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ABSTRACT Various anti-aging interventions show promise in extending lifespan, but many are ineffective or even harmful to healthspan. Ginkgolide B (GB), derived from Ginkgo biloba, reduces

aging-related morbidities such as osteoporosis, yet its effects on healthspan and longevity have not been fully understood. In this study, we found that continuous oral administration of GB

to female mice beginning at 20 months of age extended median survival and median lifespan by 30% and 8.5%, respectively. GB treatment also decreased tumor incidence; enhanced muscle quality,

physical performance and metabolism; and reduced systemic inflammation and senescence. Single-nucleus RNA sequencing of skeletal muscle tissue showed that GB ameliorated aging-associated

changes in cell type composition, signaling pathways and intercellular communication. GB reduced aging-induced Runx1+ type 2B myonuclei through the upregulation of miR-27b-3p, which

suppresses Runx1 expression. Using functional analyses, we found that Runx1 promoted senescence and cell death in muscle cells. Collectively, these findings suggest the translational

potential of GB to extend healthspan and lifespan and to promote healthy aging. Access through your institution Buy or subscribe This is a preview of subscription content, access via your

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institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS TARGETING SENESCENCE INDUCED BY AGE OR CHEMOTHERAPY WITH A POLYPHENOL-RICH

NATURAL EXTRACT IMPROVES LONGEVITY AND HEALTHSPAN IN MICE Article Open access 01 July 2024 MITOCHONDRIAL AND METABOLIC DYSFUNCTION IN AGEING AND AGE-RELATED DISEASES Article 10 February 2022

DISTINCT AND ADDITIVE EFFECTS OF CALORIE RESTRICTION AND RAPAMYCIN IN AGING SKELETAL MUSCLE Article Open access 19 April 2022 DATA AVAILABILITY The datasets generated during the current

study are available in the Gene Expression Omnibus repository (GSE218027 and GSE254739), and all other data are available from the corresponding author upon reasonable request. REFERENCES *

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National Science and Technology Council, Taiwan (111-2320-B-039-070-MY3 to C.-W.L.) and China Medical University Hospital (EXO-113-004, DMR-112-147 and DMR-113-106 to C.-W.L.). Experiments

and data analysis were performed in part through the use of the Medical Research Core Facilities Center, Office of Research & Development, at China Medical University. We thank the RNA

Technology Platform and Gene Manipulation Core Facility (RNAi core) of the National Core Facility for Biopharmaceuticals at Academia Sinica in Taiwan for providing reagents and related

services. Illustrations were created in BioRender: Lee, C. (2024) https://BioRender.com/j81t493 and Lee, C. (2020) https://BioRender.com/k43w200. AUTHOR INFORMATION Author notes * These

authors jointly supervised this work: Chien-Wei Lee, Oscar Kuang-Sheng Lee. AUTHORS AND AFFILIATIONS * Translational Cell Therapy Center, China Medical University Hospital, Taichung, Taiwan

Chien-Wei Lee, Yu-Fan Chen, Hao-Hsiang Wu, Po-Yu Cheng & Zong-Han Chou * Department of Biomedical Engineering, China Medical University, Taichung, Taiwan Chien-Wei Lee & Yu-Fan Chen

* Center for Neuromusculoskeletal Restorative Medicine, CUHK InnoHK Centres, Hong Kong Science Park, Hong Kong, China Belle Yu-Hsuan Wang & Wayne Yuk-Wai Lee * Li Ka Shing Institute of

Health Sciences, The Chinese University of Hong Kong, Hong Kong, China Belle Yu-Hsuan Wang, Allen Wei-Ting Hsiao & Wayne Yuk-Wai Lee * School of Biomedical Sciences, Faculty of Medicine,

The Chinese University of Hong Kong, Hong Kong, China Shing Hei Wong, Qin Cao, Sin-Hang Fung & Stephen Kwok Wing Tsui * Ph.D. Program for Translational Medicine, College of Medical

Science and Technology, Taipei Medical University, Taipei, Taiwan Yi-Fan Chen * Graduate Institute of Translational Medicine, College of Medical Science and Technology, Taipei Medical

University, Taipei, Taiwan Yi-Fan Chen * International Ph.D. Program for Translational Science, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan Yi-Fan

Chen * Master Program in Clinical Genomics and Proteomics, School of Pharmacy, Taipei Medical University, Taipei, Taiwan Yi-Fan Chen * Department of Orthopaedics and Traumatology, The

Chinese University of Hong Kong, Hong Kong, China Allen Wei-Ting Hsiao & Wayne Yuk-Wai Lee * SH Ho Scoliosis Research Laboratory, Joint Scoliosis Research Centre of the Chinese

University of Hong Kong and Nanjing University, The Chinese University of Hong Kong, Hong Kong, China Wayne Yuk-Wai Lee * Department of Biotechnology Medicine, MacKay Memorial Hospital,

Taipei, Taiwan Oscar Kuang-Sheng Lee Authors * Chien-Wei Lee View author publications You can also search for this author inPubMed Google Scholar * Belle Yu-Hsuan Wang View author

publications You can also search for this author inPubMed Google Scholar * Shing Hei Wong View author publications You can also search for this author inPubMed Google Scholar * Yi-Fan Chen

View author publications You can also search for this author inPubMed Google Scholar * Qin Cao View author publications You can also search for this author inPubMed Google Scholar * Allen

Wei-Ting Hsiao View author publications You can also search for this author inPubMed Google Scholar * Sin-Hang Fung View author publications You can also search for this author inPubMed 

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inPubMed Google Scholar * Po-Yu Cheng View author publications You can also search for this author inPubMed Google Scholar * Zong-Han Chou View author publications You can also search for

this author inPubMed Google Scholar * Wayne Yuk-Wai Lee View author publications You can also search for this author inPubMed Google Scholar * Stephen Kwok Wing Tsui View author publications

You can also search for this author inPubMed Google Scholar * Oscar Kuang-Sheng Lee View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS

C.-W.L. designed the study, performed the experiments and data analysis, wrote the manuscript, provided funding and supervised the study. B.Y.-H.W. performed and analyzed data of in vivo

experiments. S.H.W. and S.-H.F. performed single-nucleus RNA sequencing analysis. A.W.-T.H. performed data analyses of in vitro experiments. Y.-F.C. provided critical technical support and

feedback regarding the manuscript. Q.C. offered technical support for single-nucleus RNA sequencing analysis and manuscript editing. P.-Y.C. and Z.-H.C. performed and analyzed data of in

vitro experiments and bulk RNA sequencing. H.-H.W., Y.-F.C., W.Y.-W.L. and S.K.W.T. provided input for the manuscript. O.K.-S.L. supervised the study. All authors read and approved the final

manuscript. CORRESPONDING AUTHORS Correspondence to Chien-Wei Lee or Oscar Kuang-Sheng Lee. ETHICS DECLARATIONS COMPETING INTERESTS The authors disclose that a patent application related to

the findings presented in this paper has been submitted. The details of the patent are as follows: Patent Applicant: China Medical University; Inventors: O.K.-S.L., C.-W.L., Y.-F.C. and

H.-H.W.; Application Numbers: US14307 and 112144291; Status: Pending. The other authors declare no conflicts of interest. PEER REVIEW PEER REVIEW INFORMATION _Nature Aging_ thanks the

anonymous reviewer(s) for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in

published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 GB INCREASES MYOFIBRE SIZE AND GRIP STRENGTH IN SIX-MONTH-OLD MICE. (A) Experimental design and GB

structure: C57BL/6 female mice (six-month-old) received GB or vehicle by oral gavage for two months (6 m+Vehicle, n = 4; 6 m+GB, n = 6). Illustration was created in BioRender.com. (B)

Expression of circulating osteocalcin after GB administration for two months. (C) Representative haematoxylin and eosin staining (H&E) cross-section images of the tibialis anterior.

Scale bar, 50 μm. (D–E) Quantification and distribution of tibialis anterior (D) and soleus (E) myofibre cross-sectional area. Results are measured from 11 images from four mice for the

vehicle group and 15 images from six mice for the GB group. (F) Forelimb grip strength assay. Quantitative data are presented as the means ± SD in the histogram with data points. Statistical

analyses are performed using two-tailed Student’s t-test, with significance set at p < 0.05. (*p < 0.05; **p < 0.01; ***p < 0.001). Source data EXTENDED DATA FIG. 2 GB

ATTENUATES MUSCLE WASTING AND IMPROVES PHYSICAL ACTIVITY IN POSTMENOPAUSAL MICE. (A) Experimental design (n = 10). After OVX for one month, OVX mice received two doses of GB daily (3 and 12 

mg/kg body weight) for 2 months. Illustration was created in BioRender.com. (B) Quadriceps weight-to-body weight. (C) Representative H&E staining of quadriceps. (D–E) Quantification (D)

and distribution (E) of the myofibre cross-sectional area are measured from 30 images of 10 mice. Scale bar, 100 μm. (F–G) Healthspan was measured by grip strength (F), rotarod (G), and

balance beam test (H) (n = 5). Quantitative data are presented as the means ± SD in the histogram with the data point. Statistical analyses are performed using one-way ANOVA with Tukey’s

multiple comparison test. Means not sharing any letters are significantly different (p < 0.05). Source data EXTENDED DATA FIG. 3 GINKGOLIDE B REPRESSES H2O2-INDUCED SENESCENCE IN MUSCS.

(A) Experimental design and senescence-related assessments on MuSCs. Senescence was induced by H2O2 stimulation for 2 hours in MuSCs. Following a PBS wash, the senescent C2C12 were treated

with or without GB (5 mg/L). Sen, H2O2-induced senescence. PAX7 positive cells represent MuSCs. Scale bar, 50 μm. (B-C) Representative images (B) and quantification (C) of P21 and γ-H2AX

staining (n = 5). Scale bar, 50 μm. (D) Representative images and proportion of SA-β-gal positive cells (n = 3). Scale bar, 50 μm. Quantitative data are presented as the means ± SD in the

histogram with the data point. Statistical analyses are performed using one-way ANOVA with Tukey’s multiple comparison test. This means not share any letter are significantly different (p 

< 0.05). Source data EXTENDED DATA FIG. 4 THE EFFECT OF GB ON MULTIPLE ORGANS OF AGED MICE. (A) Organ weight-to-body weight in aged mice after GB administration for two months. (B)

H&E staining of the kidney, heart, spleen, and liver. Scale bar, 100 μm for kidney, 50 μm for heart, 400 μm for spleen, and 200 μm for liver. (C) Glomerular size (n = 5). (D)

Quantification of the cardiac myofibre cross-sectional area (n = 5). (E) Quantification of the white pulp area (n = 32-35 white pulps from 5 individual mice per group) and white pulp/red

pulp ratio of spleen (n = 5). (F) Quantification of hepatic microgranulomas (indicated by arrows) (n = 10 images from 5 individual mice per group). (G) Nile red staining reveals hepatic

lipid accumulation in aged mice (n = 5). Liver section without Nile red staining served as a negative control. Scale bar, 50 μm. (H) Masson’s trichrome staining of the kidney, heart, spleen,

and liver. Scale bar, 50 μm for kidney, 100 μm for heart, 100 μm for spleen, and 200 μm for liver. (I–L) Quantification of Masson’s trichrome staining in the kidney (I), heart (J), spleen

(K), and liver (L) (n = 5). Quantitative data are presented as the means ± SD in the histogram with data point. Statistical analyses are performed using one-way ANOVA with Tukey’s multiple

comparison test. Means not sharing any letter are significantly different (p < 0.05). Source data EXTENDED DATA FIG. 5 SINGLE-NUCLEUS LANDSCAPE OF SKELETAL MUSCLES. (A) Graphical scheme

of the experimental design. Illustration was created in BioRender.com. (B) Isolated nuclei with DAPI staining (n = 3). Scale bar, 20 μm and 5 μm from top to bottom panels. (C–D) UMAP diagram

from integrated datasets (C) and separated datasets (D) revealed the coordinates of nuclear types in GA muscle of Young+Vehicle, Aged+Vehicle, and Aged+GB groups. Type 2B myonucleus-1, Type

2B-1; Type 2B myonucleus-2, Type 2B-2; Type 2X myonucleus, Type 2X; Type 2 A myonucleus, Type 2A; myotendinous Junction, MTJ; fibro/adipogenic progenitors, FAPs; endothelial cells, EC;

pericytes, Peri; satellite cells, SatC; adipocytes, Adip; tenocytes, Teno; immune cells, IC; smooth muscles SM; Schwann cells, SchwC. (E) Venn diagrams illustrating the reversion of nuclear

types by GB administration. Percentage change in proportion over 10% was considered significant. (F) Rose diagrams revealed the numbers of aging DEGs, GB DEGs, and Rescue DEGs of various

cell types in skeletal muscles (DEG, FDR < 0.05, absolute log2FC > 0.25). (G) Rescue DEGs from various cell types and representative GO pathways in skeletal muscles. EXTENDED DATA FIG.

6 SINGLE-NUCLEUS RNA SEQUENCING ANALYSIS OF MYONUCLEI. (A–B) Volcano plot (A) and heatmap (B) of the pseudo-bulk DEGs between Aged+Vehicle vs Young+Vehicle and Aged+GB vs Aged+Vehicle

groups. Log2 fold-change (log2 FC) > 1 and an adj. p < 0.05 are used to identify DEGs. (C) Venn diagrams illustrating convergence and noncongruence of pseudo-bulk DEGs between groups.

(D) Pseudo-bulk GSEA analysis of myonuclei between Young+Vehicle vs Aged+Vehicle and Aged+GB vs Aged+Vehicle datasets. The similar pattern between the two datasets indicates GB reversed

aging-related changes in the indicative transcriptome. Gene rank metric = sign(log2FC) x –log10(adj. p-val). (E) Top 10 specific DEGs in Runx1+ myonucleus. (F) GO enrichment analysis by

biological process of top 30 specific genes in Runx1+ type2B myonuclei from integrated dataset. (G) hdWGCNA dendrograms identified 31 co-expression modules. (H) Visualization of module

eigengene value over myonucleus. (I) Visualization of turquoise module eigengene value over myonucleus. Left, all genes with turquoise module; right, top 25 hub genes within turquoise

module. (J) GO analysis of DEGs within turquoise module. (K) Volcano plot of the DEGs in Runx1+ type2B myonuclei between Aged+Vehicle vs Young+Vehicle and Aged+GB vs Aged+Vehicle groups.

Reported p-values are adjusted (p-adj) to account for multiple comparisons. EXTENDED DATA FIG. 7 EFFECT OF RUNX1 OVEREXPRESSION AND KNOCKDOWN IN C2C12. (A) The chronological series of

assessment time slots. (B) Evaluation of transfection efficiency at 24 hours post-transfection, as determined by the proportion of GFP positive C2C12 (n = 5). Control-GFP, cells transfected

with pcDNA3.1( + )-GFP. (C-D) Representative flow cytometry plot (C) and quantitation (D) of Annexin V and PI staining in C2C12 (n = 3). Control-GFP, cells transfected with pcDNA3.1(+);

Runx1 OE, cells transfected with pcDNA3.1(+)-Runx1 for 24 and 48 hours; H2O2, cells exposed to 1.5 mM H2O2 for 6 hours. (E) Detection of senescence markers by real-time PCR in Runx1

knockdown C2C12 (n = 4). (F-I) Cellular morphology, cell size (F), doubling time (G) γ-H2AX staining (H) and senescence markers (I) of Runx1 knockdown C2C12 under H2O2 stimulation (n = 5-6).

Doubling time = Duration x ln(2) / ln(Final concentration/ Initial concentration). Quantitative data are presented as the means ± SD in the histogram with the data point. Statistical

analyses are performed using two-way ANOVA or two-tailed Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001), depending on experiment design. Source data EXTENDED DATA FIG. 8

MIR-27B-3P MEDIATES THE EFFECT OF GB ON RUNX1 AND SENESCENCE. (A) Veen diagram revealed the prediction of potential Runx1 targeting human and mouse miRNAs. (B) Expression of miR-27b-3p in

aged muscle (n = 6). (C) Expression of miR-27b-3p in H2O2-induced senescent C2C12 (n = 5). Sen, H2O2-induced senescence. (D) Correlation comparisons of transcript levels of miR-27b-3p with

transcript of Runx1, IL-6 and SA-β-gal activity (n = 5). Correlations are measured by Pearson coefficient (R) with corresponding 95% confidence intervals and assessed using two-tailed

p-value analysis. (E) Expression of miR-27b-3p and Runx1 in H2O2-induced senescent C2C12 with GB and miR-27b-3p antagonist treatment (n = 3). (F) Cell viability (n = 6). (G) Representative

morphology and cell size (n = 5). Scale bar, 100 μm. (H) Representative images and quantification of SA-β-gal staining (n = 5). Scale bar, 50 μm. (I) Representative images and quantification

of ϒ-H2AX staining (n = 5). Scale bar, 50 μm. (J) Potential mechanism of action of GB. Source data EXTENDED DATA FIG. 9 SINGLE-NUCLEUS RNA SEQUENCING ANALYSIS REVEALS THE CHANGES IN

CELL-CELL COMMUNICATION. (A–B) Circle plots (A) and heatmap (B) showed the differential cell-cell communication between Young+Vehicle vs Aged+Vehicle and Aged+GB vs Aged+Vehicle datasets in

skeletal muscle. The similar signaling pattern between the two datasets indicates GB reversed aging-related changes in cell-cell communication. (C) Plot revealed the contribution of outgoing

and incoming signalling between various cells. Circle size represents the counts in each nucleus type. (D) Chord diagram showing interaction count and weight of cell-to-muscle

communication. The thickness of the line represents interaction count and weight of ligand-receptor interaction. (E) Differential cell-to-muscle signaling. SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Supplementary Figs. 1–11. REPORTING SUMMARY SUPPLEMENTARY TABLE Supplementary Tables 1–11. SOURCE DATA SOURCE DATA FIG. 1 Statistical source data. SOURCE DATA FIG.

2 Statistical source data. SOURCE DATA FIG. 3 Statistical source data. SOURCE DATA FIG. 4 Statistical source data. SOURCE DATA FIG. 5 Statistical source data. SOURCE DATA FIG. 7 Statistical

source data. SOURCE DATA FIG. 8 Statistical source data. SOURCE DATA WB Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 1 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 2

Statistical source data. SOURCE DATA EXTENDED DATA FIG. 3 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 4 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 7 Statistical

source data. SOURCE DATA EXTENDED DATA FIG. 8 Statistical source data. RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this

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such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Lee, CW., Wang, B.YH., Wong, S.H. _et al._ Ginkgolide B increases healthspan and

lifespan of female mice. _Nat Aging_ 5, 237–258 (2025). https://doi.org/10.1038/s43587-024-00802-0 Download citation * Received: 10 May 2023 * Accepted: 20 December 2024 * Published: 31

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