Phenolic extract from olive mill wastewater sustains mitochondrial bioenergetics upon oxidative insult

Abstract

In the last few years, many efforts have been devoted to the recovery and valorization of olive oil by-products because of their potentially high biological value. The olive mill wastewater (OMWW), a dark-green brown colored liquid that mainly consists of olive fruit vegetation water, is particularly exploited in this regard for its great content in phenolic compounds with strong antioxidant properties. In our previous work, we produced different OMWW fractions enriched in hydroxytyrosol- and hydroxytyrosol/oleuropein (i.e. C and OPE extracts, respectively) that exhibited considerable anti-microbial and radical-scavenging activities in vitro. Based on these findings, the present study aimed to assess the impact of C and OPE samples on mitochondrial function and oxidative stress response in mouse fibroblast-like cells (NCTC). Accordingly, OMWW phenolic extracts proved to enhance mitochondrial biogenesis and to reduce cellular sensitivity to hydrogen peroxide. Moreover, high-resolution respirometry experiments first time revealed the efficiency of OMWW phenols recovered by selective resin extraction in preventing mitochondrial respiration failure upon oxidative insult. Collected data definitely demonstrate the bioactivity of our phenolic-rich fractions, supporting the advantages of reusing the olive mill wastewater to generate, at low-cost, high added value molecules that could be useful for the improvement of health and nutrition products.

Keywords: Mitochondrial biogenesis; Mitochondrial respiration; Oil waste recovery; Oxidative stress; Phenolic compounds.

Fig. 1

A. Phase contrast images of NCTC cells treated with 1:1000 v/v C, 1:500 v/v OPE or 50 μM HT taken at time 0 and after 24 h treatment. B. Growth curves of NCTC cells treated with 1:1000 v/v C, 1:500 v/v OPE or 50 μM HT and monitored in real time by using the IncuCyte® SX1 Live-Cell imaging system over 48 h. Cell growth curves during the first 24 h, calculated using the basic analyzer module of the Incucyte software, were reported as a percentage of cell confluence. Data are expressed as mean ± SD of three independent biological experiments, each with five technical replicates (n = 5) and compared to not-treated cells (Ctrl).

Fig. 2

A-B: Cell proliferation/toxicity assay of NCTC cells pre-treated or not (Ctrl) with 1:1000 v/v C, 1:500 v/v OPE, or 50 μM HT for 24 h before being exposed to 1 mM H2O2 for 1 h (A) or 18 h (B). Results were expressed as the ratio of the phase area occupied by cells at 1 h or 18 h to the phase area occupied by cells at 0 h. C. Measurement of total ROS production via staining with CellROX™ Green Reagent. After 24 h treatment with the compound of interest, NCTC cells were loaded with 2.5 μM CellROX™ Green and subsequently exposed or not to 1 mM H₂O₂ for 1 h. The probe fluorescence was quantified at 490/516 nm following background subtraction via the surface fit method of the basic analyzer module. Results were expressed as Green Integrated Intensity per image (micron square)/phase area confluence per image (micron square). Data are expressed as mean ± SD of three independent biological experiments, each with five technical replicates (n = 5) and compared to not-treated cells (Ctrl); ** p < 0.01 and **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

A. Relative quantification of PGC-1α, NRF1 and TFAM (mitochondrial biogenesis markers) mRNAs in NCTC cells treated with 1000 v/v C or 1:500 v/v OPE samples. Data are normalized to the β-actin, expressed as mean ± SD of three independent biological experiments, each with four technical replicates (n = 4) and compared to not-treated cells (Ctrl). B. Quantification of the mitochondrial content. Western blot illustration and relative quantification of mitochondrion-specific protein SDHA level in NTCT cells treated with 1000 v/v C or 1:500 v/v OPE extracts. Data are normalized to the β-actin, expressed as means ± SD of three independent biological experiments (n = 3) and compared to not-treated cells (Ctrl). C. Measurement of mitochondrial content by using the MitoTracker® Green MGT probe. Fluorescence was measured in NCTC cells treated with1:1000 v/v C, 1:500 v/v OPE or 50 μM HT for 24 h by using the IncuCyte® Sx1 Live-Cell imaging platform. Data are expressed as mean ± SD of three independent biological experiments, each with five technical replicates (n = 5) and compared to not-treated cells (Ctrl); * p < 0.05, ** p < 0.01 and **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

A. Representative curve displaying the respirometry profile of untreated NCTC cells and the SUIT protocol applied. The respiratory states Routine, Leak, Maximal ET capacity and ROX were achieved in intact cells with the specific addition of substrates and inhibitors, as following: Omy, oligomycin; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; AZ, sodium azide. B—C. Quantitative analysis of the oxygen consumption in the analyzed states of NCTC cells pre-treated or not with 50 μM HT for 24 h and subsequently exposed to 1 mM H₂O₂ for 1 h. Data are indicated as mean ± SD Data are expressed as mean ± SD of five independent biological experiments, each with three technical replicates (n = 3); * p < 0.05, *** p < 0.001 and **** p < 0.0001.

Fig. 5

A-B. Quantitative analysis of the oxygen consumption in the analyzed states of NCTC cells pre-treated for 24 h with 1:500 v/v OPE and then exposed to 1 mM H₂O₂ for 1 h. Data are indicated as mean ± SD of five independent biological experiments, each with three technical replicates (n = 3); * p < 0.05 and ** p < 0.01.

Supplementary Fig. S1

Chemical characterization of C and OPE samples. A. Determination of pH values and total soluble solids (TSS, expressed as °Brix) of the OMWW extracts. B. Measurement of total phenols (TP, expressed as mg gallic acid equivalents (GAE)/L of sample) and quantification of HT, T and OLE. C. Evaluation of lactic acid, acetic acid and propionic acid within samples. Data are indicated as mean ± SD of three independent biological experiments, each with three technical replicates (n = 3).

Supplementary Fig. S2

Quantitative analysis of the oxygen consumption in the analyzed states of NCTC cells treated with 50 μM HT (A), 1:500 v/v OPE (B) or 1:1000 v/v C (C) for 24 h. Data are indicated as mean ± SD of four independent biological experiments, each with three technical replicates (n = 3); ** p < 0.01.