Organic synthesis and anti-influenza A virus activity of cyclobakuchiols A, B, C, and D
Fig 9
Cyclobakuchiols A–D increase the mRNA expression of NAD(P)H quinone oxidoreductase 1 in MDCK cells, and cyclobakuchiols A–C induce the nuclear factor erythroid 2-related factor 2 activation.
(A) Cyclobakuchiols A (2), B (3), C (4), and D (5) (12.5 μM), DMSO (0.125%), or (+)-(S)-bakuchiol (1) (12.5 μM) were mixed with or without A/PR/8/34 virus (MOI 0.1) and added to 1 × 105 MDCK cells for 24 h. Total RNA was extracted from the cell lysates, and the mRNA levels of NAD(P)H quinone oxidoreductase 1 (Nqo1), a nuclear factor erythroid 2-related factor 2 (Nrf2)-induced gene (n = 9 each), were determined by RT-qPCR, normalized to β-actin mRNA, and expressed relative to the DMSO-treated non-infected cells (set as 1). (B) A Nrf2 reporter assay based on the dual luciferase system was performed in MDCK cells. MDCK cells (1 × 105) were transfected with pNQO1-ARE-Fluc, expressing a Firefly luciferase gene driven by Nrf2 activation, and pRL-TK-Rluc, expressing Renilla luciferase driven by the herpes simplex viral thymidine kinase promoter. At 24 h post-transfection, the cells were treated with 2–5 (12.5 μM) (n = 6 each), DMSO (0.125%) (n = 6), or 1 (12.5μM) (n = 6). The levels of Firefly and Renilla luciferase mRNA were analyzed by RT-qPCR after 24 h and normalized to β-actin mRNA. The relative levels of Firefly per Renilla luciferase mRNAs were calculated and compared with that observed in the DMSO-treated cells (set as 1) and expressed relative to the DMSO-treated cells (set as 1). Data are presented as the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated comparisons. The results were reproducible across all experiments.