Urolithin A reduces aging caused by Doxorubicin in stem cells that support tissue repair

To determine whether urolithin A protects AD-hMSCs from doxorubicin-induced damage or exerts senolytic activity, we assessed cell proliferation using the Cell Counting Kit-8 (CCK-8) assay. Doxorubicin treatment significantly reduced proliferation, indicating cell cycle arrest. Urolithin A, however, had no effect on proliferation in either control or doxorubicin-treated cells and did not induce cell death in either condition. AD-hMSC pretreatment with urolithin A also failed to prevent doxorubicin-induced cell cycle arrest (Figure 1A). Western blot analysis showed that post-treatment with urolithin A increased the expression of p21, a checkpoint inhibitor and senescence marker (Figure 1B,C). These findings suggest that urolithin A neither reverses senescence-associated cell cycle arrest nor exhibits senolytic activity in this model.

One of the hallmarks of cellular senescence is a deregulated metabolic profile, which encompasses mitochondrial and lysosomal dysfunction [11]. In this regard, senescent cells are commonly defined by the enhanced activity of senescence-associated beta-galactosidase (SA-βGal), an enzyme present in dysfunctional lysosomes with an altered pH.

X-gal staining of AD-hMSCs revealed minimal SA-βGal activity in healthy cells, whereas doxorubicin treatment markedly increased β-galactosidase activity. Treatment of senescent AD-hMSCs with urolithin A did not attenuate the doxorubicin-induced elevation in SA-βGal activity (Figure 2). These data indicate that urolithin A does not mitigate the doxorubicin-associated increase in lysosomal β-galactosidase activity under these conditions.

Senescent cells, in addition to cell cycle arrest and dysregulated metabolism, are characterized by the acquisition of the senescence-associated secretory phenotype (SASP) formation [11]. SASP is commonly composed of various pro-inflammatory cytokines, chemokines, and matrix metalloproteinases. The detrimental effects of senescent cells are largely attributed to SASP, which sustains low-grade chronic inflammation and thereby promotes age-associated pathologies, including 'inflammaging' [12].

An emerging class of anti-senescence agents, termed «senomorphics», has been identified. In contrast to senolytics, which preferentially eliminate senescent cells by inducing cell death, senomorphics mitigate the deleterious effects of senescent cells, primarily by suppressing or reprogramming SASP [13].

Interleukin 1 beta (IL1B) and monocyte chemoattractant protein-1 (MCP1) are widely recognized as prominent components of the SASP and are included in the SenMayo gene set—a curated collection of 125 genes linked to the SASP [14]. This gene set has been validated in human bone biopsies and murine models, underscoring its value in detecting senescence and evaluating senescence-associated mechanisms. Additionally, plasminogen activator inhibitor 2 (PAI2) has been implicated in pro-inflammatory responses and the pathophysiology of cellular senescence [15,16].

To investigate whether urolithin A exhibits senomorphic activity, we performed RT-PCR and multiplex cytokine assay to evaluate its impact on key pro-senescent factors. First of all, we performed a brief RT-PCR analysis and demonstrated that urolithin A effectively reduced doxorubicin-mediated increases in pro-inflammatory factors IL1B, MCP1, and PAI2 (Figure 3).

To further evaluate the potential senomorphic activity of urolithin A, we profiled conditioned media collected from AD-hMSCs using a multiplex cytokine assay. Urolithin A attenuated the doxorubicin-induced increases in eotaxin, FMS-like tyrosine kinase 3 ligand (FLT3L), interleukin-6 (IL6), tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β; lymphotoxin-α/LTA), and MCP-1 (Figure 4). IL6, TNF-α, TNF-β, and MCP1 are well-established SASP components and are included in the SenMayo gene set [14].

Consistent with their roles in pro-inflammatory signaling, eotaxin and FLT3L have also been implicated in pathophysiology of cellular senescence [17,18]. Together with the transcriptional data, these findings further support a senomorphic effect of urolithin A in doxorubicin-treated AD-hMSCs.

Cellular senescence is accompanied by extensive chromatin remodeling, including the formation of senescence-associated heterochromatin foci (SAHF) and changes in the epigenetic landscape [19]. Reduced levels of histone methylation marks, such as H3K4me3 and H3K9me3, are among the distinguishing features reported in senescent cells [20]. In our model, we observed that doxorubicin significantly decreased H3K9me3 levels. Notably, treatment of senescent in AD-hMSCs with urolithin A restored H3K9me3 to levels comparable to those in untreated cells (Figure 5). These findings suggest that urolithin A may exert its anti-senescent effects, at least in part, through modulation of chromatin state.

Autophagy is a highly conservative fundamental cellular process that plays a dual role in the senescence pathophysiology. In particular, autophagy counteracts ER-stress and facilitates the production of SASP factors [21].

We evaluated autophagic status by Western blotting for sequestosome 1 (SQSTM1)—an adaptor protein that links ubiquitinated cargo to the autophagosome membrane—and by quantifying the ratio of microtubule-associated protein 1 light chain 3 isoforms (LC3-II/LC3-I), a commonly used marker of autophagosome maturation. Surprisingly, doxorubicin-treated AD-hMSCs exhibited changes consistent with reduced autophagic activity (Figure 6), namely, elevated SQSTM1 levels (Figure 6B) and a decreased LC3-II/LC3-I ratio (Figure 6C).

Given previous reports that urolithin A activates mitophagy, which, in turn, led to various beneficial effects [22], we next investigated whether it could restore autophagic activity in doxorubicin-treated senescent AD-hMSCs. However, urolithin A did not reverse the doxorubicin-induced impairment of autophagy, as neither SQSTM1 levels nor the LC3-II/LC3-I ratio were significantly altered by treatment (Figure 6).

These data suggest that urolithin A does not restore general autophagy in a model of doxorubicin-induced senescence.

Human adipose-derived mesenchymal stem cells (AD-hMSCs) were obtained from the human biomaterials collection of the Institute of Regenerative Medicine (Lomonosov Moscow State University, collection ID: MSU_MSC_AD; repository catalog at https://human.depo.msu.ru/↗ (accessed on 8 August 2025). AD-hMSCs were cultured in DMEM-GlutaMAX^TM medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Cytiva, Marlborough, MA, USA) and 1x antibiotic-antimycotic solution (Biowest, Nuaillé, France) and cultivated under standard conditions (5% CO₂, 37 °C). Cellular senescence was induced by treatment of AD-hMSCs with 500 nM doxorubicin (BLDpharm, Shanghai, China). Urolithin A (10 μM) was added for one day following a one-week period after the wash off of doxorubicin (post-treatment model).

To assess AD-hMSCs' proliferation rate, we used cell counting kit 8 (CCK8; Dojindo, Kumamoto, Japan). AD-hMSCs were seeded with 4000 cells per well in a 96-well plate. In the case of the pre-treatment model, cells were treated with 10 μM urolithin A (BLDpharm, Shanghai, China) for 1 day. The next day, the senescence induction was performed by two days of treatment of the cells with doxorubicin. Cells were incubated for 1 week after washing off the senescence inducer. As for the post-treatment model, the cells were then treated with 10 μM urolithin A (BLDpharm, Shanghai, China) for 1 day. After that, referring to the manufacturer's protocol, 10 μL/well CCK8 reagent was added to the cells. After incubation at 37 °C for 4 h. Optical density values in 96-well plates were measured at 450 nm in Victor™ X3 Multi Label Plate Reader (Perkin Elmer, Waltham, MA, USA). The 10 µM dose of urolithin A was selected following CCK-8 viability assays conducted across 10–100 µM, which confirmed it to be non-cytotoxic and suitable for downstream applications (). Supplementary Materials, Figure S1

SA-βGal staining was performed as described by Debacq-Chainiaux et al. [29]. Briefly, AD-hMSCs were washed twice with PBS and fixed in formaldehyde-glutaraldehyde solution. Then the cells were double-washed and stained in the mix of pH buffer with X-gal overnight at 37 °C in a CO₂-free incubator. After incubation, cells were washed twice with PBS and once with methanol, air-dried, and imaged by bright-field microscopy using an AxioObserver A2 inverted microscope (Carl Zeiss, Oberkochen, Germany). Five random images were taken for each donor within a single condition. SA-βGal staining intensity was assessed using ImageJ software version 1.54p.

AD-hMSCs were lysed in RIPA buffer with protease inhibitors (Protease Inhibitor Cocktail, HY-K0010, MedChemExpress, Junction, NJ, USA) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail II, HY-K0022, MedChemExpress, Junction, NJ, USA). Samples were resolved by 15% SDS-PAGE and electrotransferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). After blocking with 5% non-fat milk in TBST for 1 h, the membrane was incubated overnight at 4 °C with primary antibodies: anti-betaActin (#AC-15, Abcam, Cambridge, MA, USA); anti-SQSTM1 (#A19700, Abclonal, Wuhan, China); anti-LC3B (#A19665, Abclonal, Wuhan, China); and anti-p21 (#A22460, Abclonal, Wuhan, China). Appropriate horseradish peroxidase-conjugated secondary antibodies were purchased from Invitrogen, USA. Proteins were visualized using SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) by enhanced chemiluminescence in Fusion FX gel-documentation system (Vilber-Lourmat, Collégien, France) and quantified in Image Lab 6.0.1 software (BioRad, Hercules, CA, USA). Western blot data were analyzed following the Degasperi's protocol [30].

Total RNA was extracted using a RNeasy Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer's protocol. RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). First-strand cDNA was synthesized from one microgram of total RNA using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's protocol. RT-PCR was performed on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The reaction mixture contained a SYBR Green PCR Master Mix (Evrogen, Moscow, Russia), primers (10 pmol of each dNTP), and cDNA (1–5 ng cDNA). The control mixture contained all components, with the exception of the matrix, replaced by de-ionized water. After the initial denaturation stage (95 °C, 10 min), 40 amplification cycles were performed for all primer pairs with annealing and elongation at 60 °C for 60 s. The primer sequences (Evrogen, Moscow, Russia) are shown in Table 1. mRNA levels between probes were normalized by the mRNA levels of beta-actin (ACTB) as a housekeeping gene, and the 2^−ΔΔCt method was used to calculate the relative expression levels of genes. RT-qPCR using primers for TNFa, IL6 and TNFb genes was performed to validate multiplex immunoassay data (Supplementary Materials, Figure S2).

To assess cytokine secretion, doxorubicin-treated AD-hMSCs were treated with urolithin A (10 µM; BLDpharm, Shanghai, China) for 24 h. Cells were then washed three times with culture medium, and conditioned media were collected 24 h post-treatment. Supernatants were clarified by centrifugation (400× g, 5 min, then 2000× g, 10 min) and stored at −80 °C until analysis. The cytokines quantification in the conditioned media was performed using the MILLIPLEX MAP 41 Human Cytokine/Chemokine Magnetic Bead Panel (HCYTMAG-60K-PX4141, Merck, Darmstadt, Germany) in strict adherence to the manufacturer's guidelines. Initially, protein standards were serially diluted. Then, 25 µL aliquots of each standard dilution, control mixes (supplied by the manufacturer), and conditioned media samples were pipetted into a 96-well assay plate in duplicate. This was followed by the addition of fluorescent magnetic beads conjugated with cytokine-specific capture antibodies into each well. The plate was sealed and subjected to an overnight incubation at 4 °C with gentle shaking. Post-incubation, the plate was washed three times to remove unbound substances. Subsequently, a biotinylated detection antibody was added to each well and incubated for 1 h at room temperature. After a series of washes, streptavidin phycoerythrin was added, and the plate was further incubated for 30 min. Fluorescence detection and quantification were conducted using the MAGPIX^® System (Luminex-Merck, Darmstadt, Germany). The acquired data were analyzed utilizing xPONENT software 4.3.229.0, and cytokine concentrations in the conditioned media were extrapolated from the standard curves.

AD-hMSCs were grown on glass coverslips and incubated overnight with urolithin A. Cells were then fixed with 4% formaldehyde in PBS for 10 min at room temperature (RT), followed by permeabilization in PBS containing 0.25% Triton X-100 for 10 min at RT. After permeabilization, samples were blocked with 10% donkey serum and 3% BSA in PBS for 1 h at RT to prevent nonspecific binding. Coverslips were then incubated with primary antibodies against H3K9me3 (Novus Biologicals, Centennial, CO, USA) or isotype-matched controls, diluted 1:100 in 1% BSA/PBS, overnight at 4 °C. After washing three times with PBS, samples were incubated for 1 h at RT with appropriate Alexa Fluor^®-conjugated secondary antibodies donkey anti-mouse Alexa Fluor^® 594 (Thermo Fisher Scientific, USA) diluted 1:1000 in 1% BSA/PBS. Finally, nuclei were counterstained with DAPI (1 µg/mL, 10 min at RT), and coverslips were rinsed with PBS and mounted in Vectashield^® Antifade Mounting Medium (Vector Laboratories, Newark, CA, USA). Immunofluorescence images were acquired using a Stellaris 5 inverted confocal microscope (Leica Microsystems, Wetzlar, Germany). To quantify fluorescence signals, we used ImageJ software, and the corrected total cell fluorescence was calculated according to the protocol described by Gavet and Pines [31].

All the graphs, calculations, and statistical analyses were performed using GraphPad Prism software version 10.3.1 for Microsoft Windows (GraphPad Software, San Diego, CA, USA). To test whether the collected numerical data are normally distributed, the Shapiro–Wilk normality test was applied. The comparison of means between different groups of numerical variables was performed using one-way ANOVA. Homogeneity of variances was tested using Brown–Forsythe and Bartlett's tests, and in the case of unequal SDs, Brown–Forsythe and Welch ANOVA test was applied. All data were distributed normally, results are expressed as mean and standard deviation (SD) as dispersion characteristics, and p value less than 0.05 (p < 0.05) was considered as statistically significant.