# In Vitro Antioxidant Effects of Coenzyme Q10 on Cellular Metabolism in Aged Mesenchymal Stem Cells
## 3 Results
### 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 PPARy expression, with a significant reduction observed at 50 \(\upmu\)M in early passage cells (P5). In late passage cells, 10 \(\upmu\)M CoQ10 slightly increases PPARy expression compared with the control, suggesting a potential protective or compensatory effect in senescent cells.  
Figure 3: Oxygen consumption rates following CoQ10 treatment, measured using the Oroboros O2k System in early passage (P5) and late passage (P16) MSCs at concentrations of 10 \(\upmu\)M and 50 \(\upmu\)M CoQ10. The figure presents the impact of CoQ10 on routine respiration (R), leak respiration (L), maximum respiration (M), and spare respiratory capacity (SRC), (***\(p<\) 0.0001). Representative Oroboros oxygraphs, illustrating oxygen consumption and basal respiration measured by high-resolution respiratory, are provided in the Supplementary Files (Figure S3).  
CoQ10 reduces PPAR\(\gamma\) expression at 50 \(\upmu\)M, 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 \(\upmu\)M in late passage cells, potentially indicating an adaptive or protective response to oxidative stress. However, CoQ10 at 50 \(\upmu\)M 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 sittuins are shown in Figure S2.  
The expression levels of oxidative stress-related genes, including PPAR\(\gamma\), ACC, SOD, and CAT, were measured by qPCR. GAPDH was used as the reference gene for normalization. Data are presented as the mean \(\pm\) 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 sittuins were downregulated in younger cells upon CoQ10 treatment. The changes in sittuin gene expression are shown in Figure 5. The agarose gel electrophoresis of qPCR products for oxidative stress-related genes and sittuins is presented in Figure S2.  
Figure 4: Antioxidant gene expression and cellular response to CoQ10 at 10 and 50 \(\upmu\)M treatment in P5 and P16 passages, (* \(p\) < 0.05, ***_p_ < 0.0001).  
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 \(\pm\) SEM from three independent experiments. Statistical significance is denoted as follows: * \(p\) < 0.05, **_p_ < 0.001, ***_p_ < 0.0001.