Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2-independent mechanism
Open accessAbstract
Senescent cells contribute to age-related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild-type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA-β-galactosidase (β-gal) staining, senescence-associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased β-gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in β-gal staining and pro-inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP.
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Written from the paper’s full text.
TL;DR
Rapamycin inhibits senescence-associated secretory phenotype (SASP) and β-gal staining independently of Nrf2, while its effects on p16/p21 cell-cycle arrest markers require Nrf2. The paper proposes that cellular senescence has at least two separable arms—cell cycle arrest and the secretory phenotype—that can be regulated independently.
Key findings
- In wild-type mouse fibroblasts, rapamycin increased Nrf2, activated autophagy, and reduced senescence markers (p16, p21, β-gal, SASP); in Nrf2KO fibroblasts, rapamycin still decreased β-gal and SASP but did not activate autophagy or decrease p16/p21.
- In Nrf2KO mice treated with rapamycin (4 mg/kg i.p. every other day for 6 weeks), β-gal staining and pro-inflammatory cytokines decreased in fat tissue, but p16 levels were not significantly decreased; in lung tissue, rapamycin reduced p16 and p21 in WT but not Nrf2KO mice.
- Rapamycin decreased Stat3 activation in lung tissue from WT and Nrf2KO mice and in Nrf2-deficient WI38 cells, but not significantly in MEF cells.
- The authors conclude that rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP.
Methods
- In vitro models: stress-induced premature senescence (SIPS) in WT and Nrf2KO mouse embryonic fibroblasts and mouse skin fibroblasts using H2O2 (1 μM for MEF, 150 nM for skin fibroblasts) or bleomycin (1 μg/mL), and replicative senescence in human WI38 cells; rapamycin doses were 25 nM for MEF, 250 nM for skin fibroblasts, and 2 or 10 nM for WI38.
- Nrf2 manipulation: Nrf2KO MEFs, Nrf2 re-expression by transfection, and CRISPR/Cas9 knockout of NFE2L2 in WI38 cells.
- In vivo: 2-month-old WT and Nrf2KO mice (ICR background) received rapamycin 4 mg/kg i.p. every other day for 6 weeks; fat and lung tissues were analyzed.
- Main assays: SA-β-gal staining, qPCR, Western blotting, cytokine antibody array, BrdU incorporation, and one-way/two-way ANOVA or t-tests.
Limitations
- The paper does not include a dedicated limitations section; it states that the mechanism by which rapamycin increases Nrf2 is not completely elucidated and that SASP composition can vary by tissue and senescence-inducing insult.
- The text notes that rapamycin inhibits but does not reverse cellular senescence, based on similar β-gal staining in fat tissue between treated and control animals.
- Evident from the text: the study relies on mouse fibroblasts, WI38 cells, and short-term mouse tissue endpoints, with no lifespan or healthspan measurement; sex-specific effects are not analyzed despite groups containing male and female mice.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
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Cites
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Claims the paper makes
0 of 48 cited sources checked · 0 supported · 0 flagged
- Findingown data
Rapamycin decreased SASP protein levels in conditioned media from both WT and Nrf2KO MEFs, as measured by mouse cytokine array.
“using a mouse cytokine array (Fig. 3C), where rapamycin decreased significantly the SASP in cells and conditioned media from both WT and Nrf2KO MEFs”
Quote in the paperSubstantial claim - Findingown data
In WT MEFs, rapamycin inhibits H2O2-induced cellular senescence, shown by lower p16 and p21 levels.
“rapamycin inhibits cellular senescence induced by hydrogen peroxide in WT MEFs, as shown by lower levels of p16 and p21”
Quote in the paperSubstantial claim - Findingown data
In mouse skin fibroblasts, 24 h pre-incubation with rapamycin significantly decreased p16 and p21 molecular markers after H2O2-induced SIPS.
“24 h of pre‐incubation with rapamycin significantly decreased the levels of p16 and p21 molecular markers”
Quote in the paperSubstantial claim - Findingown data
In WT MEFs, rapamycin treatment correlates with lower levels of pH2AX, a marker of DNA damage.
“this correlates with lower levels of pH2AX, a marker of DNA damage”
Quote in the paperSubstantial claim - Findingown data
Rapamycin increased nuclear localization of Nrf2 in mouse skin fibroblasts.
“Activation of the Nrf2 pathway is further demonstrated by the levels of Nrf2 in the nuclear localization”
Quote in the paperNot a substantial claim
- Findingown data
In Nrf2KO MEFs, rapamycin did not affect p21 and p16 levels.
“no effects on p21 and p16 levels were observed in Nrf2KO MEFs”
Quote in the paperSubstantial claim - Findingown data
Rapamycin increased mRNA levels of Nrf2 target genes GST-Ya and NQO1 in mouse skin fibroblasts.
“by the increase in mRNA levels of down target genes such GST‐Ya and NQO1”
Quote in the paperSubstantial claim - Findingown data
Rapamycin activates autophagy only in WT cells but not in Nrf2KO MEFs, as measured by LC3I to LC3II interconversion and flux analysis.
“rapamycin activates autophagy only in WT cells but not in Nrf2KO MEFs, as measured by the interconversion of LC3I to LC3II and flux analysis”
Quote in the paperSubstantial claim - Findingown data
Re-expression of Nrf2 in Nrf2KO MEFs re-established the effect of rapamycin on p16, p21, and pH2AX expression.
“we were able to re‐establish the effect induced by rapamycin on p16, p21 and pH2AX expression”
Quote in the paperSubstantial claim - Findingown data
Rapamycin decreased SASP mRNA levels in both WT and Nrf2KO MEFs.
“The same results were observed when we measured the senescence‐associated secretory phenotype (SASP) by mRNA levels”
Quote in the paperSubstantial claim - Findingown data
Rapamycin treatment decreased the number of senescent cells in both WT and Nrf2KO MEFs, as measured by β-gal staining.
“rapamycin treatment was able to decrease the number of senescent cells in both WT and Nrf2KO MEFs”
Quote in the paperSubstantial claim - Findingown data
At basal state, MEFs deficient in Nrf2 have higher levels of senescent cells than WT cells, as measured by β-gal staining.
“MEFs deficient in Nrf2 have higher levels senescent cells than WT cells, as measured by β‐gal staining”
Quote in the paperNot a substantial claim - Findingown data
In mouse skin fibroblasts, rapamycin significantly decreased β-gal staining after H2O2-induced SIPS.
“as well as measured by the number of senescent cells measured by β‐gal staining”
Quote in the paperSubstantial claim - Findingown data
Rapamycin treatment activated autophagy in mouse skin fibroblasts, as measured by decreased p62 and increased LC3B-I to LC3B-II interconversion.
“rapamycin treatment also activated autophagy as measured by decreased levels in p62 and increased LC3B‐I to LC3B‐II interconversion”
Quote in the paperNot a substantial claim - Findingown data
In wild-type mouse fibroblasts, rapamycin lowered Keap1 levels.
“and lowered the levels of Keap1, the cytosolic inhibitor of the Nrf2 pathway”
Quote in the paperNot a substantial claim - Findingown data
The effect of rapamycin on autophagy in Nrf2KO MEFs was re-established when cells were transfected with Nrf2 expressing plasmid.
“this effect was re‐established when cells were transfected with the Nrf2 expressing plasmid”
Quote in the paperSubstantial claim - Findingown data
Knockdown of Nrf2 by CRISPR-Cas9 accelerates replicative senescence in WI38 cells compared to control cells transfected with noncoding gRNA.
“knocked down Nrf2 accelerates replicative senescence, as compared to control cells transfected with noncoding gRNA (NC)”
Quote in the paperSubstantial claim - Findingcitation
Inhibition of the inflammatory phenotype by rapamycin was correlated with inhibition of the Stat3 pathway in both cell and mouse models.
“This inflammatory status was attenuated by rapamycin treatment, and the inhibition of the inflammatory phenotype by rapamycin was correlated with an inhibition of the Stat3 pathway in both cell and mouse models (Xu et al., 2015).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Xu et al., 2015
Not checked in the free run.
- Findingown data
In WI38 model, Nrf2KO cells showed higher levels of senescence as measured by β-gal staining and cytokine levels.
“Nrf2KO cells again showed higher levels of senescence as measured by β‐gal staining and cytokine levels”
Quote in the paperSubstantial claim - Findingown data
No significant changes in the NF‐κB pathway were seen in tissues from Nrf2KO mice, measured by p65/p50 translocation.
“however, we did not see significant changes in the NF‐κB pathway in tissues from Nrf2KO mice (measured by p65/p50 translocation).”
Quote in the paperSubstantial claim - Findingown data
WI38 cells deficient in Nrf2 showed increased basal levels of p-Stat3.
“WI38 cells deficient in Nrf2, which showed increased basal levels of the phosphorylated form of Stat3 (p‐Stat3)”
Quote in the paperSubstantial claim - Findingown data
No significant change in p-Stat3 levels was observed in MEF cells with rapamycin.
“we did not observe a significant change when we used MEF cells”
Quote in the paperNot a substantial claim - Findingcitation
Rapamycin treatment attenuated the inflammatory status in Nrf2KO mice.
“This inflammatory status was attenuated by rapamycin treatment, and the inhibition of the inflammatory phenotype by rapamycin was correlated with an inhibition of the Stat3 pathway in both cell and mouse models (Xu et al., 2015).”
Quote in the paperSubstantial claim - Findingown data
Rapamycin treatment decreased p-Stat3 levels in WI38 cells deficient in Nrf2.
“rapamycin treatment was able to decrease these levels”
Quote in the paperSubstantial claim - Findingown data
In fat tissue from Nrf2KO mice at basal state, β-gal staining and proinflammatory cytokines (mRNA) were higher than WT controls.
“in fat tissue from Nrf2KO mice at basal state showed higher levels of β‐gal staining and proinflammatory cytokines (measured by mRNA) than WT controls”
Quote in the paperSubstantial claim - Findingown data
A significant decrease in pro-inflammatory cytokine levels was observed.
“We did observe a significant decrease in pro‐inflammatory cytokine levels, suggesting that another pro‐inflammatory pathway may be inhibited by rapamycin even in the absence of Nrf2.”
Quote in the paperSubstantial claim - Findingown data
Rapamycin treatment reduced p-Stat3 levels in lung tissue from Nrf2KO mice.
“rapamycin treatment reduced these levels”
Quote in the paperSubstantial claim - Findingown data
In lung tissue, rapamycin reduced p16 and p21 levels in WT but not in Nrf2KO mice.
“rapamycin reduced p16 and p21 levels in WT but not in Nrf2KO mouse”
Quote in the paperSubstantial claim - Findingown data
Tissue and plasma from Nrf2KO mice present higher levels of inflammation at 4 months of age.
“Our data show that tissue and plasma from Nrf2KO mice present higher levels of inflammation at 4 month of age (measured by the cytokine levels), corroborating previous reports (Ishii et al., 2005; Khor et al., 2008).”
Quote in the paperNot a substantial claim0 of 2 sources checked - Findingown data
Pre-incubation with rapamycin for 24 h before SIPS (H2O2) had better results in inhibiting cellular senescence than co-incubation with SIPS.
“This explains why pre‐incubation with rapamycin for 24 h before SIPS (H2O2) had better results in the inhibition of cellular senescence (Fig. S1, Supporting information) than co‐incubation with SIPS.”
Quote in the paperNot a substantial claim - Findingown data
Rapamycin treatment delayed replicative senescence in both control and Nrf2KO WI38 cells.
“Rapamycin treatment was able to delay the replicative senescence in both control and Nrf2KO WI38 cells”
Quote in the paperSubstantial claim - Findingown data
Rapamycin treatment decreased β-gal staining in fat tissue from both WT and Nrf2KO mice.
“rapamycin treatment decreased β‐gal staining in fat tissue from both WT and Nrf2KO mice”
Quote in the paperSubstantial claim - Findingown data
Rapamycin reduced the number of senescent cells in both WT and Nrf2KO WI38 cells, as measured by β-gal staining.
“rapamycin reduced the number of senescent cells in both WT and Nrf2KO WI38 cells”
Quote in the paperSubstantial claim - Findingown data
Rapamycin reduced pro-inflammatory cytokines mainly in Nrf2KO mice, but much less in WT mice (fat tissue).
“rapamycin reduced the pro‐inflammatory cytokines mainly in the Nrf2KO mice, but much less in WT mice”
Quote in the paperSubstantial claim - Findingown data
Levels of β‐gal staining in fat tissue are similar between rapamycin-treated animals and their controls.
“Our data also confirm that rapamycin inhibits but does not reverse cellular senescence, because the levels of β‐gal staining in fat tissue are similar between animals treated with rapamycin and their controls.”
Quote in the paperSubstantial claim - Findingown data
Lung tissue from Nrf2KO mice have increased basal levels of p-Stat3.
“tissue from Nrf2KO mice have increased basal levels of p‐Stat3”
Quote in the paperSubstantial claim - Findingown data
Rapamycin did not reduce significantly the levels of p16 (mRNA and immunohistochemistry) in fat tissue from Nrf2KO mice.
“rapamycin did not reduce significantly the levels of p16 (measured by mRNA and immunohistochemistry) in fat tissue from Nrf2KO mice”
Quote in the paperSubstantial claim - Findingown data
Rapamycin did not increase Nrf2 mRNA levels.
“rapamycin did not increase Nrf2 mRNA levels, but decreased Keap1, a protein that triggers Nrf2 degradation (Kobayashi et al., 2004).”
Quote in the paperSubstantial claim - Findingcitation
Rapamycin decreased Keap1.
“rapamycin did not increase Nrf2 mRNA levels, but decreased Keap1, a protein that triggers Nrf2 degradation (Kobayashi et al., 2004).”
Quote in the paperNot a substantial claim - Premisecitation
Rapamycin inhibits cell senescence in vitro.
“Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species.”
Quote in the paperSubstantial claim0 of 3 sources checked - Premisecitation
Inhibition of MK2 by rapamycin results in a decrease in cytokine expression.
“inhibition of MK2 by rapamycin results in a concomitant decrease in cytokine expression”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Herranz et al., 2015 source
Not checked in the free run.
- Premisecitation
mTOR activates translation of MAPKAPK2 (MK2).
“Herranz et al. showed that mTOR activates the translation of MAPKAPK2 (MK2), a Ser/Thr‐kinase downstream of the MAP kinase p38”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Herranz et al., 2015 source
Not checked in the free run.
- Interpretationown data
Inhibition of the SASP by rapamycin is independent of the Nrf2 pathway.
“In summary, our data show that inhibition of the SASP by rapamycin is independent of the Nrf2 pathway and does not require a cell cycle arrest (see our current model in Fig. S7, Supporting information).”
Quote in the paperSubstantial claim - Interpretationown data
Inhibition of the SASP by rapamycin does not require a cell cycle arrest.
“In summary, our data show that inhibition of the SASP by rapamycin is independent of the Nrf2 pathway and does not require a cell cycle arrest (see our current model in Fig. S7, Supporting information).”
Quote in the paperSubstantial claim - Interpretationown data
JAK/Stat could at least partially explain the inhibitory effect of rapamycin on SASP induction in Nrf2KO mice.
“These data suggest that JAK/Stat could at least partially explain the inhibitory effect of rapamycin on SASP induction in Nrf2KO mice.”
Quote in the paperSubstantial claim- Rests on an assumption: Low-grade sterile inflammation is linked to activation of the JAK/Stat pathway.
- Interpretationcitation
Inhibition of the SASP is dependent on mTOR activation in a manner that is independent of Nrf2.
“Based on recently published data, we believe that while cell cycle arrest is mediated by Nrf2 (and inhibited by rapamycin), the inhibition of SASP is dependent on mTOR activation in a manner that is independent of Nrf2.”
Quote in the paperSubstantial claim - Premisecitation
Translation of the mRNA for IL1α is highly dependent on mTOR activity.
“Similarly, the studies by Laberge et al. found that the translation of the mRNA for IL1α is highly dependent on mTOR activity.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Laberge et al.
Not checked in the free run.
- Interpretationown data
β-gal staining is linked to the SASP rather than cell cycle arrest.
“Our data also showed that β‐gal staining is linked to the SASP rather than cell cycle arrest.”
Quote in the paperSubstantial claim- Rests on an assumption: Concurrent occurrence of elevated p16 protein together with β-gal staining and SASP is a reliable marker of cell senescence.
- Interpretationown data
Another pro-inflammatory pathway may be inhibited by rapamycin even in the absence of Nrf2.
“We did observe a significant decrease in pro‐inflammatory cytokine levels, suggesting that another pro‐inflammatory pathway may be inhibited by rapamycin even in the absence of Nrf2.”
Quote in the paperSubstantial claim - Interpretationcitation
Inhibition of the SASP probably requires activation of MAPKAPK2.
“Inhibition of the SASP probably requires activation of MAPKAPK2, which would explain the decrease in pro‐inflammatory cytokines.”
Quote in the paperNot a substantial claim - Interpretationcitation
The mechanism by which rapamycin increases Nrf2 levels seems to occur at the level of protein stabilization rather than increasing Nrf2 gene expression.
“The actual mechanism by which rapamycin increases Nrf2 levels is not completely elucidated; however, this seems to occur at the level of protein stabilization rather than increasing Nrf2 gene expression because rapamycin did not increase Nrf2 mRNA levels, but decreased Keap1, a protein that triggers Nrf2 degradation (Kobayashi et al., 2004).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Kobayashi et al., 2004 source
Not checked in the free run.
- Interpretationown data
Rapamycin prevents cell senescence by reducing DNA damage.
“suggesting that rapamycin prevents cell senescence by reducing DNA damage”
Quote in the paperSubstantial claim - Interpretationown data
Rapamycin activates autophagy in a Nrf2-dependent manner.
“suggesting that rapamycin activates autophagy in a Nrf2‐dependent manner”
Quote in the paperSubstantial claim - Interpretationown data
Nrf2 is necessary for rapamycin to inhibit expression of senescent markers p16 and p21.
“Together, our data indicate that Nrf2 is necessary for rapamycin to inhibit expression of the senescent markers p16 and p21.”
Quote in the paperSubstantial claim - Premisecitation
Removal of senescent cells has some negative effects, such as in wound healing.
“However, because it has also been shown that removal of senescent cells has some negative effects, such as in wound healing (Demaria et al., 2014), it seems that suppression of the SASP by rapamycin, but without the actual removal of senescent cells may have broader beneficial effects as a therapy against age‐related diseases.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Demaria et al., 2014 source
Not checked in the free run.
- May combine two claims. 26
- The quote only partly states this claim. 1
- Classified as probably not a substantial claim. 28
May combine two claims.
FindingcitationIn WI38 human cells, rapamycin delays replicative senescence and inhibits cell cycle arrest.
“rapamycin delays replicative senescence and inhibit cell cycle arrest”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe effect of rapamycin on WI38 replicative senescence is dose-dependent and correlates with increased levels of Nrf2 and its downstream target gene NQO1, and a diminution in p16 levels.
“the effect of rapamycin is dose‐dependent, which correlates with increased levels of Nrf2 and its downstream target gene NQO1, and a diminution in p16 levels”
Quote in the paperNot a substantial claimThe quote only partly states this claim.
Findingown dataIn wild-type mouse fibroblasts, rapamycin increased Nrf2 levels in a dose-dependent manner.
“Pre‐incubation of mouse skin fibroblasts with rapamycin for 24 h increased the levels of Nrf2 in a dose‐dependent manner”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataIn WI38 cells, p16 and p21 levels decreased with rapamycin only in WT cells, not in Nrf2 knockdown cells.
“We observed a decreased in the levels of p16 and p21, only in WT cells, not in Nrf2 knockdown cells”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataRapamycin inhibited the NF‐κB pathway only in MEF from WT but not from Nrf2KO MEF.
“By measuring the levels of p65/p50 in the nucleus vs. cytosol (Fig. S6, Supporting information), we also observed that rapamycin inhibited the NF‐κB pathway only in MEF from WT but not from Nrf2KO MEF.”
Quote in the paperSubstantial claimMay combine two claims.
PremisecitationMK2 stabilizes mRNAs and upregulates expression of many cytokines.
“It has been observed that MK2 stabilizes the mRNAs and upregulate the expression of many cytokines”
Quote in the paperSubstantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationNrf2 levels decrease with aging.
“Studies have found an inverse correlation between aging and Nrf2 expression, both in mouse tissues and in vitro during replicative senescence”
Quote in the paperNot a substantial claim0 of 3 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationInhibition of Nrf2 by caveolin-1 promotes stress-induced premature senescence.
“inhibition of Nrf2 by caveolin‐1 promotes stress‐induced premature senescence”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Volonte et al., 2013 source
Not checked in the free run.
May combine two claims.
PremisecitationInhibition of mTOR by rapamycin selectively inhibits SASP and SA β-gal staining of senescent cells without affecting cell cycle arrest.
“inhibition of mTOR by rapamycin selectively inhibits the SASP and SA β‐gal staining of senescent cells without affecting their cell cycle arrest”
Quote in the paperSubstantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationSilencing the Nrf2 pathway in human embryonic fibroblasts leads to premature senescence.
“silencing the Nrf2 pathway in human embryonic fibroblasts leads to premature senescence”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Kapeta et al., 2010 source
Not checked in the free run.
May combine two claims.
PremisecitationInhibition of JAK pathway by JAK inhibitors rescues in part the SASP in senescent pre-adipocytes in vivo, correlated with reduction in phosphorylation of Stat3.
“inhibition of the JAK pathway by JAK inhibitors (Xu et al., 2015) rescues in part the SASP in senescent pre‐adipocytes in vivo. This effect was correlated with a significant reduction in the phosphorylation of Stat3.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Xu et al., 2015
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationNrf2 is a key mediator of the beneficial effects of calorie restriction.
“Nrf2 is a pro‐longevity signaling pathway (Harman, 2006) and also a key mediator of the beneficial effects of calorie restriction.”
Quote in the paperNot a substantial claim0 of 3 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe Stat3 pathway is linked to production of SASP.
“Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Xu et al., 2015
Not checked in the free run.
Classified as probably not a substantial claim. May combine two claims.
PremisecitationSenescent cells contribute to age-related pathology and loss of function.
“Senescent cells contribute to age‐related pathology and loss of function, and their selective removal improves physiological function and extends longevity.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Baker et al., 2011, 2016
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationSelective removal of senescent cells improves physiological function and extends longevity.
“Senescent cells contribute to age‐related pathology and loss of function, and their selective removal improves physiological function and extends longevity.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Baker et al., 2011, 2016
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationIn C. elegans and Drosophila, Nrf2 activation induces a significant increase in longevity.
“In both C. elegans and Drosophila, Nrf2 activation induces a significant increase in longevity”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites Sykiotis & Bohmann, 2008
Not checked in the free run.
- cites Tullet et al., 2008 source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationNrf2−/− MEFs are prone to immortalization but also show a short lifespan.
“Nrf2−/− MEFs were prone to immortalization but also showed a short lifespan”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Jodar et al., 2011 source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationNrf2 is a pro-longevity signaling pathway.
“Nrf2 is a pro‐longevity signaling pathway (Harman, 2006) and also a key mediator of the beneficial effects of calorie restriction.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Harman, 2006 source
Not checked in the free run.
Classified as probably not a substantial claim.
PremisecitationSilencing Nrf2 gene induces premature senescence.
“silencing Nrf2 gene induces premature senescence”
Quote in the paperSubstantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationIn C. elegans, resveratrol, metformin, and rapamycin activate Nrf2 signaling as part of their mechanism of lifespan extension.
“in C. elegans, drugs believed to be ‘calorie restriction mimetics’ (resveratrol, metformin, and rapamycin) activate the Nrf2 signaling pathway as part of their mechanism of lifespan extension”
Quote in the paperNot a substantial claim0 of 3 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationSA β‐gal staining is associated with an increase in the mTOR pathway and activation of lysosome activity, but is not necessarily linked to activation of autophagy.
“Although it is not known how the inhibition of mTOR ameliorates the SA β‐gal, some evidence suggests that SA β‐gal staining is associated with an increase in the mTOR pathway and activation of lysosome activity, but it is not necessary linked to an activation of autophagy, which seems to be down regulated in cell senescence (Pena‐Llopis & Brugarolas, 2011).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Pena‐Llopis & Brugarolas, 2011
Not checked in the free run.
May combine two claims.
InterpretationreasoningRapamycin may suppress cellular senescence and uncouple it from cell division.
“This can explain why Nrf2KO mice (that have high levels of oxidative stress and abnormal mitochondria) showed enhanced p16/p21 pathway and also suggest a new mechanism by which rapamycin may suppress cellular senescence and uncouple it from cell division.”
Quote in the paperSubstantial claim- Rests on an assumption: Nrf2KO mice have high levels of oxidative stress and abnormal mitochondria.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationAutophagy seems to be downregulated in cell senescence.
“Although it is not known how the inhibition of mTOR ameliorates the SA β‐gal, some evidence suggests that SA β‐gal staining is associated with an increase in the mTOR pathway and activation of lysosome activity, but it is not necessary linked to an activation of autophagy, which seems to be down regulated in cell senescence (Pena‐Llopis & Brugarolas, 2011).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Pena‐Llopis & Brugarolas, 2011
Not checked in the free run.
May combine two claims.
Interpretationown dataRapamycin required Nrf2 to mediate the inhibition of cell cycle arrest, but did not require Nrf2 to inhibit SASP and β-gal staining.
“We showed that rapamycin required Nrf2 to mediate the inhibition of cell cycle arrest, but did not require the Nrf2 to inhibit the SASP and β‐gal staining.”
Quote in the paperSubstantial claim- Rests on an assumption: Concurrent occurrence of elevated p16 protein together with β-gal staining and SASP is a reliable marker of cell senescence.
Probably not a substantial claim: may not pass S2 (checkable).
InterpretationcitationSuppression of the SASP by rapamycin without the actual removal of senescent cells may have broader beneficial effects as a therapy against age-related diseases.
“However, because it has also been shown that removal of senescent cells has some negative effects, such as in wound healing (Demaria et al., 2014), it seems that suppression of the SASP by rapamycin, but without the actual removal of senescent cells may have broader beneficial effects as a therapy against age‐related diseases.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Demaria et al., 2014 source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationIncreased levels of Nrf2 can directly activate transcription of the mTOR gene and activate the PI3K signaling pathway, but only in cells with normal physiological PI3K activity.
“However, recently an interesting study has shown that increased levels of Nrf2 can directly activate transcription of the mTOR gene and activate the PI3K signaling pathway. However, this happens only in cells that have a normal physiological PI3K activity (Bendavit et al., 2016).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Bendavit et al., 2016 source
Not checked in the free run.
May combine two claims.
Interpretationown dataCell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP.
“Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP.”
Quote in the paperSubstantial claim- Rests on an assumption: Concurrent occurrence of elevated p16 protein together with β-gal staining and SASP is a reliable marker of cell senescence.
Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).
InterpretationreasoningThe mTOR pathway is involved in a very complex network, which will change depending on the metabolic state of the cells.
“All these studies confirm that the mTOR pathway is involved in a very complex network, which will change depending on the metabolic state of the cells.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataInhibition of β-gal-positive cells and the inflammatory phenotype (SASP) by rapamycin is Nrf2 independent, while inhibition of p16 and p21 induction is Nrf2 dependent.
“Together, these data suggest that the inhibition of both β‐gal‐positive cells and the inflammatory phenotype that escorts cellular senescence are Nrf2 independent, while the inhibition of the induction of p16 and p21 is Nrf2 dependent.”
Quote in the paperSubstantial claim- Rests on an assumption: Concurrent occurrence of elevated p16 protein together with β-gal staining and SASP is a reliable marker of cell senescence.
May combine two claims.
InterpretationcitationRapamycin can prevent the induction of cell senescence by either genotoxic agents or by replicative exhaustion.
“Our study confirmed that rapamycin can prevent the induction of cell senescence by either genotoxic agents or by replicative exhaustion”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe SASP can vary significantly in terms of the quantity and quality of secreted proteins and other factors, depending on the insult used to trigger cell senescence.
“However, we must to understand that the SASP can vary significantly in terms of the quantity and quality of secreted proteins and other factors, depending on the insult used to trigger cell senescence (Maciel‐Baron et al., 2016; Wiley et al., 2016); therefore, the composition of the SASP may be vary depending on the tissue and the insult.”
Quote in the paperNot a substantial claim0 of 2 sources checkedMay combine two claims.
Interpretationown dataRapamycin inhibits but does not reverse cellular senescence.
“Our data also confirm that rapamycin inhibits but does not reverse cellular senescence, because the levels of β‐gal staining in fat tissue are similar between animals treated with rapamycin and their controls.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationNrf2 is repressed by the mTOR pathway; therefore, inhibition of mTOR by rapamycin would increase the activity of Nrf2.
“Based on previous studies in the aging field, it is believed that Nrf2 is repressed by mTOR pathway (downstream target); therefore, an inhibition of mTOR by rapamycin would increase the activity of Nrf2 (Johnson et al., 2013).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Johnson et al., 2013 source
Not checked in the free run.
May combine two claims.
Interpretationown dataRapamycin was able to reduce SASP and β-gal staining, but not cell cycle arrest in Nrf2KO mice.
“Again, rapamycin was able to reduce SASP and β‐gal staining, but not cell cycle arrest in this animal model.”
Quote in the paperSubstantial claim- Rests on an assumption: Concurrent occurrence of elevated p16 protein together with β-gal staining and SASP is a reliable marker of cell senescence.
Probably not a substantial claim: may not pass S3 (load-bearing).
InterpretationcitationThe composition of the SASP may vary depending on the tissue and the insult.
“However, we must to understand that the SASP can vary significantly in terms of the quantity and quality of secreted proteins and other factors, depending on the insult used to trigger cell senescence (Maciel‐Baron et al., 2016; Wiley et al., 2016); therefore, the composition of the SASP may be vary depending on the tissue and the insult.”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationcitationCell cycle arrest and the SASP are not coupled and can be regulated independently.
“These findings are novel and imply that in cell senescence, cell cycle arrest and the SASP are not coupled and therefore can be regulated independently.”
Quote in the paperSubstantial claim- Rests on an assumption: Concurrent occurrence of elevated p16 protein together with β-gal staining and SASP is a reliable marker of cell senescence.
May combine two claims.
InterpretationreasoningCell senescence involves at least two arms, cell cycle arrest and the secretory phenotype, and these can be regulated independently at many levels.
“Therefore, cell senescence is a very complex process that involves at least two arms, cell cycle arrest and the secretory phenotype, and these can be regulated independently at many levels (from transcriptional to paracrine/autocrine signaling pathways).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationReduction in mitochondrial content by mTORC1 inhibition prevents cellular senescence.
“Interestingly, they showed that the reduction in mitochondrial content by mTORC1 inhibition prevents cellular senescence.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Correia‐Melo et al. (2016) source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.
Interpretationnone givenCell cycle arrest is mediated by Nrf2 and inhibited by rapamycin.
“Based on recently published data, we believe that while cell cycle arrest is mediated by Nrf2 (and inhibited by rapamycin), the inhibition of SASP is dependent on mTOR activation in a manner that is independent of Nrf2.”
Quote in the paperNot a substantial claimMay combine two claims.
PremisecitationIL1α, when expressed and secreted, triggers transcription of NF‐κB in both paracrine and autocrine fashions, which activates multiple cytokine-encoding genes.
“When IL1α is expressed and secreted, it triggers the transcription of the master inflammatory transcription factor NF(B in both paracrine and autocrine fashions, which ultimately will activate multiple cytokine‐encoding genes.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Laberge et al.
Not checked in the free run.
Classified as probably not a substantial claim. May combine two claims.
PremisecitationDNA damage response induced by stress or cell replication activates mitochondrial biogenesis, which mediates cell cycle arrest.
“Recently, Correia‐Melo et al. (2016) reported that DNA damage response (DDR) [induced by stress (i.e., ROS) or cell replication] activates mitochondrial biogenesis which will mediate a cell cycle arrest.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Correia‐Melo et al. (2016) source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataCells were verified to be GFP positive with 80% transfection efficiency 24 h following transfection.
“24 h following transfection cells were verified to be GFP positive (80% transfection efficiency)”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenThis study received prior approval from Oregon State University Institutional Animal Care and Use Committee.
“This study received prior approval from Oregon State University Institutional Animal Care and Use Committee.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAll in vitro experiments were repeated at least three times.
“All in vitro experiments were repeated at least three times.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim. May combine two claims.
PremisecitationThe secretory phenotype of senescent cells is a major starting point in chronic sterile inflammation that correlates with several age-related diseases, as well as the anti-aging effects observed upon removal of senescent cells.
“This is important because it is believed that the secretory phenotype of senescent cells is a major starting point in the chronic sterile inflammation that correlates with several age‐related diseases, as well as the anti‐aging effects observed upon removal of senescent cells (Baker et al., 2016).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Baker et al., 2016 source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductcitationThe β‐gal assay was performed in subconfluent cultures displaying comparable cell density to avoid staining due to cell confluence rather than proliferative senescence.
“To avoid staining due to cell confluence rather than to proliferative senescence (Severino et al., 2000), the assay was performed in subconfluent cultures displaying comparable cell density.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Severino et al., 2000 source
Not checked in the free run.
The paper relies on this without support.
Concurrent occurrence of elevated p16 protein together with β-gal staining and SASP is a reliable marker of cell senescence.
“concurrent occurrence of elevated levels of p16 protein together with β‐gal staining and SASP has been widely used as reliable markers of cell senescence”
The paper relies on this without support.
Low-grade sterile inflammation is linked to activation of the JAK/Stat pathway.
“Evidence from the literature has shown that low grade, sterile inflammation is linked to activation of the JAK/Stat pathway.”
The paper relies on this without support.
Nrf2 regulates NF‐κB activation.
“This is consistent with the notion that Nrf2 regulates NF‐κB activation; however, we did not see significant changes in the NF‐κB pathway in tissues from Nrf2KO mice (measured by p65/p50 translocation).”
The paper relies on this without support.
SA β‐gal is more closely linked to the SASP than to cell cycle arrest.
“Several recent observations also demonstrated that SA β‐gal is more closely linked to the SASP than to cell cycle arrest.”
The paper relies on this without support.
Nrf2KO mice have high levels of oxidative stress and abnormal mitochondria.
“This can explain why Nrf2KO mice (that have high levels of oxidative stress and abnormal mitochondria) showed enhanced p16/p21 pathway and also suggest a new mechanism by which rapamycin may suppress cellular senescence and uncouple it from cell division.”
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?
Accumulation of senescent cells in tissues contributes to age-related diseases.
“Accumulation of senescent cells in tissues contributes to age-related diseases [[3], [4], [5], [6], [7]].”
From Fibrates as drugs with senolytic and autophagic activity for osteoarthritis therapy (2019) · cites this paper as [3]
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.
Discussion reverses the reported effect on fat-tissue β-gal staining
MajorThe Results say rapamycin decreased fat-tissue β-gal staining in both WT and Nrf2KO mice. The Discussion instead says staining was similar in treated animals and their controls, and uses that claim to support its conclusion that rapamycin does not reverse senescence.
“Our data also confirm that rapamycin inhibits but does not reverse cellular senescence, because the levels of β‐gal staining in fat tissue are similar between animals treated with rapamycin and their controls.”
“We observed that rapamycin treatment decreased β‐gal staining in fat tissue from both WT and Nrf2KO mice;”
Figure 5D is identified as lung rather than fat tissue
MinorThe Results cite Figure 5D for p16 measurements in fat tissue, but the Figure 5 caption identifies panel D as protein measurements in lung tissue.
“In contrast, rapamycin did not reduce significantly the levels of p16 (measured by mRNA and immunohistochemistry) in fat tissue from Nrf2KO mice (Fig. 5C,D).”
“(D) p16 and p21 protein levels in lung tissues from WT and Nrf2KO mice with or without rapamycin treatment.”
Figure 2 caption reverses the stated direction of the cell-cycle effect
MinorThe Figure 2 title describes rapamycin-induced cell cycle arrest, whereas the Discussion says rapamycin inhibits cell cycle arrest through Nrf2.
“Figure: Figure 2 Rapamycin‐induced cell cycle arrest and autophagy activation is dependent on Nrf2.”
“We showed that rapamycin required Nrf2 to mediate the inhibition of cell cycle arrest, but did not require the Nrf2 to inhibit the SASP and β‐gal staining.”
Statistical key assigns two thresholds to the # symbol
MinorFigure 1 defines # as P < 0.001, while the Statistics section defines it as P < 0.01. The two keys give different interpretations for results marked #.
“Statistical significance is indicated at *P < 0.05 **P < 0.01 and # P < 0.001.”
“Data are presented as mean ± SEM, and statistical significance is indicated at P‐value less than *P < 0.05; **P < 0.01; and #P < 0.01.”
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“However, we must to understand that the SASP can vary significantly in terms of the quantity and quality of secreted proteins and other factors, depending on the insult used to trigger cell senescence (Maciel-Baron et al., 2016; Wiley et al., 2016); therefore, the composition of the SASP may be vary depending on the t…”
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Elsayed, R., Elashiry, M., Liu, Y., Morandini, A. C., El-Awady, A., Elashiry, M. M., Hamrick, M., & Cutler, C. W. (2023) · Aging and disease · doi:10.14336/ad.2022.0623 · Cited by 40 · see citing works
- Authors
- Wang, R., Yu, Z., Sunchu, B., Shoaf, J., Dang, I., Zhao, S., Caples, K., Bradley, L., Beaver, L. M., Ho, E., Löhr, C. V., & Perez, V. I.
- Affiliations
- Oregon State University (US)
- Linus Pauling Institute; Oregon State University; Corvallis OR 97331 USA
- ORCID
- Venue
- Aging Cell
- Publisher
- Wiley
- Published
- 2017-03-31
- Volume
- 16
- Issue
- 3
- Pages
- 564-574
- ISSN
- 1474-9718 (ISSN-L)
- PMC
- PMC5418203
- OpenAlex
- W2604477713
- Semantic Scholar
- d07eb6b731e16bdf0c9353fd572ed07226a20ac7
- Cited by
- 360
- References
- 38
- Citations by year
- 2026: 43 · 2025: 41 · 2024: 45 · 2023: 40 · 2022: 57 · 2021: 38 · 2020: 43 · 2019: 23 · 2018: 25 · 2017: 5
- FWCI
- 19.42
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Aging Cell (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, other-oa)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- American Federation for Aging Research
- The Linus Pauling Institute
- Biochemistry and Biophysics Department
- Oregon State University
- Topics
- Health Sciences › Medicine › Physiology › Telomeres, Telomerase, and Senescence
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
- Health Sciences › Medicine › Geriatrics and Gerontology › Sirtuins and Resveratrol in Medicine
- Keywords
- rapamycin, Nrf2, cellular senescence, senescence-associated secretory phenotype, mTOR inhibition, autophagy, STAT3 signaling, p16, p21, Nrf2 knockout mice, fibroblasts, aging, longevity, inflammatory cytokines, cell cycle arrest
- MeSH terms
- Aging, Animals, Autophagy, beta-Galactosidase, Female, Fibroblasts, Gene Expression Regulation, Humans, Male, Phenotype, Signal Transduction, Cytokines, Cellular Senescence, Mice, Knockout, Cyclin-Dependent Kinase Inhibitor p16, Sirolimus, Cell Proliferation, Cyclin-Dependent Kinase Inhibitor p21, STAT3 Transcription Factor, NF-E2-Related Factor 2, Mice, TOR Serine-Threonine Kinases, Primary Cell Culture
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