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Cited by 397Telomeres, Telomerase, and SenescenceGenetics, Aging, and Longevity in Model OrganismsAntioxidants, Aging, Portulaca oleracea

Elimination of senescent cells by β-galactosidase-targeted prodrug attenuates inflammation and restores physical function in aged mice

Open access
Cai, Yusheng, Zhou, Huanhuan, Zhu, Yinhua, Sun, Qi, Ji, Yin, Xue, Anqi, Wang, Yuting, Chen, Wenhan +6 more·2020·Cell Research
DOI 10.1038/s41422-020-0314-9PMC PMC7184167
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Abstract

Cellular senescence, a persistent state of cell cycle arrest, accumulates in aged organisms, contributes to tissue dysfunction, and drives age-related phenotypes. The clearance of senescent cells is expected to decrease chronic, low-grade inflammation and improve tissue repair capacity, thus attenuating the decline of physical function in aged organisms. However, selective and effective clearance of senescent cells of different cell types has proven challenging. Herein, we developed a prodrug strategy to design a new compound based on the increased activity of lysosomal β-galactosidase (β-gal), a primary characteristic of senescent cells. Our prodrug SSK1 is specifically activated by β-gal and eliminates mouse and human senescent cells independently of senescence inducers and cell types. In aged mice, our compound effectively cleared senescent cells in different tissues, decreased the senescence- and age-associated gene signatures, attenuated low-grade local and systemic inflammation, and restored physical function. Our results demonstrate that lysosomal β-gal can be effectively leveraged to selectively eliminate senescent cells, providing a novel strategy to develop anti-aging interventions.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper reports SSK1, a β-galactosidase-activated gemcitabine prodrug that selectively kills senescent cells across mouse and human cell types and senescence inducers. In aged and lung-injured mice, SSK1 reduced senescent-cell markers and SASP/inflammation and improved physical function without obvious systemic toxicity.

Key findings

  • SSK1 is cleaved by lysosomal β-gal to release cytotoxic gemcitabine; it selectively killed mouse and human senescent cells independent of senescence inducer and cell type, and GLB1 knockdown impaired this killing. Its mechanism involved p38 MAPK activation and apoptosis.
  • In bleomycin-injured mice, SSK1 (0.5 mg/kg) reduced lung SA-β-gal-positive cells by 3.8-fold versus vehicle, decreased senescence markers and fibrosis genes, and improved treadmill and grip strength performance.
  • In 20–22-month-old C57BL/6 mice, SSK1 (0.5 mg/kg, 3 days every 2 weeks for 8 weeks) reduced SA-β-gal-positive cells in liver, kidney, and lung, decreased p16/p21 and SASP-associated genes, lowered serum IL1α, IL6, CXCL1, and TNFα, and improved rotarod, treadmill, grip strength, rearing, and balance beam performance; serum biochemistry and routine blood analysis showed no obvious systemic toxicity.
  • SSK1 showed broader specificity/potency than ABT263, dasatinib plus quercetin, and fisetin in tested human cell types and improved all four tested physical-function assays in aged mice, whereas the other senolytics had limited effects.

Methods

  • Cell-based assays used primary mouse and human fibroblasts, HUVECs, and human preadipocytes; senescence was induced by replication, irradiation, KrasG12V, etoposide, or H2O2. Viability was measured by Hoechst 33342/PI staining, with flow cytometry, western blot, RT-qPCR, LC-MS/MS metabolism analysis, and RNA-seq.
  • Mouse models included bleomycin-induced lung injury in 3–6-month-old mice and aged 20–22-month-old C57BL/6 mice treated intraperitoneally with SSK1 (0.5 mg/kg), gemcitabine (0.5 mg/kg), or vehicle for 3 days every 2 weeks for 8 weeks. Physical function was assessed by rotarod, treadmill, grip strength, balance beam, and rearing tests; toxicity testing used SSK1 at 3–100 mg/kg.
  • Statistical analysis used t-tests, one-way or two-way ANOVA, GSEA, GO enrichment, and xCell; RNA-seq reads were aligned to mm10.

Limitations

  • Stated: elevated β-gal activity can occur in some non-senescent cells, including activated macrophages and epithelial cells, so off-target effects are possible; the authors report only a few apoptotic kidney cells and no obvious reduction of endogenous acidic β-gal-positive non-senescent cells after SSK1.
  • Stated: SA-β-gal-positive macrophages can be harmful and accumulate in injured and aged tissues; SSK1 also reduces them, which may contribute to anti-inflammatory effects but complicates attribution of benefits solely to senescent-cell clearance.
  • Evident: the findings are from mouse models and cultured cells; no human clinical efficacy or long-term safety data are reported, and the main aged-mouse functional data are in female mice, with some male data shown in supplementary figures.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41422-020-0314-9

Cites

73 of 73 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 59 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    SSK1 decreased senescence-associated markers compared with vehicle treatment in bleomycin-injured mice.

    “RT-qPCR results revealed that SSK1 also decreased senescence-associated markers compared with vehicle treatment (Fig. 3c).”
    Quote in the paperSubstantial claim
  • Findingown data

    SSK1 selectively killed senescent HUVECs and human preadipocytes compared with non-senescent counterparts.

    “We also treated human umbilical vein endothelial cells (HUVECs) and human preadipocytes with SSK1, and found that senescent cells were selectively killed by SSK1 when compared with their non-senescent counterparts (Fig. 2d; Supplementary information, Figs. S2g–i and S3e).”
    Quote in the paperSubstantial claim
  • Findingown data

    SSK1 treatment activates p38 MAPK and MKK3/MKK6 by phosphorylation in senescent cells.

    “After SSK1 treatment, both p38 MAPK and MKK3/MKK6 were activated by phosphorylation in senescent cells (Fig. 1f; Supplementary information, Fig. S2a, b)”
    Quote in the paperSubstantial claim
  • Findingown data

    SSK1 entered senescent cells independent of nucleoside transporters.

    “Nucleoside transporter inhibitor was unable to block the killing effect of SSK1 indicating that SSK1 entered the senescent cells independent of transporter (Supplementary information, Fig. S1e).”
    Quote in the paperSubstantial claim
  • Findingown data

    SSK1 significantly reduced the percentage of SA-β-gal-positive cells in lung by 3.8-fold compared with vehicle in bleomycin-injured mice.

    “SSK1 significantly reduced the percentage of SA-β-gal-positive cells in lung by 3.8-fold compared with that in vehicle-treated lung-injured mice (Fig. 3b).”
    Quote in the paperSubstantial claim
  • The quote only partly states this claim. 4
  • May combine two claims. 39
  • Classified as probably not a substantial claim. 21

  • The quote only partly states this claim.

    Findingown data

    In aged mice, SSK1 attenuated low-grade chronic inflammation.

    “attenuated low-grade chronic inflammation”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    In aged mice, SSK1 decreased senescence- and age-associated gene signatures.

    “decreased senescence- and age-associated gene signatures”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    The authors designed a new prodrug that is specifically cleaved by lysosomal β-gal into cytotoxic gemcitabine and induces apoptosis in senescent cells.

    “Here, we designed a new prodrug that was specifically cleaved by lysosomal β-gal into cytotoxic gemcitabine and induced apoptosis in senescent cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SSK1 is specifically cleaved to release cytotoxic gemcitabine in senescent cells but not in non-senescent cells.

    “We also validated that the prodrug SSK1 was specifically cleaved to release cytotoxic gemcitabine in senescent cells but not in non-senescent cells (Fig. 1c; Supplementary information, Fig. S1d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SSK1 induces apoptosis in senescent cells, as shown by annexin V/PI double-positive cells and caspase 3 activation.

    “flow cytometry analysis showed that SSK1 induced senescent cells into annexin V and propidium iodide double-positive cells, and western blot result showed SSK1 could activate caspase 3, which indicated that SSK1 killed senescent cells by inducing apoptosis (Fig. 1g; Supplementary information, Fig. S2b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Senescent cells accumulated in different tissues of aged mice, consistent with observation that SA-β-gal-positive cells increased in aged livers, kidneys and lungs.

    “Senescent cells accumulated in different tissues of aged mice,4,40,41 which was consistent with our observation that SA-β-gal-positive cells increased in aged livers, kidneys and lungs (Supplementary information, Fig. S5a–c).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [4] van Deursen, J. M. The role of senescent cells in ageing. Nature 509, 439–446 (2014). source

      Not checked in the free run.

    • cites [40] Wang, C. et al. DNA damage response and cellular senescence in tissues of aging mice. Aging Cell 8, 311–323 (2009). source

      Not checked in the free run.

    • cites [41] Krishnamurthy, J. et al. Ink4a/Arf expression is a biomarker of aging. J. Clin. Investig. 114, 1299–1307 (2004). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    SSK1 reduced lung fibrosis and expression of fibrosis genes Col1a1, Col3a1 and Fn1 in bleomycin-injured mice.

    “We further found that lung fibrosis and the expression of fibrosis genes, including Col1a1, Col3a1 and Fn1, an outcome mediated by the accumulation of senescence,36,39 were reduced by SSK1 treatment (Fig. 3d; Supplementary information, Fig. S4c, d).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [36] Schafer, M. J. et al. Cellular senescence mediates fibrotic pulmonary disease. Nat. Commun. 8, 14532 (2017). source

      Not checked in the free run.

    • cites [39] Yanai, H. et al. Cellular senescence-like features of lung fibroblasts derived from idiopathic pulmonary fibrosis patients. Aging 7, 664–672 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    SSK1 is toxic to senescent cells and non-toxic to non-senescent cells.

    “These results suggested that SSK1 was toxic to senescent cells and non-toxic to non-senescent cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Knockdown of GLB1 reduced SA-β-gal activity and showed little effect on other senescence markers p16, p21 and Il1α.

    “Knockdown of GLB1 reduced SA-β-gal activity (Supplementary information, Fig. S1m) and showed little effect on other senescence markers, such as p16, p21 and Il1α (Supplementary information, Fig. S1n).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SSK1 selectively cleared human senescent cells induced by different stresses in a dose-dependent manner, without obvious effect on non-senescent counterparts.

    “The results showed that SSK1 selectively cleared human senescent cells induced by different stresses in a dose-dependent manner, without an obvious effect on non-senescent counterparts (Fig. 2c; Supplementary information, Fig. S2e, f and Table S2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SSK1 selectively and potently eliminated β-gal-positive senescent cells within a wide therapeutic window.

    “We found that SSK1 selectively and potently eliminated β-gal-positive senescent cells within a wide therapeutic window (Fig. 1d; Supplementary information, Fig. S1f–i).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    The prodrug SSK1 eliminates both mouse and human senescent cells independent of senescence inducers and cell types.

    “This prodrug eliminated both mouse and human senescent cells independent of the senescence inducers and cell types.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SSK1 selectively killed mouse and human senescent cells induced by various stimuli, while non-senescent cells were largely unaffected.

    “These senescent cells induced by various stimuli were selectively killed by SSK1, while non-senescent cells were largely unaffected (Fig. 2a; Supplementary information, Table S2).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    In aged mice, SSK1 improved physical function.

    “and improved physical function.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    F4/80-positive macrophages accumulated in livers of aging mice and SSK1 attenuated macrophage infiltration.

    “We performed immunofluorescence analysis of F4/80-positive macrophages, and found that these cells accumulated in livers of aging mice and that SSK1 could attenuate the macrophage infiltration (Supplementary information, Fig. S6g).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In a mouse lung injury model, SSK1 cleared stress-induced senescent cells in vivo and alleviated associated symptoms.

    “In a mouse lung injury model, SSK1 cleared stress-induced senescent cells in vivo and alleviated associated symptoms.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SSK1 eliminated stress-induced senescent cells effectively and decreased different senescence markers in the bleomycin-induced lung injury model.

    “SSK1 eliminated stress-induced senescent cells effectively and decreased different senescence markers in bleomycin-induced lung injury model”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Deletion of senescent cells by a genetic approach attenuates age-related deterioration and extends health-span in aged mice.

    “In contrast, deletion of senescent cells by a genetic approach attenuated age-related deterioration and extended the health-span in aged mice.8,9”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [8] Baker, D. J. et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479, 232–236 (2011). source

      Not checked in the free run.

    • cites [9] Baker, D. J. et al. Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan. Nature 530, 184–189 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Cellular senescence drives chronic low-grade inflammation, exacerbating aging.

    “Cellular senescence impairs the ability of tissue regeneration and drives chronic low-grade inflammation, which exacerbates the aging process.5,6”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [5] Coppe, J. P., Desprez, P. Y., Krtolica, A. & Campisi, J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu. Rev. Pathol. 5, 99–118 (2010). source

      Not checked in the free run.

    • cites [6] Childs, B. G. et al. Senescent cells: an emerging target for diseases of ageing. Nat. Rev. Drug Discov. 16, 718–735 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Transplantation of senescent cells into young mice is sufficient to drive age-related pathology and cause persistent physical dysfunction.

    “Importantly, transplantation of senescent cells into young mice was sufficient to drive age-related pathology and cause persistent physical dysfunction.7”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [7] Xu, M. et al. Senolytics improve physical function and increase lifespan in old age. Nat. Med. 24, 1246–1256 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Senescence is one of the major drivers of aging and clearing senescent cells is a promising approach to treat age-related diseases and improve physical function.

    “These studies demonstrated that senescence is one of the major drivers of aging and that clearing senescent cells is a promising approach to treat age-related diseases and improve physical function.6,10”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [6] Childs, B. G. et al. Senescent cells: an emerging target for diseases of ageing. Nat. Rev. Drug Discov. 16, 718–735 (2017). source

      Not checked in the free run.

    • cites [10] He, S. & Sharpless, N. E. Senescence in Health and Disease. Cell 169, 1000–1011 (2017). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Reported senolytics target anti-apoptotic pathways up-regulated to inhibit apoptosis in senescent cells.

    “Reported senolytics target anti-apoptotic pathways, which are up-regulated to inhibit apoptosis in senescent cells.11,14”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [11] Zhu, Y. et al. The Achilles’ heel of senescent cells: from transcriptome to senolytic drugs. Aging Cell 14, 644–658 (2015). source

      Not checked in the free run.

    • cites [14] Kirkland, J. L., Tchkonia, T., Zhu, Y., Niedernhofer, L. J. & Robbins, P. D. The clinical potential of senolytic drugs. J. Am. Geriatr. Soc. 65, 2297–2301 (2017). source

      Not checked in the free run.

  • May combine two claims. The quote only partly states this claim.

    Interpretationown data

    SSK1 has higher specificity and potency to eliminate senescent cells than ABT263, dasatinib plus quercetin, and fisetin.

    “These results demonstrated that SSK1 has higher specificity and potency to eliminate senescent cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    SSK1 is activated by lysosomal β-gal and selectively kills senescent cells through activation of p38 MAPK and induction of apoptosis.

    “These results suggested that our prodrug SSK1 was activated by lysosomal β-gal and selectively killed senescent cells through the activation of p38 MAPK and induction of apoptosis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    SSK1 eliminates SA-β-gal-positive cells and decreases senescence-associated markers in vivo.

    “Taken together, our results indicated that SSK1 could eliminate SA-β-gal-positive cells and decrease senescence-associated markers in vivo.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    SA-β-gal in diverse types of senescent cells is a widely used marker for identifying senescence in vitro and in vivo, linked to increased lysosome content.

    “Notably, SA-β-gal in diverse types of senescent cells is one widely used marker for identifying senescence in vitro and in vivo, which is linked to the increased content of lysosomes.20–23”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [20] Childs, B. G., Durik, M., Baker, D. J. & van Deursen, J. M. Cellular senescence in aging and age-related disease: from mechanisms to therapy. Nat. Med. 21, 1424–1435 (2015). source

      Not checked in the free run.

    • cites [21] Sharpless, N. E. & Sherr, C. J. Forging a signature of in vivo senescence. Nat. Rev. Cancer 15, 397–408 (2015). source

      Not checked in the free run.

    • cites [22] Hernandez-Segura, A., Nehme, J. & Demaria, M. Hallmarks of cellular senescence. Trends Cell Biol. 28, 436–453 (2018). source

      Not checked in the free run.

    • cites [23] Gorgoulis, V. et al. Cellular senescence: defining a path forward. Cell 179, 813–827 (2019). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    SSK1 could overcome limitations including cell-type dependency, high toxicity on non-senescent cells, and low efficiency on senescent cells.

    “Most importantly, SSK1 could overcome these limitations, including cell-type dependency, high toxicity on non-senescent cells, and low efficiency on senescent cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    SSK1 is robust and specific in reducing senescent cells.

    “These results demonstrated the robustness and specificity of our prodrug in reducing senescent cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    SSK1-induced clearance of SA-β-gal-positive senescent cells is a robust and safe approach to ameliorate age-related functional decline.

    “Collectively, our results suggested that SSK1-induced clearance of SA-β-gal-positive senescent cells to be a robust and safe approach to ameliorate age-related functional decline.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Absence of apparent toxicity in mice at the tested doses predicts safety in humans.

  • May combine two claims.

    Interpretationown data

    SSK1 reduced naturally accumulated senescent cells and decreased senescence markers in mice.

    “These results indicated that SSK1 reduced naturally accumulated senescent cells and decreased senescence markers in mice.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Gemcitabine is a widely used FDA-approved drug with proven safety and short plasma circulation time.

    “gemcitabine is a widely used FDA-approved drug with proven safety and short plasma circulation time.25,26”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [25] Mini, E., Nobili, S., Caciagli, B., Landini, I. & Mazzei, T. Cellular pharmacology of gemcitabine. Ann. Oncol. 17(Suppl 5), v7–v12 (2006). source

      Not checked in the free run.

    • cites [26] Plunkett, W. et al. Gemcitabine: metabolism, mechanisms of action, and self-potentiation. Semin. Oncol. 22, 3–10 (1995).

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    SSK1 treatment improved motor function, balance ability, exercise endurance, skeletal muscle capacity and spontaneous exploration in aged mice.

    “These results of mouse functional assays demonstrated an improvement in motor function, balance ability, exercise endurance, skeletal muscle capacity and spontaneous exploration.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    SSK1 efficiently alleviated SASP both locally and systemically, probably due to elimination of accumulated senescent cells and macrophages in aged organisms.

    “Collectively, these results showed that SSK1 efficiently alleviated SASP both locally and systemically, which were probably due to elimination of accumulated senescent cells and macrophages in aged organisms.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    SASP contributes to local and systemic low-grade inflammation during aging, age-related degenerative phenotypes, and impairment of physical function.

    “SASP contributes to local and systemic low-grade inflammation during aging, to age-related degenerative phenotypes, and to impairment of physical function.5,6,43”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [5] Coppe, J. P., Desprez, P. Y., Krtolica, A. & Campisi, J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu. Rev. Pathol. 5, 99–118 (2010). source

      Not checked in the free run.

    • cites [6] Childs, B. G. et al. Senescent cells: an emerging target for diseases of ageing. Nat. Rev. Drug Discov. 16, 718–735 (2017). source

      Not checked in the free run.

    • cites [43] Franceschi, C. & Campisi, J. Chronic inflammation (inflammaging) and its potential contribution to age-associated diseases. J. Gerontol. A Biol. Sci. Med. Sci. 69(Suppl 1), S4–S9 (2014). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    During virus-induced acute inflammation, macrophages produce pro-inflammatory factors and trigger initiation of cytokine storms.

    “During virus-induced acute inflammation, macrophages produce pro-inflammatory factors and trigger initiation of cytokine storms.57”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [57] Liu, Q., Zhou, Y. H. & Yang, Z. Q. The cytokine storm of severe influenza and development of immunomodulatory therapy. Cell Mol. Immunol. 13, 3–10 (2016). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Mice were randomized to SSK1, gemcitabine or vehicle delivered by intraperitoneal injection.

    “Mice were randomized to SSK1, gemcitabine or vehicle delivered by intraperitoneal injection.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    For quantification of p21-positive cells, images were randomly taken at 20× magnification at the same exposure using confocal laser endomicroscopy and then analyzed by ImageJ.

    “For quantification of p21-positive cells, images were randomly taken at 20× magnification at the same exposure using confocal laser endomicroscopy (LSM 710 NLO &DuoScan System) and then analyzed by ImageJ.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Human samples were collected from consenting donors according to ethically approved procedures.

    “Samples were collected from consenting donors according to ethically approved procedures at China–Japanese Friendship Hospital.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The study demonstrates the superiority and safety of the prodrug strategy targeting β-gal to selectively remove senescent cells of multiple cell types.

    “our study demonstrates the superiority and safety of this prodrug strategy by targeting β-gal to selectively remove senescent cells of multiple cell types”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Absence of apparent toxicity in mice at the tested doses predicts safety in humans.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For the statistics of SA-β-gal-positive area of lung, the regions of lung NBFs were randomly selected to be photographed, avoiding analysis of larger pulmonary blood vessels and trachea.

    “For the statistics of SA-β-gal-positive area of lung, the regions of lung NBFs were randomly selected to be photographed, avoiding analysis of larger pulmonary blood vessels and trachea.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Low-passage proliferative cells were used as normal controls to avoid replication-induced senescent cells.

    “To avoid replication-induced senescent cells, low-passage proliferative NBFs (<3 passages), MEFs (<3 passages), mouse lung fibroblasts (<3 passages), HEFs (<10 passages), HUVECs (<6 passages), and human preadipocytes (<5 passages) were used as normal controls.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Activated macrophages play crucial roles in acute inflammation and cytokine storm, especially those induced by virus infection.

    “activated macrophages play crucial roles in acute inflammation and cytokine storm,56 especially those induced by virus infection.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [56] Hamidzadeh, K., Christensen, S. M., Dalby, E., Chandrasekaran, P. & Mosser, D. M. Macrophages and the recovery from acute and chronic inflammation. Annu. Rev. Physiol. 79, 567–592 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The short duration of SSK1 treatment might avoid significant impairment in related tissues and further minimize its side effects.

    “the short duration of SSK1 treatment might avoid significant impairment in related tissues and further minimize its side effects”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    SSK1 ameliorated impaired motor function, balance, exhausted exercise, muscle strength, and spontaneous exploration in aged mice.

    “SSK1 ameliorated the impaired motor function, balance, exhausted exercise, muscle strength, and spontaneous exploration in aged mice”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Gemcitabine is a nucleoside analog that potently affects rapidly dividing cells.

    “gemcitabine is a nucleoside analog that potently affects rapidly dividing cells”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    GAPDH was used as a control to normalize the expression of target genes in RT-qPCR.

    “GAPDH was used as a control to normalize the expression of target genes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All functional assays were conducted at least 5 days after the last dose of drug was administered.

    “All functional assays were conducted at least 5 days after the last dose of drug was administered.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Eliminating macrophage accumulation by SSK1 might reduce chronic inflammation and benefit aged organisms.

    “Therefore, eliminating macrophage accumulation by SSK1 might reduce chronic inflammation and benefit aged organisms.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Protein concentrations were normalized using the BCA Protein Assay Kit.

    “Protein concentrations were normalized using the BCA Protein Assay Kit (Thermo Fisher Scientific, 23225).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Depletion of macrophages could protect mice from coronavirus-induced lethal infection.

    “the depletion of macrophages could protect mice from coronavirus-induced lethal infection.58”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [58] Channappanavar, R. et al. Dysregulated type I interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in SARS-CoV-infected mice. Cell Host Microbe 19, 181–193 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The present study was approved by the Research Ethics Committee of China-Japan Friendship Hospital and the Institute of Review Board in Peking University.

    “the present study was approved by the Research Ethics Committee of China-Japan Friendship Hospital (Ethical approval No: 2009-50) and the Institute of Review Board in Peking University (IRB 00001052-1508719070)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For the statistics of SA-β-gal-positive area of liver, the regions were randomly selected to be photographed.

    “For the statistics of SA-β-gal-positive area of liver, the regions were randomly selected to be photographed.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For the statistics of SA-β-gal-positive area of kidney, the regions of renal cortex were randomly selected to be photographed.

    “For the statistics of SA-β-gal-positive area of kidney, the regions of renal cortex were randomly selected to be photographed.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Interpretationreasoning

    These findings open a new avenue for the treatment of age-associated diseases and provide a clinical opportunity for intervention into the aging process.

    “These findings open a new avenue for the treatment of age-associated diseases and provide a clinical opportunity for intervention into the aging process.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The aged mouse model and functional assays are valid proxies for human age-related physical decline and clinical outcomes.
    • Rests on an assumption: Absence of apparent toxicity in mice at the tested doses predicts safety in humans.

  • May combine two claims.

    Premisecitation

    SA-β-gal-positive macrophages can be harmful and accumulate in injured and aged tissues contributing to chronic inflammation.

    “These SA-β-gal-positive macrophages can be harmful and have been found to accumulate in injured and aged tissues contributing to chronic inflammation.44,45”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [44] Hall, B. M. et al. Aging of mice is associated with p16(Ink4a)- and β-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells. Aging 8, 1294–1315 (2016). source

      Not checked in the free run.

    • cites [45] Oishi, Y. & Manabe, I. macrophages in age-related chronic inflammatory diseases. NPJ Aging Mech. Dis. 2, 16018 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    β-tubulin or β-actin protein level was determined as the loading control.

    “The β-tubulin or β-actin protein level was also determined as the loading control by using the β-tubulin antibody (Cell Signaling Technology, 2128) or β-actin antibody (Cell Signaling Technology, 4970).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    SA-β-gal is widely used as a marker of cellular senescence.

    “While SA-β-gal is widely used as a marker of cellular senescence,20–22”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [20] Childs, B. G., Durik, M., Baker, D. J. & van Deursen, J. M. Cellular senescence in aging and age-related disease: from mechanisms to therapy. Nat. Med. 21, 1424–1435 (2015). source

      Not checked in the free run.

    • cites [21] Sharpless, N. E. & Sherr, C. J. Forging a signature of in vivo senescence. Nat. Rev. Cancer 15, 397–408 (2015). source

      Not checked in the free run.

    • cites [22] Hernandez-Segura, A., Nehme, J. & Demaria, M. Hallmarks of cellular senescence. Trends Cell Biol. 28, 436–453 (2018). source

      Not checked in the free run.

57 of 57

  • The paper relies on this without support.

    The aged mouse model and functional assays are valid proxies for human age-related physical decline and clinical outcomes.

    “These findings open a new avenue for the treatment of age-associated diseases and provide a clinical opportunity for intervention into the aging process.”

  • The paper relies on this without support.

    Absence of apparent toxicity in mice at the tested doses predicts safety in humans.

    “This provides strong evidence for the in vivo safety of SSK1.”

  • The paper relies on this without support.

    Bleomycin induces lung senescence and fibrosis in mice.

    “To induce lung senescence and fibrosis, male and female mice (3–6-month-old) were subjected to transtracheal injection of bleomycin (1.5 mg/kg) as previously reported.68”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • 20-month-old mice treated for 8 weeks with SSK1 exhibited greater exercise capacity, coordination and balance, and muscle strength compared to vehicle-treated mice.

    “Similarly, 20-month-old mice treated for 8 weeks with an innovative SA-βgal-responsive prodrug, senescence-specific killing compound 1 (SSK1), which takes advantage of the heightened activity of this lysosomal enzyme in many senescent cell to promote drug specificity, exhibited greater exercise capacity, coordination and balance, and muscle strength compared to vehicle-treated mice (Cai et al., 2”

    From Skeletal muscle aging, cellular senescence, and senotherapeutics: current knowledge and future directions (2021) · cites this paper as Cai et al., 2020

  • The impact of SSK1 on features of the molecular and morphological phenotype of skeletal muscle or its resident cells were not assessed.

    “As above, the impact of the drug on features of the molecular and morphological phenotype of skeletal muscle or its resident cells were not assessed.”

    From Skeletal muscle aging, cellular senescence, and senotherapeutics: current knowledge and future directions (2021) · cites this paper as Cai et al., 2020

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Two separately labeled GLB1 shRNAs have identical sequences

    Minor

    The sequences listed for shGLB1#1 and shGLB1#3 are identical, despite being presented as separate knockdown reagents.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Zhu, Y. et al. New agents that target senescent cells: the flavone, fisetin, and the BCL-X-L inhibitors, A1331852 and A1155463. Aging 9, 955–963 (2017). · DOI · Paper page

Cited by

Sorted by citations

Related

Authors
Cai, Y., Zhou, H., Zhu, Y., Sun, Q., Ji, Y., Xue, A., Wang, Y., Chen, W., Yu, X., Wang, L., Chen, H., Li, C., Luo, T., & Deng, H.
Affiliations
Peking University (CN)
Corresponding author
  • Luo, Tuoping
  • Deng, Hongkui
ORCID
Venue
Cell Research
Publisher
Springer Science and Business Media LLC
Published
2020-04-27
Volume
30
Issue
7
Pages
574-589
ISSN
  • 1001-0602 (ISSN-L)
  • 1748-7838
DOI
10.1038/s41422-020-0314-9
PMC
PMC7184167
OpenAlex
W3019747905
Semantic Scholar
2b59b72c648e8a39f99b74719e621264c96b547f
Cited by
397
References
73
Citations by year
2026: 57 · 2025: 69 · 2024: 85 · 2023: 60 · 2022: 58 · 2021: 47 · 2020: 21
FWCI
28.11
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Research (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
National Natural Science Foundation of China (31521004)
Topics
  • Health Sciences › Medicine › Physiology › Telomeres, Telomerase, and Senescence
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Antioxidants, Aging, Portulaca oleracea
Keywords
cellular senescence, senolytics, lysosomal beta-galactosidase, prodrugs, SSK1, senescent cell clearance, low-grade inflammation, anti-aging interventions, aged mice
MeSH terms
Aging, Animals, beta-Galactosidase, Cell Survival, Disease Models, Animal, Fibroblasts, Humans, Inflammation, Mice, Inbred C57BL, Prodrugs, Drug Design, Cellular Senescence, Cell Death, Lung Injury

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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