In Vitro Antioxidant Effects of Coenzyme Q10 on Cellular Metabolism in Aged Mesenchymal Stem Cells

Abstract

(1) Background: this study investigates the short-term effects of coenzyme Q10 (CoQ10) on mitochondrial respiration, fatty acid metabolism, oxidative stress gene expression, and sirtuin activity in young (passage 5, P5) and aged (passage 16, P16) mesenchymal stem cells (MSCs). (2) Methods: Mitochondrial respiration was assessed by measuring oxygen consumption after 24 h of treatment. Gas chromatography–mass spectrometry (GC-MS) analysis assessed cellular fatty acid methyl ester profiles. Quantitative polymerase chain reaction (qPCR) demonstrated the passage-dependent expression of oxidative stress-related genes and sirtuins in response to CoQ10 treatment. (3) Results: CoQ10 enhanced basal respiration and spare respiratory capacity (SRC), particularly in older senescent cells. CoQ10 improved basal respiration and ATP-linked oxygen consumption in young MSCs and partially restored these functions in aged MSCs. Moreover, CoQ10 increased saturated fatty acids, particularly in young cells, and decreased monounsaturated fatty acids in aged cells. qPCR analysis revealed passage-dependent modifications in oxidative stress-related genes and sirtuin expression; CoQ10 exposure significantly influenced SIRT1 and SIRT3 activity, leading to an increase in PPARγ and CAT expression. (4) Conclusions: these results highlight CoQ10’s potential to alleviate mitochondrial dysfunction and metabolic shifts associated with cellular aging, underscoring its therapeutic value for age-related mitochondrial and metabolic disorders.

1. Introduction

Stem cell aging is a key process in understanding organismal aging. Adult stem cells found in various tissues are essential for organ-specific regeneration, self-renewal, and tissue maintenance, particularly as the body ages [1]. Their functionality plays a vital role in organ-specific regeneration and self-renewal, particularly as the body ages or upon exposure to tissue damage causing agents [2]. Tissue maintenance and regeneration rely heavily on the presence and functionality of stem cells. Consequently, age-related declines in stem cell numbers or functionality can significantly impair the body’s regenerative capacity, emphasizing their critical role in sustaining tissue health and repair throughout life [3]. Over the years, mesenchymal stem cells (MSCs) have emerged as a promising resource for stem cell-based therapies, with particular emphasis on their potential applications in regenerative medicine as anti-aging treatments. Their exceptional clinical attributes, such as ease of accessibility, simple isolation procedures, robust self-renewal and proliferation capacity, multilineage differentiation potential, and immunomodulatory properties, make them highly promising [1]. However, numerous studies have demonstrated that MSCs experience functional decline and progressive loss of stemness with advancing age in vivo or during extended in vitro culture [3,4,5]. These age-related changes significantly constrain their therapeutic applications. While the mechanisms underlying MSC senescence remain incompletely understood, considerable progress has been made in identifying age-associated phenotypic alterations and uncovering the potential molecular pathways contributing to MSC aging. This growing body of knowledge offers valuable insights in overcoming the limitations of MSC-based therapies and enhancing their clinical usability [1].

Aging is associated with a pro-oxidizing shift in the cellular redox state, coupled with the accumulation of oxidatively damaged biomolecules. Together, these factors are believed to contribute causally to cellular senescence [6]. This concept, often referred to as the oxidative stress hypothesis, posits that aging involves an imbalance between pro-oxidant production and antioxidant cellular defenses, leading to an increased oxidant load or stress. Mitochondria are considered to play a central role in this dysregulation, as they are major sources of reactive oxygen species (ROS). During aging, mitochondrial ROS production (primarily O₂⁻ and H₂O₂) increases, accompanied by oxidative damage to mitochondrial DNA, proteins, and lipids [6,7]. A critical consequence of this damage is a self-perpetuating cycle in which oxidative injury accelerates the rate of ROS generation, a phenomenon first described by Sohal and colleagues [8]. Another significant age-related mitochondrial alteration is the decline in ADP-stimulated (state 3) and maximal (uncoupled) rates of mitochondrial oxygen consumption [9]. These changes, combined with increased oxidative stress and damage from elevated O₂⁻/H₂O₂ production and a diminished mitochondrial capacity to synthesize ATP, are believed to progressively impair the functional capacity of various physiological systems [6].

Coenzyme Q10 (CoQ10) is a lipophilic redox-active molecule primarily localized within the inner mitochondrial membrane, where it plays a pivotal role in cellular energy production. It is also integrated into the phospholipid bilayer of various cellular membranes, where it helps modulate their physicochemical properties [10]. CoQ10 is particularly concentrated in the mitochondria, highlighting its essential function in oxidative phosphorylation and electron transport processes [4]. As a vital component of the electron transport chain, CoQ10 directly participates in oxidative phosphorylation, enabling efficient energy production [11].

In addition to its central role in energy metabolism, CoQ10 enhances mitochondrial mass [12], modulates the permeability transition pore [10], improves mitochondrial function [13], and reduces the generation of ROS [14]. Its critical involvement in mitochondrial health [15,16], coupled with its potent antioxidant properties, positions CoQ10 as a promising therapeutic agent, especially in clinical trials targeting neurodegenerative diseases [17,18].

CoQ10 levels typically increase during early life, but they decline with aging [10,19]. CoQ10 plays an essential role in protecting the neurological system from degeneration by facilitating electron transport, supporting energy metabolism, and enhancing oxygen utilization, particularly in the musculoskeletal and nervous systems. Previous research has highlighted the therapeutic potential of CoQ10 in preventing and managing a range of neurological, cardiovascular, and metabolic disorders [10,20,21,22,23].

This study investigates the short-term effects of exposure to CoQ10 on mitochondrial respiration, fatty acid metabolism, oxidative stress-related gene expression, and sirtuin expression in young (P5) and aged (P16) MSCs. For these experiments, MSCs were derived from stem cells of human exfoliated deciduous teeth (SHEDs). The Oroboros system was used to assess the mitochondrial function. Gas chromatography–mass spectrometry (GC-MS) analysis evaluated the fatty acid profiles in young and aged MSC cells. Further, quantitative polymerase chain reaction (qPCR) demonstrated passage-dependent changes in the expression of oxidative stress-related genes and sirtuins in response to CoQ10 treatment. These findings highlight, for the first time, CoQ10’s passage-specific effects on oxidative stress metabolism, suggesting its potential to mitigate mitochondrial dysfunction and metabolic shifts associated with cellular aging.

2. Materials and Methods

2.1. Cell Culture and CoQ10 Treatment

In these experiments, mesenchymal stem cells derived from human exfoliated deciduous teeth of children aged between 7 and 12 years were utilized and prepared according to protocols described in previous studies [24,25]. Parental consent was obtained following strict ethical standards, adhering to the principales outlined in the 1964 Declaration of Helsinki. The dental pulp tissue was carefully extracted, washed with phosphate-buffered saline (PBS) (Gibco, Life Technologies, Bleiswijk, Netherlands), and cut into 0.5–1 mm³ fragments to facilitate MSC isolation from tissue explants. SHEDs were cultured in alpha minimum essential medium (Gibco, Thermofisher, Paisley, UK). The medium was supplemented with 10% penicillin/streptomycin (Life Technologies, Grand Island, NY, USA) and refreshed twice a week. Cells were passaged when they reached 80–90% confluence to ensure optimal growth conditions. Cells from passages 5 (P5) and 16 (P16) were used in the experiments. To minimize passage-dependent effects and avoid confounding influences from culture conditions or donor variability, all MSCs from the dental pulp were grown under identical conditions, including the same basal medium, supplements, and passage protocols to ensure consistency. To minimize donor-specific variations, young and aged cells from the same donor were consistently compared, while replicating the experiment with at least two additional donors. Cells were always plated at the same density, using consistent culture conditions before treatment, to reduce variability in cell cycle distribution among experimental groups.

Coenzyme Q10 (Sigma–Aldrich, St. Louis, MO, USA) was prepared as a 10 mM stock solution in ethanol (Sigma–Aldrich, Ayrshire, UK). The stock solution was diluted in cell culture medium to final concentrations of 10 µM, 25 µM, 50 µM, and 70 µM. MSCs were incubated with these concentrations for 24 h to assess the effects of the chosen dosages. The selection of CoQ10 doses (10 and 50 µM) for the further assessment of metabolic and gene expression changes was based on their distinct effect on cell viability and their frequent use in the literature as effective low-to-medium concentrations for modulating stem cell metabolism, providing a foundation for future studies investigating long-term effects and minimizing the potential toxic effects of higher concentrations [4,26]. Control cells were grown under similar conditions as the test cells, adding the corresponding ethanol concentration to the culture medium, without CoQ10.

2.2. Viability Assay

To evaluate the effects of CoQ10 on cell proliferation, the xCELLigence system (Santa Clara, CA, USA) was used. This advanced platform employs specialized microtiter plates with interdigitated gold microelectrodes to noninvasively monitor cell viability in real-time, with electrical impedance serving as the primary readout. The system’s real-time cell monitoring enables the distinction between different factors affecting cell viability, including cellular senescence, and cell cycle arrest. For the assay, cells were seeded at a density of 10⁴ cells per well and allowed to adhere overnight. CoQ10 was then administered at concentrations of 10 µM, 25 µM, 50 µM, and 70 µM, and cell viability was assessed after 24 h of incubation. Data analysis was performed using RTCA Software Pro from Agilent Technologies (Santa Clara, CA, USA), and statistical comparisons between experimental groups were performed using one-way ANOVA, followed by post hoc t-tests for pairwise comparisons. Results are expressed as mean ± standard deviation (SD), with statistical significance set at p < 0.05.

2.3. SA-β-Gal Staining

Cellular senescence was evaluated using the senescence-associated β-galactosidase (SA-β-Gal) staining method, employing the senescence cell histochemical staining kit (Sigma-Aldrich, St. Louis, MO, USA). For the histochemical identification of senescent cells, 2 × 10⁴ cells per well were seeded in 8 well plates and cultured in complete MEM alpha medium. After an overnight incubation to allow cell attachment, cells were treated with CoQ10 at final concentrations of 10 µM and 50 µM for 24 h. Following treatment, cells were stained according to the manufacturer’s instructions. The staining solution was applied and incubated overnight at 37 °C in a CO₂-free environment for optimal staining development. Senescence was assessed by counting blue-stained cells, a marker of SA-β-gal activity, using a phase-contrast inverted microscope (Zeiss, Axio Observer Z1, Oberkochen, Germany) at 200× magnification. The percentage of senescent cells was determined by analyzing five distinct fields per well. The proportion of β-galactosidase-positive (blue-stained) cells relative to the total cell count was calculated using the following equation:

$$\%\mathsf{\beta }-\mathrm{g}\mathrm{a}\mathrm{l}-\mathrm{p}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}=100\times (\mathrm{b}\mathrm{l}\mathrm{u}\mathrm{e}-\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{d} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}/\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s})$$

2.4. Mitochondrial Respiration and Energy Metabolism Evaluation

To evaluate changes in energy metabolism, oxygen consumption using the Oxygraph-2K system (Oroboros Instruments, Innsbruck, Austria) was measured. Trypsinized cells were resuspended in culture medium and introduced into the chamber of the system for analysis. Following the sealing of the chambers, baseline routine respiration was recorded. To assess non-ATP-linked respiration, ATP synthase activity was inhibited by the addition of oligomycin (2.5 μM). Subsequently, to evaluate the non-phosphorylating capacity of the electron transport system (ETS), mitochondria were uncoupled using a stepwise titration of carbonyl cyanide 4- (trifluoromethoxy) phenylhydrazone (FCCP), by administering increments of 0.5 μM. The maximum respiratory capacity before sequentially adding rotenone (0.5 μM) and antimycin A (2.5 μM) was determined. All reagents used were obtained from Sigma Aldrich (St Louis, MO, USA). Therefore, complexes I and III were inhibited, allowing to measure residual oxygen consumption. Prior to these measurements, cells were pre-incubated with 10 μM and 50 μM CoQ10 for 24 h.

2.5. Antioxidant and Sirtuin Gene Expression Analysis

Total RNA was extracted from the control and experimental groups using Trizol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol. The RNA concentration was precisely quantified with a Nanodrop ND-1000 spectrophotometer, ensuring accurate measurement. Before sample measurements, RNase-free water was used as a blank. RNA purity was assessed using 260/280 absorbance ratios, consistently falling between 1.9 and 2.02, indicating high-quality RNA suitable for downstream applications.

For the synthesis of complementary DNA (cDNA), 1 µg of total RNA was reverse-transcribed in a 20 µL reaction using the RevertAid First Strand cDNA Kit (Thermo Scientific, EU, Lithuania), adhering to the manufacturer’s guidelines to ensure reliable results. Quantitative real-time PCR (qPCR) was conducted on the synthesized cDNA using KiCqStart SYBR Green qPCR ReadyMix (Sigma, St. Louis, MO, USA) on a LightCycler 480II instrument (Roche, Basel, Switzerland). Specific primers were designed to target the genes of interest related to oxidative stress and sirtuin pathways. The analyzed oxidative stress response genes included PPARγ, Acetyl-CoA, SOD, and CAT. Additionally, the expression of sirtuins (SIRT1, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7) was examined to understand their roles in cellular survival, metabolic regulation, and longevity in response to CoQ10 treatment. Relative gene expression levels were normalized to GAPDH as a housekeeping gene. Detailed primer sequences and corresponding information are provided in Supplementary Table S1.

2.6. Assessment of Fatty Acid Profile

To evaluate the fatty acid composition following short-term treatment with CoQ10, 1 × 10⁶ cells per experimental condition were processed. The protocols of extraction used in this method were adapted after previous publications of the group [25,27]. All solvents and reagents used in the extraction, derivatization, and analysis were GC-grade and obtained from Sigma-Aldrich (St. Louis, MO, USA).

Cells were washed with ice-cold PBS to remove residual media and centrifuged at 1500 rpm for 10 min to form a pellet used further for lipid extraction. Lipid extraction was performed by resuspending the cell pellet in 400 µL of a chloroform/methanol solution (2:1 v/v), and the mixture was centrifuged at 4000 rpm for 5 min. The lower (chloroform) layer was transferred to a clean tube, and 200 µL of PBS and 100 µL of chloroform were added, followed by vortexing and a second centrifugation at 4000 rpm for 10 min. The lower (chloroform) phase was collected, evaporated to dryness under a nitrogen stream, and stored for subsequent analysis. The dried residue was subjected to saponification by adding 0.45 M sodium hydroxide and heated at 65 °C for 1 h. The resulting solution was neutralized with 0.45 M hydrochloric acid, and 2% acetic acid in hexane was added for extraction two times. The upper (hexane) layers were pooled and dried under nitrogen gas. The resulted residue was derivatized with 100 µL of acetyl chloride in 5 mL methanol under continuous agitation at room temperature for 45 min. The reaction was stopped by adding 3 mL of 0.25 M potassium carbonate. Then, 1 mL of hexane was used to extract the resulting fatty acid methyl esters (FAMEs) under continuous agitation at room temperature for 1 h. The resulted upper (hexane) layer was also dried under inert nitrogen gas and stored at −20 °C for further GC-MS analysis.

The resulting residue containing FAMEs was resuspended in 100 µL hexane and was analyzed using an Agilent 6890 gas chromatograph coupled with a 5973 MSD quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Separation was achieved using a DB-WAX capillary column (30 m × 0.25 mm × 0.25 µm) with helium as the carrier gas at a flow rate of 1 mL/min. The oven temperature started from 50 °C to 250 °C, with a rate of 6 °C/min. Then, 1 µL of each sample was injected into an inlet in splitless mode, the vaporization temperature being 230 °C and the mass spectrometer source being set at 150 °C.

The mass spectra of the compounds were monitored in full-scan mode after a solvent delay of 4 min, and the mass range was set between 50 and 600 Da. The ionization energy was set to 70 eV. The identification of the compounds was made by comparing their spectra to the NIST11 mass spectral library using ChemStation software, version B.01.00 (Agilent Technologies, Palo Alto, CA, USA). Solvent blanks and control samples were analyzed under the same GC-MS conditions to exclude contamination and ensure data accuracy. The area percentage of each FAME was calculated as follows: the peak area for a FAME was divided by the summed areas of all the FAME peaks in the chromatogram.

2.7. Statistical Analyses

Differences among experimental groups were evaluated using a one-way analysis of variance (ANOVA), with subsequent pairwise post hoc t-tests employed to identify specific group distinctions. Statistical analysis was carried out using Microsoft Excel (Office Pro Plus, 2021). For parametric data, results are presented as mean ± standard deviation (SD). A p-value of less than 0.05 was considered statistically significant. To ensure reproducibility and statistical reliability, three biological replicates were analyzed for each experiment. Each biological replicate consisted of independently cultured cells, which were subjected to the same procedures.

3. Results

3.1. Cell Viability

Cell viability was assessed using the xCELLigence system. The cell index was measured at 24 h for both young (P5) and old (P16) MSCs under control conditions (C) and treatment with four concentrations of CoQ10 (10 µM, 25 µM, 50 µM, and 70 µM) (Figure 1). P16 cells consistently exhibited a higher cell index compared with P5 cells across all conditions, indicating enhanced adhesion or a distinct response to CoQ10 treatment in older cells. Furthermore, CoQ10 showed a concentration-dependent effect on cell viability, enhancing it at 10 µM and 25 µM, but reducing it at 50 µM and 70 µM. Older MSCs (P16) were more resilient and responded more favorably to CoQ10 treatment than younger MSCs (P5).

3.2. SA-β-Gal Staining

When evaluated for senescence-associated β-galactosidase (SA-β-gal) activity, early-passage cells (P5) exhibited minimal weak staining, indicative of a low senescence level. In contrast, MSCs at late passages (P16) displayed a significant increase in β-gal staining, signaling a transition to a senescent phase. This shift was evident from the markedly higher number of β-gal-positive cells observed at P16 compared with P5 (Figure 2). The pronounced increase in β-gal-positive cells at late passages underscores the replicative exhaustion of SHEDs as they approach higher passage numbers. This behavior aligns with the characteristic decline in regenerative potential seen in stem cells during prolonged in vitro expansion. CoQ10 treatment demonstrated efficacy in mitigating the rise in β-gal-positive (senescent) cells at advanced passages. However, this protective effect was accompanied by a reduction in total cell number at the higher CoQ10 concentration of 50 µM, suggesting a dose-dependent impact on cellular proliferation.

Senescent cells exhibited blue staining due to β-gal activity, enabling visual quantification under a Zeiss Axio Observer Z1 inverted microscope, at 200× magnification. The percentage of senescent cells (C) was calculated by determining the ratio of β-gal-positive cells to the total cell count, expressed as a percentage for early passage (P5) and late passage (P16) cells.

3.3. Mitochondrial Respiration and Changes in Energy Metabolism

Oxygen consumption following CoQ10 treatment was assessed in live non-permeabilized MSCs. In early passage cells (P5), basal respiration was relatively high (10.6643 pmol O₂/min), reflecting robust metabolic activity. In contrast, basal respiration significantly declined in late passage cells (P16) to 6.79 pmol O₂/min, indicating a reduction in metabolic function associated with cellular aging. CoQ10 treatment exhibited dose-dependent effects on MSC metabolism, with the most pronounced improvements observed at 50 µM. This concentration notably enhanced mitochondrial function and spare respiratory capacity (SRC), particularly in older senescent cells. SRC, which represents the difference between maximal and routine respiration, reflects a cell’s ability to respond to energetic stress and serves as a critical indicator of cellular fitness. A reduction in SRC may compromise a cell’s ability to manage stressors, potentially leading to mitochondrial dysfunction. In early passage cells (P5), CoQ10 at 50 µM significantly boosted both routine (R) and maximum respiration (M) compared with the control and the 10 µM treatment, indicating enhanced mitochondrial efficiency. In late passage cells (P16), maximum respiration reached its peak at 50 µM, suggesting improved mitochondrial reserve capacity in senescent cells. However, an increase in leak (L) respiration was observed in older cells treated with 50 µM CoQ10, potentially indicating mild uncoupling or an adaptive mitochondrial response to stress. These findings underscore the potential of CoQ10 to support mitochondrial function and improve the metabolic fitness of both young and senescent cells. Oxygen consumption rates of early and late passage cells in response to CoQ10 treatment were measured using the Oroboros O2k system (Figure 3).

3.4. Analysis of Antioxidant and Sirtuin Gene Expression Response to CoQ10

Antioxidant gene expression is influenced by passage number, and cells exhibit a dose-dependent response to CoQ10 treatment. CoQ10 induces a dose-dependent decrease in PPARγ expression, with a significant reduction observed at 50 µM in early passage cells (P5). In late passage cells, 10 µM CoQ10 slightly increases PPARγ expression compared with the control, suggesting a potential protective or compensatory effect in senescent cells. CoQ10 reduces PPARγ expression at 50 µM, but maintains higher levels than the control, indicating a concentration- and age-dependent dual effect. CoQ10 also decreases ACC expression in both early and late passage cells, with a more pronounced effect in early passage cells. Similarly, CoQ10 suppresses SOD expression in early passage cells, possibly reflecting reduced oxidative stress due to its antioxidant properties. In senescent cells, where SOD levels are already low, CoQ10 further downregulates expression, though to a lesser extent than in young cells. CoQ10 exhibits a dose-dependent inhibitory effect on CAT activity and expression in early passage cells. CoQ10 enhances CAT expression at 10 µM in late passage cells, potentially indicating an adaptive or protective response to oxidative stress. However, CoQ10 at 50 µM no longer enhances CAT expression, reverting to baseline levels, suggesting a threshold beyond which its effects plateau or reverse. The changes in oxidative stress gene expression are shown in Figure 4. The agarose gel electrophoresis of qPCR products for oxidative stress-related genes and sirtuins are shown in Figure S2.

The expression levels of oxidative stress-related genes, including PPARγ, ACC, SOD, and CAT, were measured by qPCR. GAPDH was used as the reference gene for normalization. Data are presented as the mean  ±  SEM from three independent experiments. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.001, *** p < 0.0001.

Quantitative PCR analysis revealed a significant reduction in SIRT3 and SIRT4 expression in older MSCs, consistent with the decline in mitochondrial function and metabolic regulation typically associated with cellular aging. Treatment with CoQ10 induced a robust dose-dependent upregulation of SIRT1 and SIRT3 expression in both early passage (P5) and late passage (P16) cells. In late passage cells, a modest increase in SIRT4 expression was also noted following CoQ10 treatment. In contrast, SIRT5, SIRT6, and SIRT7 exhibited an inhibitory response to CoQ10 in older cells. Interestingly, apart from SIRT1, all other sirtuins were downregulated in younger cells upon CoQ10 treatment. The changes in sirtuin gene expression are shown in Figure 5. The agarose gel electrophoresis of qPCR products for oxidative stress-related genes and sirtuins is presented in Figure S2.

The expression levels of sirtuin-related genes, including SIRT1, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7, were measured by qPCR. GAPDH was used as the reference gene for normalization. Data are presented as the mean  ±  SEM from three independent experiments. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.001, *** p < 0.0001.

3.5. Assessment of Fatty Acid Content

Following CoQ10 treatment, the fatty acid profiles exhibited notable changes in both early and late passage cells. In early passage cells (P5), CoQ10 induced a dose-dependent increase in the majority of fatty acids, particularly lauric, myristic, pentadecanoic, and palmitic acids, but elaidic acid levels decreased with higher concentrations. In late -passage cells (P16), CoQ10 exhibited variable effects. Palmitic acid levels consistently increased with treatment, mirroring the trend observed in P5 cells, though a slight decline from the peak was noted at higher CoQ10 doses. Stearic acid and elaidic acid levels showed a dose-dependent increase in response to CoQ10 supplementation. Pentadecanoic acid levels exhibited a modest increase in both P5 and P16 cells. In contrast, lauric acid, myristic acid, and oleic acid levels decreased following CoQ10 treatment. These findings are summarized in Figure 6, while representative chromatograms highlighting the primary methylated fatty acids are presented in Figure S3.

4. Discussion

Coenzyme Q10 is an essential component of the respiratory chain within the inner mitochondrial membrane, playing a pivotal role in mitochondrial function. It acts as a cofactor for uncoupling proteins and modulates the permeability transition pore, as highlighted by Rauchová et al. (2021) [10]. Reactive oxygen species are major contributors to mesenchymal stem cell aging, primarily through the activation of the Wnt/β-catenin pathway and disruption of the balance between pro-oxidants and antioxidants [28]. As a potent antioxidant, CoQ10 plays a critical role in mitochondrial respiration, maintaining cellular membrane integrity, and regulating gene expression to mitigate oxidative damage [28,29,30,31]. Evidence of its bioenergetic importance includes studies showing reduced CoQ10 levels and compromised antioxidant defenses in aged animals, leading to heightened oxidative stress [32].

This study explores the impact of CoQ10 on MSC metabolism, demonstrating its capacity to reduce β-galactosidase expression, enhance mitochondrial respiration, and strengthen antioxidant defenses. These effects are mediated by upregulating PPARγ and CAT gene expression, with pronounced benefits observed in late passage cells. These findings underscore CoQ10’s robust antioxidant and protective role in counteracting ROS-driven MSC aging. In the context of aging and stem cell proliferation, a concentration-dependent effect of CoQ10 on MSC viability was observed. Late passage cells exhibited a more favorable response to CoQ10 treatment compared with younger cells. This may be due to differences in mitochondrial function, oxidative stress levels, and gene expression profiles between young and old MSCs. Older cells, with higher baseline oxidative stress, are likely more responsive to CoQ10’s antioxidant effects. In contrast, younger cells (P5) are more sensitive to higher CoQ10 concentrations.

The effects of CoQ10 on oxygen consumption in MSCs provide valuable insights into its role in mitochondrial function across aging stages. CoQ10 is essential in the mitochondrial respiratory chain, transferring electrons between complexes I (NADH dehydrogenase), II (succinate dehydrogenase), and III (cytochrome bc1 complex). Its reduction in metabolic activity in older cells highlights the need for interventions to restore mitochondrial efficiency [33]. A dose-dependent effect following CoQ10 treatment was observed, with 50 µM being the most effective concentration, enhancing the maximum respiration in late-passage cells, improving mitochondrial efficiency, resilience, and reserve capacity, and restoring SRC (a key indicator of cellular response to energetic stress).

Supporting evidence suggests that CoQ10 can reverse mitochondrial dysfunction and reduce ROS production, highlighting its potential to mitigate MSC dysfunction and delay aging [13,28,34,35]. CoQ10 supplementation significantly restored mitochondrial function, decreased ROS and lipid peroxide generation, inhibited lipid droplet accumulation, prevented NLRP3 inflammasome formation, and reduced both interleukin-1β release and cell death [36]. However, an increase in leak respiration indicates mild uncoupling or an adaptive response to oxidative stress. This suggests a trade-off between improved metabolic activity and increased mitochondrial stress handling, particularly in younger cells.

The effects of CoQ10 on antioxidant gene expression are dose- and age-dependent. In early passage cells (P5), CoQ10 exhibits a strong inhibitory response, with a dose-dependent decrease in PPARγ, ACC, SOD, and CAT expression. The pronounced suppression at 50 µM suggests a shift in cellular energy balance. As CoQ10 is abundant in the mitochondrial inner membrane, this inhibitory effect in young cells may represent a protective mechanism to maintain redox balance and prevent excessive antioxidant activity. This is consistent with in vivo findings, where CoQ10 supplementation (1200 mg/kg/day for 60 days) improved oxidative stress markers in transgenic mice [37]. Similarly, in vitro treatment (2.5 µM) reduced mitochondrial superoxide levels in CoQ10-deficient neurons [38].

In aged cells (P16), CoQ10’s effects vary by dose. At 10 µM, increased PPARγ and CAT expression suggests a compensatory response to oxidative stress. However, at 50 µM, expression levels return to baseline, indicating a dual-phase response. Antioxidant enzymes, such as CAT, play a vital role in neutralizing free radicals and mitigating oxidative stress. CoQ10 not only directly scavenges free radicals, but also enhances the activity of these enzymes by upregulating their gene expression, thereby preserving their function and reinforcing cellular defense mechanisms [39,40]. Our study offers valuable insights into cellular metabolic processes following CoQ10 supplementation, based on gene expression levels and cellular proliferation, viability, and senescence. However, while CoQ10 is known to regulate antioxidant and chronic inflammatory gene activation or repression, future studies should investigate protein level changes, using proteomic and other protein analysis methods. Also, various other cell types (stem and non-stem cells) should be employed in order to acquire a more comprehensive metabolic profile [41].

CoQ10 differentially regulates sirtuin gene expression in MSCs across early (P5) and late (P16) passages, suggesting a nuanced impact on cellular aging and metabolism. In both young and older cells, CoQ10 robustly upregulates SIRT1 in a dose-dependent manner. As a NAD⁺-dependent deacetylase, SIRT1 promotes mitochondrial biogenesis, stress resistance, and metabolic regulation, indicating that CoQ10 enhances cellular resilience and metabolic homeostasis, particularly in aged MSCs [42]. In late passage cells, CoQ10 also strongly increases SIRT3 expression, which is essential for mitochondrial protein deacetylation and oxidative stress reduction. This regulation of SIRT3 is influenced by SIRT1 activity and partially mediated through PPARγ-dependent pathways [42,43].

In late passage MSCs, CoQ10 modestly increases SIRT4 expression, while significantly downregulating this sirtuin in early passage MSCs. SIRT4, regulates oxygen consumption and fatty acid metabolism via ANT2-dependent coupling, and shows reduced baseline expression in P16 cells, reflecting mitochondrial dysfunction associated with aging. The modest upregulation of SIRT4 by CoQ10 in older cells suggests a restoration of mitochondrial pathways. In contrast, CoQ10 downregulates the expression of SIRT5, SIRT6, and SIRT7, which are involved in mitochondrial function and cellular stress responses [44]. This suppression may represent a compensatory shift favoring SIRT1-, SIRT3-, and SIRT4-dependent pathways for maintaining mitochondrial health and stress adaptation. The differential regulation of sirtuins by CoQ10 underscores its potential as an anti-aging therapeutic, particularly for mitigating mitochondrial dysfunction in aged cells. However, the observed suppression of specific sirtuins raises important questions about the balance within the sirtuin network, highlighting the need for further investigation to elucidate potential trade-offs and optimize therapeutic benefits.

Fatty acids (FAs) are vital for energy storage, membrane structure, and the synthesis of signaling molecules. They serve as a primary energy source through mitochondrial β-oxidation [45]. CoQ10, in its reduced form, acts as a potent antioxidant, improving lipid packaging and cellular membrane stability. This protection safeguards biological membranes against oxidative damage, enhances resistance to rupture, and slows the release of hydrophilic components [46]. CoQ10 also supports fatty acid oxidation by upregulating peroxisome proliferator-activated receptors (PPARs) and CAT, which are key regulators of β-oxidation and lipolysis [33]. Fatty acid β-oxidation predominantly occurs in mitochondria, with peroxisomes playing a complementary role, particularly for very long-chain fatty acids. Unlike mitochondrial β-oxidation, which generates ATP, peroxisomal β-oxidation directly transfers high-energy electrons to oxygen, producing hydrogen peroxide (H₂O₂) and releasing heat. Catalase, a peroxisomal enzyme, converts H₂O₂ into water and oxygen, mitigating oxidative damage. Intracellularly, H₂O₂ also facilitates the degradation of unwanted proteins and defends against foreign particles [47]. CoQ10 supplementation appears to enhance fatty acid β-oxidation in an age-dependent manner, as evidenced by increased PPAR-γ and catalase activity. This mechanism likely reduces oxidative stress, particularly in older cells, where oleic acid serves as a primary substrate. In younger cells, CoQ10 supplementation elevates the levels of lauric acid, myristic acid, and palmitic acid. Conversely, late passage cells exhibit significant increases in palmitic acid, stearic acid, and elaidic acid, suggesting a shift from unsaturated fatty acids, such as oleic and elaidic acids, toward saturated fatty acids. This shift may enhance membrane fluidity and rigidity, as saturated fatty acids, with their straight chains, pack more efficiently within bilayer membranes [47,48]. The observed increase in stearic acid, known for its antioxidant-enhancing properties, highlights its role in reducing oxidative stress and improving cellular resilience. Additionally, CoQ10 supplementation increases pentadecanoic acid (C15:0, PDA), a rare odd-chain fatty acid with a low synthesis rate in humans [49]. PDA is associated with improved health span and longevity through mechanisms involving mTOR inhibition and AMPK activation, pathways shared with rapamycin and metformin [50]. PDA can be fully β-oxidized, yielding six acetyl-CoA molecules and one propionyl-CoA [49].

5. Conclusions

Our findings suggest that CoQ10 enhances cellular metabolism through its potent antioxidant properties and by modulating membrane stability. It upregulates PPAR activity, facilitating β-oxidation and mitigating oxidative damage, especially in aging cells. CoQ10 shows promise in improving mitochondrial function and enhancing metabolic fitness in both youthful and senescent cells. Supplementation with CoQ10 enhances mitochondrial respiration and efficiency, partially restoring these parameters in aged cells. Additionally, CoQ10 upregulates PPARγ and catalase, promoting fatty acid oxidation and reducing oxidative stress, particularly via peroxisomal β-oxidation. The resulting shift in the fatty acid profile may improve cellular membrane rigidity and enzymatic stability, increasing resilience against oxidative damage. However, our current study was focused only on the short-term effects of CoQ10 addition (up to 24 h) The long-term effects of CoQ10 in mitigating age-related oxidative stress, modulating inflammatory processes, and maintaining genomic stability should be investigated, along with biosafety and bioavailability studies. A better understanding of the mechanistic interactions between CoQ10 supplementation, stem cell metabolism, and age-associated cellular alterations could ultimately lead to targeted interventions for age-related pathologies, including cancer. Exploring dose-dependent responses, optimal delivery strategies, and potential synergistic interactions with other compounds will be crucial for a deeper understanding of CoQ10’s potential to enhance cellular health, support longevity, and counteract age-related metabolic decline.