Research Paper Volume 14, Issue 19 pp 7662—7691

Figure 7. Neferine and several flavonoids play a central role in the antiaging effects of lotus germ extract. (A and B) Neferine treatment induced autophagy and suppressed SA-β-gal activity. Aging NB1RGB cells were treated with DMSO (−) or the indicated concentration of lotus germ extract or neferine for 24 h and subjected to immunoblot (A) or SA-β-gal activity (B) assays. Lotus germ extract at 50 μg/mL included 1 μM neferine. (C and D) Several polyphenols included in lotus germ extract did not induce autophagy or suppress SA-β-gal activity. Aging NB1RGB cells were treated with DMSO (-) or the indicated compounds at 10 μM for 24 h and subjected to immunoblot (C) or SA-β-gal activity (D) assays. The polyphenol mixture (0.11 μM orientin and 0.76 μM vicenin-1) stimulated neferine-dependent induction of autophagy and suppressed SA-β-gal activity. Aging NB1RGB cells were treated with DMSO (−) or 50 μg/mL lotus germ extract or 1 μM neferine with or without a mixture of 0.76 μM vicenin-1 and 0.11 μM orientin for 24 h and subjected to immunoblotting (E) or SA-β-gal activity (F) assays. (G–J) Neferine rejuvenates aging fibroblasts. Aging NB1RGB cells were treated with DMSO (−), 50 μg/mL lotus germ extract, neferine at the indicated concentration (G–I, K), or 1 μM (J) for 24 h. (G) Cell viability was determined using an MTT assay. (H) ΔΨm was determined by the TMRM red fluorescence intensity, indicating activated mitochondria. (I) ATP levels were determined using a CellTiter-Glo assay. (J) Mitochondrial complex I activity was determined. (K) Collagen productions were determined. (L) Aging NB1RGB cells were treated with 1 μM neferine for the indicated times and subjected to immunoblotting using the indicated antibodies. Data are presented as the mean ± SD of three simultaneous experiments (B, D, F–K). Each P value was calculated using two-way ANOVA; ^*P < 0.05, ^**P < 0.01.