Fig. 4: Eye-drops of NAD⁺ precursors restore 3β-HSD activity and improve meibomian gland function in aged mice.

a, Chromatograms showing NAD⁺-dependent 3β-HSD enzymatic activity in H295R cells. Veh, vehicle. b, 3β-HSD activities in a. n = 3 biologically independent cell samples for each treatment. c, Intracellular NAD⁺ levels in a. n = 4 cell samples, except n = 3 for NMN. d, Schematic of eye-drop regimen. Left eye was untreated. e, Meibomian 3β-HSD activities in d. Veh, n = 5; NR, n = 5; NMN, n = 6 mice. f, BrdU⁺ acinar cells in d. Veh, n = 7; NR, n = 6; NMN, n = 6 mice. For comparison, Hsd3b6^−/− mice were treated with NMN (n = 4, 2–3 mo). g, Schematic of daytime versus night-time regimen. h, Relative ratio between left and right eye BrdU⁺ cells in g. n = 11 (daytime) or 9 (night-time) mice. Arrows indicate BrdU⁺ acinar cells. i, Meibomian gland morphology after 90-d instillation of Veh, NR or NMN. Scale bars, 1 mm. The graph shows fold increases in gland area relative to that in untreated left eyes. Veh, NMN, n = 8; NR, n = 10 mice. j, EDE test after 90-d instillation. NR, n = 9; NMN, n = 8 mice. Left eye was treated with Veh. Arrows indicate area of the epithelial defect. Scale bars, 0.5 mm. The graphs represent Δscore before and after desiccation (iD). k, Meibomian NAD⁺ levels after single eye-drop (n = 8 eyes per time point, except n = 7 for 60 min, NMN, right, n = 6 for 150 min, NMN (right) and n = 6 for NMN (left)). ^**P = 0.0072, two-way ANOVA and Bonferroni test; ^##P = 0.0068, unpaired two-sided Student’s t-test. l, Relative mRNA levels of Wnt2 and Adamts17 after 14-d instillation of Veh, NR or NMN. Values are mean ± variance of two biological replicates. Error bars in b,c,e,f,i,k indicate s.e.m. Statistics in b,c,i were one-way ANOVA and Bonferroni test; statistics in e,f (right) were one-way ANOVA and Holm–Sidak test; statistics in e,f (left), h,j were paired, two-sided Student’s t-tests.