natural antioxidants derived from diet could be a strategic tool to alleviate both of them, at the same time enhancing the defense system [2,3,4].

In the present study, the effects of a polyphenol-rich strawberry extract were assessed on hepatoma cancer cells (HepG2). HepG2 cells were treated with 100 µg/mL strawberry extract for 24 h and then were stressed alternatively with 10 mM AAPH for 24 hours to induce oxidative stress or with 20 µg/mL tunicamycin for 6 hours to induce ER stress.

By the other hand, taking into account the essential role of mitochondria in ROS production and in cellular stress, mitochondrial function has been analyzed. Oxygen consumption rate (OCR) in HepG2 was measured in real-time using a XF-24 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica MA, USA) and after an OCR baseline measurement, a profiling of mitochondrial function was performed by sequential injection of three compounds that affect bioenergetics, as follows: Oligomycin (final concentration 2.5 µg/ml) at injection A, FCCP (final concentration 3μM) at injection B and rotenone/Antimycin (final concentration 10 µM/1µM) at injection C.

Strawberry extract showed remarkable protective effects on oxidative and ER stresses at cellular and mitochondrial levels. A significant improvement in cell viability and apoptosis rate (Figure 1a) was found in cells treated with strawberry and stressed with AAPH or tunicamycin, as well as a relevant decrease in intracellular ROS (Figure 1b) and NO levels.

As shown in Figure 2a, basal OCR -an indicator of a damaged mitochondrial functionality- was markedly affected in cells treated with AAPH or tunicamycin, while cells pre-incubated with strawberry extract and stressed with AAPH or tunicamycin showed an OCR similar to that of control group, highlighting a considerable protection on mitochondria against oxidative and ER stress. At the same time, spare respiratory capacity (SRC) and maximal respiratory capacity (MRC) were affected in cells stressed with AAPH or tunicamycin compared to control cells (Figure 2b), while after pre-treatment with strawberries a significant improvement was found, demonstrating again the beneficial and protective effect of the extract on mitochondrial functionality. The maintenance of some respiratory capacity, even under conditions of maximal physiological or pathophysiological stimulus, is a major factor defining the vitality and/or survival of the cells. The ability of cells to respond to stress under conditions of increased energy demand is influenced by the bioenergetics capacity of mitochondria. This bioenergetic capacity is determined by several factors, including the ability of the cell to deliver substrate to the mitochondria, and the functional capacity of the enzymes involved in electron transport.


Bioblast editor: Kandolf G


Labels: MiParea: Respiration, Pharmacology;toxicology 


Organism: Human  Tissue;cell: Liver 


Coupling state: LEAK, ET  Pathway: ROX 

Event: D2, Poster 


Affiliations

Reboredo-Rodríguez P(1), Giampieri F(2), Forbes-Hernández TY(2), Gasparrini M(2), Afrin S(2), Cianciosi D(2), Varela-López A(2), Battino M(2,3)
  1. Dept Analytical Food Chemistry, Univ Vigo, Ourense, Spain
  2. Dept DISCO, Sez Biochimica, Fac Medicina, UNIVPM, Ancona, Italy
  3. Centre Nutrition Health, Univ Europea Atlantico (UEA), Santander, Spain.- preboredo@uvigo.es

Figures

Rodriguez Figure1 MiPschool Obergurgl 2017.jpg

Figure 1. (a) Percentage of live, dead and apoptotic cells after each treatment and (b) Intracellular ROS generation in HepG2 after each treatment. Results are reported as mean ± SD of three experiments. Mean values belonging to the same set of data with different superscript letters are significantly different (P < 0.05).



Rodriguez Figure2 MiPschool Obergurgl 2017.jpg

Figure 2. Strawberry improved OCR. Mitochondria oxygen consumption was monitored after sequential injection of different compounds that affect bioenergetics at the indicated time points into each well, after baseline rate measurement. Values are means ± SE. Mean values belonging to the same set of data with different superscript letters are significantly different (P < 0.05). SRC: spare respiratory capacity; MRC: maximal respiratory capacity.



References and acknowledgements

  1. Cao SS, Kaufman RJ (2014) Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease. Antioxid Redox Signal 21:396-413.
  2. Giampieri F, Alvarez-Suarez JM, Battino M (2014) Strawberry and human health: Effects beyond antioxidant activity. J Agricult Food Chem 62:3867-76.
  3. Giampieri F, Alvarez-Suarez JM, Mazzoni L, Forbes-Hernandez TY, Gasparrini M, González-Paramás AM, Santos-Buelga C, Quiles JL, Bompadre S, Mezzetti B, Battino M (2014) An anthocyanin-rich strawberry extract protects against oxidative stress damage and improves mitochondrial functionality in human dermal fibroblasts exposed to an oxidizing agent. Food Function 5:1939-48.
  4. Tulipani S, Armeni T, Giampieri F, Alvarez-Suarez JM, Gonzalez-Paramás AM, Santos-Buelga C, Busco F, Principato G, Bompadre S, Quiles JL, Mezzetti B, Battino M (2014) Strawberry intake increases blood fluid, erythrocyte and mononuclear cell defenses against oxidative challenge. Food Chemistry 156:87-93.
Reboredo-Rodríguez P acknowledges Xunta de Galicia for their post-doctoral contract.