2020 Volume 43 Issue 7 Pages 1104-1110
Latifolin, a natural flavonoid found in Dalbergia odorifera T. Chen, has been reported to exhibit anti-inflammatory and anticarcinogenic activities in vitro. However, the anti-aging effects of latifolin are unknown. In this study, we selected a model in vitro system, hydrogen peroxide (H2O2)-induced senescence in human dermal fibroblasts (HDFs), to examine the protective effects of latifolin against senescence and the detailed molecular mechanisms involved. Latifolin reversed the senescence-like phenotypes of the oxidant-challenged model, including senescence-associated β-galactosidase (SA-β-gal) staining, cell proliferation, and the expression of senescence-related proteins, such as caveolin-1, ac-p53, p21Cip1/WAF1, p16Ink4α, pRb, and cyclinD1. We also found that latifolin induced the expression of silent information regulator 1 (SIRT1) in a concentration- and time-dependent manner, and the anti-senescence effect of latifolin was abrogated by SIRT1 inhibition. Latifolin also suppressed the activation of Akt and S6K1 and attenuated the increase in SA-β-gal activity after H2O2 exposure. Our results indicate that latifolin exerts protective effects against senescence in HDFs and that induction of SIRT1 and inhibition of the mammalian target of rapamycin (mTOR) pathway are key mediators of its anti-aging effects.
Dalbergia odorifera T. Chen possesses a wide range of biological activities, including anti-osteoporotic, anti-inflammatory, antioxidant, and neuroprotective effects, in diverse cell types.1–3) Latifolin, is isolated from the heartwood of Dalbergia odorifera T. Chen.4) A previous study showed that latifolin attenuates inflammatory responses by inhibiting nuclear factor-kappaB (NF-κB) activation through induction of nuclear factor-E2-related factor 2 (Nrf2)-regulated heme oxygenase-1.5) However, the anti-aging effects of latifolin have not been reported.
Aging is the most important risk factor for the development of numerous diseases, including cancer, osteoarthritis, dementia, atherosclerosis, and infection.6) Cellular aging, also known as senescence, is closely related to the accumulation of reactive oxygen species (ROS).7) Hydrogen peroxide (H2O2) is a major ROS in cells and a key source of DNA damage which is closely associated with cellular senescence. During senescence, cells exhibit increased activity of β-galactosidase (β-gal), which serves as a biomarker of cellular senescence and changes in senescence-related proteins.6) The regulation of cellular senescence is complex. Silent information regulator 1 (SIRT1) plays a central role in regulating cellular senescence and is an important determinant of longevity.8) SIRT1 regulates lifespan extension and prevents cellular senescence by negatively regulating the acetylation of p53.9) In addition, the Cdk inhibitor p21Cip1/WAF1 progressively accumulates in senescent cells, and the Cdk4–Cdk6 inhibitor p16 Ink4α maintains senescent cell cycle arrest, leading to downregulation of the phosphorylation of cyclin D and retinoblastoma protein.10) Caveolin-1 expression negatively regulates cell cycle progression via the p53/p21Cip1/WAF1 pathway,11) and the mammalian target of rapamycin (mTOR) pathway plays a central role in aging.12)
In this study, we investigated the mechanism by which latifolin protected cells against H2O2-included senescence and the related intracellular signaling pathways.
Latifolin (Fig. 1A) was obtained from the Standardized Material Bank for New Botanical Drugs (no. NNMBP026), Wonkwang University (Republic of Korea). H2O2 and 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (X-Gal) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Antibodies against caveolin-1, cyclin D1, p21waf/Cip1 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Anti-sirtuin 1 (-SIRT1) antibody was purchased from Merck Millipore (Life Science business of Merck KGaA, Darmstadt, Germany). Anti-acetylated-p53 (-ac-p53), -p16Ink4α, -phospho-Rb (-p-Rb), -phospho-Akt (-p-Akt), -phospho-S6 (-p-S6) antibodies were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). All other chemicals were obtained from Sigma-Aldrich .
(B) HDFs were incubated for 12 h with different concentration of latifolin (0–160 µM). Cell viability was determined by MTS assay. All of the data are presented as the means ± S.D. of 3 three independent experiments. * p < 0.05 versus control.
Human dermal fibroblasts (HDFs) were purchased from Korea Cell Line Bank (Republic of Korea) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS) (10% (v/v)), antibiotics and antimycotics (1% (v/v)) at 37°C in a humidified incubator with 5% CO2. The cells within passage numbers 12–18 were used in all experiments. For determination of cell viability, a total of 10 µL of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] was added to each well (7 × 104 cells per well in 48-well) according to the manufacturer’s protocol, and then recorded the absorbance at 490 nm. The percentage of cell survival was determined.
Senescence-Associated β-Galactosidase (SA-β-gal) Staining AssayCells were stained for SA-β-gal solution as described previously.13) Briefly, cells were washed with phosphate buffered saline (PBS), fixed in 3% formaldehyde for 5 min, and then stained at 37°C in SA-β-gal solution containing 1 mg/mL X-gal, 40 mM citric acid/sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM sodium chloride and 2 mM magnesium chloride. After 24 h, the stained cells were photographed and counted.
Western Blot AnalysisBriefly, cells were harvested, washed with PBS, and resuspended in pre-cold radio immunoprecipitation assay (RIPA) buffer (Cell Signaling, Beverly, MA, U.S.A.) with protease inhibitors. Protein concentration was determined with the Bio-Rad protein assay kit. Samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel and were transferred to polyvinylidene difluoride (PVDF) membranes. Each membrane was incubated with primary antibody at 4°C overnight, followed by 2 h in secondary antibody. Proteins bands were visualized using enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Piscataway, NJ, U.S.A.).
Cell CountingCells were seeded in 12-well dishes and incubated overnight. The cells were given the drug treatment as indicated, harvested and then counted on a haemocytometer by inverted microscope.
SIRT1 Activity AssaySIRT1 activity assay was performed using a SIRT1 Fluorometric Drug Discovery Kit according to the manufacturer’s protocol (Enzo Life Sciences International, Inc., Plymouth Meeting, U.S.A.) as previously described.14) Briefly, HDFs were harvested and lysed with RIPA buffer. Lysates (50 µg) were incubated in SIRT1 assay buffer (100 µM Fluor de Lys-SIRT1 substrate, 5 µM TSA and 200 µM oxidized form of nicotinamide adenine dinucleotide (NAD+)) for 45 min at 37°C, the reaction was stopped by 2 × SIRT1 Developer (contains 2 M Nicotinamide) for 15 min at room temperature. Fluorescence of the samples were read using a SpectraMax M3 instrument with an excitation set to 355 nm and emission set to 460 nm.
RT-Quantitative (q) PCRHDFs were washed twice with PBS and the total RNA was extracted by easy-Blue™ kit, according to the manufacturer’s protocol. RNA concentration was read using a GeneQuant pro RNA/DNA calculator (Amersham Biosciences, Uppsala, Sweden). According to the manufacture’s protocol, cDNA was synthesized with total RNA using a High capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, U.S.A.). The cDNA was mixed with SIRT1 primer (4331182) (Applied Biosystems; Thermo Fisher Scientific, Inc.) and then performed using CFX96™ Real-Time System (Bio-Rad). Cycling conditions performed as follow: preparation at 50°C for 2 min, denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 10 s and at 60°C for 30 s. The data were analyzed using StepOne™ software.
Transfection with Small Interfering RNAs (siRNAs)Instructions for using SIRT1 siRNA were provided by Santa Cruz Biotechnology (Santa Cruz). Cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, U.S.A.) with 80 nM SIRT1 siRNA, according to the manufacture’s protocol (Invitrogen). After overnight transfection, the medium was changed, and the cells were continued to another experiment.
Statistical AnalysisResults are expressed as mean ± standard deviation (S.D.). One-way ANOVA was used for comparisons involving more than two groups. In the case of two sample comparisons, a Student’s t-test assuming unequal variance was used. Statistical significance was defined as the conventional p < 0.05.
First, latifolin was tested for possible cytotoxic effects in HDFs by evaluating the cell viability in the presence of latifolin, using the MTS assay. HDFs were cultured for 12 h with different concentrations of latifolin (0–160 µM). The results showed that latifolin, at concentrations up to ≤40 µM, did not affect cell viability (Fig. 1B). Exposure of various cell types to sub-lethal concentrations of hydrogen peroxide (H2O2) has been shown to induce senescence.15–17) To assess the anti-senescence effect of latifolin, HDFs were exposed to H2O2 with or without latifolin. As expected, exposure of the HDFs to H2O2 (at 200 µM for 3 d) resulted in an enlarged and flattened cell morphology (Fig. 2A) and an increase in senescence-associated β-galactosidase (SA-β-gal) activity (Fig. 2B). The addition of latifolin (10–40 µM) significantly reduced this senescent phenotype. We also found that pretreatment with latifolin effectively suppressed the increases in caveolin-1, ac-p53, p21Cip1/WAF1, and p16Ink4α triggered by H2O2 (Fig. 2C). In contrast, the levels of cyclin D1 and p-Rb were increased by latifolin pretreatment (Fig. 2C). Latifolin pretreatment also increased the growth rate of HDFs compared to that of cells treated with only H2O2 (Fig. 2D).
(A) Morphological changes and SA-β-gal staining following 200 µM H2O2 for 72 h with or without latifolin. (B) The percentages of SA-β-gal positive cells were calculated from three randomly chosen fields. (C) The expression of caveolin-1, ac-p53, p53, p21Cip1/WAF1, p16Ink4α, cyclin D1, p-Rb and β-actin were detected by Western blotting. The proteins were quantified by densitometry based on immunoblot images. (D) The cell number was examined by cell counting. All of the data are presented as the means ± S.D. of three independent experiments. * p < 0.05 versus control. # p < 0.05 versus H2O2-treated cells. The bar represents 100 µm.
SIRT1 is a longevity-related protein that plays an important role in the regulation of aging. To determine if SIRT1 expression in HDFs is induced by latifolin, the cells were treated with different concentrations of latifolin (10–40 µM) for 12 h. The effect of latifolin on SIRT1 expression is shown in Fig. 3A. Latifolin induced SIRT1 expression in a concentration-dependent manner (Fig. 3A), and SIRT1 expression peaked at 40 µM latifolin. SIRT1 expression was also induced by 40 µM latifolin in a time-dependent manner (Fig. 3B). Next, we examined the effect of latifolin on SIRT1 activity and found that SIRT1 activity was increased by latifolin in a concentration- and time-dependent manner (Figs. 3C and D, respectively). Furthermore, the mRNA level of SIRT1 also increased following treatment with latifolin (Figs. 3E and F, respectively).
(A, C, F) SIRT1 expression was measured 12 h after treatment with indicated concentrations of latifolin. (B, D, F) Cells were treated with 40 µM latifolin and harvested at indicated time points. (A, B) The expression of SIRT1 and β-actin were detected by Western blotting. The proteins were quantified by densitometry based on immunoblot images. (C, D) SIRT1 activity was performed using a SIRT1 fluorometric kit. (E, F) SIRT1 mRNA was determined using RT-qPCR. All of the data are presented as the means ± S.D. of three independent experiments. * p < 0.05 versus control.
According to the above-mentioned results, we then examined whether latifolin could protect against H2O2-induced cellular senescence by SIRT1 induction. As shown in Figs. 4A–C, HDFs were exposed with H2O2, the expression level and activities of SIRT1 significantly decreased, but the effect of H2O2 was reversed as HDFs pretreated with latifolin. Furthermore, to investigate the involvement of SIRT1 in the protective effect of latifolin, SIRT1 inhibition was determined using an siRNA. As shown in Figs. 4D and E, inhibition of SIRT1 abrogated the effects of latifolin on the senescence-specific morphological changes and increase in SA-β-gal activity. Likewise, the decreases in caveolin-1, ac-p53, p21Cip1/WAF1, and p16Ink4α and the increases in cyclin D1 and p-Rb were not observed (Fig. 4F). These results indicate that SIRT1 may play a vital role in the protective effect of latifolin against a senescent phenotype.
HDFs were treated with DMF (40 µM, 12 h) then H2O2 (200 µM, 3 d) after transfection with or without SIRT1 siRNA (80 nM, 18 h). (A) The expression of SIRT1 and β-actin were detected by Western blotting. The proteins were quantified by densitometry based on immunoblot images. (B) SIRT1 activity was performed using a SIRT1 fluorometric kit. (C) SIRT1 mRNA was determined using RT-qPCR. (D) Senescent cells were stained by SA-β-gal (blue). (E) The percentages of SA-β-gal positive cells were calculated from three randomly chosen fields. (F) The expression of caveolin-1, ac-p53, p53, p21Cip1/WAF1, p16Ink4α, cyclinD1, p-Rb and β-actin were detected by Western blotting. The proteins were quantified by densitometry based on immunoblot images. All of the data are presented as the means ± S.D. of three independent experiments. * p < 0.05 versus control. # p < 0.05 versus H2O2-treated cells. The bar represents 100 µm.
Exposure of HDFs to H2O2 induces the activation of various kinases involved in the induction of senescence.18,19) Evidence has shown that the mTOR/S6K1 signaling plays an important role in aging,20) and increased S6K1 activation is associated with an enhanced Akt activity.12) In our study, we assessed Akt and S6K1 activity by evaluating the phosphorylation levels of Akt-S473 and the S6K1 substrate S6 at serine 240/224 (S6-S240/224), respectively, at different time points after H2O2 treatment. As shown in Fig. 5A, the activation of Akt and S6 were evident as early as 1 h; the augmentation lasted for an additional 5 h, and then decreased after 12 h of H2O2 treatment. The induction of Akt and S6 protein were mostly activated after 2 h of H2O2 treatment. Furthermore, we found that latifolin or LY294002 (Akt inhibitior21)) treatment significantly downregulated p-Akt and p-S6 (at 2 h H2O2 treatment), suggesting that latifolin modulated H2O2-induced Akt/S6K1 signaling (Fig. 5B). In addition, we also found that inhibition of the Akt/S6K1 signaling pathway induced by pretreatment with latifolin or LY294002 significantly reduced SA-β-gal activity (Fig. 5C). These results suggested that latifolin could attenuate H2O2-induced senescence by blocking the Akt/S6K1 pathway. Next, we examined whether induction of SIRT1 by latifolin could protect against H2O2-induced cellular senescence via inhibiting Akt/S6K1 signaling pathway. As shown in Fig. 5D, latifolin pretreatment decreased the activation of Akt and S6 after 2 h H2O2 exposure, but knockdown of SIRT1 by siRNA abolished the inhibition of latifolin for Akt/S6K1 signaling.
(A) HDFs were incubated in 200 µM H2O2 and harvested with indicated time points. The expression of phospho-Akt (p-Akt), Akt, phosphor-S6 ribosomal protein (p-S6), S6 and β-actin were detected by Western blotting. The proteins were quantified by densitometry based on immunoblot images. (B) HDFs were pretreated with LY294002 (2.5, 5, 10 µM, 30 min), latifolin (40 µM, 12 h), and then incubated in 200 µM H2O2 for 2 h. The expression of p-Akt, Akt, p-S6, S6 and β-actin were detected by Western blotting. The proteins were quantified by densitometry based on immunoblot images. (C) HDFs were pretreated with LY294002 (2.5, 5, 10 µM, 30 min), latifolin (40 µM, 12 h), and then incubated in H2O2 (200 µM, 3 d). The percentages of SA-β-gal positive cells were calculated from three randomly chosen fields. (D) HDFs were treated with DMF (40 µM, 12 h) then H2O2 (200 µM, 2 h) after transfection with or without SIRT1 siRNA (80 nM, 18 h). The expression of p-Akt, Akt, p-S6, S6 and β-actin were detected by Western blotting. The proteins were quantified by densitometry based on immunoblot images. All of the data are presented as the means ± S.D. of three independent experiments. * p < 0.05 versus control. # p < 0.05 versus H2O2-treated cells.
Senescence is a complex process that is regulated by various stresses, including oncogene activity, telomere uncapping, and oxidative activity, among others. During senescence, cells exhibit certain characteristics, including irreversible growth arrest, altered protein expression, enlarged and flattened morphology, and increased SA-β-gal activity.6) Recently, researchers identified several compounds that can significantly reverse stress-induced senescence by improving the above-mentioned senescence-related phenotypes.15,17) In our study, we found that latifolin protected HDFs from senescence induced by H2O2 (Fig. 2). Consistent with our findings, Mao et al. found that salidroside protects 2BS cells from H2O2-induced senescence by reducing DNA damage, SA-β-gal activity, and the expression of related proteins, including p53, p21, and p16.15) Another study reported that cilostazol inhibits H2O2-induced senescent phenotypes in human umbilical vein endothelial cells (HUVECs) by downregulating the acetylation of p53 at lysine 373/382.17)
Next, we aimed to determine the cytoprotective-related molecular mechanism of latifolin, with a focus on the SIRT1 and Akt/mTOR pathways.
SIRT1 plays important roles in cellular senescence and aging.16,22) Zhou et al. reported that SIRT1 alleviates the senescence of degenerative human intervertebral disc cartilage endo-plate cells by inhibiting the p53/p21 pathway.23) Yao et al. reported that SIRT1 protects against emphysema via FOXO3-mediated reduction of premature senescence in mice.24) However, H2O2 causes a reduction in SIRT1 protein levels,16,17) and Ota et al. found that inhibition of SIRT1 leads to increased SA-β-gal activity, a senescent phenotype, and endothelial dysfunction.25) These reports suggest that SIRT1 is closely related to both senescence and oxidative stress. SIRT1 is upregulated by several plant-derived compounds, such as salidroside,15) the ginsenoside Rb1,16) and cilostazol.17) In the present study, we also found that latifolin induced SIRT1 expression in HDFs (Fig. 3). However, knockdown of SIRT1 by SIRT1 siRNA abolished the protective effect of latifolin against H2O2-induced senescence (Fig. 4), which is consistent with previous reports using other cells.16,17,25)
Here, we speculated that the Akt/mTOR signaling pathway could be involved in the observed anti-aging effects of latifolin, as Akt is upstream of mTOR/S6 signaling and Akt/mTOR signaling pathway has been shown to play important roles in cellular senescence.12,26) Rajapakse et al. reported that an increased phosphorylation of Akt can highly induce the S6K1 activation in senescence/aging.12) Nacarelli et al. reported that aberrant ROS accumulation during senescence is closely related to activation of the mTOR signaling pathway.27) Persistent ROS can stimulate Akt activation, which directly induces senescence through mTORC1-dependent SA-β-gal activity and increased p53 expression.28,29) In agreement with these previous findings, in the present study, we observed that H2O2-induced senescence was accompanied by immediate activation of p-Akt and p-S6 (Fig. 5A). Previous studies have shown that inhibition of the Akt/mTOR signaling pathway prolongs lifespan and prevents cellular senescence,30) and this signaling is downregulated by several plant-derived compounds, including resveratrol12) and epigallocatechin gallate.26) Here, we showed that latifolin suppressed the Akt/mTOR signaling pathway and reduced SA-β-gal activity (Figs. 5B and C, respectively). Moreover, previous studies have reported that up-regulation of SIRT1 plays an important role in prevention of neuronal cell growth through inhibiting the mTOR signaling pathway.31) In present study, we demonstrated that latifolin treatment stimulated the SIRT1 expression in a concentration- and time-dependent manner. So, we examined the role of Akt/mTOR signaling in SIRT1 expression. The inhibition effect of latifolin for H2O2-induced Akt/mTOR activation was abolished as SIRT1 gene silencing (Fig. 5D).
In summary, we showed that latifolin inhibited H2O2-induced senescence and that upregulation of SIRT1 activation and downregulation of the mTOR pathway played important roles in the latifolin-mediated prevention of senescence in HDFs. These findings demonstrate that latifolin has the potential to ameliorate the aging process and attenuate age-related diseases in humans.
This study was supported by Wonkwang University in 2018.
The authors declare no conflict of interest.
