Effects of NADH and NADH + NMN Co-Formulation on Human Dermal Fibroblasts: Preliminary In Vitro Analysis

Background/Objectives: Depletion of nicotinamide adenine dinucleotide (NAD+) impairs dermal fibroblast function and accelerates skin aging. NAD+ precursors such as nicotinamide mononucleotide (NMN) and the reduced form, NADH, are therefore candidate cosmetic actives, but their comparative and combined effects in skin cells remain poorly explored. This in vitro study compared NMN alone, NADH alone, and an NMN + NADH co-formulation. Methods: Human dermal fibroblasts (HDFs) and human mesenchymal stromal cells (hMSCs) were treated with 10 mg/mL of NMN, NADH, or NMN + NADH. Expression of FOXO3, SIRT1, and SIRT3 (HDFs) and NRF2, NOG, and WNT5A (hMSCs) was measured by RT-qPCR, and cellular senescence was quantified by β-galactosidase staining. Results: All treatments increased gene expression relative to untreated controls (p < 0.05), except WNT5A in NMN-treated hMSCs. Increases ranged from 15.4% to 52.3% with NMN, 22.3% to 60.7% with NADH, and 30.5% to 78.1% with NMN + NADH. The co-formulation produced the highest mean expression in every panel and was significantly higher than both single compounds for FOXO3, NRF2, NOG, and WNT5A and higher than NMN for SIRT1; NADH exceeded NMN for NRF2 and WNT5A. The proportion of β-galactosidase-positive HDFs fell from 15.6% in controls to 4.8% (NMN), 3.4% (NADH), and 3.0% (NMN + NADH) (p < 0.01), with no significant differences among treatments. Conclusions: NMN, NADH, and their co-formulation each reduced senescence and modulated longevity-, antioxidant-, and tissue-homeostasis-related genes, with the co-formulation showing the largest transcriptional effects. Because the amount of each active differed between the single compounds and the co-formulation, additive, synergistic, and dose-related effects cannot be distinguished.