Optimizing Se- methylselenocysteine concentration to enhance glutathioneperoxidase 1 expression and mitigate oxidative stress in senescent humanfibroblast

Glutathione peroxidase 1 (GPx1) activity, gene expression, and several oxidative stress (OS) marker levels were investigated in the senescent passage (P) 20, 25, and 30 fibroblasts cultured in media supplemented with increasing Se-Methylselenocysteine (MSC) increments. While GPx1 activity slightly increased in cells grown in standard culture medium (CM1) compared to primary P5 cells, the enzyme exhibited significant MSC-dose-dependent elevations in cells cultured in MSC-supplemented media (CM3-CM6) compared to CM1 (p<0.001). GPx1 activity in CM5-incubated P30, P25, and P20 cells equaled 5.99±0.62, 4.72±0.48, and 4.06±0.36 µmoles/min/mg protein respectively (p<0.001), with percentage increases of 250% in P30 cells compared to 190% in P20 cells when cultured with CM1. Similarly, GPx1 expression was markedly upregulated in CM2, CM4, and CM6-incubated cells compared to primary P5 cells (p<0.001), with fold change values of 1.51±0.12, 1.99±0.16, and 2.31±0.19 in P20 cells. Percentage upregulations were 50.0±3.68%, 89.5±7.11%, and 126.5±9.74% in CM2, CM4, and CM6-incubated P20 cells respectively, and reached 248.0±18.6% in P30 cells at the highest MSC concentration. Concurrently, OS marker levels were substantially higher in CM1-cultured P25 and P30 senescent cells compared to primary P5 cells (p<0.001). Furthermore, hydrogen peroxide levels were significantly reduced in CM3-incubated cells compared to CM1 (p<0.01), reaching the lowest values in CM6 (p<0.001), with reductions of approximately 11.5%, 40%, 57%, and 58% in P30 CM3, CM4, CM5, and CM6-incubated cells respectively. MSC-Km values for GPx1 were 0.87, 1.13, and 1.92 µM in P20, P25, and P30 cells, respectively, with corresponding Vmax values of 4.59, 5.68, and 7.94 µmole/min/mg protein. These findings suggest that senescent cells utilize higher amounts of MSC to upregulate GPx1 expression and maximize its activity, supporting using Se supplements to combat OS.