Oligonol promotes anti-aging pathways via modulation of SIRT1-AMPK-Autophagy Pathway

Abstract

Background/objectives: Oligonol, mainly found in lychee fruit, is an antioxidant polyphenolic compound which has been shown to have anti-inflammatory and anti-cancer properties. The detailed mechanisms by which oligonol may act as an anti-aging molecule have not been determined.

Materials/methods: In this study, we evaluated the ability of oligonol to modulate sirtuin (SIRT) expression in human lung epithelial (A549) cells. Oligonol was added to A549 cells and reactive oxygen species production, mitochondrial superoxide formation, and p21 protein levels were measured. Signaling pathways activated upon oligonol treatment were also determined by western blotting. Furthermore, the anti-aging effect of oligonol was evaluated ex vivo in mouse splenocytes and in vivo in Caenorhabditis elegans.

Results: Oligonol specifically induced the expression of SIRT1, whose activity is linked to gene expression, metabolic control, and healthy aging. In response to influenza virus infection of A549 cells, oligonol treatment significantly up-regulated SIRT1 expression and down-regulated viral hemagglutinin expression. Oligonol treatment also resulted in the activation of autophagy pathways and the phosphorylation of AMP-activated protein kinase (AMPK). Furthermore, oligonol-treated spleen lymphocytes from old mice showed increased cell proliferation, and mRNA levels of SIRT1 in the lungs of old mice were significantly lower than those in the lungs of young mice. Additionally, in vivo lethality assay revealed that oligonol extended the lifespan of C. elegans infected with lethal Vibrio cholerae.

Conclusions: These data demonstrated that oligonol may act as an anti-aging molecule by modulating SIRT1/autophagy/AMPK pathways.

Keywords: Oligonol; SIRT1; aging; senescence.

Fig. 1. Oligonol suppressed reactive oxygen species (ROS) production in human lung epithelial cells.

(A) A549 cells were treated with oligonol at 1, 10, 50, or 100 µg/mL and cell viability was determined by the MTT assay. Data are shown as the mean ± SD of three independent experiments. (B) High-passage A549 cells were treated with oligonol at a concentration of 1, 10, or 50 µg/mL for 24 h and the total ROS level was determined using dicholorofluorescein diacetate (DCF-DA). Data are shown as the mean ± SD of three independent experiments and are presented as the percentage of control (dimethyl sulfoxide [DMSO]-treated). (C) Mitochondrial superoxide production was evaluated by detecting MitoSOX-positive cells using confocal microscopy. (D). Interleukin-8 (IL-8) levels were measured by enzyme-linked immunosorbent assay after oligonol treatment. Data are shown as the mean ± SD of three independent experiments and are presented as the percentage of control (DMSO-treated). ^* P < 0.05 vs. the DMSO control cells.

Fig. 2. Oligonol up-regulated sirtuin 1 (SIRT1) expression.

(A) High-passage A549 cells were treated with 10 µg/mL oligonol for 2 h, total cellular RNA was isolated, and SIRT1-7 mRNA levels were measured by reverse transcription PCR (RT-PCR). (B) The mRNA levels of SIRT1 were determined by quantitative real-time PCR (qRT-PCR). The value obtained for control (dimethyl sulfoxide [DMSO]-treated) cells was arbitrarily set to 1, and relative expression is shown in the graph. Expression of target genes was normalized to that of β-actin. The qRT-PCR experiments were performed in duplicate and data are shown as the mean ± SD of three independent experiments. ^* P < 0.05 vs. DMSO control cells. (C) High-passage A549 cells were treated with 10 µg/mL oligonol for 2 h before infection with PR8 influenza virus at a multiplicity of infection of 0.1 and incubation for 24 h prior to RNA isolation. Expression of a major influenza antigen, hemagglutinin (HA) (C) and SIRT1 (D) were measured and normalized to that of β-actin. The qRT-PCR experiments were performed in duplicate and data are shown as the mean ± SD of three independent experiments.

Fig. 3. Oligonol induced the AMP-activated protein kinase (AMPK)/autophagy pathway.

(A) For the measurement of autophagy, high-passage A549 cells were treated with control or 10 µg/mL oligonol for 24 h and cells were stained with anti-LC3 antibody. Cells were examined by fluorescence confocal microscopy. Magnification × 400. (B) High-passage cells were treated with 10 µg/mL oligonol for various times (0, 0.5, 1, 1.5, 4, or 8 h) and total cellular lysates were collected for western blotting. Expression of the indicated proteins were determined using the relevant antibodies. Representative images are shown from three independent experiments. (C) Phosphorylation of AMPK was measured using antibodies detecting total AMPK and phospho-AMPK (active form, phosphorylated at Thr172). Protein bands were quantified using densitometry, and the normalized densitometric units were plotted against the relevant treatment (R.E.: Relative Expression).

Fig. 4. Oligonol induced splenocyte proliferation proliferation in old mice

Spleens were obtained from young (3-4 months) (A) and old (18-24 months) (B) C57BL/6 mice. Lymphocytes were incubated with interleukin-2 (IL-2) for 48 h and then with various concentrations (1, 5, 10, 50 µg/mL) of oligonol for another 48 h. Colorimetric determination of MTT reduction was made at 540nm and % cell viability is shown in the graph. The assays were performed in duplicate and data are shown as the mean ± SD of three mice. (C) Lung tissues from young and old mice. (D) Lung tissues from senescence marker protein-30 (SMP30) knock-out (SMP30^Y/-) and wild-type (SMP30^Y/+) mice were collected and SIRT1 mRNA levels were examined by quantitative real-time PCR (qRT-PCR). Target gene expression was normalized to that of mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The qRT-PCR experiments were performed in duplicate and each symbol represents the value for an individual mouse. The bar indicates the mean for the study group. ^* P < 0.05 for the comparison with the control group, Kruskal-Wallis test (one-way analysis of variance).

Fig. 5. Oligonol delayed lethality during Vibrio cholerae infection in Caenorhabditis elegans.

Lethality analysis was performed in C. elegans fer-15(b26)II; fem-1(hc17) that were fed with V. cholerae. Worms were treated with medium supplemented with 1, 10, or 100 µg/mL of oligonol in 6-well plates (30 worms per well). Controls were maintained in medium supplemented with dimethyl sulfoxide (DMSO). Assay plates were kept at 25℃ and scored for survivors at 24-h intervals. Data were plotted using the Kaplan-Meier method and survival curves were compared using the log-rank test. P values for oligonol: 1 µg/mL = 0.0501, 10 µg/mL = 0.3570, and 100 µg/mL = 0.0001.

Fig. 6. Schematic representation of oligonol-mediated activation of sirtuin 1 (SIRT1)- AMP-activated protein kinase (AMPK)-autophagy pathways.

In to the presence of oxidative stress or viral infection, oligonol treatment can enhance SIRT1 expression and downstream signaling pathways, leading to activation of autophagy and AMPK pathways. As a result, this may contribute to up-regulation of antioxidant formation and inhibition of viral replication.