Rhapontigenin Alleviates Cellular Senescence and Physiological Aging by Upregulating Sirt1 and Promoting Autophagy

Rhapontigenin Alleviates Cellular Senescence and Physiological Aging by Upregulating Sirt1 and Promoting Autophagy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Rhapontigenin Alleviates Cellular Senescence and Physiological Aging by Upregulating Sirt1 and Promoting Autophagy Shuang Liu, Wendi Chen, Guoqiang Xu, Xin Liu, yuxuan shi, Guolong Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7162814/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Jan, 2026 Read the published version in Chinese Medicine → Version 1 posted 9 You are reading this latest preprint version Abstract Background Aging is characterized by cellular senescence, inflammation, and physiological decline. Currently available antiaging therapies often have limitations due to their toxicity and off-target effects. However, natural compounds derived from Chinese herbal medicine, such as Rhapontigenin (Rhap), have shown potential as safer antiaging agents. Purpose This study aimed to evaluate the potential of Rhap to be used as an antiaging agent by investigating its effects on cellular senescence, physical function, immune modulation, and autophagy in both in vitro and in vivo aging models. Methods NIH3T3 and IMR90 cells were subjected to oxidative or genotoxic stress to induce senescence and then treated with Rhap. Senescence markers, cell viability, and autophagy-related protein levels were assessed. Aged mice were treated with Rhap for 8 weeks, and physical performance, immune modulation, and organ health were evaluated. Mechanistic studies were conducted to determine the role of Sirt1 in mediating the effects of Rhap. Results Rhap treatment significantly reduced cellular senescence marker (p16 and p21) levels and senescence-associated β-galactosidase (SA-β-Gal) activity in stressed cells. In aged mice, Rhap improved physical performance, such as grip strength and motor coordination, and reduced depressive-like behaviors. Rhap also decreased liver senescence, lipid accumulation, and fibrosis and increased immune function by reducing proinflammatory cytokine production and enhancing T-cell homeostasis. Mechanistically, Rhap upregulated Sirt1 and promoted autophagy, both of which contributed to its antiaging effects. Sirt1 knockdown abolished the effects of Rhap on autophagy and senescence, indicating the importance of Sirt1 in mediating these beneficial effects. Conclusion Rhap is a promising candidate for mitigating age-related cellular and physiological decline by reducing cellular senescence, promoting autophagy, and modulating immune function. However, further studies are needed to explore the long-term effects and therapeutic potential of Rhap in human populations. Rhapontigenin Cellular senescence Antiaging Autophagy Sirt1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Aging is a complex biological process that is characterized by a progressive decline in physiological function and increased susceptibility to age-related diseases ( 1 , 2 ). Cellular senescence, which is a state of irreversible cell cycle arrest, is a key hallmark of aging and contributes to tissue dysfunction through the secretion of proinflammatory cytokines, chemokines, and matrix-degrading enzymes; collectively, these phenomena are known as the senescence-associated secretory phenotype (SASP) ( 3 , 4 ). The accumulation of senescent cells has been implicated in various age-related conditions, including cardiovascular diseases, neurodegeneration, and metabolic disorders ( 5 – 7 ). The antiaging therapeutic strategies that are currently available target cellular senescence, reduce systemic inflammation, and promote cellular repair mechanisms ( 8 , 9 ). Drugs, such as senolytics, which selectively eliminate senescent cells, have shown promise in extending healthspans in preclinical models ( 10 , 11 ). However, the off-target effects and potential toxicity of these compounds limit their clinical applicability ( 12 ). Natural compounds, particularly polyphenols, have emerged as attractive candidates for use in antiaging therapeutic strategies because of their ability to modulate multiple aging-related pathways and relatively low toxicity ( 13 ). Despite these advances, effective and safe antiaging agents that can simultaneously address multiple aspects of aging are needed. Rhapontigenin (Rhap; 3,3’,5-trihydroxy-4’-methoxy-stilbene, C 15 H 14 O 4 ) is a hydroxylated and methoxylated stilbene compound that is derived mainly from the rhizomes of the Chinese herbal medicinal Rheum rhaponticum and Rheum undulatum , both of which have been extensively utilized in traditional Asian medicine for the treatment of inflammation, digestive disorders, and systemic imbalances ( 14 , 15 ). Rhap has been reported to scavenge intracellular reactive oxygen species (ROS), inhibit DNA, RNA, and protein synthesis, and induce the apoptosis of certain pathogens ( 16 , 17 ). Moreover, Rhap has diverse pharmacological activities, including antioxidant, anti-inflammatory, anticancer, cardioprotective, lipid-lowering, and antimicrobial activities ( 18 – 20 ). Given these properties, Rhap has potential for use as an agent to treat oxidative stress, inflammation, and cellular damage, all of which are major contributors to aging. In this study, we aimed to evaluate the potential of Rhap to be used as an antiaging agent. We investigated the effects of Rhap on cellular senescence, physical performance, immune modulation, and autophagy in both in vitro and in vivo models of aging. Our findings suggest that Rhap may be a promising candidate for mitigating age-related cellular and physiological decline, highlighting its potential for use as a therapeutic agent for age-associated conditions. Materials and Methods Chemicals Rhapontigenin (Rhap, C 15 H 14 O 4 , purity ≥ 99.92%) was purchased from Selleck (S9163, Selleck, Shanghai, China).Resveratrol (Resv, C 14 H 12 O 3 , purity ≥ 99.99%) was purchased from Selleck (S1396, Selleck, Shanghai, China). Etoposide (Etop, C 29 H 32 O 13 , purity ≥ 99.96%) was purchased from Selleck (S1225, Selleck, Shanghai, China). Hydrogen peroxide (H 2 O 2 ) and sodium carboxymethyl cellulose (CMC-NA) were obtained from Sigma‒Aldrich (323381 and 419273, Sigma‒Aldrich, Merck, Germany). Dimethyl sulfoxide (DMSO) was purchased from Solarbio (D8371, Solarbio, Beijing, China). Cell Culture NIH3T3 cells were authenticated by STR profiling (March 2025) and tested negative for mycoplasma contamination, as certified by Wuhan Pricella Biotechnology Co., Ltd. (Wuhan, China). The cells were cultured in DMEM/F12 medium (CD0001, SparkJade, Wuhan, China) supplemented with 10% fetal bovine serum (FBS, Biological Industries) and 1% penicillin/streptomycin (P/S, 10378016, Gibco). IMR90 cells were also purchased from Wuhan Pricella Biotechnology Co., Ltd. and authenticated by STR. They were cultured in Minimum Essential Medium (MEM) supplemented with 10% FBS and 1% P/S. Both cell lines were maintained in a humidified incubator at 37°C with 5% CO₂. To induce senescence, NIH3T3 cells were treated with 400 µM H 2 O 2 for 3 hours, washed three times with phosphate-buffered saline (PBS), and then cultured in fresh medium for 24 or 48 hours. To evaluate the anti-senescence effects of Rhap, the cells were first treated with 400 µM H 2 O 2 for 3 hours, washed with PBS, and incubated overnight in fresh medium. Then, the medium was replaced with medium supplemented with different concentrations of Rhap. IMR90 cells were exposed to 50 µM Etop for 48 hours to induce senescence, followed by incubation in normal medium for 3 days. For anti-senescence experiments, IMR90 cells were treated with Etop, and then, the medium was replaced with medium supplemented with different concentrations of Rhap. Animals Male C57BL/6JNifdc mice, aged 19 months (old) or 5 weeks (young), were purchased from Weitong Lihua Experimental Animal Technical Co., Ltd. (Beijing, China). The mice were housed in a specific pathogen-free (SPF) environment at the Experimental Animal Center of Shandong University with a 12:12 light/dark cycle, a temperature of 23 ± 1°C, a humidity of 40–70%, and the mice were given free access to food and water. After one week of acclimatization, the old mice were randomly assigned to three groups (n = 6/group) and received either vehicle or Rhap (50, 100 mg/kg) via oral gavage every two days for two months. These doses were selected based on the previous literature ( 41 ). Behavioral tests and tissue collection were conducted two weeks after the final gavage. All the animal experiments complied with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication 86 − 23) and approved by the Animal Ethical and Welfare Committee (AEWC) of Shandong University Cheeloo College of Medicine (No. 21172). Behavioral Tests: Grip Strength, Rotarod, and Tail Suspension Tests Grip strength was assessed with a Grip Strength Meter (SA417, Sansbio, Jiangsu, China). The mice were lifted by the tail and allowed to grasp a bar with their front paws. Then, the mice were pulled horizontally until they released their grip on the bar. The force exerted was recorded, and three measurements per mouse were averaged. Motor coordination and balance were evaluated using the rotarod test (LE8205, Panlab Harvard Apparatus). The mice were trained for three days at rotation speeds of 4, 6, and 8 revolutions per minute (rpm) for 200 seconds in each session. On the test day, the speed of the rod was increased from 4 to 40 rpm over the course of five minutes. Each mouse was subjected to the test three times, and the average latency to fall and rotation speed were recorded. The tail suspension test (TST) was used to assess depressive-like behavior. The mice were suspended by the tail from a hook at the top of a wooden frame, 50 cm above the floor, for six minutes. Mouse behavior was recorded and analyzed with TopScan software. The immobility time was defined as the period during which the mice remained motionless and did not actively attempt to escape. Senescence-Associated β-Galactosidase (SA-β-Gal) Staining SA-β-Gal activity was measured with a Senescence β-Galactosidase Staining Kit (C0602, Beyotime, Shanghai, China) according to the manufacturer's protocol. Cells or tissue sections were fixed with β-Gal staining fixative for 15 minutes at room temperature (RT), washed with PBS, and then incubated with β-Gal staining solution for 16–18 hours at 37°C in a CO₂-free incubator. Stained cells or sections were observed under a light microscope, and the number of SA-β-Gal-positive cells or area of positive staining was quantified using ImageJ software. Western Blotting Western blotting was performed as previously described ( 21 ). Cultured cells were lysed with RIPA buffer (PC101, Epizyme, Shanghai, China) supplemented with protease and phosphatase inhibitors (78442, Thermo Fisher Scientific, MA, USA). The protein concentrations were measured with a BCA Protein Assay Kit (23227, Thermo). Equal amounts of proteins were subjected to SDS‒PAGE and transferred to PVDF membranes (ISEQ00010, Millipore, USA). After being blocked with Protein-Free Rapid Blocking Buffer (PS108, EpiZyme, Shanghai, China), the membranes were incubated overnight at 4°C with primary antibodies, followed by incubation with secondary antibodies for 1 hour at RT. The protein bands were visualized using Immobilon Western HRP Substrate (WBKLS0500, Millipore) and quantified using Image Lab Software (Bio-Rad) and ImageJ. The primary antibodies used are as follows: anti-p16 ink4a antibody (1:1000, ab211542, Abcam), anti-p21 cip1 antibody (1:1000, ab188224, Abcam), anti-Sirt1 antiboody (1:1000, ab110304, Abcam), anti-p-AMPK antibody (1:2000, ab133448, Abcam), anti-AMPK antibody (1:1000, ab207442, Abcam), anti-p62 antibody (1:1000, 5114S, CST), anti-LC3B antibody (1:1000, NB100-2220SS, Novus), anti-Beta Actin antibody (1:10000, 20536-1-AP, Proteintech). Hematoxylin and Eosin (H&E) Staining H&E staining was performed as previously described( 22 ). Liver tissues were dissected, fixed in 4% paraformaldehyde for 48 hours, processed in an embedding box, and embedded in paraffin. Sections (5 µm) were cut and stained with an H&E Stain Kit (G1120, Solarbio) according to the manufacturer's instructions. Stained sections were examined under a light microscope, and pathology was assessed by a blinded veterinary pathologist. Oil Red O Staining Lipid accumulation in liver tissues was assessed with Oil Red O staining. Liver tissues were dissected, washed with PBS, embedded in optimal cutting temperature (OCT) compound, and frozen at -80°C. Frozen sections (10 µm) were stained with an Oil Red O Stain Kit (ab150678, Abcam) according to the manufacturer's instructions. The relative lipid content in 10 random fields from three mice was quantified using ImageJ. Masson Staining To assess liver fibrosis, Masson's trichrome staining was performed with a Masson’s Trichrome Stain Kit (G1340, Solarbio) according to the manufacturer's instructions. Paraffin-embedded sections were dewaxed, rehydrated, and stained with Weigert’s iron hematoxylin, acid fuchsin, phosphomolybdic acid, and aniline blue. Fibrosis was quantified as the ratio of the blue collagen-positive area to the red normal area. Quantitative Polymerase Chain Reaction (qPCR) qPCR was performed as previously described ( 23 ), with slight modifications. Total RNA was extracted from frozen spleen and liver tissues using TRIzol Reagent (15596026, Invitrogen). cDNA synthesis was carried out using the SPARKscript II RT Plus Kit (AG0304, SparkJade). qPCR was performed using 2× SYBR Green qPCR Mix (AH0101, SparkJade) on a Roche LightCycler 480 instrument. Actin-β was used as the internal reference gene, and the comparative quantification method was 2^(-ΔΔCt). The sequences of the primers that were used are listed in Supplementary Table S1 . Immunofluorescence Analysis Cryosections of liver and spleen tissues were incubated in PBS for 10 minutes to remove the OCT compound. Antigens were retrieved by boiling and heating for 10 minutes in 1× Tris-EDTA antigen repair solution (pH 9.0, C1038, Solarbio). The sections were then permeabilized with 0.5% Triton X-100 for 20 minutes at RT. After being blocked with QuickBlock Blocking Buffer for Immunol Staining (P0260, Beyotime), the sections were incubated in a wet box with primary antibodies against p21 (1:200, ab188224, Abcam), p16 (1:200, ab211542, Abcam) or Sirt1 (1:200, ab110304, Abcam) at 4°C overnight. Then, the sections were washed three times in PBS, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG H&L (1:500, ab150077, Abcam), Alexa Fluor 594-conjugated goat anti-rabbit IgG H&L (1:500, ab150080, Abcam) or Alexa Fluor 594-conjugated goat anti-mouse IgG H&L (1:500, ab150116, Abcam) secondary antibodies for 2 h at RT in the wet box in the dark. After being washed with PBS, the sections were mounted with Mounting Medium, antifading (with DAPI) (S2110, Solarbio). Finally, images of randomly selected fields were captured at 20× magnification by fluorescence microscopy (Olympus BX53). Images were randomly captured of 3 fields per mouse from 3 mice. The fluorescence intensity was analyzed with ImageJ. Flow Cytometry Analysis The spleen and peripheral blood were harvested from mice, and single-cell suspensions were prepared according to previously described methods ( 24 ). Blood was collected from anesthetized mice by heart puncture and transferred to EDTA-coated tubes. Moreover, spleens were harvested from euthanized mice and placed in cold PBS. After the spleens were pierced with 1-ml syringes, spleen cells were removed with 1× PBS and filtered through a 70-µm cell strainer. Red blood cells in the peripheral blood and splenocytes samples were lysed using ACK lysis buffer (Cat. No. A1049201, Gibco) according to the manufacturer’s instructions. Then, the cells were washed with cold stain buffer (FBS) (BD Biosciences, Cat No. 554656) and incubated with an anti-mouse CD16/32 mAb (BD Biosciences, 553141) for 5 minutes on ice to block the Fc receptor. The cells were subsequently incubated with fluorescently labeled primary antibodies for 30 minutes on ice in the dark. The antibodies that were used to stain the blood or spleen single-cell suspensions were as follows: PE-conjugated anti-CD3 (Clone No. 145-2C11, 1:100, BD Biosciences, Cat No. 553063), PerCP-conjugated anti-CD4 (RM4-5, 1:100, BD Biosciences, 553052), BV421-conjugated anti-CD8 (53 − 6.7, 1:100, eBioscience, 48-0081-82), FITC-conjugated anti-NK1.1 (PK136, 1:100, BD Biosciences, 553164). After staining, the cells were washed, resuspended in staining buffer and detected with a BD LSRFortessa flow cytometer. Data analysis was performed with FlowJo (v10.7.2). Clinical Chemistries At the end of the experiments with aged mice, the mice were anesthetized with isoflurane, and blood was collected into EDTA anticoagulation tubes. Plasma was obtained by centrifugation (3000 rpm) at 4°C for 15 minutes, and then, it was aliquoted and stored at -80°C until analysis. The concentrations of plasma biochemical parameters, such as ALT, AST, CHE, UREA, CREA, and UA, were measured by a fully automated biochemical analyzer according to the manufacturer’s instructions. Plasma samples from 5 mice per group were analyzed. Luminex Plasma samples were collected and stored as described above. The levels of cytokines in the plasma samples were measured on a Luminex X-200 instrument with a Bio-Plex Pro Mouse Cytokine 23-plex Assay (M60009RDPD, Bio-Rad) according to the manufacturer’s instructions. Cell Counting Kit-8 Assay Cell viability was determined with a Cell Counting Kit-8 (CCK8, Dojindo, Japan). A total of 1×10 4 cells were seeded in each well of a 96-well plate and treated with vehicle (DMSO) or 5, 10, 15, or 20 µM Rhap for 24 h after the cells had attached to the wells. After washing with PBS, 100 µl of medium supplemented with 10 µl of CCK8 reagent was added. The absorbance at 450 nm was measured after 2 hours of incubation in a cell culture incubator. To calculate the half maximal inhibitory concentration ( IC 50 ), 5×10 3 cells were seeded in each well of a 96-well plate and treated with vehicle (DMSO) or 0.01, 0.1, 1, 10, 50, 100, 500, 1000 µM Rhap for 48 h after the cells had attached to the wells. The absorbance at 450 nm was measured after 1 hours of incubation with CCK8 reagent. IC 50 was determined by fitting the concentration-response data to a nonlinear regression model using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). EdU cell proliferation assay A total of 1×10 4 NIH3T3 cells were seeded in each well of 96-well plates and treated with (5, 10, 15, or 20 µM) or without Rhap for 24 h after the cells had attached to the wells. EdU-based cell proliferation was measured with a Cell-LightTM EdU Apollo In Vitro Kit (C10310-3, RiboBio) following the manufacturer's instructions. EdU signals were measured by flow cytometry, and the results were analyzed with FlowJo. Molecular Docking Molecular docking analysis was conducted using AutoDock Vina ( 25 ). The 3D protein structure of Sirt1 (PDB ID: 4if6, 4kxq, 4i5i and 4zzj) was downloaded from the RCSB PDB website ( https://www.rcsb.org/ ), and the compound structure of Rhap (PubChem CID: 5320954) was obtained from PubChem ( http://pubchem.ncbi.nlm.nih.gov/ ). The binding affinity was used to predict the optimal binding between the compound and the protein. 3D structural diagrams showing the interaction between the compound and protein were generated using PyMol software. The binding sites were identified on the PLIP website ( http://projects.biotec.tu-dresden.de/plip-web/plip/index ) ( 26 ) and visualized using PyMol software. Microscale Thermophoresis (MST) To determine the binding affinity between SIRT1 and Rhapontigenin, MST analysis was performed using a Monolith NT.115 instrument (Zoonbio Biotechnology Co., Ltd., Nanjing, China). Purified SIRT1 protein was fluorescently labeled with the RED-NHS 2nd Generation Kit (MO-L011, NanoTemper Technologies, Munich, Germany) according to the manufacturer's protocol, yielding a final concentration of 0.1 µM. For ligand dilution, Rhapontigenin was serially diluted twofold in PBST buffer containing 5% DMSO, spanning a concentration range from 0.25 mM to 7.63 nM (to avoid precipitation at high concentrations). For binding measurements, 10 µL of gradient-diluted ligand was mixed with 10 µL of labeled SIRT1 and incubated for 5 min at room temperature. Samples were loaded into standard capillaries (MO-K022) and analyzed under optimized MST conditions. Data processing was performed using MO. Control software to calculate the dissociation constant (K d ) with signal-to-noise ratios maintained at > 5 to ensure reliability. Transmission Electron Microscopy (TEM) Autophagosomes and autolysosomes in the cells were observed and photographed by TEM, which was performed by Wuhan Servicebio Technology Co., Ltd. In brief, NIH3T3 cells were collected after they were treated with or without 400 µM H 2 O 2 for 3 h and then cultured in medium supplemented with or without 20 µM Rhap for 48 h. Then, cell pellets were fixed for 2 h in electron microscope fixative supplemented with 2.5% glutaraldehyde (G1102, Servicebio). After being washed three times with 0.1 M phosphate buffer (pH 7.4), the cell pellets were encapsulated in 1% agarose and then fixed with 1% osmic acid in 0.1 M phosphate buffer (pH 7.4) for 2 h at room temperature in the dark. The samples were dehydrated with gradient alcohol and acetone. The samples were subsequently infiltrated and embedded with 812 embedding agent before being allowed to polymerize in an oven at 60°C for 48 h. The resin blocks were sectioned at a thickness of 60 nm with an ultrathin microtome, and the pieces were harvested with 150-mesh square Chinese membrane copper mesh. Then, ultrathin sections in the copper mesh were stained with 2% uranyl acetate saturated with alcohol solution and 2.6% lead citrate. The samples were subsequently observed and imaged by TEM (Hitachi HT7800). Finally, the numbers of autophagic vacuoles (AVs, including both autophagosomes and autolysosomes) were counted and analyzed. RNA-seq and Data Analysis Total RNA was isolated from snap-frozen liver tissues with a TRIzol RNA extraction kit (TIANGEN, Cat. No. DP424), yielding > 2 µg of total RNA per sample. The RNA quality was examined by 0.8% agarose gel electrophoresis and spectrophotometry, and high-quality RNA with a 260/280 ratio ranging from 1.8–2.2 was used for library construction and sequencing. The library was constructed following the Illumina manufacturer's instructions (Illumina, USA). Oligo-dT primers were utilized to transcribe mRNA into cDNA (APExBIO, Cat. No. K1159). cDNA was amplified for the synthesis of the second chain of cDNA. The cDNA products were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). After library construction, the library fragments underwent enrichment by PCR amplification and were selected on the basis of a fragment size of 350–550 bp. Next, the library was quality-assessed with an Agilent 2100 Bioanalyzer (Agilent, USA). Then, the library was sequenced on the Illumina NovaSeq 6000 platform (paired-end 150) to produce raw reads. Raw data have been deposited to National Center for Biotechnology Information (NCBI) under the BioProject number PRJNA1235652. Raw paired-end fastq reads were filtered by TrimGalore to discard the adapters and low-quality bases via calling the Cutadapt tool. The obtained clean reads were subsequently aligned to the mm10 mouse genome using HISAT2 ( 27 ), followed by reference genome-guided transcriptome assembly and gene expression quantification with StringTie ( 28 ). Differentially expressed genes (DEGs) of samples with replications were identified using DEseq2 ( 29 ) or edgeR ( 30 ) for samples with no replication, with thresholds of |fold-change|>1.5 and p < 0.05. The functional enrichment analysis of the annotated significant DEGs and the potential genes in the identified modules based on Gene Ontology (GO) and KEGG pathway categories was performed using the clusterProfiler ( 31 ). Terms with p values < 0.05 were considered significant. Cell Transfection with siRNAs The mouse negative control siRNA (si-NC) and siRNA targeting Sirt1 (si-Sirt1) were purchased from GeneChem (Shanghai, China). NIH3T3 cells were transfected with siRNAs using LipofectamineTM 2000 (11668019, Invitrogen, CA, USA) following the manufacturer’s instructions. We used two si-Sirt1 sequences to prevent off-target effects. The sequences of the si-Sirt1 that were used were as follows: si-Sirt1-1 sense 5’-GCCAUGUUUGAUAUUGAGUAUdTdT-3’ and antisense 5’-AUACUCAAUAUCAAACAUGGCdTdT-3’; si-Sirt1-2 sense 5’-AGUGAGACCAGUAGCACUAAUdTdT-3’ and antisense 5’-AUUAGUGCUACUGGUCUCACUdTdT-3’ Statistical Analysis All the experimental data are presented as the mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 8.0 software. Comparisons between two groups were performed using Student's t test, whereas comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. The significance level was set to p < 0.05. C. elegans survival analyses were conducted using the log rank (Mantel‒Cox) test. Results Rhap Reduces Oxidative Stress-Induced Cellular Senescence in Vitro To determine the effect of Rhap on cellular senescence, NIH3T3 and IMR90 cells were subjected to oxidative or genotoxic stress, followed by treatment with Rhap. Supplementary Figure S1 shows that NIH3T3 cells that were treated with increasing concentrations of H 2 O 2 exhibited a dose-dependent increase in senescence, as indicated by SA-β-Gal staining and increased expression of the senescence markers p16 and p21 (Fig. S1 A-E). In NIH3T3 cells, exposure to 400 µM H 2 O 2 led to increased senescence, whereas subsequent treatment with Rhap (5 or 10 µM) significantly reduced the number of senescent cells (Fig. 1 A, C). Similarly, in IMR90 cells, Etop-induced senescence was attenuated by Rhap treatment (Fig. 1 B, D). Western blotting analysis further demonstrated that Rhap treatment reduced the expression of the senescence-associated markers p16 and p21 in NIH3T3 cells (Fig. 1 E-G). These findings suggest that Rhap effectively mitigates oxidative and genotoxic stress-induced cellular senescence. Cell viability was assessed to determine whether Rhap exerted any cytotoxic effects at the concentrations used. The CCK-8 assay revealed no significant reduction in cell viability after treatment with a range of Rhap concentrations (Fig. 1 H). Moreover, EdU staining and flow cytometry revealed that Rhap did not impair cell proliferation, indicating that its anti-senescence effects did not occur due to reduced cell viability or proliferation (Fig. 1 I, J). The 50% inhibitory concentration ( IC 50 ) of Rhap inhibiting NIH3T3 cell viability or proliferation after 48 hours was 116 µM ( 95 % CI , 107.9-124.8 µM). Furthermore, Rhap did not affect cell viability at concentrations up to 50 µM, which aligns with our previous observations (Fig. 1 K, H). In addition, the impact of Rhap on organismal longevity was evaluated with N2 nematodes. Survival analysis revealed a significant extension in lifespan after Rhap treatment. Compared to the vehicle group, the median lifespan of nematodes treated with 0.2 mM Rhap was extended by 21.7% and the maximum lifespan by 15%; the median lifespan of nematodes treated with 1 mM Rhap was extended by 30.4% and the maximum lifespan by 20%. (Fig. S2 and Table S2). The results suggests that the potential benefits of Rhap beyond those observed in cellular models. Rhap Increases Physical Performance and Maintains Organ Function in Aged Mice To evaluate the effects of Rhap on aging, 19-month-old mice were treated with Rhap or vehicle for 8 weeks (Fig. 2 A). The mice treated with Rhap had smoother fur than the control group (Fig. 2 B). Rhap-treated mice exhibited significantly improved physical performance, as indicated by increased grip strength normalized to body weight (Fig. 2 C, D) and enhanced motor coordination in the rotarod test (Fig. 2 E). Furthermore, Rhap-treated mice demonstrated shorter immobility times in the TST, suggesting improved mood or reduced depressive-like behavior (Fig. 2 F). Rhap treatment did not significantly affect body weight or organ weights (liver, heart, spleen, lung, and kidney weights), indicating that it did not have adverse effects on overall health (Fig. 2 G-L). Plasma biochemical analysis revealed a significant reduction in AST levels in Rhap-treated mice, suggesting improved liver function, whereas other markers (ALT, CHE, UREA and CREA) remained unchanged (Fig. 2 M-R). Histological analysis by HE staining revealed no adverse effects in liver and kidney pathology due to Rhap treatment (Fig. 3 A, B). These results indicate that Rhap increases physical function and maintains organ health in aged mice and lacks liver or kidney toxicity at the concentrations used, highlighting its potential to mitigate age-related declines. Rhap Attenuates Liver Senescence, Lipid Accumulation, and Fibrosis in Aged Mice To assess the impact of Rhap on aging, SA-β-Gal staining was performed on the tissues of the heart, liver, spleen, lung and kidney. There were no significant differences observed in the tissues of the heart, lungs and kidneys (data not shown). In particular, SA-β-Gal staining revealed a significant increase in the number of senescent cells in the livers of vehicle-treated aged mice, and this effect was markedly suppressed by Rhap treatment (Fig. 3 C, D). qPCR analysis revealed that Rhap reduced the expression of senescence markers (p21 and p53) and SASP factors (Fig. 3 E). Immunofluorescence staining confirmed that p21 expression was significantly decreased in the livers of Rhap-treated aged mice (Fig. 3 F, G). Oil red O staining revealed reduced lipid accumulation in the livers of Rhap-treated aged mice, suggesting improved lipid metabolism (Fig. 3 H, I). Additionally, Masson staining revealed a significant reduction in liver fibrosis in Rhap-treated aged mice compared with control mice (Fig. 3 J, K). These findings indicate that Rhap effectively attenuates liver senescence, lipid accumulation, and fibrosis in aged mice, highlighting its potential for use as an agent for treating age-related liver dysfunction. Rhap Attenuates Spleen Senescence and Modulates Immune Function in Aged Mice Among the tissues assessed, spleen aging also displayed marked changes following Rhap treatment. Specifically, SA-β-Gal staining revealed a significant decrease in the numbers of senescent cells in the spleens of Rhap-treated aged mice compared with those in the spleens of vehicle-treated control mice (Fig. 4 A, B). qPCR analysis revealed that Rhap treatment significantly decreased the expression of senescence markers (p16 and p21) and SASP factors (Fig. 4 C). Immunofluorescence staining for p16 and p21 further confirmed that expression of these markers was reduced in the spleens of Rhap-treated aged mice (Fig. 4 D-G). Moreover, Luminex analysis of plasma samples revealed that compared with vehicle, Rhap significantly reduced the levels of proinflammatory cytokines, including IFNγ, TNFα, IL-1α, IL-17a, and MCP-1, in aged mice, indicating that Rhap exerts an anti-inflammatory effect (Fig. 5 A). To evaluate the effects of Rhap on the immune function, the immune cell subsets were detected using flow cytometry. This analysis revealed that compared with vehicle treatment, Rhap treatment increased the proportion of CD3 + T cells in aged mice (Fig. 5 B, C). Furthermore, Rhap treatment significantly increased the proportion of CD4 + T cells, whereas the proportion of CD8 + T cells was not significantly different; these effects resulted in an elevated CD4+/CD8 + T-cell ratio, which suggests a better T-cell balance (Fig. 5 D-G). It has been shown that mature NK cells are reduced in the peripheral tissues of aged mice compared with young mice ( 32 ). In our study, we found that the proportion of NK cells (NK1.1 + CD3-) in peripheral blood and spleen of aged mice was lower than that of young mice, but there was a trend of increase in Rhap-treated aged mice compared with vehicle-treated group, but the difference was not statistically significant, indicating that Rhap may have an effect on NK cells (Fig. S3A-D ). These findings indicate that Rhap effectively reduces splenic senescence and modulates immune function, promoting an anti-inflammatory immune profile in aged mice. Rhap Upregulates Sirt1 Expression and Promotes Autophagy in the liver tissue of aged mice Rhap (3,3’,5-trihydroxy-4’-methoxy-stilbene, C 15 H 14 O 4 ) is a naturally occurring stilbenoid compound that is characterized by its unique chemical structure featuring a trans-stilbene backbone with hydroxyl groups at the 3, 3’, 5 positions and a methoxy group at the 4’ position (Fig. 6 A). Methoxylated stilbenes have been suggested to be more suitable options for oral administration than hydroxylated stilbenes, such as resveratrol, because methoxylated stilbenes have increased bioavailability and similar biological activities compared with hydroxylated stilbenes ( 33 ). To elucidate the molecular mechanisms underlying the antiaging effects of Rhap, we performed a comprehensive analysis of its targets and their roles in aging and senescence-related pathways. A Venn diagram analysis identified 28 genes as shared targets of Rhap, senescence, and aging (Fig. 6 B). KEGG pathway enrichment of shared targets indicated involvement in the cellular senescence pathway (Fig. 6 C). Protein-protein interaction (PPI) network constructed via STRING database identified core hub proteins including TP53, AKT1, MAPK1, SIRT1 that functionally converge on cellular senescence regulation (Fig. 6 D). Network pharmacology analysis revealed Rhapontigenin (Rhap) as a multi-target agent against senescence and aging. Transcriptomic analysis of liver tissues revealed significant changes in gene expression following Rhap treatment. Volcano plot analysis identified 2072 differentially expressed genes (DEGs) between vehicle- and Rhap-treated aged mice, with 994 upregulated and 1078 downregulated genes (Fig. 7 A). Hierarchical clustering of select DEGs demonstrated clear separation between treatment groups, with distinct expression patterns across biological replicates. Quantitative analysis revealed that Rhap treatment significantly upregulated Sirt1 expression while suppressing transcriptional levels of cell cycle inhibitors Cdkn1a compared with vehicle controls (Fig. 7 B). KEGG and GO enrichment analyses of DEGs revealed that Rhap treatment was associated with pathways related to Longevity, Cellular senescence, AMPK signaling, FoxO signaling, NF-κB signaling and autophagy (Fig. 7 C, D). Venn analysis identified two critical genes Sirt1 and Sod2 at the shared genes of network pharmacology predictions (Fig. 6 B) and DEGs of transcriptomic profiling (Fig. 7 E). Considering that Sirt1 is implicated in the regulation of multiple signaling pathways related to aging, such as cellular senescence, longevity regulation, NF-κB, AMPK, FoxOs and Autophagy ( 34 ), and exhibits a high degree of connectivity within the PPI network (Fig. 6 D), coupled with its upregulated expression in tissues of Rhap-treated aged mice (Fig. 7 A, B), we propose the hypothesis that Sirt1 plays a crucial role in mediating the anti-aging effects of Rhap. Molecular docking analysis confirmed that Rhap may interact with the active site of Sirt1 (Fig. 7 F, G and Table S3). MST experiments determining the dissociation constant (K d ) between Rhap and SIRT1 yielded a value of 2.0572E-06 M (Fig. 7 H), indicating a strong binding affinity. These findings suggest that Rhap plays a role in modulating Sirt1 activity. Immunofluorescence staining revealed that Sirt1 expression was significantly greater in the livers of Rhap-treated aged mice than in those of vehicle-treated control mice, suggesting a role of Rhap in promoting Sirt1 activity (Fig. 7 I, J). Furthermore, TEM analysis revealed an increase in the number of AVs, including autophagosomes and autolysosomes, in Rhap-treated aged mice, indicating increased autophagy (Fig. 7 K, L). These findings suggest that Rhap exerts its antiaging effects, at least in part, by upregulating Sirt1 and promoting autophagy, thereby contributing to improved cellular health in aged mice. Rhap Reduces Cellular Senescence by Upregulating Sirt1 and Promoting Autophagy To investigate the molecular mechanisms by which Rhap reduces cellular senescence, we examined the role of Sirt1 and autophagy in NIH3T3 cells that were treated with H 2 O 2 and Rhap. Western blotting analysis revealed that Rhap treatment significantly increased the expression of Sirt1 and the ratio of p-AMPK to AMPK in a dose-dependent manner, indicating AMPK signaling activation (Fig. 8 A-C). Additionally, Rhap treatment increased the LC3BII/LC3BI ratio and decreased the p62 levels, suggesting an increase in the autophagic flux (Fig. 8 D-F). TEM confirmed that the number of AVs, including autophagosomes and autolysosomes, was increased in Rhap-treated cells compared with control cells (Fig. 8 G, H). These findings indicate that Rhap promotes autophagy in response to oxidative stress. To determine whether Sirt1 is required for the anti-senescence effects of Rhap, NIH3T3 cells were transfected with siRNA targeting Sirt1 (si-Sirt1) or a negative control (si-NC). Western blotting analysis revealed that Sirt1 knockdown abolished the Rhap-induced increase in the LC3BII/LC3BI ratio and the reduction in p21 and p16 expression, indicating that Sirt1 is necessary for the Rhap-mediated attenuation of autophagy and senescence (Fig. 8 I-K). These findings suggest that Rhap alleviates cellular senescence by activating Sirt1 and promoting autophagy, highlighting its potential for use as an agent for treating age-related cellular dysfunction. Discussion In this study, we demonstrated that Rhap, which is a stilbene aglycone metabolite that is derived from rhaponticin ( 15 ), effectively attenuates cellular senescence, increases physical function, reduces inflammation, and promotes autophagy in both in vitro and in vivo models of aging. These findings provide new insights into the potential of Rhap to be used as an antiaging therapeutic agent and contribute to the growing body of literature suggesting a role for natural compounds in mitigating age-associated pathologies. Rhap is extracted from medicinal plants such as rhubarb rhizomes ( 35 ), and exhibit various biological activities, including anti-inflammatory, antioxidant, anticancer, antihyperlipidemic, antiallergic, and antibacterial activities ( 20 , 33 , 36 – 39 ). Additionally, Rhap has been reported to scavenge ROS ( 17 ) and inhibit DNA, RNA, and protein synthesis in C. albicans ( 16 ). Emerging evidence suggests that Rhap exhibits therapeutic potential in mitigating pathological features of Alzheimer's disease (AD), positioning it as a promising therapeutic candidate for AD ( 40 , 41 ). Our study extends these findings by highlighting the ability of Rhap to counteract oxidative and genotoxic stress-induced cellular senescence, which is a hallmark of aging ( 42 ). The anti-senescence effects of Rhap that were observed in our study are consistent with the findings of previous research on polyphenolic compounds, such as resveratrol and quercetin, which reduce senescence marker levels and improve cellular health by activating Sirt1 and promoting autophagy ( 43 , 44 ). Our data show that Rhap significantly reduces the activity of SA-β-Gal and the expression of the key senescence markers p16 and p21 in NIH3T3 and IMR90 cells. Interestingly, the effects of Rhap were observed even at lower concentrations compared to other polyphenols and displayed markedly lower toxicity at higher concentrations, suggesting that Rhap may be a more potent antiaging compound with fewer off-target effects. In vivo, Rhap-treated aged mice exhibited significant improvements in physical performance, including enhanced grip strength, improved motor coordination, and reduced depressive-like behaviors. These findings are consistent with those of studies showing that polyphenols can enhance motor function and mitigate mood disturbances during aging ( 45 , 46 ). Furthermore, Rhap increased liver health by attenuating hepatic senescence, lipid accumulation, and fibrosis, highlighting its potential for use as an agent for treating age-related liver dysfunction. Our study also highlights the role of Rhap in modulating immune function. Rhap treatment significantly reduced the levels of proinflammatory cytokines, including IFNγ, TNFα, IL-1α, IL17a and MCP1, which are key components of the SASP. The decrease in the levels of these SASP factors suggests that Rhap can alleviate systemic inflammation and promote a more balanced immune profile. Mechanistically, our data indicate that the antiaging effects of Rhap are mediated, at least in part, by the upregulation of Sirt1 and the promotion of autophagy. Sirt1 is a well-established regulator of aging, and it is involved in processes such as DNA repair, inflammation, and mitochondrial function ( 47 , 48 ). Rhap-treated aged mice exhibited increased Sirt1 expression and autophagy, which support the hypothesis that Rhap promotes cellular homeostasis via autophagy. This finding was further confirmed in vitro, where Sirt1 knockdown abolished the beneficial effects of Rhap on autophagy and senescence. The dual targeting of the Sirt1 and AMPK signaling pathways by Rhap suggests that Rhap has a broader and more versatile mechanism of action than other inducers of autophagy, suggesting that Rhap is a potent antiaging compound. Despite these promising results, our study has several limitations. We relied primarily on the NIH3T3 and IMR90 cell lines, which may not fully capture the complexity of cellular senescence in vivo. Future studies should include primary cells from aged organisms to confirm the anti-senescence effects of Rhap. Additionally, while our study demonstrated the efficacy of Rhap in aged mice, the translation of these findings to humans remains uncertain. Clinical studies are needed to evaluate the safety, bioavailability, and therapeutic potential of Rhap in human populations. Moreover, a detailed pharmacokinetic analysis of Rhap would provide crucial insights into its metabolism and distribution, which are essential for understanding its therapeutic applicability. Conclusion In conclusion, Rhap is a promising candidate for mitigating age-related cellular and physiological decline. By activating Sirt1, promoting autophagy, and attenuating inflammation, Rhap exerts multifaceted antiaging effects with significant therapeutic implications. Future studies should focus on understanding the long-term effects of Rhap treatment, its pharmacokinetics, and its potential for use as an agent for treating age-related diseases in humans. Abbrevations ALT, alanine aminotransferase; AST, aspartate transaminase; CHE, cholinesterase; CREA, creatinine; IFNγ, Interferon gamma; IL-10, Interleukin-10; IL-1α, Interleukin-1 alpha; IL-1β, Interleukin-1 beta; IL-6, Interleukin-6; MCP-1, Monocyte Chemoattractant Protein-1; Rhap, Rhapontigenin; SA-β-Gal, senescence-associated β-galactosidase; SASP, senescence-associated secretory phenotype; Sirt1, Sirtuin 1; TGF-β, Transforming Growth Factor beta; TNFα, Tumor Necrosis Factor alpha; UA, uric acid Declarations Acknowledgements The authors thank Xiaowen Liu and Wenbin Chen in Central Laboratory, Shandong Provincial Hospital Affiliated to Shandong University for the donation of anaesthetic drugs. We acknowledge the State Key Laboratory of Reproductive Medicine and Offspring Health for supporting the experiment. Consent for publication Not applicable. Conflict of Interest The authors declare that they have no competing interests. Ethics statement All the animal experiments complied with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication 86-23) and approved by the Animal Ethical and Welfare Committee (AEWC) of Shandong University Cheeloo College of Medicine (No. 21172). Data Availability Statement The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Authors' contributions Shuang Liu: Writing – original draft, Visualization, Validation, Investigation. Wendi Chen: Validation, Methodology, Investigation. Guoqiang Xu: Validation. Xin Liu: Investigation, Formal analysis. Yuxuan Shi: Formal analysis, Data curation. Guolong Wang: Software, Data curation. Yongzhi Cao: Visualization, Resources. Yunna Ning: Validation, Supervision. Ming Li: Writing – original draft, Supervision, Formal analysis. Yueran Zhao: Writing – review and editing, Supervision, Resources, Funding acquisition, Conceptualization. All authors read and approved the final manuscript. Funding This research was funded by The National Key Research and Development Program of China (No. 2022YFC2702905) and Taishan Scholars Program of Shandong Province (tsqnz20240852). References López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G. Hallmarks of aging: An expanding universe. Cell. 2023;186(2):243-78. Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell. 2013;153(6):1194-217. Gorgoulis V, Adams PD, Alimonti A, Bennett DC, Bischof O, Bishop C, et al. Cellular Senescence: Defining a Path Forward. Cell. 2019;179(4):813-27. 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The role of sirtuin 1 in ageing and neurodegenerative disease: A molecular perspective. Ageing Res Rev. 2024;102:102545. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Published Journal Publication published 09 Jan, 2026 Read the published version in Chinese Medicine → Version 1 posted Editorial decision: Revision requested 18 Oct, 2025 Reviews received at journal 15 Oct, 2025 Reviewers agreed at journal 09 Oct, 2025 Reviewers agreed at journal 29 Jul, 2025 Reviewers agreed at journal 28 Jul, 2025 Reviewers invited by journal 28 Jul, 2025 Editor assigned by journal 23 Jul, 2025 Submission checks completed at journal 23 Jul, 2025 First submitted to journal 19 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7162814","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":492613939,"identity":"f07bb829-280b-4911-a004-1badda105202","order_by":0,"name":"Shuang Liu","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Liu","suffix":""},{"id":492613942,"identity":"e707ae6e-a38d-4e74-bf57-57ff79a90f37","order_by":1,"name":"Wendi Chen","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Wendi","middleName":"","lastName":"Chen","suffix":""},{"id":492613943,"identity":"a1317756-5cb1-4145-956c-9ea65a99829b","order_by":2,"name":"Guoqiang Xu","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Guoqiang","middleName":"","lastName":"Xu","suffix":""},{"id":492613944,"identity":"828452ee-58c3-49ef-9c20-d8ffb06437b4","order_by":3,"name":"Xin Liu","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Liu","suffix":""},{"id":492613945,"identity":"022ae68a-8c33-4dfe-97b2-c209da88d974","order_by":4,"name":"yuxuan shi","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"yuxuan","middleName":"","lastName":"shi","suffix":""},{"id":492613946,"identity":"6122e6eb-f175-4731-81ac-b8087efb5824","order_by":5,"name":"Guolong Wang","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Guolong","middleName":"","lastName":"Wang","suffix":""},{"id":492613947,"identity":"9886d84d-8674-471e-87a6-c9bb84ac25d7","order_by":6,"name":"Yunna Ning","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Yunna","middleName":"","lastName":"Ning","suffix":""},{"id":492613948,"identity":"413790bb-d632-43f0-9579-c4fecfb7ca48","order_by":7,"name":"Yongzhi Cao","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Yongzhi","middleName":"","lastName":"Cao","suffix":""},{"id":492613949,"identity":"94680475-3f2d-4c0b-bd9f-840617c10903","order_by":8,"name":"Ming Li","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Li","suffix":""},{"id":492613950,"identity":"66f9230c-e4d6-422b-b084-0020fc0b62c9","order_by":9,"name":"Yueran Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYHACNiC2YWA4AKR4SNCSRrqWwyRoMbh9/NmDHxXn7fluJDA+eNvGIG9OUMu5hHTDnjO3E2feSGA2nNvGYLizgZCWMwzHJHjbbicY3Ehgk+ZtY0gwOEBQC2Ob5N+2c/ZALey/idTCDDL8AOMGoC3MRGmRPMPGJi1zJjlx5pmHzZJzzkkYbiCkhe8M+zPJNxV29nzHkw9+eFNmI0/QFgWEAsYGICFBQD0QyDcQVjMKRsEoGAUjHQAAWDtA/0txVUQAAAAASUVORK5CYII=","orcid":"","institution":"Shandong University","correspondingAuthor":true,"prefix":"","firstName":"Yueran","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2025-07-19 07:53:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7162814/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7162814/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13020-025-01319-3","type":"published","date":"2026-01-09T15:58:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88002507,"identity":"5f002eb4-6aba-4279-80fc-e700ad0b62d3","added_by":"auto","created_at":"2025-07-31 10:27:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1171292,"visible":true,"origin":"","legend":"\u003cp\u003eRhapontigenin (Rhap) attenuates cellular senescence in vitro. (A, C) NIH3T3 cells treated with 400 μM H2O2 for 3 hours followed by Rhap (5 or 10 μM) for 48 hours exhibited reduced senescence, as indicated by SA-β-Gal staining. Resveratrol (Resv, 20 μM) was used as a positive control of efficacy. Scale bar: 100 μm. (B, D) IMR90 cells treated with 50 μM etoposide for 48 hours followed by Rhap (5 or 10 μM) for 72 hours exhibited decreased senescence. Resv (20 μM) was used as a positive control of efficacy. Scale bar: 100 μm. (E-G) Western blotting analysis of p16 and p21 expression in NIH3T3 cells treated with H2O2 and Rhap. β-actin served as the loading control. (H) Viability of NIH3T3 cells treated with various Rhap concentrations was assessed with the CCK-8 assay. (I, J) EdU staining and flow cytometry revealed that Rhap had no significant effect on NIH3T3 cell proliferation. (K) IC50 of Rhap in NIH3T3 cells after 48 h treatment (CCK-8 assay, nonlinear regression fit). The data are presented as the means ± SDs. Statistical analysis was performed with one-way ANOVA with Tukey's multiple comparisons. ## p \u0026lt; 0.01 vs. the Control group, #### p \u0026lt; 0.0001 vs. the Control group, * p \u0026lt; 0.05 vs. the H2O2 group, ** p \u0026lt; 0.01 vs. the H2O2 group, **** p \u0026lt; 0.0001 vs. the H2O2 group, ns = not significant.\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7162814/v1/7069d933c1b96d2285037f91.jpg"},{"id":88002511,"identity":"87d370b1-c585-46b3-8d6e-5d28fe72e82e","added_by":"auto","created_at":"2025-07-31 10:27:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1430449,"visible":true,"origin":"","legend":"\u003cp\u003eRhapontigenin (Rhap) increases physical performance and organ health in aged mice. (A) Schematic representation of the experimental design. Aged mice (19 months) were treated with 50, 100 mg/kg Rhap or vehicle every two days for 8 weeks, followed by tissue collection at 10 weeks. (B) Representative images of aged mice treated with vehicle or Rhap. (C) Grip strength analysis. (D) Grip strength normalized to body weight. (E) Rotarod performance. (F) Tail suspension test results. (G-L) Body and organ weights, including liver, heart, spleen, lung, and kidney weights. (M-R) Plasma biochemical parameters, including ALT, AST, CHE, UREA, CREA, and UA. The data are presented as the means ± SDs. Statistical analysis was performed with one-way ANOVA with Tukey's multiple comparisons or Student's t test. ### p \u0026lt; 0.001 vs. the Young group, * p \u0026lt; 0.05 vs. the Vehicle group, ** p \u0026lt; 0.01 vs. the Vehicle group, *** p \u0026lt; 0.001 vs. the Vehicle group, ns = not significant. R50, Rhapontigenin 50 mg/kg; R100, Rhapontigenin 100 mg/kg; ALT, alanine aminotransferase; AST, aspartate transaminase; CHE, cholinesterase; CREA, creatinine; UA, uric acid.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7162814/v1/0d6771a096db74d6a418e578.jpg"},{"id":88002508,"identity":"f232bc0e-51a9-4096-b337-305333c875a2","added_by":"auto","created_at":"2025-07-31 10:27:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2892294,"visible":true,"origin":"","legend":"\u003cp\u003eAdministration of Rhap attenuates liver senescence in aged mice. (A) HE staining was used to assess pathological features of liver tissues (scale bar, 100 μm). (B) Pathological characteristics of kidney tissues as assessed by HE staining (scale bar, 100 μm). (C) SA-β-Gal staining in liver sections from young mice and old mice treated with vehicle or Rhap (20× magnification). (D) Percentage of the total area that was positive for SA-β-Gal staining in (C). (E) The transcript levels of the senescence-associated markers p16, p21, and p53 and the SASP factors IL-6, IL1-β, MCP-1, TNFα, IL-10, IFNγ, IL-1α, and TGF-β in the livers of the mice were measured by qPCR. (F) Representative immunofluorescence images of immunofluorescence staining for p21 in liver tissues (scale bar = 50 μm). (G) Quantification of the fluorescence intensities of p21. (H) Representative images of Oil Red O-stained liver sections (10× original magnification). (I) Quantification of the Oil Red O-positive area. (J) Representative images of Masson-stained liver tissues (10× original magnification). (K) Quantification of the fibrotic area as a percentage of the entire field of view. n = 10 fields from 3 mice. The data are presented as the means ± SDs, n = 3–5 mice/group. Statistical analysis was performed with Student’s t test or one-way ANOVA. #### p \u0026lt; 0.0001 vs. the Young group, * p \u0026lt; 0.05 vs. the Vehicle group, ** p \u0026lt; 0.01 vs. the Vehicle group, **** p \u0026lt; 0.0001 vs. the Vehicle group, ns, no significance. SASP, senescence-associated secretory phenotype.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7162814/v1/141c19e3af811352af001f2d.jpg"},{"id":88004179,"identity":"77662b8d-696f-48bb-8e4d-397c43fae792","added_by":"auto","created_at":"2025-07-31 10:35:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1126212,"visible":true,"origin":"","legend":"\u003cp\u003eAdministration of Rhap reduces the levels of senescence-related markers in the spleens of aged mice. (A) SA-β-Gal staining of spleen sections from young mice and aged mice treated with vehicle or Rhap (5× or 20× magnification). (B) Percentage of the total area that was positive for SA-β-Gal staining in (A). (C) The transcript levels of senescence-associated markers and SASP factors in the spleens of the mice were measured by qPCR. (D and F) Representative images of immunofluorescence staining for p16 and p21 in spleen tissues (scale bar = 50 μm). (E and G) Quantification of the fluorescence intensities of p16 and p21 in (D) and (F) respectively. The data are presented as the means ± SEM, n = 3–5 mice/group. Statistical analysis was performed with Student’s t test for comparisons between two groups and one-way ANOVA for comparisons among three groups. # p \u0026lt; 0.05 vs. the Young group, * p \u0026lt; 0.05 vs. the Vehicle group, ** p \u0026lt; 0.01 vs. the Vehicle group, *** p \u0026lt; 0.001 vs. the Vehicle group, ns, no significance.\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7162814/v1/164c86360d579ccaa2d51746.jpg"},{"id":88005530,"identity":"fd77364c-1091-48ac-9db8-f671c5051b37","added_by":"auto","created_at":"2025-07-31 10:43:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":899413,"visible":true,"origin":"","legend":"\u003cp\u003eRhap affects immune function. (A) Cytokine levels in plasma samples from young and aged mice treated with vehicle or Rhap were measured by Luminex. Bar charts showing the plasma cytokine concentrations. (B and C) Representative flow cytometric plots and quantification of the proportion of CD3+ T cells in blood samples from the mice. (D-G) Representative flow cytometric plots (D) and quantification of the proportions of CD4+ T cells (E) and CD8+ T cells (F) and the ratio of CD4+ T cells/CD8+ T cells (G) in blood samples from the mice. The data are presented as the means ± SEM, n = 3–5 mice/group. Statistical analysis was performed with one-way ANOVA.\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7162814/v1/f958a7480b50a474ecc24965.jpg"},{"id":88002516,"identity":"82e09e65-6aea-49b1-9af7-d8c1049e2b7e","added_by":"auto","created_at":"2025-07-31 10:27:43","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1555374,"visible":true,"origin":"","legend":"\u003cp\u003eNetwork pharmacology analysis identifies that Rhapontigenin targets genes related to senescence and aging. (A) Chemical structure of Rhap (PubChem CID 5320954). (B) Venn diagram of senescence targets (relevance score\u0026gt;3.0, from GeneCards), aging targets (relevance score\u0026gt;7.0, from GeneCards) and Rhap targets (from the traditional Chinese medicine databases TCMSP, ETCM, BATMAN-TCM, HERB, PubChem and the compound-target prediction websites STITCH, SEA, TargetNet, PharmMapper). (C) KEGG enrichment pathways of shared targets between Rhap, senescence and aging. (D) Construction of the PPI network for shared targets using STRING.\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7162814/v1/bca3622856c6c64a17f87ce5.jpg"},{"id":88004188,"identity":"20bebd8c-aa14-4e02-a894-e8b21be39de6","added_by":"auto","created_at":"2025-07-31 10:35:43","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2305722,"visible":true,"origin":"","legend":"\u003cp\u003eRhap upregulates Sirt1 expression and promotes autophagy. (A) The volcano plot shows the differentially expressed genes (DEGs) in liver tissues between old mice treated with vehicle and Rhap. (B) Heatmap showing some of the DEGs between old mice treated with vehicle and Rhap. (C) KEGG pathway enrichment of DEGs. (D) GO enrichment in biological process (BP), cellular component (CC), molecular function (MF) of DEGs. (E) Venn Diagram of Overlap between Shared Genes (intersection of Rhap targets, Senescence-related genes, and aging-related Genes) and DEGs. (F) Molecular docking of the Sirt1 protein (PDB ID 4IF6) and Rhap was performed using AutoDock Vina. Docking poses of Rhap (red) in the active site of Sirt1 (blue). The figures were generated by PyMOL. (G) The binding sites of the Sirt1 protein (PDB ID: 4IF6) and Rhap were identified by the PLIP website. The amino acid residues are labeled as indicated. The blue line segments indicate hydrogen bonds. The gray dashed lines indicate hydrophobic interactions. ASP, aspartic acid; ARG, arginine; VAL, valine; GLU, glutamic acid; ASN, asparagine; GLY, glycine; CYS, cystine. (H) The binding capability of Rhap with SIRT1 was examined via MST technology. (I) Representative images of immunofluorescence staining for Sirt1 in liver tissues (scale bar = 100 μm). (J) Quantification of the fluorescence intensity of Sirt1. (K) Autophagosomes and autolysosomes (black arrows) in hepatocytes were observed by transmission electron microscopy (TEM). Representative TEM images of autophagic vacuoles (AVs) are shown. (L) The bar chart shows the number of AVs, including autophagosomes and autolysosomes. The data are presented as the means ± SEM, n = 3–5 mice/group. Statistical analysis was performed with Student’s t test to compare two groups. * p \u0026lt; 0.05, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7162814/v1/12cc7eccf7c90c86ab8df9bf.jpg"},{"id":88004182,"identity":"37664f0e-601e-41f5-8d9c-9d48546db735","added_by":"auto","created_at":"2025-07-31 10:35:43","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1626106,"visible":true,"origin":"","legend":"\u003cp\u003eRhap attenuates cellular senescence in part by upregulating Sirt1 and promoting autophagy. (A-C) NIH3T3 cells were cultured without or with 400 μM H2O2 for 3 h. The next day, the medium was replaced with medium supplemented with 5, 10, 15, or 20 μM Rhap, and the cells were incubated for 48 h. The protein expression of p-Ampk, Ampk, and Sirt1 was measured by Western blotting, and the levels of p-Ampk/Ampk and Sirt1 were quantified. β-actin was used as the reference protein. (D-F) The protein expression of LC3B and p62 was measured by Western blotting, and the levels of LC3BⅡ/LC3BI and p62 were quantified. β-actin was used as the reference protein. (G) Autophagosomes and autolysosomes (black arrows) in cells were observed by transmission electron microscopy. (H) The numbers of autophagic vacuoles (AVs), including autophagosomes and autolysosomes, were counted. (I) NIH3T3 cells were transfected with siRNA negative control (si-NC) or an siRNA against sirt1 (si-Sirt1). The protein expression of Sirt1 was measured by Western blotting. The representative results from three repetitions are shown in the figures, and the relative quantities are shown in the scatter bar chart. (J) Western blotting analysis of Sirt1, p62, LC3B, p21, and p16 protein expression in NIH3T3 cells transfected with si-NC or si-Sirt1 followed by treatment with or without H2O2 and Rhap at the concentrations shown in the figure. (K) Bar graph of the relative quantification of the gray values of the protein bands shown in Figure (J).\u003c/p\u003e","description":"","filename":"Figure8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7162814/v1/db835b32f2c32d22bb68f4a2.jpg"},{"id":100069424,"identity":"faa32e3f-4926-418e-8bac-20cf34ea2257","added_by":"auto","created_at":"2026-01-12 16:13:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14075149,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7162814/v1/23a7a21d-3058-49ef-900b-57b76abe0521.pdf"},{"id":88004181,"identity":"f49cb2c3-1eef-4071-9fff-ce0bfc1c92ad","added_by":"auto","created_at":"2025-07-31 10:35:43","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2554724,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7162814/v1/54f591b0dccd078b4d6dec15.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rhapontigenin Alleviates Cellular Senescence and Physiological Aging by Upregulating Sirt1 and Promoting Autophagy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAging is a complex biological process that is characterized by a progressive decline in physiological function and increased susceptibility to age-related diseases (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Cellular senescence, which is a state of irreversible cell cycle arrest, is a key hallmark of aging and contributes to tissue dysfunction through the secretion of proinflammatory cytokines, chemokines, and matrix-degrading enzymes; collectively, these phenomena are known as the senescence-associated secretory phenotype (SASP) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The accumulation of senescent cells has been implicated in various age-related conditions, including cardiovascular diseases, neurodegeneration, and metabolic disorders (\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe antiaging therapeutic strategies that are currently available target cellular senescence, reduce systemic inflammation, and promote cellular repair mechanisms (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Drugs, such as senolytics, which selectively eliminate senescent cells, have shown promise in extending healthspans in preclinical models (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). However, the off-target effects and potential toxicity of these compounds limit their clinical applicability (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Natural compounds, particularly polyphenols, have emerged as attractive candidates for use in antiaging therapeutic strategies because of their ability to modulate multiple aging-related pathways and relatively low toxicity (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Despite these advances, effective and safe antiaging agents that can simultaneously address multiple aspects of aging are needed.\u003c/p\u003e\u003cp\u003eRhapontigenin (Rhap; 3,3\u0026rsquo;,5-trihydroxy-4\u0026rsquo;-methoxy-stilbene, C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) is a hydroxylated and methoxylated stilbene compound that is derived mainly from the rhizomes of the Chinese herbal medicinal \u003cem\u003eRheum rhaponticum\u003c/em\u003e and \u003cem\u003eRheum undulatum\u003c/em\u003e, both of which have been extensively utilized in traditional Asian medicine for the treatment of inflammation, digestive disorders, and systemic imbalances (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Rhap has been reported to scavenge intracellular reactive oxygen species (ROS), inhibit DNA, RNA, and protein synthesis, and induce the apoptosis of certain pathogens (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Moreover, Rhap has diverse pharmacological activities, including antioxidant, anti-inflammatory, anticancer, cardioprotective, lipid-lowering, and antimicrobial activities (\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Given these properties, Rhap has potential for use as an agent to treat oxidative stress, inflammation, and cellular damage, all of which are major contributors to aging.\u003c/p\u003e\u003cp\u003eIn this study, we aimed to evaluate the potential of Rhap to be used as an antiaging agent. We investigated the effects of Rhap on cellular senescence, physical performance, immune modulation, and autophagy in both in vitro and in vivo models of aging. Our findings suggest that Rhap may be a promising candidate for mitigating age-related cellular and physiological decline, highlighting its potential for use as a therapeutic agent for age-associated conditions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eChemicals\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRhapontigenin (Rhap, C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e, purity\u0026thinsp;\u0026ge;\u0026thinsp;99.92%) was purchased from Selleck (S9163, Selleck, Shanghai, China).Resveratrol (Resv, C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, purity\u0026thinsp;\u0026ge;\u0026thinsp;99.99%) was purchased from Selleck (S1396, Selleck, Shanghai, China). Etoposide (Etop, C\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e13\u003c/sub\u003e, purity\u0026thinsp;\u0026ge;\u0026thinsp;99.96%) was purchased from Selleck (S1225, Selleck, Shanghai, China). Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) and sodium carboxymethyl cellulose (CMC-NA) were obtained from Sigma‒Aldrich (323381 and 419273, Sigma‒Aldrich, Merck, Germany). Dimethyl sulfoxide (DMSO) was purchased from Solarbio (D8371, Solarbio, Beijing, China).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell Culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNIH3T3 cells were authenticated by STR profiling (March 2025) and tested negative for mycoplasma contamination, as certified by Wuhan Pricella Biotechnology Co., Ltd. (Wuhan, China). The cells were cultured in DMEM/F12 medium (CD0001, SparkJade, Wuhan, China) supplemented with 10% fetal bovine serum (FBS, Biological Industries) and 1% penicillin/streptomycin (P/S, 10378016, Gibco). IMR90 cells were also purchased from Wuhan Pricella Biotechnology Co., Ltd. and authenticated by STR. They were cultured in Minimum Essential Medium (MEM) supplemented with 10% FBS and 1% P/S. Both cell lines were maintained in a humidified incubator at 37\u0026deg;C with 5% CO₂.\u003c/p\u003e\u003cp\u003eTo induce senescence, NIH3T3 cells were treated with 400 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 3 hours, washed three times with phosphate-buffered saline (PBS), and then cultured in fresh medium for 24 or 48 hours. To evaluate the anti-senescence effects of Rhap, the cells were first treated with 400 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003efor 3 hours, washed with PBS, and incubated overnight in fresh medium. Then, the medium was replaced with medium supplemented with different concentrations of Rhap. IMR90 cells were exposed to 50 \u0026micro;M Etop for 48 hours to induce senescence, followed by incubation in normal medium for 3 days. For anti-senescence experiments, IMR90 cells were treated with Etop, and then, the medium was replaced with medium supplemented with different concentrations of Rhap.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimals\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMale C57BL/6JNifdc mice, aged 19 months (old) or 5 weeks (young), were purchased from Weitong Lihua Experimental Animal Technical Co., Ltd. (Beijing, China). The mice were housed in a specific pathogen-free (SPF) environment at the Experimental Animal Center of Shandong University with a 12:12 light/dark cycle, a temperature of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, a humidity of 40\u0026ndash;70%, and the mice were given free access to food and water. After one week of acclimatization, the old mice were randomly assigned to three groups (n\u0026thinsp;=\u0026thinsp;6/group) and received either vehicle or Rhap (50, 100 mg/kg) via oral gavage every two days for two months. These doses were selected based on the previous literature (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Behavioral tests and tissue collection were conducted two weeks after the final gavage. All the animal experiments complied with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication 86\u0026thinsp;\u0026minus;\u0026thinsp;23) and approved by the Animal Ethical and Welfare Committee (AEWC) of Shandong University Cheeloo College of Medicine (No. 21172).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBehavioral Tests: Grip Strength, Rotarod, and Tail Suspension Tests\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGrip strength was assessed with a Grip Strength Meter (SA417, Sansbio, Jiangsu, China). The mice were lifted by the tail and allowed to grasp a bar with their front paws. Then, the mice were pulled horizontally until they released their grip on the bar. The force exerted was recorded, and three measurements per mouse were averaged.\u003c/p\u003e\u003cp\u003eMotor coordination and balance were evaluated using the rotarod test (LE8205, Panlab Harvard Apparatus). The mice were trained for three days at rotation speeds of 4, 6, and 8 revolutions per minute (rpm) for 200 seconds in each session. On the test day, the speed of the rod was increased from 4 to 40 rpm over the course of five minutes. Each mouse was subjected to the test three times, and the average latency to fall and rotation speed were recorded.\u003c/p\u003e\u003cp\u003eThe tail suspension test (TST) was used to assess depressive-like behavior. The mice were suspended by the tail from a hook at the top of a wooden frame, 50 cm above the floor, for six minutes. Mouse behavior was recorded and analyzed with TopScan software. The immobility time was defined as the period during which the mice remained motionless and did not actively attempt to escape.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSenescence-Associated β-Galactosidase (SA-β-Gal) Staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSA-β-Gal activity was measured with a Senescence β-Galactosidase Staining Kit (C0602, Beyotime, Shanghai, China) according to the manufacturer's protocol. Cells or tissue sections were fixed with β-Gal staining fixative for 15 minutes at room temperature (RT), washed with PBS, and then incubated with β-Gal staining solution for 16\u0026ndash;18 hours at 37\u0026deg;C in a CO₂-free incubator. Stained cells or sections were observed under a light microscope, and the number of SA-β-Gal-positive cells or area of positive staining was quantified using ImageJ software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern Blotting\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWestern blotting was performed as previously described (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Cultured cells were lysed with RIPA buffer (PC101, Epizyme, Shanghai, China) supplemented with protease and phosphatase inhibitors (78442, Thermo Fisher Scientific, MA, USA). The protein concentrations were measured with a BCA Protein Assay Kit (23227, Thermo). Equal amounts of proteins were subjected to SDS‒PAGE and transferred to PVDF membranes (ISEQ00010, Millipore, USA). After being blocked with Protein-Free Rapid Blocking Buffer (PS108, EpiZyme, Shanghai, China), the membranes were incubated overnight at 4\u0026deg;C with primary antibodies, followed by incubation with secondary antibodies for 1 hour at RT. The protein bands were visualized using Immobilon Western HRP Substrate (WBKLS0500, Millipore) and quantified using Image Lab Software (Bio-Rad) and ImageJ. The primary antibodies used are as follows: anti-p16\u003csup\u003eink4a\u003c/sup\u003e antibody (1:1000, ab211542, Abcam), anti-p21\u003csup\u003ecip1\u003c/sup\u003e antibody (1:1000, ab188224, Abcam), anti-Sirt1 antiboody (1:1000, ab110304, Abcam), anti-p-AMPK antibody (1:2000, ab133448, Abcam), anti-AMPK antibody (1:1000, ab207442, Abcam), anti-p62 antibody (1:1000, 5114S, CST), anti-LC3B antibody (1:1000, NB100-2220SS, Novus), anti-Beta Actin antibody (1:10000, 20536-1-AP, Proteintech).\u003c/p\u003e\u003cp\u003e\u003cb\u003eHematoxylin and Eosin (H\u0026amp;E) Staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eH\u0026amp;E staining was performed as previously described(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Liver tissues were dissected, fixed in 4% paraformaldehyde for 48 hours, processed in an embedding box, and embedded in paraffin. Sections (5 \u0026micro;m) were cut and stained with an H\u0026amp;E Stain Kit (G1120, Solarbio) according to the manufacturer's instructions. Stained sections were examined under a light microscope, and pathology was assessed by a blinded veterinary pathologist.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOil Red O Staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLipid accumulation in liver tissues was assessed with Oil Red O staining. Liver tissues were dissected, washed with PBS, embedded in optimal cutting temperature (OCT) compound, and frozen at -80\u0026deg;C. Frozen sections (10 \u0026micro;m) were stained with an Oil Red O Stain Kit (ab150678, Abcam) according to the manufacturer's instructions. The relative lipid content in 10 random fields from three mice was quantified using ImageJ.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMasson Staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess liver fibrosis, Masson's trichrome staining was performed with a Masson\u0026rsquo;s Trichrome Stain Kit (G1340, Solarbio) according to the manufacturer's instructions. Paraffin-embedded sections were dewaxed, rehydrated, and stained with Weigert\u0026rsquo;s iron hematoxylin, acid fuchsin, phosphomolybdic acid, and aniline blue. Fibrosis was quantified as the ratio of the blue collagen-positive area to the red normal area.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantitative Polymerase Chain Reaction (qPCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eqPCR was performed as previously described (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), with slight modifications. Total RNA was extracted from frozen spleen and liver tissues using TRIzol Reagent (15596026, Invitrogen). cDNA synthesis was carried out using the SPARKscript II RT Plus Kit (AG0304, SparkJade). qPCR was performed using 2\u0026times; SYBR Green qPCR Mix (AH0101, SparkJade) on a Roche LightCycler 480 instrument. Actin-β was used as the internal reference gene, and the comparative quantification method was 2^(-ΔΔCt). The sequences of the primers that were used are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunofluorescence Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCryosections of liver and spleen tissues were incubated in PBS for 10 minutes to remove the OCT compound. Antigens were retrieved by boiling and heating for 10 minutes in 1\u0026times; Tris-EDTA antigen repair solution (pH 9.0, C1038, Solarbio). The sections were then permeabilized with 0.5% Triton X-100 for 20 minutes at RT. After being blocked with QuickBlock Blocking Buffer for Immunol Staining (P0260, Beyotime), the sections were incubated in a wet box with primary antibodies against p21 (1:200, ab188224, Abcam), p16 (1:200, ab211542, Abcam) or Sirt1 (1:200, ab110304, Abcam) at 4\u0026deg;C overnight. Then, the sections were washed three times in PBS, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG H\u0026amp;L (1:500, ab150077, Abcam), Alexa Fluor 594-conjugated goat anti-rabbit IgG H\u0026amp;L (1:500, ab150080, Abcam) or Alexa Fluor 594-conjugated goat anti-mouse IgG H\u0026amp;L (1:500, ab150116, Abcam) secondary antibodies for 2 h at RT in the wet box in the dark. After being washed with PBS, the sections were mounted with Mounting Medium, antifading (with DAPI) (S2110, Solarbio). Finally, images of randomly selected fields were captured at 20\u0026times; magnification by fluorescence microscopy (Olympus BX53). Images were randomly captured of 3 fields per mouse from 3 mice. The fluorescence intensity was analyzed with ImageJ.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFlow Cytometry Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe spleen and peripheral blood were harvested from mice, and single-cell suspensions were prepared according to previously described methods (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Blood was collected from anesthetized mice by heart puncture and transferred to EDTA-coated tubes. Moreover, spleens were harvested from euthanized mice and placed in cold PBS. After the spleens were pierced with 1-ml syringes, spleen cells were removed with 1\u0026times; PBS and filtered through a 70-\u0026micro;m cell strainer. Red blood cells in the peripheral blood and splenocytes samples were lysed using ACK lysis buffer (Cat. No. A1049201, Gibco) according to the manufacturer\u0026rsquo;s instructions. Then, the cells were washed with cold stain buffer (FBS) (BD Biosciences, Cat No. 554656) and incubated with an anti-mouse CD16/32 mAb (BD Biosciences, 553141) for 5 minutes on ice to block the Fc receptor. The cells were subsequently incubated with fluorescently labeled primary antibodies for 30 minutes on ice in the dark. The antibodies that were used to stain the blood or spleen single-cell suspensions were as follows: PE-conjugated anti-CD3 (Clone No. 145-2C11, 1:100, BD Biosciences, Cat No. 553063), PerCP-conjugated anti-CD4 (RM4-5, 1:100, BD Biosciences, 553052), BV421-conjugated anti-CD8 (53\u0026thinsp;\u0026minus;\u0026thinsp;6.7, 1:100, eBioscience, 48-0081-82), FITC-conjugated anti-NK1.1 (PK136, 1:100, BD Biosciences, 553164). After staining, the cells were washed, resuspended in staining buffer and detected with a BD LSRFortessa flow cytometer. Data analysis was performed with FlowJo (v10.7.2).\u003c/p\u003e\u003cp\u003e\u003cb\u003eClinical Chemistries\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt the end of the experiments with aged mice, the mice were anesthetized with isoflurane, and blood was collected into EDTA anticoagulation tubes. Plasma was obtained by centrifugation (3000 rpm) at 4\u0026deg;C for 15 minutes, and then, it was aliquoted and stored at -80\u0026deg;C until analysis. The concentrations of plasma biochemical parameters, such as ALT, AST, CHE, UREA, CREA, and UA, were measured by a fully automated biochemical analyzer according to the manufacturer\u0026rsquo;s instructions. Plasma samples from 5 mice per group were analyzed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLuminex\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePlasma samples were collected and stored as described above. The levels of cytokines in the plasma samples were measured on a Luminex X-200 instrument with a Bio-Plex Pro Mouse Cytokine 23-plex Assay (M60009RDPD, Bio-Rad) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell Counting Kit-8 Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCell viability was determined with a Cell Counting Kit-8 (CCK8, Dojindo, Japan). A total of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells were seeded in each well of a 96-well plate and treated with vehicle (DMSO) or 5, 10, 15, or 20 \u0026micro;M Rhap for 24 h after the cells had attached to the wells. After washing with PBS, 100 \u0026micro;l of medium supplemented with 10 \u0026micro;l of CCK8 reagent was added. The absorbance at 450 nm was measured after 2 hours of incubation in a cell culture incubator. To calculate the half maximal inhibitory concentration (\u003cem\u003eIC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e), 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells were seeded in each well of a 96-well plate and treated with vehicle (DMSO) or 0.01, 0.1, 1, 10, 50, 100, 500, 1000 \u0026micro;M Rhap for 48 h after the cells had attached to the wells. The absorbance at 450 nm was measured after 1 hours of incubation with CCK8 reagent. \u003cem\u003eIC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e was determined by fitting the concentration-response data to a nonlinear regression model using GraphPad Prism software (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEdU cell proliferation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e NIH3T3 cells were seeded in each well of 96-well plates and treated with (5, 10, 15, or 20 \u0026micro;M) or without Rhap for 24 h after the cells had attached to the wells. EdU-based cell proliferation was measured with a Cell-LightTM EdU Apollo In Vitro Kit (C10310-3, RiboBio) following the manufacturer's instructions. EdU signals were measured by flow cytometry, and the results were analyzed with FlowJo.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular Docking\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMolecular docking analysis was conducted using AutoDock Vina (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The 3D protein structure of Sirt1 (PDB ID: 4if6, 4kxq, 4i5i and 4zzj) was downloaded from the RCSB PDB website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the compound structure of Rhap (PubChem CID: 5320954) was obtained from PubChem (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"http://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The binding affinity was used to predict the optimal binding between the compound and the protein.\u003c/p\u003e\u003cp\u003e3D structural diagrams showing the interaction between the compound and protein were generated using PyMol software. The binding sites were identified on the PLIP website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://projects.biotec.tu-dresden.de/plip-web/plip/index\u003c/span\u003e\u003cspan address=\"http://projects.biotec.tu-dresden.de/plip-web/plip/index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) and visualized using PyMol software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicroscale Thermophoresis (MST)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine the binding affinity between SIRT1 and Rhapontigenin, MST analysis was performed using a Monolith NT.115 instrument (Zoonbio Biotechnology Co., Ltd., Nanjing, China). Purified SIRT1 protein was fluorescently labeled with the RED-NHS 2nd Generation Kit (MO-L011, NanoTemper Technologies, Munich, Germany) according to the manufacturer's protocol, yielding a final concentration of 0.1 \u0026micro;M. For ligand dilution, Rhapontigenin was serially diluted twofold in PBST buffer containing 5% DMSO, spanning a concentration range from 0.25 mM to 7.63 nM (to avoid precipitation at high concentrations). For binding measurements, 10 \u0026micro;L of gradient-diluted ligand was mixed with 10 \u0026micro;L of labeled SIRT1 and incubated for 5 min at room temperature. Samples were loaded into standard capillaries (MO-K022) and analyzed under optimized MST conditions. Data processing was performed using MO. Control software to calculate the dissociation constant (K\u003csub\u003ed\u003c/sub\u003e) with signal-to-noise ratios maintained at \u0026gt;\u0026thinsp;5 to ensure reliability.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTransmission Electron Microscopy (TEM)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAutophagosomes and autolysosomes in the cells were observed and photographed by TEM, which was performed by Wuhan Servicebio Technology Co., Ltd. In brief, NIH3T3 cells were collected after they were treated with or without 400 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 3 h and then cultured in medium supplemented with or without 20 \u0026micro;M Rhap for 48 h. Then, cell pellets were fixed for 2 h in electron microscope fixative supplemented with 2.5% glutaraldehyde (G1102, Servicebio). After being washed three times with 0.1 M phosphate buffer (pH 7.4), the cell pellets were encapsulated in 1% agarose and then fixed with 1% osmic acid in 0.1 M phosphate buffer (pH 7.4) for 2 h at room temperature in the dark. The samples were dehydrated with gradient alcohol and acetone. The samples were subsequently infiltrated and embedded with 812 embedding agent before being allowed to polymerize in an oven at 60\u0026deg;C for 48 h. The resin blocks were sectioned at a thickness of 60 nm with an ultrathin microtome, and the pieces were harvested with 150-mesh square Chinese membrane copper mesh. Then, ultrathin sections in the copper mesh were stained with 2% uranyl acetate saturated with alcohol solution and 2.6% lead citrate. The samples were subsequently observed and imaged by TEM (Hitachi HT7800). Finally, the numbers of autophagic vacuoles (AVs, including both autophagosomes and autolysosomes) were counted and analyzed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA-seq and Data Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was isolated from snap-frozen liver tissues with a TRIzol RNA extraction kit (TIANGEN, Cat. No. DP424), yielding\u0026thinsp;\u0026gt;\u0026thinsp;2 \u0026micro;g of total RNA per sample. The RNA quality was examined by 0.8% agarose gel electrophoresis and spectrophotometry, and high-quality RNA with a 260/280 ratio ranging from 1.8\u0026ndash;2.2 was used for library construction and sequencing. The library was constructed following the Illumina manufacturer's instructions (Illumina, USA). Oligo-dT primers were utilized to transcribe mRNA into cDNA (APExBIO, Cat. No. K1159). cDNA was amplified for the synthesis of the second chain of cDNA. The cDNA products were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). After library construction, the library fragments underwent enrichment by PCR amplification and were selected on the basis of a fragment size of 350\u0026ndash;550 bp. Next, the library was quality-assessed with an Agilent 2100 Bioanalyzer (Agilent, USA). Then, the library was sequenced on the Illumina NovaSeq 6000 platform (paired-end 150) to produce raw reads. Raw data have been deposited to National Center for Biotechnology Information (NCBI) under the BioProject number PRJNA1235652.\u003c/p\u003e\u003cp\u003eRaw paired-end fastq reads were filtered by TrimGalore to discard the adapters and low-quality bases via calling the Cutadapt tool. The obtained clean reads were subsequently aligned to the mm10 mouse genome using HISAT2 (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), followed by reference genome-guided transcriptome assembly and gene expression quantification with StringTie (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Differentially expressed genes (DEGs) of samples with replications were identified using DEseq2 (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) or edgeR (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) for samples with no replication, with thresholds of |fold-change|\u0026gt;1.5 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The functional enrichment analysis of the annotated significant DEGs and the potential genes in the identified modules based on Gene Ontology (GO) and KEGG pathway categories was performed using the clusterProfiler (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Terms with p values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell Transfection with siRNAs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe mouse negative control siRNA (si-NC) and siRNA targeting Sirt1 (si-Sirt1) were purchased from GeneChem (Shanghai, China). NIH3T3 cells were transfected with siRNAs using LipofectamineTM 2000 (11668019, Invitrogen, CA, USA) following the manufacturer\u0026rsquo;s instructions. We used two si-Sirt1 sequences to prevent off-target effects. The sequences of the si-Sirt1 that were used were as follows: si-Sirt1-1 sense 5\u0026rsquo;-GCCAUGUUUGAUAUUGAGUAUdTdT-3\u0026rsquo; and antisense 5\u0026rsquo;-AUACUCAAUAUCAAACAUGGCdTdT-3\u0026rsquo;; si-Sirt1-2 sense 5\u0026rsquo;-AGUGAGACCAGUAGCACUAAUdTdT-3\u0026rsquo; and antisense 5\u0026rsquo;-AUUAGUGCUACUGGUCUCACUdTdT-3\u0026rsquo;\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll the experimental data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 8.0 software. Comparisons between two groups were performed using Student's t test, whereas comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. The significance level was set to p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. \u003cem\u003eC. elegans\u003c/em\u003e survival analyses were conducted using the log rank (Mantel‒Cox) test.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eRhap Reduces Oxidative Stress-Induced Cellular Senescence in Vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine the effect of Rhap on cellular senescence, NIH3T3 and IMR90 cells were subjected to oxidative or genotoxic stress, followed by treatment with Rhap. Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e shows that NIH3T3 cells that were treated with increasing concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exhibited a dose-dependent increase in senescence, as indicated by SA-β-Gal staining and increased expression of the senescence markers p16 and p21 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-E). In NIH3T3 cells, exposure to 400 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e led to increased senescence, whereas subsequent treatment with Rhap (5 or 10 \u0026micro;M) significantly reduced the number of senescent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, C). Similarly, in IMR90 cells, Etop-induced senescence was attenuated by Rhap treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, D). Western blotting analysis further demonstrated that Rhap treatment reduced the expression of the senescence-associated markers p16 and p21 in NIH3T3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G). These findings suggest that Rhap effectively mitigates oxidative and genotoxic stress-induced cellular senescence.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCell viability was assessed to determine whether Rhap exerted any cytotoxic effects at the concentrations used. The CCK-8 assay revealed no significant reduction in cell viability after treatment with a range of Rhap concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Moreover, EdU staining and flow cytometry revealed that Rhap did not impair cell proliferation, indicating that its anti-senescence effects did not occur due to reduced cell viability or proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, J). The 50% inhibitory concentration (\u003cem\u003eIC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e) of Rhap inhibiting NIH3T3 cell viability or proliferation after 48 hours was 116 \u0026micro;M (\u003cem\u003e95\u003c/em\u003e% \u003cem\u003eCI\u003c/em\u003e, 107.9-124.8 \u0026micro;M). Furthermore, Rhap did not affect cell viability at concentrations up to 50 \u0026micro;M, which aligns with our previous observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, H).\u003c/p\u003e\u003cp\u003eIn addition, the impact of Rhap on organismal longevity was evaluated with N2 nematodes. Survival analysis revealed a significant extension in lifespan after Rhap treatment. Compared to the vehicle group, the median lifespan of nematodes treated with 0.2 mM Rhap was extended by 21.7% and the maximum lifespan by 15%; the median lifespan of nematodes treated with 1 mM Rhap was extended by 30.4% and the maximum lifespan by 20%. (Fig. S2 and Table S2). The results suggests that the potential benefits of Rhap beyond those observed in cellular models.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRhap Increases Physical Performance and Maintains Organ Function in Aged Mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the effects of Rhap on aging, 19-month-old mice were treated with Rhap or vehicle for 8 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The mice treated with Rhap had smoother fur than the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Rhap-treated mice exhibited significantly improved physical performance, as indicated by increased grip strength normalized to body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D) and enhanced motor coordination in the rotarod test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Furthermore, Rhap-treated mice demonstrated shorter immobility times in the TST, suggesting improved mood or reduced depressive-like behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRhap treatment did not significantly affect body weight or organ weights (liver, heart, spleen, lung, and kidney weights), indicating that it did not have adverse effects on overall health (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-L). Plasma biochemical analysis revealed a significant reduction in AST levels in Rhap-treated mice, suggesting improved liver function, whereas other markers (ALT, CHE, UREA and CREA) remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM-R). Histological analysis by HE staining revealed no adverse effects in liver and kidney pathology due to Rhap treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). These results indicate that Rhap increases physical function and maintains organ health in aged mice and lacks liver or kidney toxicity at the concentrations used, highlighting its potential to mitigate age-related declines.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRhap Attenuates Liver Senescence, Lipid Accumulation, and Fibrosis in Aged Mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the impact of Rhap on aging, SA-β-Gal staining was performed on the tissues of the heart, liver, spleen, lung and kidney. There were no significant differences observed in the tissues of the heart, lungs and kidneys (data not shown). In particular, SA-β-Gal staining revealed a significant increase in the number of senescent cells in the livers of vehicle-treated aged mice, and this effect was markedly suppressed by Rhap treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). qPCR analysis revealed that Rhap reduced the expression of senescence markers (p21 and p53) and SASP factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Immunofluorescence staining confirmed that p21 expression was significantly decreased in the livers of Rhap-treated aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G).\u003c/p\u003e\u003cp\u003eOil red O staining revealed reduced lipid accumulation in the livers of Rhap-treated aged mice, suggesting improved lipid metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, I). Additionally, Masson staining revealed a significant reduction in liver fibrosis in Rhap-treated aged mice compared with control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ, K). These findings indicate that Rhap effectively attenuates liver senescence, lipid accumulation, and fibrosis in aged mice, highlighting its potential for use as an agent for treating age-related liver dysfunction.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRhap Attenuates Spleen Senescence and Modulates Immune Function in Aged Mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAmong the tissues assessed, spleen aging also displayed marked changes following Rhap treatment. Specifically, SA-β-Gal staining revealed a significant decrease in the numbers of senescent cells in the spleens of Rhap-treated aged mice compared with those in the spleens of vehicle-treated control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). qPCR analysis revealed that Rhap treatment significantly decreased the expression of senescence markers (p16 and p21) and SASP factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eImmunofluorescence staining for p16 and p21 further confirmed that expression of these markers was reduced in the spleens of Rhap-treated aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-G). Moreover, Luminex analysis of plasma samples revealed that compared with vehicle, Rhap significantly reduced the levels of proinflammatory cytokines, including IFNγ, TNFα, IL-1α, IL-17a, and MCP-1, in aged mice, indicating that Rhap exerts an anti-inflammatory effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the effects of Rhap on the immune function, the immune cell subsets were detected using flow cytometry. This analysis revealed that compared with vehicle treatment, Rhap treatment increased the proportion of CD3\u0026thinsp;+\u0026thinsp;T cells in aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). Furthermore, Rhap treatment significantly increased the proportion of CD4\u0026thinsp;+\u0026thinsp;T cells, whereas the proportion of CD8\u0026thinsp;+\u0026thinsp;T cells was not significantly different; these effects resulted in an elevated CD4+/CD8\u0026thinsp;+\u0026thinsp;T-cell ratio, which suggests a better T-cell balance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-G).\u003c/p\u003e\u003cp\u003eIt has been shown that mature NK cells are reduced in the peripheral tissues of aged mice compared with young mice (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In our study, we found that the proportion of NK cells (NK1.1\u0026thinsp;+\u0026thinsp;CD3-) in peripheral blood and spleen of aged mice was lower than that of young mice, but there was a trend of increase in Rhap-treated aged mice compared with vehicle-treated group, but the difference was not statistically significant, indicating that Rhap may have an effect on NK cells (Fig. S3A-D ). These findings indicate that Rhap effectively reduces splenic senescence and modulates immune function, promoting an anti-inflammatory immune profile in aged mice.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRhap Upregulates Sirt1 Expression and Promotes Autophagy in the liver tissue of aged mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRhap (3,3\u0026rsquo;,5-trihydroxy-4\u0026rsquo;-methoxy-stilbene, C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) is a naturally occurring stilbenoid compound that is characterized by its unique chemical structure featuring a trans-stilbene backbone with hydroxyl groups at the 3, 3\u0026rsquo;, 5 positions and a methoxy group at the 4\u0026rsquo; position (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Methoxylated stilbenes have been suggested to be more suitable options for oral administration than hydroxylated stilbenes, such as resveratrol, because methoxylated stilbenes have increased bioavailability and similar biological activities compared with hydroxylated stilbenes (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). To elucidate the molecular mechanisms underlying the antiaging effects of Rhap, we performed a comprehensive analysis of its targets and their roles in aging and senescence-related pathways. A Venn diagram analysis identified 28 genes as shared targets of Rhap, senescence, and aging (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). KEGG pathway enrichment of shared targets indicated involvement in the cellular senescence pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Protein-protein interaction (PPI) network constructed via STRING database identified core hub proteins including TP53, AKT1, MAPK1, SIRT1 that functionally converge on cellular senescence regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Network pharmacology analysis revealed Rhapontigenin (Rhap) as a multi-target agent against senescence and aging.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTranscriptomic analysis of liver tissues revealed significant changes in gene expression following Rhap treatment. Volcano plot analysis identified 2072 differentially expressed genes (DEGs) between vehicle- and Rhap-treated aged mice, with 994 upregulated and 1078 downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Hierarchical clustering of select DEGs demonstrated clear separation between treatment groups, with distinct expression patterns across biological replicates. Quantitative analysis revealed that Rhap treatment significantly upregulated Sirt1 expression while suppressing transcriptional levels of cell cycle inhibitors Cdkn1a compared with vehicle controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). KEGG and GO enrichment analyses of DEGs revealed that Rhap treatment was associated with pathways related to Longevity, Cellular senescence, AMPK signaling, FoxO signaling, NF-κB signaling and autophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D). Venn analysis identified two critical genes Sirt1 and Sod2 at the shared genes of network pharmacology predictions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and DEGs of transcriptomic profiling (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Considering that Sirt1 is implicated in the regulation of multiple signaling pathways related to aging, such as cellular senescence, longevity regulation, NF-κB, AMPK, FoxOs and Autophagy (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), and exhibits a high degree of connectivity within the PPI network (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), coupled with its upregulated expression in tissues of Rhap-treated aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B), we propose the hypothesis that Sirt1 plays a crucial role in mediating the anti-aging effects of Rhap. Molecular docking analysis confirmed that Rhap may interact with the active site of Sirt1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, G and Table S3). MST experiments determining the dissociation constant (K\u003csub\u003ed\u003c/sub\u003e) between Rhap and SIRT1 yielded a value of 2.0572E-06 M (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH), indicating a strong binding affinity. These findings suggest that Rhap plays a role in modulating Sirt1 activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eImmunofluorescence staining revealed that Sirt1 expression was significantly greater in the livers of Rhap-treated aged mice than in those of vehicle-treated control mice, suggesting a role of Rhap in promoting Sirt1 activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI, J). Furthermore, TEM analysis revealed an increase in the number of AVs, including autophagosomes and autolysosomes, in Rhap-treated aged mice, indicating increased autophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK, L). These findings suggest that Rhap exerts its antiaging effects, at least in part, by upregulating Sirt1 and promoting autophagy, thereby contributing to improved cellular health in aged mice.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRhap Reduces Cellular Senescence by Upregulating Sirt1 and Promoting Autophagy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the molecular mechanisms by which Rhap reduces cellular senescence, we examined the role of Sirt1 and autophagy in NIH3T3 cells that were treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and Rhap. Western blotting analysis revealed that Rhap treatment significantly increased the expression of Sirt1 and the ratio of p-AMPK to AMPK in a dose-dependent manner, indicating AMPK signaling activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-C). Additionally, Rhap treatment increased the LC3BII/LC3BI ratio and decreased the p62 levels, suggesting an increase in the autophagic flux (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTEM confirmed that the number of AVs, including autophagosomes and autolysosomes, was increased in Rhap-treated cells compared with control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG, H). These findings indicate that Rhap promotes autophagy in response to oxidative stress.\u003c/p\u003e\u003cp\u003eTo determine whether Sirt1 is required for the anti-senescence effects of Rhap, NIH3T3 cells were transfected with siRNA targeting Sirt1 (si-Sirt1) or a negative control (si-NC). Western blotting analysis revealed that Sirt1 knockdown abolished the Rhap-induced increase in the LC3BII/LC3BI ratio and the reduction in p21 and p16 expression, indicating that Sirt1 is necessary for the Rhap-mediated attenuation of autophagy and senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI-K). These findings suggest that Rhap alleviates cellular senescence by activating Sirt1 and promoting autophagy, highlighting its potential for use as an agent for treating age-related cellular dysfunction.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated that Rhap, which is a stilbene aglycone metabolite that is derived from rhaponticin (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), effectively attenuates cellular senescence, increases physical function, reduces inflammation, and promotes autophagy in both in vitro and in vivo models of aging. These findings provide new insights into the potential of Rhap to be used as an antiaging therapeutic agent and contribute to the growing body of literature suggesting a role for natural compounds in mitigating age-associated pathologies.\u003c/p\u003e\u003cp\u003eRhap is extracted from medicinal plants such as rhubarb rhizomes (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), and exhibit various biological activities, including anti-inflammatory, antioxidant, anticancer, antihyperlipidemic, antiallergic, and antibacterial activities (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Additionally, Rhap has been reported to scavenge ROS (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) and inhibit DNA, RNA, and protein synthesis in \u003cem\u003eC. albicans\u003c/em\u003e (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Emerging evidence suggests that Rhap exhibits therapeutic potential in mitigating pathological features of Alzheimer's disease (AD), positioning it as a promising therapeutic candidate for AD (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Our study extends these findings by highlighting the ability of Rhap to counteract oxidative and genotoxic stress-induced cellular senescence, which is a hallmark of aging (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe anti-senescence effects of Rhap that were observed in our study are consistent with the findings of previous research on polyphenolic compounds, such as resveratrol and quercetin, which reduce senescence marker levels and improve cellular health by activating Sirt1 and promoting autophagy (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Our data show that Rhap significantly reduces the activity of SA-β-Gal and the expression of the key senescence markers p16 and p21 in NIH3T3 and IMR90 cells. Interestingly, the effects of Rhap were observed even at lower concentrations compared to other polyphenols and displayed markedly lower toxicity at higher concentrations, suggesting that Rhap may be a more potent antiaging compound with fewer off-target effects.\u003c/p\u003e\u003cp\u003eIn vivo, Rhap-treated aged mice exhibited significant improvements in physical performance, including enhanced grip strength, improved motor coordination, and reduced depressive-like behaviors. These findings are consistent with those of studies showing that polyphenols can enhance motor function and mitigate mood disturbances during aging (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Furthermore, Rhap increased liver health by attenuating hepatic senescence, lipid accumulation, and fibrosis, highlighting its potential for use as an agent for treating age-related liver dysfunction.\u003c/p\u003e\u003cp\u003eOur study also highlights the role of Rhap in modulating immune function. Rhap treatment significantly reduced the levels of proinflammatory cytokines, including IFNγ, TNFα, IL-1α, IL17a and MCP1, which are key components of the SASP. The decrease in the levels of these SASP factors suggests that Rhap can alleviate systemic inflammation and promote a more balanced immune profile.\u003c/p\u003e\u003cp\u003eMechanistically, our data indicate that the antiaging effects of Rhap are mediated, at least in part, by the upregulation of Sirt1 and the promotion of autophagy. Sirt1 is a well-established regulator of aging, and it is involved in processes such as DNA repair, inflammation, and mitochondrial function (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Rhap-treated aged mice exhibited increased Sirt1 expression and autophagy, which support the hypothesis that Rhap promotes cellular homeostasis via autophagy. This finding was further confirmed in vitro, where Sirt1 knockdown abolished the beneficial effects of Rhap on autophagy and senescence. The dual targeting of the Sirt1 and AMPK signaling pathways by Rhap suggests that Rhap has a broader and more versatile mechanism of action than other inducers of autophagy, suggesting that Rhap is a potent antiaging compound.\u003c/p\u003e\u003cp\u003eDespite these promising results, our study has several limitations. We relied primarily on the NIH3T3 and IMR90 cell lines, which may not fully capture the complexity of cellular senescence in vivo. Future studies should include primary cells from aged organisms to confirm the anti-senescence effects of Rhap. Additionally, while our study demonstrated the efficacy of Rhap in aged mice, the translation of these findings to humans remains uncertain. Clinical studies are needed to evaluate the safety, bioavailability, and therapeutic potential of Rhap in human populations. Moreover, a detailed pharmacokinetic analysis of Rhap would provide crucial insights into its metabolism and distribution, which are essential for understanding its therapeutic applicability.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, Rhap is a promising candidate for mitigating age-related cellular and physiological decline. By activating Sirt1, promoting autophagy, and attenuating inflammation, Rhap exerts multifaceted antiaging effects with significant therapeutic implications. Future studies should focus on understanding the long-term effects of Rhap treatment, its pharmacokinetics, and its potential for use as an agent for treating age-related diseases in humans.\u003c/p\u003e"},{"header":"Abbrevations","content":"\u003cp\u003eALT, alanine aminotransferase; AST, aspartate transaminase; CHE, cholinesterase; CREA, creatinine; IFNγ, Interferon gamma; IL-10, Interleukin-10; IL-1α, Interleukin-1 alpha; IL-1β, Interleukin-1 beta; IL-6, Interleukin-6; MCP-1, Monocyte Chemoattractant Protein-1; Rhap, Rhapontigenin; SA-β-Gal, senescence-associated β-galactosidase; SASP, senescence-associated secretory phenotype; Sirt1, Sirtuin 1; TGF-β, Transforming Growth Factor beta; TNFα, Tumor Necrosis Factor alpha; UA, uric acid\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Xiaowen Liu and Wenbin Chen in Central Laboratory, Shandong Provincial Hospital Affiliated to Shandong University for the donation of anaesthetic drugs. We acknowledge the State Key Laboratory of Reproductive Medicine and Offspring Health for supporting the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the animal experiments complied with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication 86-23) and approved by the Animal Ethical and Welfare Committee (AEWC) of Shandong University Cheeloo College of Medicine (No. 21172).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShuang Liu: Writing \u0026ndash; original draft, Visualization, Validation, Investigation. Wendi Chen: Validation, Methodology, Investigation. Guoqiang Xu: Validation. Xin Liu: Investigation, Formal analysis. Yuxuan Shi: Formal analysis, Data curation. Guolong Wang: Software, Data curation. Yongzhi Cao: Visualization, Resources. Yunna Ning: Validation, Supervision. Ming Li: Writing \u0026ndash; original draft, Supervision, Formal analysis. Yueran Zhao: Writing \u0026ndash; review and editing, Supervision, Resources, Funding acquisition, Conceptualization.\u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by The National Key Research and Development Program of China (No. 2022YFC2702905) and Taishan Scholars Program of Shandong Province (tsqnz20240852).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eL\u0026oacute;pez-Ot\u0026iacute;n C, Blasco MA, Partridge L, Serrano M, Kroemer G. 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Ageing Res Rev. 2024;102:102545.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Rhapontigenin, Cellular senescence, Antiaging, Autophagy, Sirt1","lastPublishedDoi":"10.21203/rs.3.rs-7162814/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7162814/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eAging is characterized by cellular senescence, inflammation, and physiological decline. Currently available antiaging therapies often have limitations due to their toxicity and off-target effects. However, natural compounds derived from Chinese herbal medicine, such as Rhapontigenin (Rhap), have shown potential as safer antiaging agents.\u003c/p\u003e\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eThis study aimed to evaluate the potential of Rhap to be used as an antiaging agent by investigating its effects on cellular senescence, physical function, immune modulation, and autophagy in both in vitro and in vivo aging models.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eNIH3T3 and IMR90 cells were subjected to oxidative or genotoxic stress to induce senescence and then treated with Rhap. Senescence markers, cell viability, and autophagy-related protein levels were assessed. Aged mice were treated with Rhap for 8 weeks, and physical performance, immune modulation, and organ health were evaluated. Mechanistic studies were conducted to determine the role of Sirt1 in mediating the effects of Rhap.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eRhap treatment significantly reduced cellular senescence marker (p16 and p21) levels and senescence-associated β-galactosidase (SA-β-Gal) activity in stressed cells. In aged mice, Rhap improved physical performance, such as grip strength and motor coordination, and reduced depressive-like behaviors. Rhap also decreased liver senescence, lipid accumulation, and fibrosis and increased immune function by reducing proinflammatory cytokine production and enhancing T-cell homeostasis. Mechanistically, Rhap upregulated Sirt1 and promoted autophagy, both of which contributed to its antiaging effects. Sirt1 knockdown abolished the effects of Rhap on autophagy and senescence, indicating the importance of Sirt1 in mediating these beneficial effects.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eRhap is a promising candidate for mitigating age-related cellular and physiological decline by reducing cellular senescence, promoting autophagy, and modulating immune function. However, further studies are needed to explore the long-term effects and therapeutic potential of Rhap in human populations.\u003c/p\u003e","manuscriptTitle":"Rhapontigenin Alleviates Cellular Senescence and Physiological Aging by Upregulating Sirt1 and Promoting Autophagy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 10:27:38","doi":"10.21203/rs.3.rs-7162814/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-18T10:25:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-15T07:08:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210899417377648606539667924662740337612","date":"2025-10-09T11:35:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123149116350780396381139788137033585018","date":"2025-07-29T12:57:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125736410575056231272378054832801592312","date":"2025-07-29T02:58:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-29T02:18:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-23T14:35:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-23T14:35:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chinese Medicine","date":"2025-07-19T07:47:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"541f264b-9c87-42c0-bca1-93c2d74aa9d4","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:05:28+00:00","versionOfRecord":{"articleIdentity":"rs-7162814","link":"https://doi.org/10.1186/s13020-025-01319-3","journal":{"identity":"chinese-medicine","isVorOnly":false,"title":"Chinese Medicine"},"publishedOn":"2026-01-09 15:58:50","publishedOnDateReadable":"January 9th, 2026"},"versionCreatedAt":"2025-07-31 10:27:38","video":"","vorDoi":"10.1186/s13020-025-01319-3","vorDoiUrl":"https://doi.org/10.1186/s13020-025-01319-3","workflowStages":[]},"version":"v1","identity":"rs-7162814","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7162814","identity":"rs-7162814","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}