Researchers developed a 3-dimensional human epidermal equivalent model and treated it with two concentrations of vitamin C—0.1 mM and 1.0 mM—for 7 and 14 days, reflecting physiologic blood and skin tissue levels. Sodium ascorbate was used to avoid pH fluctuations, and L-ascorbic acid was confirmed as the active intracellular form.
In vitamin C–treated samples, the epidermal cell layer significantly increased in thickness. There was also a rise in the number of actively dividing cells, as shown by Ki-67 protein staining. Cell viability improved compared with untreated samples.
This increase in epidermal thickness was linked to changes in DNA methylation. Vitamin C raised levels of 5-hydroxymethylcytosine (5-hmC), a marker of DNA demethylation, by up to 7.1-fold, while 5-methylcytosine (5-mC) levels remained stable. These epigenetic changes were not observed in sodium-only controls, confirming vitamin C as the active agent.
To verify the role of DNA demethylation, researchers co-treated samples with the ten–eleven translocation (TET) enzyme inhibitor Bobcat339. TET enzymes convert 5-mC to 5-hmC in the presence of vitamin C. Blocking TET activity significantly reduced vitamin C–induced changes in skin thickness, proliferation, and 5-hmC levels.
Gene expression profiling showed that vitamin C upregulated 283 genes involved in DNA replication and cell proliferation and downregulated 110 genes related to terminal differentiation. A total of 393 genes were differentially expressed.
Whole-genome bisulfite sequencing identified 10,279 differentially methylated regions, of which 10,138 were hypomethylated in vitamin C–treated samples. Of the 81 genes that were both upregulated and hypomethylated, 12—including ROS1, SOX9, RUNX2, and CYP1B1—had known links to cell proliferation. Quantitative PCR confirmed that all 12 genes were upregulated by 1.6- to 75.2-fold.
Markers of skin differentiation, including keratin 14, filaggrin, and loricrin, were not significantly altered, suggesting that vitamin C’s main effect was to enhance proliferation rather than induce differentiation.
Although sodium ascorbate was used in the culture system, control experiments demonstrated that sodium alone did not affect skin thickness, cell viability, or epigenetic markers. The authors noted that protein expression and histone modifications were not examined and may warrant further study.
These findings suggest that vitamin C may support therapies to reverse or prevent age-related epidermal thinning. Through epigenetic modulation of gene expression, vitamin C may help maintain skin health beyond its antioxidant effects.
Full disclosures can be found in the study.