Abstract

   The aging population is steadily increasing, with aging and age-related
   diseases serving as major risk factors for morbidity, mortality, and
   economic burden. Peanuts, known as the “longevity nut” in China, have
   been shown to offer various health benefits, with peanut skin extract
   (PSE) emerging as a key compound of interest. This study investigates
   the bioactive compound in PSE with anti-aging potential and explores
   its underlying mechanisms of action. Procyanidin A1 (PC A1) was
   isolated from PSE, guided by the K6001 yeast replicative lifespan
   model. PC A1 prolonged the replicative lifespan of yeast and the
   yeast-like chronological lifespan of PC12 cells. To further confirm its
   anti-aging effect, cellular senescence, a hallmark of aging, was
   assessed. In senescent cells induced by etoposide (Etop), PC A1
   alleviated senescence by reducing ROS levels, decreasing the percentage
   of senescent cells, and restoring proliferative capacity.
   Transcriptomics analysis revealed that PC A1 induced apoptosis, reduced
   senescence-associated secretory phenotype (SASP) factors, and modulated
   the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)
   signaling pathway. The antioxidative capacity of PC A1 was also
   evaluated, showing enhanced resistance to oxidative stress in PC12
   cells by reducing reactive oxygen species (ROS) and malondialdehyde
   (MDA) levels and increasing superoxide dismutase (SOD) activity.
   Moreover, PC A1 induced autophagy, as evidenced by an increase in
   fluorescence-labeled autophagic compartments and confirmation via
   Western blot analysis of autophagy-related proteins. In addition, the
   treatment of an autophagy inhibitor abolished the antioxidative stress
   and senescence-alleviating effects of PC A1. These findings reveal that
   PC A1 extended lifespans and alleviated cellular senescence by
   enhancing oxidative stress resistance and inducing autophagy,
   positioning it as a promising candidate for further exploration as a
   geroprotective agent.

   Keywords: aging, peanut skin, procyanidin A1, cell senescence,
   antioxidative stress, autophagy, PI3K/Akt signaling pathway

1. Introduction

   Aging is a gradual and irreversible biological process characterized by
   a progressive loss of physiological integrity, which leads to declining
   function and increased susceptibility to age-related diseases such as
   cancer, cardiovascular disorders, and neurodegenerative diseases. With
   a global population that is living longer and becoming older,
   age-related diseases have emerged as significant contributors to
   morbidity, mortality, and social and economic burdens [[28]1]. Despite
   the inevitability of death, the rate of aging can be modulated, thus
   making healthy aging attainable. Numerous compounds have demonstrated
   significant anti-aging activity, particularly natural products, which
   often exhibit superior safety and efficacy [[29]2]. Therefore, the
   exploration of anti-aging molecules derived from natural products
   presents great potential for the prevention and treatment of aging and
   age-related diseases.

   During the screening of active compounds with anti-aging potential,
   aging models play a crucial role in evaluating the efficacy of
   potential therapeutics and understanding their interactions with
   biological systems. The yeast Saccharomyces cerevisiae has been a
   prominent model organism for studying pathways relevant to aging. Two
   different paradigms of aging have been established using yeast: the
   replicative lifespan (RLS) and chronological lifespan (CLS). The RLS of
   a yeast cell, which is based on the replicative potential of the cells,
   is defined as the number of daughter cells produced from a single
   mother cell before death. This metric may correlate with the aging of
   mitotically active cells in multicellular organisms [[30]3]. The K6001
   strain, a genetically modified strain of yeast derived from W303, is
   particularly useful for conducting RLS assays, as only the mother cells
   can reproduce offspring in glucose medium, whereas daughter cells
   cannot [[31]4]. The second paradigm, CLS, measures the duration of
   survival for non-dividing yeast cells and may parallel the aging of
   non-dividing cells in higher organisms [[32]5]. However, the yeast
   chronological senescence does not perfectly align with cellular
   senescence in mammals. To address this, a yeast-like CLS assay for
   mammalian cells has been developed to evaluate chronological senescence
   [[33]6]. Significantly, the same signal transduction pathways that
   drive yeast-like chronological senescence also influence aging in other
   organisms. Inhibitors that attenuate the senescent phenotype are found
   to decelerate yeast-like chronological senescence, establishing this
   model as a valuable tool for drug discovery focused on anti-aging
   effects [[34]6].

   Cell senescence is a hallmark of aging [[35]7]. Senescence cells
   accumulate in aged organisms and contribute to the progression of aging
   and age-related diseases. Cell senescence is a state characterized by
   irreversible cell cycle arrest. In addition to cell cycle blockade and
   proliferation defects, senescent cells also have the following
   features: increased cell size and flattening, impaired mitochondrial
   function and membrane integrity, elevated levels of reactive oxygen
   species (ROS), heightened activity of senescence-associated
   β-galactosidase (SA-β-gal) at a pH of 6, the development of a
   senescence-associated secretory phenotype (SASP), and nuclear
   alterations such as DNA damage. Numerous stressors can induce cellular
   senescence, including replicative stress, genotoxic agents, oncogene
   activation, oxidative stress, and metabolic stress [[36]8]. Cell
   senescence has attracted increasing attention, and it is considered a
   potential target for preventing or treating age-related diseases and
   extending healthspan. Senotherapeutic strategies that target senescent
   cells can be classified into two categories: senolytic treatments,
   which involve the clearance of senescent cells, and senomorphic
   treatments, which aim to reduce the effects of SASP [[37]9].

   ROS are generally small, short-lived, and highly reactive molecules
   formed by the incomplete one-electron reduction in oxygen. ROS include
   superoxide, peroxide, and hydroxyl radicals and singlet oxygen. When
   tightly controlled, ROS serve as signaling molecules. However, when the
   redox balance is disrupted, excess ROS can lead to oxidative stress,
   resulting in damage to lipids, proteins, DNA, and carbohydrates.
   Antioxidants are substances that play a crucial role in delaying,
   preventing, or removing oxidative damage to target molecules. A variety
   of antioxidants are present in biological systems, including enzymes
   (such as superoxide dismutase (SOD)) and various small molecules. The
   supplementation of antioxidants has been advocated as a strategy to
   reduce cellular oxidative stress and potentially extend lifespans in
   different organisms [[38]10,[39]11].

   Autophagy is a cellular process that delivers cytoplasmic substrates,
   including proteins, DNA, and organelles, to lysosomes for degradation.
   This process can be broadly divided into several stages: initiation,
   nucleation, elongation, fusion, degradation, and recycling. Autophagy
   promotes cellular growth and development, protects cells from metabolic
   stress and oxidative damage, and plays a crucial role in maintaining
   cellular homeostasis, as well as the synthesis, degradation, and
   recycling of cellular products [[40]12,[41]13]. Research has shown that
   autophagy capacity declines with aging, and the regulation of autophagy
   is vital for lifespan maintenance [[42]14]. Several pharmacological
   autophagy inducers, such as rapamycin (Rapa), spermidine, flavonoid
   4,4′-dimethoxychalcone, and urolithin A, have been shown to prolong
   lifespans [[43]15,[44]16,[45]17,[46]18].

   Arachis hypogaea Linn., commonly known as peanut or groundnut, is a
   significant oilseed and food crop in many tropical and subtropical
   regions. It is extensively processed into oil, snacks, and peanut
   paste. China stands as the largest producer of peanuts, accounting for
   approximately 38% of global production [[47]19]. In Chinese culture,
   peanuts are referred to as the “longevity nut”. The skin of the peanut,
   a by-product of the roasting process, has been utilized in Traditional
   Chinese Medicine (TCM) for centuries to treat various disorders,
   including hemophilia, hemorrhage, primary and secondary
   thrombocytopenic purpura, ulcers, inflammation, kidney issues, and
   hypertension. Recent studies have demonstrated that peanut skin
   extracts (PSEs) exhibit anti-obesity, anti-atherosclerotic,
   anti-inflammation, and antioxidant effects in mice. Additionally, PSE
   contributes to the maintenance of gut microbiota
   [[48]20,[49]21,[50]22,[51]23]. In this study, a compound that is
   capable of prolonging the yeast RLS was isolated from the skin of
   Arachis hypogaea Linn. This compound was identified as procyanidin A1
   (PC A1) through spectral analysis, with comparisons made to published
   data. The anti-aging effects and underlying mechanisms of PC A1 were
   further explored, with findings indicating that it alleviates cell
   senescence, enhances oxidative stress resistance, and induces
   autophagy.

2. Materials and Methods

2.1. General

   Analytical pure reagents (methanol, ethanol, dichloromethane) were
   acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
   Chromatographic-grade methanol was obtained from the TEDIA Company,
   LLC. (Fairfield, OH, USA). Silica gel (200–300 mesh) was purchased from
   the Yantai Research Institute of Chemical Industry (Yantai, China).
   Reversed-phase C18 (Octadecylsilyl, ODS) silica gel (Cosmosil
   75C18-OPN) was from Nacalai Tesque, Inc. (Kyoto, Japan). Develosil
   ODS-UG-5 (Nomura Chemical Co., Ltd., Aichi, Japan) and Supersil Phenyl
   (Dalian Elite Analytical Instruments Co., Ltd., Dalian, China) packed
   columns were used for the isolation and purification of the natural
   products. Thin-layer chromatography (TLC) analysis was performed using
   the TLC silica gel plates (Yantai Jiangyou Silicone Development Co.,
   Ltd., Yantai, China) and TLC silica gel 60 RP-18 F254s 25 glass plates
   (0.25 mm) (Merck KGaA, Darmstadt, Germany). CD[3]OD (Cambridge Isotope
   Laboratories Inc., Andover, WA, USA) was used as the solvent for ^1H
   NMR. ^1H NMR spectra and HR ESI-TOF-MS data were obtained using a
   Bruker AV III-500 spectrometer (Bruker Corporation, Karlsruhe, Germany)
   and Agilent 6224A LC/MS (Agilent Technologies Inc., Beijing, China),
   respectively.

   The following reagents and compounds were purchased from the indicated
   manufacturer and used in biological experiments: dimethyl sulfoxide
   (DMSO) from Merck KGaA (Darmstadt, Germany). Resveratrol (Res),
   rapamycin (Rapa), etoposide (Etop), and chloroquine (CQ) were obtained
   from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).
   SBI-0206965 was from MedChemExpress LLC. (Shanghai, China).

2.2. Isolation and Structure Identification of PC A1

   The PSE was prepared according to the previous literature [[52]20].
   Under the guidance of K6001 yeast replicative lifespan assay, 1 g of
   PSE was subjected to ODS open-column chromatography with a
   methanol/water solvent system (10/90, 30/70, 50/50, 70/30, 100/0).
   Fractions were combined into 11 fractions according to TLC analysis,
   and the active fraction (348.1 mg) was obtained from a 30% aqueous
   methanol elution. Then, 100 mg of this fraction was taken out and
   subjected to silica open-column chromatography with a
   dichloromethane/methanol elution system (100/0, 90/10, 70/30, 50/50,
   30/70, 10/90, 0/100). Then, 7 samples were obtained. Sample 1 (15.4
   mg), obtained from dichloromethane/methanol (90/10), was purified first
   through HPLC (Develosil ODS-UG-5 (ϕ·10 × 250 mm), methanol: water =
   18:100, 100 min, 3 mL/min, 280 nm) to obtain sample 2 (5 mg, t[R] = 75
   min). Sample 2 was then purified again by another HPLC (Supersil Phenyl
   (ϕ·10 × 200 mm), methanol: water = 20:100, 20 min, 3 mL/min, 280 nm) to
   obtain PC A1 (3 mg, t[R] = 16 min) as colorless needles. The isolation
   scheme of PC A1 from PSE is shown in [53]Supplementary Figure S1. The
   chemical structure of PC A1 was identified by comparing the ^1H NMR
   spectra and MS with the literature [[54]24]: ^1H NMR (500 MHz,
   CD[3]OD): δ[H] = 7.13 (1H, d, J = 2.1 Hz), 7.01 (1H, dd, J = 8.3, 2.1
   Hz), 6.91 (1H, s), 6.81 (3H, m), 6.08 (1H, s), 6.06 (1H, d, J = 2.3
   Hz), 5.95 (1H, d, J = 2.3 Hz), 4.73 (1H, d, J = 7.8 Hz), 4.23 (1H, d, J
   = 3.5 Hz), 4.15 (1H, m), 4.07 (1H, d, J = 3.5 Hz), 2.94 (1H, dd, J =
   16.5, 5.6 Hz), and 2.57 (1H, dd, J = 16.4, 8.3 Hz). The ^1 H NMR
   spectrum of PC A1 is shown in [55]Supplementary Figure S2.
   High-resolution ESI-TOF-MS m/z 577.1344 was calculated for
   C[30]H[25]O[12] [M+H]^+ 577.1341. The high-resolution ESI-MS
   chromatograms of PC A1 is shown in [56]Supplementary Figure S3.

2.3. Cell Lines

   The PC12 cell line was purchased from the Cell Bank of the Chinese
   Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s
   Modified Eagle Medium (DMEM, Cellmax Cell Technology (Beijing) Co.,
   Ltd. (Beijing, China)) supplemented with 10% horse serum (Gibco
   (Herndon, VA, USA)), 7.5% fetal bovine serum (Cellmax Cell Technology
   (Beijing) Co., Ltd. (Beijing, China)), and 1% antibiotic–antimycotic
   solution (Beijing Solarbio Science & Technology Co., Ltd. (Beijing,
   China)). The NIH/3T3 cell line was generously given from MeilunBio
   (Dalian, China) and cultured in DMEM containing 10% fetal bovine serum
   and 1% antibiotic–antimycotic solution. All cells were cultured at 37
   °C in a humidified incubator of 5% CO[2]. Etoposide-induced senescence
   (ETIS) was triggered in NIH/3T3 and PC12 cells after culturing for 2 d
   in the presence of different concentrations of Etop (0.3 and 7.5 µM,
   respectively). Oxidative stress was triggered in PC12 cells after
   culturing for 2 h in the presence of 0.7 mM H[2]O[2]. All cell lines
   tested negative for microbial contamination and were routinely
   authenticated with STR assays.

2.4. Yeast Replicative Lifespan Assay

   K6001 yeast was cultured in galactose liquid medium, which comprised 3%
   galactose, 2% hipolypeptone, and 1% yeast extract. Following incubation
   for 24 h with continuous shaking (180 rpm, 28 °C), a total of 4000
   cells were washed and then evenly distributed on yeast peptone dextrose
   (YPD) agar plates, which were formulated with 2% glucose, 2%
   hipolypeptone, 1% yeast extract, and 2% agar and supplemented with Res
   or varying concentrations of PC A1. After incubation for 48 h at 28 °C,
   forty microcolonies from the agar plate were randomly selected for
   observation under an optical microscope (Olympus, Tokyo, Japan), and
   the number of daughter cells produced by one mother cell was counted.

2.5. Yeast-like Chronological Lifespan Assay

   The yeast-like chronological lifespan (CLS) assay was performed
   according to the reference [[57]6]. The principle of this assay is
   based on the observation that, over time, a densely overgrown cell
   culture loses its ability to survive and re-enter the growth phase when
   transferred to a fresh nutrient-rich medium. This loss of viability
   occurs in a density-dependent and time-dependent (chronological)
   manner, reflecting the aging process of the cells. In detail, PC12
   cells were seeded at a high density of 8 × 10^4 cells per well in
   96-well plates, with each well containing 0.2 mL of culture medium.
   After 24 h, the culture medium was replaced with serum-free medium
   supplemented with either 0.5% DMSO (vehicle control), 200 nM of
   rapamycin (Rapa, positive control), or procyanidin A1 (PC A1) at
   concentrations of 1, 3, and 10 µM. After 4 days, the medium, including
   any floating cells, was carefully aspirated. The adherent cells were
   then trypsinized using 0.2 mL of trypsin solution. Subsequently, an
   equivalent volume of the cell culture (4 µL of aliquot), representing
   approximately 2% of the total adherent (viable) cells, was transferred
   into 6-well plates containing 4 mL of fresh medium per well. The cells
   were cultured for an additional 15 days for colony formation. To assess
   cellular viability, the resulting colonies were fixed with 4%
   paraformaldehyde for 15 min and subsequently stained with a 0.1% (w/v)
   crystal violet solution for 20 min. Following staining, the cells were
   thoroughly rinsed with water and allowed to air dry. The number of
   viable colonies, which serves as a quantitative indicator of
   chronological lifespan, was enumerated and subsequently analyzed for
   statistical significance.

2.6. Cell Viability Assay

   An MTT assay was performed to measure the cell viability. In general,
   5000 cells were seeded into each well of a 96-well plate and cultured
   under the indicated treatment. Then, 100 µL of serum-free DMEM
   containing 500 µg/mL of MTT (Richu BioScience Co., Ltd. (Shanghai,
   China)) was added and incubated for 4 h followed by the removal of the
   medium carefully and an addition of 100 µL of DMSO. The plates were
   read with the absorbance at 570 nm using a plate reader (BioTek Synergy
   H1, Agilent, Winooski, VT, USA).

2.7. Senescence-Associated β-Galactosidase (SA-β-gal) Assay

   SA-β-gal staining was performed using the senescence β-galactosidase
   staining kit (Beyotime Biotechnology Inc., Shanghai, China), adhering
   strictly to the manufacturer’s protocols. For the assessment of
   SA-β-gal activity, approximately 50,000 PC12 cells or 20,000 NIH/3T3
   cells were seeded into each well of a 24-well plate. Then, cells were
   exposed to various test samples; 0.5% dimethyl sulfoxide (DMSO) was
   employed as the negative control, while Rapa served as the positive
   control. After 24 h, cells were treated with Etop for 2 days. After the
   treatment, cells were gently washed, fixed, and stained with the
   provided dyeing solution as directed. Cells were examined under a
   bright-field microscope (BX63, Olympus, Japan, 20× objective).
   SA-β-gal^+ percentages were quantified by counting the number of
   SA-β-gal-positive cells per visual field.

2.8. Cell Proliferation Assay

   The 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay was conducted
   to evaluate cellular proliferation. For this purpose, the BeyoClick™
   EdU Cell Proliferation Kit with Alexa Fluor 488, supplied by Beyotime
   Biotechnology Inc. in Shanghai, China, was utilized, and the assay was
   carried out in accordance with the manufacturer’s guidelines. Following
   treatment, the cells were incubated with EdU at a concentration of 10
   µM for 2 h and subsequently fixed and permeabilized, then cells were
   treated with the click reaction additive solution for 30 min. Following
   this step, the cells were counterstained with Hoechst 33342 for 10 min.
   After a thorough washing process, the EdU-positive cells were
   visualized using a fluorescence microscope (BX63, Olympus, Japan)
   equipped with a 20× objective lens. The proliferating cells were then
   quantified based on the fluorescence signals observed.

2.9. RNA Extract, RNA-Seq, and Data Analysis

   NIH/3T3 cells (4 × 10^5 cells) at passage 5 were seeded into each 10 cm
   dish. The next day, the cells were treated with or without 10 µM of PC
   A1 for 2 days before exposure to 0.3 µM of Etop for 2 days. The control
   group was treated with the same medium containing an equal amount of
   DMSO. Total RNA was extracted using the TRIzon reagent (Jiangsu Cowin
   Biotech Co., Ltd. (Taizhou, China)).

   The processes of quality control, cDNA library construction, and
   sequencing were expertly handled by ShenZhen BGI Genomics Co., Ltd.
   (Shenzhen, China). Three biological replicates were used for each
   group. Quality control of the RNA samples was conducted using a
   Fragment Analyzer, while sequencing was performed on the DNBSEQ
   platform. The raw data obtained from sequencing were filtered using
   SOAPnuke to obtain clean data. The clean reads were then aligned to the
   reference genome using HISAT and to the gene set using Bowtie2. The
   reference species used for this study was Mus musculus, and the
   specific reference genome version is GCF_000001635.27_GRCm39, sourced
   from the NCBI database. Differential gene expression analysis was
   conducted using the DESeq2 method, identifying genes with a Q value
   (adjusted p value) ≤ 0.05 and a log[2](Fold Change (FC)) ≥ 0.5 or ≤−0.5
   as differentially expressed genes (DEGs). For comprehensive data
   analysis and visualization, including the Kyoto Encyclopedia of Genes
   and Genomes (KEGG) pathway enrichment analysis and the Gene Ontology
   (GO) biological process enrichment analysis, the online bioinformatic
   platform tool Dr. Tom provided by BGI ([58]https://biosys.bgi.com
   (accessed on 18 November 2024)) was utilized. Additionally, volcano
   plots and heatmaps were carried out using another online platform,
   CNSKnowall ([59]https://cnsknowall.com (accessed on 20 February 2025)).

2.10. ROS Assay

   A ROS assay was performed using a ROS Assay Kit (Beyotime Biotechnology
   Inc., Shanghai, China), following the manufacturer’s instructions.
   Generally, 50,000 cells were seeded into each well of a 24-well plate.
   Then, cells were treated with PC A1 (1, 3, 10, and 30 µM) for 18 h, and
   then with H[2]O[2] for 2 h or Etop for 2 days. Then, DCFH-DA, at a
   final concentration of 10 µM, was added and incubated with cells for 30
   min. After washing with phosphate-buffered saline (PBS), fluorescence
   was observed using a fluorescence microscope (IX53, Olympus, Japan, 20×
   objective).

2.11. MDA Quantifications and SOD Assay

   Approximately 10^6 of the PC12 cells were seeded in a 60 mm culture
   dish. Then, cells were treated with Res (10 µM) or PC A1 (1, 3, 10, and
   30 µM) for 18 h, and then with 0.7 mM H[2]O[2] for another 2 h. The MDA
   quantification and SOD assay were determined using the MDA assay kit
   and SOD assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing,
   China) in accordance with the manufacturer’s instructions,
   respectively.

2.12. Autophagy Detection

   At first, approximately 80,000 PC12 cells were seeded in each well of a
   24-well plate. After 24 h, cells were treated with CQ (10 µM, as a
   negative control), Rapa (500 nM) plus CQ (as a positive control), and
   PC A1 (at doses of 0, 3, 10, and 30 µM) plus CQ. After 18 h, the cells
   were stained with the CYTO-ID^® Autophagy Detection Kit (Enzo Life
   Sciences, Inc., New York, NY, USA), according to the manufacturer’s
   instructions. Briefly, the culture medium was removed, and the cells
   were rinsed twice with an assay buffer supplemented with 5% fetal
   bovine serum. Subsequently, 250 µL of assay buffer, containing 0.2%
   green detection reagent and 0.1% Hoechst 33342 nuclear stain, was added
   to each well. Following a 30 min incubation, the cells were fixed,
   washed with the assay buffer, and then examined under a confocal
   microscope (BX61, Olympus, Japan) with a 20× objective lens.

2.13. Western Blot Analysis

   Whole-cell lysates were prepared using the RIPA lysis buffer containing
   a 1% complete protease inhibitor cocktail (Jiangsu Cowin Biotech Co.,
   Ltd. (Taizhou, China)), 1% phosphatase inhibitor cocktail II, and 1%
   phosphatase inhibitor cocktail III (Abcam Limited. (Cambridge, UK)) and
   homogenized and centrifuged at 12,000× g for 20 min at 4 °C. The
   protein concentration of cell lysates was determined by the BCA protein
   assay kit. Cell lysates were added with thr SDS-PAGE sample loading
   buffer and heated for 10 min at 100 °C. Then, 20 µg of protein of each
   sample was separated by electrophoresis on sodium dodecyl sulfate (SDS)
   polyacrylamide gels and transferred to an Immun-Blot PVDF membrane
   (Bio-Rad Laboratories Inc., Hercules, CA, USA). Membranes were
   incubated with primary and then secondary antibodies. Primary
   antibodies against phosphoinositide 3-kinase (PI3K) (#4249, 1:1000),
   phospho-PI3K (#4228, 1:1000), mammalian target of rapamycin (mTOR,
   #2983, 1:1000), phospho-mTOR (#5536, 1:1000), unc-51 like autophagy
   activating kinase 1 (ULK1, #8054, 1:1000), phospho-ULK1 (Ser757,
   #14202, 1:1000), p62 (#5114, 1:1000), microtubule-associated protein 1
   light chain 3 (LC3B, #2775, 1:1000), protein kinase B (Akt, #9272,
   1:1000), and phospho-Akt (#9271, 1:1000) were procured from Cell
   Signaling Technology, Inc. (Boston, MA, USA). Beclin-1 (#HA721216,
   1:1000) was purchased from Hangzhou HuaAn Biotechnology Co., Ltd.
   (Hangzhou, China). p21 (#AP021, 1:200) was from Beyotime Biotechnology
   Co., Ltd. (Shanghai, China). β-Actin (#CW0096, 1:1000) was from Jiangsu
   Cowin Biotech Co., Ltd. (Taizhou, China). HRP-conjugated goat
   anti-rabbit IgG (#CW0103S, 1:5000) and anti-mouse IgG (#CW0102S,
   1:5000) were from Jiangsu Cowin Biotech Co., Ltd. (Taizhou, China).
   Finally, antigens were visualized using the SuperPico ECL
   Chemiluminescence Kit (Nanjing Vazyme Biotech Co., Ltd., Nanjing,
   China). The Bio-Rad ChemiDoc^TM MP Imaging System (Bio-Rad
   Laboratories, Inc., Hercules, CA, USA) was used for the detection of
   proteins of interest, and the blot density was quantified utilizing
   Image Lab software (Version 6.1, Bio-Rad Laboratories, Inc., Hercules,
   CA, USA).

2.14. Statistical Analysis

   Statistical analyses were performed using the GraphPad Prism software
   (Version 9.0, GraphPad Software, LLC, San Diego, CA, USA). To assess
   differences among multiple groups, ordinary one-way ANOVA was
   conducted, followed by Dunnett’s multiple comparisons test for post hoc
   analysis. For pairwise comparisons between the treatment group and the
   control group in the replicative lifespan assay, two-tailed, unpaired
   Student’s t-tests were employed. Statistical significance was defined
   as p < 0.05. Each experiment was repeated three times, and data for
   each experiment are shown as mean ± SEM.

3. Results

3.1. PC A1 Extended the Replicative Lifespan of K6001 Yeast and Yeast-like
Chronological Lifespan of PC12 Cells

   Due to its convenience and speed, the K6001 yeast RLS assay was
   utilized as a guide to isolate compounds with potential anti-aging
   activity. In this study, PC A1 was isolated and purified from PSE under
   the guidance of the yeast RLS system. The isolation scheme and the
   chemistry structure of PC A1 are presented in [60]Supplementary Figure
   S1 and [61]Figure 1a, respectively. In the yeast RLS assay, Res was
   employed as a positive control for its stimulating effects on SIRTI and
   its ability to prolong the lifespan in yeast [[62]25]. The results in
   [63]Figure 1b demonstrate that PC A1 (0.1, 0.3, 1, 3, and 10 µM) and
   Res significantly slowed the replicative aging of yeast. However, PC A1
   at 30 µM exhibited toxicity to the yeast. Furthermore, yeast-like CLS
   assays were conducted on PC12 cells to evaluate the lifespan-extending
   effect of PC A1 at the mammalian cell level. The results in [64]Figure
   1c,d indicate that PC A1 (1 and 3 µM) and Rapa (as a positive control)
   increased the number of colonies, demonstrating that PC A1 could
   prevent the senescence-mediated decline of clonogenic survival in
   highly confluent PC12 cells [[65]6]. Collectively, these findings
   suggest that PC A1 prolongs both yeast RLS and yeast-like CLS in
   mammalian cells.

Figure 1.

   [66]Figure 1
   [67]Open in a new tab

   Procyanidin A1 (PC A1) isolated and purified from peanut skin extended
   the replicative lifespan of K6001 yeast and the yeast-like
   chronological lifespan of PC12 cells. (a) The chemical structure of PC
   A1. (b) The effect of PC A1 on the replicative lifespan of K6001 yeast.
   Resveratrol (Res) at 10 µM was used as the positive control. The
   experiment was conducted three independent times, with each replicate
   involving 40 individual mother cells per treatment group. The
   replicative lifespan was determined by counting the number of daughter
   cells produced by each mother cell. To assess the significance of the
   results, a two-sided Student’s t-test was used to compare the number of
   daughter cells in each treatment group to the control group. (c,d) The
   colony formation of PC12 cells treated with PC A1 (1, 3, and 10 µM) and
   rapamycin (Rapa, 200 nM) for testing the yeast-like chronological
   lifespan of PC12 cells. The representative images of colony-forming
   units (CFUs) (c) and quantification (d) are shown. Rapamycin (Rapa) was
   used as the positive control. Experiments were repeated three times.
   Data represent mean ± SEM. Significant difference was obtained with
   ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test.
   *, **, and *** indicate significant differences at p < 0.05, p < 0.01,
   and p < 0.001 compared with the control (C) group, respectively.

3.2. PC A1 Alleviated Cellular Senescence Induced by Etoposide

3.2.1. PC A1 Alleviated Cellular Senescence Induced by Etoposide in PC12
Cells

   The evaluation of cellular senescence as a hallmark of aging was
   conducted. Cellular senescence is characterized by elevated levels of
   ROS, an increased expression of SA-β-gal, and arrested cell
   proliferation. Etoposide (Etop), a chemotherapy agent, inhibits
   topoisomerase II, destroys the rejoining of DNA after superhelical
   unwinding, induces DNA damage, and subsequently leads to cell cycle
   arrest and senescence. Therefore, Etop was employed to model the
   pathological conditions associated with cellular senescence. The PC12
   cell line, derived from a pheochromocytoma of the rat adrenal medulla,
   is widely utilized in neuroscience research, including studies on
   neuroprotection, neurosecretion, neuroinflammation, and synaptogenesis
   [[68]26]. Recent studies also demonstrated that PC12 cells are valuable
   in simulating the cellular changes associated with nervous system aging
   [[69]27]. Initially, the viability of PC12 cells following treatment
   with PC A1 was assessed. The results indicated that PC A1 (at
   concentrations of 1, 3, 10, and 30 µM) did not exhibit any significant
   cytotoxic effects on normal PC12 cells, whereas the treatment of PC A1
   at 100 µM resulted in a 50% reduction in cellular viability ([70]Figure
   2a). Subsequently, PC12 cells were pre-treated with PC A1 (at 1, 3, 10,
   and 30 µM) for one day and then exposed to Etop (7.5 µM) for 2 days to
   evaluate the protective effect of PC A1 against cell senescence. Rapa
   at 500 nM was used as a positive control due to its known
   geroprotective effects [[71]15]. Cell viability was assessed using the
   MTT assay, and the results demonstrated that PC A1 (at 1, 3, and 10 µM)
   and Rapa (500 nM) significantly mitigated the loss of cellular
   viability caused by Etop ([72]Figure 2b). Furthermore, the level of ROS
   was found to be elevated by Etop; however, treatment with PC A1 (1, 3,
   10, and 30 µM) and Rapa (500 nM) significantly reduced ROS levels
   ([73]Figure 2c,d). Subsequently, SA-β-gal staining was performed, as
   shown in [74]Figure 2e,f, and quantitative analysis revealed that over
   70% of Etop-treated cells exhibited a senescent phenotype characterized
   by SA-β-gal-positive cells. In contrast, the pre-treatment of PC A1 (at
   1, 3, 10, and 30 µM) significantly decreased the number of
   SA-β-gal-positive PC12 cells. Cell cycle arrest, another hallmark of
   cell senescence, was assessed using the EdU cell proliferation kit.
   EdU, a thymidine analog, was incorporated into DNA during synthesis,
   followed by a click reaction with a green fluorescence-labeled azide
   probe, thereby labeling newly synthesized DNA for the detection of
   proliferating cells. To visualize all cells, nuclei were stained with
   Hoechst 33342. As shown in [75]Figure 2g,h, nearly 25% of the PC12
   cells in the control group were EdU positive, exhibiting green
   fluorescence, whereas this percentage dropped to 4% in Etop-induced
   senescent cells. The pre-treatment of PC A1 (1, 3, and 10 µM) and Rapa
   (500 nM) protected against the proliferative impairment caused by Etop.
   These experimental findings suggest that PC A1 effectively prevented
   cellular viability loss, reduced ROS levels, decreased the number of
   SA-β-gal-positive senescent cells, and restored proliferative capacity,
   thereby alleviating Etop-induced senescence in PC12 cells.

Figure 2.

   [76]Figure 2
   [77]Open in a new tab

   Procyanidin A1 (PC A1) alleviated Etop-induced senescence in PC12
   cells. (a) The cell viability of PC12 cells after treatment with PC A1
   (1, 3, 10, 30, and 100 µM) for 24 h. (b–h) PC12 cells were pre-treated
   with PC A1 (1, 3, 10, and 30 µM) for 24 h followed by 7.5 µM of
   etoposide (Etop) for 48 h. The cellular viability was determined (b),
   and ROS level (c,d), senescence-associated β-galactosidase (SA-β-gal)
   staining (e,f), and cell proliferation (g,h) are pictured and
   quantified. Proliferating cells were labeled with green fluorescence,
   and nuclei were stained with Hoechst 33342. Scale bar: 100 µm. Data
   represent mean ± SEM, n = 3 for each group. ** and *** indicate
   significant differences at p < 0.01 and p < 0.001 compared with the
   control (C) group, respectively; #, ##, and ### indicate significant
   differences at p < 0.05, p < 0.01, and p < 0.001 compared with the
   etoposide (Etop) group, respectively.

3.2.2. PC A1 Alleviated Cell Senescence Induced by Etoposide in NIH/3T3 Cells

   To further confirm the cell senescence alleviation effect of PC A1, we
   then induced cell senescence using Etop in the NIH/3T3 cell line. The
   NIH/3T3 mouse embryonic fibroblast cell line serves as a vital model
   for in vitro research, particularly in the fields of cellular
   senescence and cell cycle studies. Considering that NIH/3T3 cells
   exhibit greater sensitivity to Rapa and Etop, the concentration of Rapa
   was adjusted to 50 nM to serve as a positive control, whereas the dose
   of Etop was reduced to 0.3 µM. The effect of PC A1 on cellular
   viability was also assessed. As shown in [78]Figure 3a, PC A1 (at 1, 3,
   10, and 30 µM) demonstrated no toxicity to NIH/3T3 cells. Following
   this, NIH/3T3 cells were pre-treated with PC A1, subsequently inducing
   cell senescence with Etop for 2 days, and then we evaluated cellular
   viability, the expression of p21, ROS levels, and SA-β-gal activity.
   The results in [79]Figure 3b show that the pre-treatment of PC A1 (1,
   3, 10, and 30 µM) and Rapa (50 nM) significantly protected NIH/3T3
   cells from the loss of viability. The expression levels of p21, a
   biomarker of cellular senescence, were then assessed. As shown in
   [80]Figure 3c, PC A1 at 30 µM and Rapa reduced p21 levels elevated by
   Etop. The results of ROS assays ([81]Figure 3d,e) and SA-β-gal staining
   ([82]Figure 3f,g) were consistent with those obtained from the PC12
   cells, demonstrating that both PC A1 and Rapa significantly decreased
   ROS levels and the number of senescent cells, as indicated by
   SA-β-gal-positive cells. In conclusion, PC A1 alleviated cellular
   senescence induced by Etop in both PC12 and NIH/3T3 cells.

Figure 3.

   [83]Figure 3
   [84]Open in a new tab

   Procyanidin A1 (PC A1) alleviated Etop-induced senescence in NIH/3T3
   cells. (a) The cell viability of NIH/3T3 cells after treatment with PC
   A1 (1, 3, 10, and 30 µM) for 24 h. (b–g) NIH/3T3 cells were pre-treated
   with PC A1 (1, 3, 10, and 30 µM) for 24 h followed by 0.3 µM of
   etoposide (Etop) for 48 h. The cellular viability was determined (b),
   the expression levels of p21 were evaluated (c), and ROS levels (d,e)
   and senescence-associated β-galactosidase (SA-β-gal) staining (f,g)
   were pictured and quantified. Scale bar: 100 µm. The experimental
   samples and controls used for the comparative analysis in (c) were run
   on the same blot/gel. Data represent mean ±SEM, n = 3. *** indicates
   significant differences at p < 0.001 compared with the control (C)
   group; #, ##, and ### indicate significant differences at p < 0.05, p <
   0.01, and p < 0.001, compared with the etoposide (Etop) group,
   respectively.

3.2.3. The PI3K/Akt Signaling Pathway Played a Role in the Alleviation Effect
of PC A1 on Cell Senescence

   To further understand how PC A1 mitigates cell senescence, gene
   expression profiling of NIH/3T3 cells was performed through RNA
   sequencing. As shown in [85]Figure 4a–c, a total of 5724 differentially
   expressed genes (DEGs) (log[2]FoldChange (FC) ≥ 0.5 or ≤−0.5, Q value ≤
   0.05) were identified between the Etop-treated group and control group,
   with 3175 genes upregulated and 2549 genes downregulated in
   Etop-treated cells compared to the control group. Additionally, 181
   DEGs were found between the Etop + PC A1 group and the Etop-only group,
   with 113 genes upregulated and 68 downregulated in the PC A1
   pre-treatment group compared to the Etop group.

Figure 4.

   [86]Figure 4
   [87]Open in a new tab

   RNA sequencing analysis on senescent NIH/3T3 cells treated with
   procyanidin A1 (PC A1). NIH/3T3 cells were pre-treated with PC A1 (10
   µM) for 24 h followed by 0.3 µM of etoposide (Etop) for 48 h. RNA was
   extracted from cells lysis for RNA sequencing analysis. (a) Volcano
   plot of gene expression in the Etop group compared to the control
   group. (b) Volcano plot of gene expression in the PC A1 + Etop group
   compared to the Etop group. In the volcano plot, the two vertical
   dashed lines indicate log2(FoldChange) values of −0.5 and 0.5, while
   the horizontal dashed line represents a Q-value threshold of 0.05.
   Genes with log2(FoldChange) ≥ 0.5 or ≤−0.5 and a Q value ≤ 0.05 were
   identified as differentially expressed. (c) Venn diagram showing the
   number of differentially expressed genes (DEGs, with log[2]FoldChange
   (FC) ≥ 0.5 or ≤−0.5, Q value ≤ 0.05, calculated by raw count value) and
   overlapped genes between A and B (A. Etop group versus control group;
   B. PC A1 + Etop group versus Etop group). (d) KEGG pathway enrichment
   analysis of the overlapped 165 common differentially expressed genes.
   (e) GO biological processes associated with the 165 common
   differentially expressed genes. (f) Heatmaps of genes associated with
   negative regulation of the apoptosis process. (g) Heatmaps of genes
   associated with senescence associated secretory phenotype (SASP)
   factors in different groups. (h) Heatmaps of cell senescence-related
   genes according to the CellAge database. n = 3 for each group. n stands
   for the number of samples in a group.

   The 165 genes that were differentially expressed in both comparisons
   were analyzed using the KEGG pathways and GO enrichment analysis. As
   shown in [88]Figure 4d, the KEGG pathway analysis identified several
   significantly enriched pathways associated with the effects of PC A1
   treatment on Etop-induced senescence. Among these pathways, the
   PI3K/Akt signaling pathway, focal adhesion, ECM–receptor interaction,
   and pathways in cancer were prominently represented. Notably, the
   PI3K/Akt signaling pathway exhibited the highest number of enriched
   genes and a low Q value. Therefore, we propose that the PI3K/Akt
   signaling pathway plays a crucial role in the anti-aging effect of PC
   A1.

   The DEGs were annotated using the GO terms from the GO database to
   enhance our understanding of their molecular characteristics. As shown
   in [89]Figure 4e, the DEGs were annotated using the GO terms from the
   GO database to further discover their molecular characterization. The
   DEGs were categorized into three main areas: biological processes,
   molecular functions, and cellular components. Within the biological
   process category, the DEGs were implicated in the positive regulation
   of cell proliferation, the cell cycle, and the negative regulation of
   the apoptotic process. We further analyzed the transcriptomic
   expression profile. As shown in [90]Figure 4f, 17 genes were associated
   with the negative regulation of the apoptotic process. We observed that
   the expression of eight genes associated with apoptosis resistance
   increased with Etop treatment but was reduced upon treatment with PC
   A1. These genes included Ptgs2, Ier3, Tcim, Angpt14, Plaur, Cth, Plk3,
   and Pim. Additionally, we noted a significant upregulation of several
   SASP factors (including Cxcl2, Plau, Csf3, Ereg, Mmp3, Mmp10, Mmp13,
   Ngf, Plaur, Pgf, and Il11) during cellular senescence, which were
   substantially downregulated by PC A1. Four genes (Col16a1, Col1a2,
   Col4a5, and Col4a6), encoding members of the collagen family, along
   with Tnfrsf11b, were found to be downregulated in senescent cells but
   substantially upregulated by treatment with PC A1 ([91]Figure 4g).
   Furthermore, a search of gene expression databases, specifically
   CellAge [[92]28], revealed 16 genes associated with cell senescence
   that were regulated by Etop but alleviated by PC A1 treatment
   ([93]Figure 4h). Overall, these findings confirm that PC A1 ameliorates
   cell senescence induced by Etop by promoting apoptosis in senescent
   cells, regulating the gene transcription levels of SASP factors and
   restoring proliferative capacity. Moreover, the PI3K/Akt signaling
   pathway appears to play a role in the protective effect of PC A1
   against cellular senescence.

3.3. PC A1 Remedied Oxidative Stress in H[2]O[2]-Exposed PC12 Cells

   In organisms, the aging process is associated with progressive
   mitochondrial dysfunction, leading to an increased production of ROS.
   The pathological level of ROS contributes to further mitochondrial
   deterioration and widespread cellular damage, including harm to DNA,
   proteins, and lipids, which are significant factors in aging and
   age-related diseases [[94]29,[95]30]. Therefore, we investigated the
   ability of PC A1 to reduce ROS and protect PC12 cells from oxidative
   stress. H[2]O[2], the most commonly used endogenous source of cellular
   oxidative stress, was employed to induce this condition. Initially, the
   appropriate concentration of H[2]O[2] was pre-tested. After incubating
   cells with varying doses of H[2]O[2] for 2 h, cell viability was
   assessed using the MTT assay. As shown in [96]Figure 5a, H[2]O[2] at
   0.7 mM resulted in a 50% reduction in cell viability compared to the
   control and was chosen as an appropriate concentration for further
   experiments. Cells were subsequently treated with different doses of PC
   A1 for 24 h, followed by exposure to 0.7 mM H[2]O[2] for 2 h. The
   findings in [97]Figure 5b indicate that the reduction in cell viability
   caused by H[2]O[2] was alleviated by the pre-treatment with PC A1 (1,
   3, and 10 µM). Res was utilized as a positive control due to its
   well-established antioxidant properties [[98]31]. The level of MDA, a
   common indicator in oxidative stress studies, was also assessed. As
   shown in [99]Figure 5c, pre-treatment with Res (10 µM) and PC A1 (1, 3,
   10, and 30 µM) significantly lowered the elevated MDA levels induced by
   H[2]O[2]. Furthermore, ROS levels were evaluated using DCFH-DA, a probe
   that undergoes hydrolysis and oxidation to produce fluorescent DCF. As
   shown in [100]Figure 5d,e, H[2]O[2] produced a marked increase in ROS
   levels, which were significantly reduced by pre-treatment with Res and
   PC A1 (at 1, 3, 10, and 30 µM). SOD is a crucial endogenous antioxidant
   enzyme that scavenges superoxide anion free radicals and protects cells
   from oxidative damage. The activities of total SOD, SOD1, and SOD2 were
   measured. The results in [101]Figure 5f–h demonstrate the PC A1
   enhanced SOD2 activity but did not affect SOD1 activity. In conclusion,
   PC A1 protects PC12 cells from oxidative damage induced by H[2]O[2] by
   increasing SOD2 activity and reducing both ROS and MDA levels.

Figure 5.

   [102]Figure 5
   [103]Open in a new tab

   Antioxidative stress effect of procyanidin A1 (PC A1). (a) PC12 cell
   viability change caused by different doses of H[2]O[2]. (b–h) PC A1
   reduced H[2]O[2]-induced toxicity and attenuated oxidative stress. PC12
   cells were pre-treated with PC A1 (1, 3, 10, and 30 µM) for 24 h
   followed by 0.7 mM H[2]O[2] for 2 h. Then, the cellular viability (b),
   MDA level (c), ROS level (d), and its digital result (e) and total SOD
   (f), SOD1 (g), and SOD2 activities (h) of PC12 cells were assessed.
   Scale bar: 100 µm. Data represent mean ± SEM, n = 3 for each group. ns,
   *, **, and *** indicate significant differences at p > 0.05, p < 0.05,
   p < 0.01, and p < 0.001 compared with the control (C) group,
   respectively; ns, #, ##, and ### indicate significant differences at p
   > 0.05, p < 0.05, p < 0.01, and p < 0.001 compared with the H[2]O[2]
   group, respectively.

3.4. PC A1 Induced Autophagy in PC12 Cells

   Impaired microautophagy has been proposed as a hallmark of aging
   [[104]7]. Autophagy is the cellular process by which portions of the
   cell, including macromolecules and entire organelles, are degraded
   within lysosome. CYTO-ID^® green dye, a fluorescent probe, serves as an
   effective marker for vesicles produced during autophagy. To assess the
   capacity of PC A1 to induce autophagy, CQ was employed to block the
   conversion from autophagosomes to autolysosomes by elevating the
   lysosomal/vacuolar pH. Rapa was used as a positive control for its
   known capacity to induce autophagy by inhibiting TOR (in particular,
   TOR complex 1) activity [[105]15]. As shown in [106]Figure 6a,b, Rapa
   (500 nM) and PC A1 (3 and 10 µM) significantly increased the
   fluorescent intensity of autophagic vacuoles. Given that the conversion
   from autophagosomes to autolysosomes was blocked by CQ, we conclude
   that PC A1 effectively induces autophagy in PC12 cells.

Figure 6.

   [107]Figure 6
   [108]Open in a new tab

   Procyanidin A1 (PC A1) induced autophagy in PC12 cells. (a) Staining of
   the autophagic vesicles with a fluorescent dye in PC12 cells after
   treatment with rapamycin (Rapa) and PC A1. Scale bar: 40 µm. (b) The
   fluorescence intensity quantification results of (a). (c) The Western
   blot results of p-mTOR (Ser2448), mTOR, p-ULK1 (Ser757), ULK1,
   Beclin-1, p62, and LC3 compared with β-Actin in PC12 cells after
   treatment with 500 nM of Rapa and different doses of PC A1 for 18 h.
   (d) The Western blot results of p-PI3K (p85 (Tyr458)/p55 (Tyr199)),
   PI3K, p-Akt (Ser473), and Akt in PC12 cells after treatment with 500 nM
   of Rapa and different doses of PC A1 for 18 h. (e–i) The digital
   Western blot results of p-mTOR (Ser2448)/mTOR (e), p-ULK1 (Ser757)/ULK1
   (f), Beclin-1 (g), p62 (h), and LC3II/I (i). (j–l) The digital Western
   blot results of p-PI3K p85 (Tyr458)/PI3K (j), p-PI3K p55 (Tyr199)/PI3K
   (k), and p-Akt (Ser473)/Akt (l). The samples used for the Western blot
   analysis in (c,d) on different proteins are derived from the same
   experiment or parallel experiments and the blots are processed in
   parallel. *, **, and *** represent significant differences compared
   with the negative control (p < 0.05, p < 0.01, and p < 0.001,
   respectively). The experiments were repeated three times, and data from
   each experiment are displayed as mean ± SEM.

   Then, the expressions of autophagy-associated proteins mTOR, ULK1,
   Beclin-1, p62, and LC3 were evaluated through Western blot analysis to
   confirm the intracellular autophagy. PC12 cells were incubated with
   varying doses of PC A1 (0.3, 1, 3, 10, and 30 µM) for 18 h to assess
   dose dependence. As illustrated in [109]Figure 6c, the levels of
   phospho-mTOR (Ser2448) were reduced by both PC A1 and Rapa. Meanwhile,
   the levels of Beclin-1 and the ratio of LC3II/LC3I were upregulated,
   whereas the p62 levels were decreased in response to PC A1. The digital
   results of [110]Figure 6c are shown in [111]Figure 6e–i. Additionally,
   PC12 cells were incubated with 3 µM of PC A1 for varying time periods
   to assess time dependence, and the Western blot results for mTOR, ULK1,
   Beclin-1, p62, and LC3 are shown in [112]Supplementary Figure S4a.
   These results further confirm that PC A1 induces autophagy in a dose-
   and time-dependent manner.

   Then, the PI3K/Akt signaling pathway, serving as an upstream signaling
   pathway in the induction of autophagy, was also explored. As shown in
   [113]Figure 6d, PC A1 decreased the level of the phosphorylated form of
   PI3K p85 (Tyr458) and p55 (Tyr199), as well as Akt (Ser473), in a
   dose-dependent manner. The digital results are presented in [114]Figure
   6j–l. Time dependence was similarly assessed, and the Western blot
   results are shown in [115]Supplementary Figure S4b. These findings
   suggest that PC A1 enhances autophagy, in which the PI3K/Akt signaling
   pathway was involved.

3.5. Autophagy Inhibitor Abolished Antioxidative Stress and Cell Senescence
Alleviation Effects of PC A1

   Studies have indicated that many features of aging are interconnected.
   The impairment of autophagy or genetic defects in autophagy in young
   cells can lead to a loss of proteostasis, heightened mitochondrial
   dysfunction, and increased oxidative stress, ultimately resulting in
   cellular senescence [[116]12]. To test whether the antioxidative stress
   and cell senescence alleviation effects of PC A1 could be negated by
   impaired autophagy, SA-β-gal staining and ROS assays were conducted.
   The selective ULK1 kinase inhibitor SBI-0206965, used to inhibit
   autophagy in vitro, was administered [[117]32]. The results in
   [118]Figure 7a indicated that PC A1 at 10 µM distinctly reduced the
   SA-β-gal^+ cell percentage promoted by Etop. However, SBI-0206965
   negated this effect of PC A1. The evaluation of ROS yielded results
   consistent with the SA-β-gal assay. As shown in [119]Figure 7b, Etop at
   0.3 µM induced elevated levels of ROS in NIH/3T3 cells; pre-treatment
   with Rapa (50 nM) and PC A1 (10 µM) reduced ROS levels. Conversely,
   SBI-0206965 abrogated the ROS scavenging effect of PC A1. The results
   confirm that there is a crosstalk between autophagy and oxidative
   stress, as well as cellular senescence. The inhibition of autophagy can
   obstruct the ROS scavenging and cell senescence elimination effects of
   PC A1.

Figure 7.

   [120]Figure 7
   [121]Open in a new tab

   The autophagy inhibitor SBI-0206965 abolished antioxidative stress and
   the cell senescence alleviation effect of procyanidin A1 (PC A1). (a–d)
   NIH-3T3 cells were pre-treated with PC A1 at 10 µM with or without
   SBI-0206965 for 24 h followed by 0.3 µM of etoposide (Etop) for 48 h.
   Rapa (50 nM) was used as a positive control. The senescence-associated
   β-galactosidase (SA-β-gal) staining (a,b) and ROS level (c,d) are
   pictured and quantified. Scale bar: 100 µm. Data represent mean ± SEM,
   n = 3 for each group. ns, **, and *** indicate significant differences
   at p > 0.05, p < 0.01, and p < 0.001 compared with the designated
   group, respectively.

4. Discussion

   The aging population is steadily increasing, highlighting the
   importance of identifying phytochemical senotherapeutics compounds with
   significant potential. In China, peanuts are known as the “longevity
   nut” and PSE is recognized as a form of TCM. Modern pharmacological
   studies have demonstrated that PSE offers multiple benefits against
   various age-related vulnerabilities. However, the potential anti-aging
   effects of the bioactive molecules from PSE remain not fully
   understood. In this study, we utilized K6001 yeast RLS as a guiding
   model to isolate PC A1 from PSE. Our research findings indicate that PC
   A1 can extend the RLS, as well as the CLS of mammalian cells.
   Additionally, we observed that PC A1 provides a protective effect
   against cellular senescence and oxidative stress and induces autophagy.

   PC A1 is classified as an A-type procyanidin dimer, which is a part of
   the proanthocyanidin class of flavonoids. The constituent units of
   procyanidins are catechin and/or epicatechin. A-type procyanidins
   (linked via C[4]-C[6] or C[4]-C[8] and C[2]-O-C[7]) and B-type
   procyanidins (linked via C[4]-C[6] or C[4]-C[8]) are categorized based
   on the specific interflavan linkages among their constituent units.
   Previous studies have identified peanut skin as a rich source of
   procyanidins, including monomers, dimers, trimers, and tetramers, with
   a particular abundance of A-type procyanidins [[122]33]. Studies have
   shown that A-type procyanidins demonstrate prebiotic-like,
   antioxidative, anti-inflammation, anti-diabetic, antiviral,
   neuroprotective, and autophagy- and apoptosis-inducing effects
   [[123]34]. Notably, some studies have also indicated that procyanidins
   may have the potential to extend lifespans and alleviate age-related
   pathologies [[124]35,[125]36]. However, different procyanidins may
   exhibit inverse phenotypes. For example, procyanidin C1 was shown to
   increase ROS in senescent cells, but not for procyanidin B2 [[126]36].
   Thereby, the anti-aging mechanism of PC A1 was further investigated.

   A fundamental aging mechanism that has attracted increasing attention
   is cellular senescence. Senescent cells accumulate with age, and if
   persistent, can adversely affect tissue function due to the SASP that
   they develop. Despite the cytotoxic microenvironment they create,
   senescent cells evade death by regulating pro-survival and
   anti-apoptotic pathways, such as the PI3K/Akt signaling pathway.
   Senolytic agents, which selectively eliminate senescent cells, and
   senomorphic agents, which reduce the SASP, have shown promise as
   interventions for aging and treating age-related diseases [[127]9]. In
   this study, Etop, a DNA topoisomerase inhibitor, was utilized to induce
   cellular senescence. This treatment elevated the expression of p21,
   increased ROS production, resulted in a rise in the number of
   SA-β-gal-positive cells, and caused cell cycle arrest, while the
   treatment of PC A1 alleviated these changes ([128]Figure 2 and
   [129]Figure 3). To further investigate the mechanisms of PC A1, we
   conducted RNA-sequencing analysis. The results revealed that PC A1
   regulates the PI3K/Akt signaling pathway. Furthermore, PC A1
   downregulated the expression of anti-apoptosis genes that had been
   increased by Etop ([130]Figure 4f) and reduced many SASP factors
   produced by senescent cells ([131]Figure 4g). Thus, it is believed that
   PC A1 exhibited both senolytic and senomorphic functions, with the
   PI3K/Akt signaling pathway playing a significant role in the
   senotherapeutic effects of PC A1.

   Oxidative stress is a prevalent theme among the key features associated
   with the aging process. It can lead to various hallmarks of aging,
   including the accumulation of damaged proteins, telomere attrition,
   epigenetic alterations, cellular senescence, and mitochondrial
   dysfunction, contributing to aging and age-related diseases. While
   antioxidants may act as scavengers of ROS to maintain the biological
   redox homeostasis, they may play a protective role in aging and
   age-related diseases [[132]29]. In this study, H[2]O[2] was used to
   induce oxidative stress in PC12 cells, and the treatment of PC A1
   significantly improved cell viability under oxidative stress and
   reduced ROS levels. MDA, a harmful end product of lipid peroxidation,
   was also assessed as an indicator of oxidative damage. The results
   indicated that PC A1 decreased MDA production. As a primary defense
   against ROS-mediated damage, SODs were also evaluated. We observed an
   increase in total SOD and SOD2 activity following the treatment with PC
   A1; however, SOD1 activity remained unchanged ([133]Figure 5). Notably,
   elevated ROS levels were also observed in senescent cells, and
   pre-treatment with PC A1 significantly reduced ROS levels in both
   senescent PC12 cells and NIH/3T3 cells ([134]Figure 2c and [135]Figure
   3d).

   Disabled macroautophagy is one of the hallmarks of aging [[136]7]. The
   term ‘autophagic flux’ refers to the entire process of autophagic
   degradation, including the formation of autophagosome and the
   subsequent degradation of the cargo within lysosomes. To assess the
   ability to induce autophagy, we blocked the fusion of lysosome and
   autophagosome using CQ and evaluated the autophagy flux by labeling
   autophagic compartments with a CYTO-ID fluorescence dye [[137]37]. The
   results indicated that PC A1 increased autophagy ([138]Figure 6a,b).
   Consistently, the expression levels of autophagy-related proteins,
   including p62, Beclin-1, and LC3II/I, also confirmed the activation of
   autophagy ([139]Figure 6c). Furthermore, the upstream signaling pathway
   that regulates autophagy was tested through Western blot analysis,
   which revealed that PC A1 downregulated the levels of phosphorylated
   PI3K and Akt ([140]Figure 6d).

   Studies have demonstrated that impaired autophagy leads to oxidative
   stress and cellular senescence [[141]12]. In this study, we observed
   that Rapa, as an autophagy inducer, also reduced ROS levels in
   senescent cells ([142]Figure 2c and [143]Figure 3d). Conversely,
   SBI-0206965, a highly selective ULK1 kinase inhibitor and autophagy
   inhibitor, exacerbated the senescence phenotype. Cells treated with
   both SBI-0206965 and Etop were even bigger and flatter than those
   treated with Etop alone ([144]Figure 7a). Additionally, SBI-0206965
   elevated ROS levels in senescent cells. Notably, the ability of PC A1
   to scavenge ROS and decrease the number of SA-β-gal-positive cells was
   diminished by SBI-0206965 ([145]Figure 7). These findings support the
   notion that autophagy deficiency plays a critical role in oxidative
   stress and cellular senescence. Meanwhile, ULK1, a conserved kinase
   involved in autophagy initiation, emerges as a necessary component in
   the anti-aging mechanism of PC A1.

   The PI3K/Akt signaling pathway regulates signal transduction and
   biological processes such as cell growth, proliferation, survival,
   apoptosis, autophagy, and metabolism [[146]38,[147]39]. Previous
   research has shown that PI3K inhibition can alleviate aging in
   Drosophila and reduce cardiac aging and immune senescence in older
   adults [[148]40,[149]41,[150]42]. In this work, the levels of
   phosphorylated PI3K and Akt were reduced by PC A1 ([151]Figure 3d).
   Furthermore, RNA-seq analysis indicated that the PI3K/Akt signaling
   pathway is implicated in the senescence-alleviating effects of PC A1
   ([152]Figure 6d). Therefore, we propose that the PI3K/Akt signaling
   pathway plays a role in the anti-aging effects of PC A1. However,
   further data are needed to substantiate this in our future work.
   Additionally, this investigation into the anti-aging effects of PC A1
   is currently limited to the cellular level and requires validation at
   the animal level. Given the low concentration of PC A1 in peanut skin,
   alternative sources must be explored for its isolation.

5. Conclusions

   Overall, our findings highlight the potential of PC A1 as an anti-aging
   agent. Isolated from PSE, PC A1 demonstrates significant efficacy,
   including an extension of the RLS of yeast and the CLS of mammalian
   cells, as well as alleviating cell senescence, mitigating oxidative
   stress, and inducing autophagy. Furthermore, the PI3K/Akt signaling
   pathway plays a role in the anti-aging effects of PC A1. Given these
   promising results, further investigation into PC A1 as a potential
   geroprotective agent is warranted.

Acknowledgments