Figure 4.

The cardioprotective effect of NMN in oxidative stress is SIRT3-dependent. NRVMs with shRNA knockdown of scrambled RNA (sh-Sc) or Klf15 (sh-Klf15) were transfected with 25 nM Sirt1, Sirt3 siRNA, or negative control siRNA for 48 h. (A) qPCR analysis of Sirt1 and Sirt3 levels in NRVMs (n = 4, *: p < 0.001 vs. NRVM transfected with negative control siRNA and sh-Sc; #: p < 0.001 vs. NRVM transfected with negative control siRNA and sh-Klf15). (B) NRVMs were pretreated with 2 mM NMN or PBS for 24 h prior to exposure to 100 μM H₂O₂ for 90 min. (B) NAD⁺ level. (C) NAD⁺/NADH ratio. (D) ROS was measured by DHE fluorescence intensity in a microplate reader, with excitation at 485 nm and emission at 590 nm. The fluorescence intensity was normalized to total protein in each sample. (E) Percentage of cytotoxicity was derived from LDH assay. (n = 3–4, *: p < 0.01 vs. sh-Sc without treatment; #: p < 0.05 vs. sh-Sc treated with H₂O₂; &: p < 0.01 vs. sh-Klf15 without treatment; $: p < 0.05 vs. sh-Klf15 treated with H₂O₂). Two-way ANOVA with Bonferroni correction was used to determine statistic difference. Data are presented as mean ± SEM.