Microwave-treated propolis extract reduces cell aging and stress in human skin cells by activating protective and growth pathways

Raw Propolis Material: Raw propolis was obtained from the Jeju Honey Agricultural Association Corporation (Jeju-si, Republic of Korea). All samples were stored in a desiccated state at 4 °C until further processing.

Microwave-Assisted Extraction (MAE): MAPE, a standardized Jeju propolis extract, was prepared by microwave-assisted extraction to maximize the recovery of bioactive polyphenols while minimizing thermal degradation. Ground propolis (25 g) was mixed with butylene glycol at a solvent-to-solid ratio of 20:1 (w/w) in a borosilicate beaker. The mixture was subjected to microwave irradiation using a microwave system (LG Electronics, Seoul, Republic of Korea) at a power of 1000 W for 30 s, followed by a 30-s cooling interval. This heating–cooling cycle was repeated seven times (total microwave irradiation time: 210 s). During microwave irradiation, the extraction temperature was monitored and reached approximately 150 °C. After completion of the extraction, the mixture was allowed to cool to room temperature and was then filtered through qualitative filter paper (Whatman No. 1; Cytiva, Marlborough, MA, USA) to obtain the liquid extract, designated as MAPE.

Phytochemical Profiling and Chemical Fingerprinting (HPLC Analysis): High-performance liquid chromatography (HPLC) with photodiode array (PDA) detection was performed to compare the chemical profiles of the different propolis extracts, including MAPE. To establish a comprehensive phytochemical fingerprint of the selected extract, MAPE was screened for 20 common phenolic compounds (gallic acid, D-(-)-Salicin, protocatechuic acid, scopolin, chlorogenic acid, puerarin, caffeic acid, vanillin, p-coumaric acid, ferulic acid, rutin hydrate, narirutin, hesperidin, rosmarinic acid, myricetin, quercetin, (±)-naringenin, apigenin, kaempferol, and formononetin). In addition, three representative propolis flavonoids—chrysin, pinocembrin, and galangin—were selected as marker compounds for extract standardization, and their contents were quantified. The analysis was performed using a Waters 2695 Separation Module equipped with a Waters 2996 PDA Detector (Waters Corporation, Milford, MA, USA). Chromatographic separation was achieved on a Luna C18 column (Phenomenex, Torrance, CA, USA) (4.6 mm × 250 mm, 5 μm) maintained at room temperature. The mobile phase consisted of solvent A (0.1 trifluoroacetic acid in water) and solvent B (acetonitrile), delivered at a flow rate of 1.0 mL/min. The gradient elution program was as follows: 0–50 min, 38% B; 50–52 min, linear increase to 100% B; 52–55 min, 100% B; 55–58 min, linear decrease to 38% B; 58–70 min, 38% B. The injection volume was 10 μL, and detection was performed at a wavelength of 280 nm. Three independent batches of MAPE were analyzed to confirm the reproducibility of the extraction process. Key compounds were identified by comparing their UV spectra (200–400 nm) and retention times with those of the corresponding standards. Quantification was performed using calibration curves generated from the standards.

Comparative Extraction Screening: To compare the efficiency of different extraction approaches, propolis was extracted using several solvents and extraction conditions prior to microwave-assisted extraction. Conventional thermal extraction was performed using ethanol, dipropylene glycol, water, and butylene glycol at 80 °C for 3 h with a solvent-to-solid ratio of 20:1 (w/w). The extraction efficiency of these methods was evaluated by determining total phenolic content (TPC). Microwave-assisted butylene glycol extraction (MAPE) showed the highest phenolic recovery and was therefore selected for subsequent phytochemical characterization and biological evaluation. Total phenolic content (TPC) was determined using the Folin–Ciocalteu colorimetric assay and expressed as gallic acid equivalents (GAE) [22].

Primary human neonatal dermal fibroblasts (HDFs; PCS-201-010, ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Welgene, Daegu, Republic of Korea) supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/mL and 50 μg/mL, respectively) at 37 °C in a humidified atmosphere containing 5% CO₂. Oxidative stress-induced senescence was generated by continuously exposing HDFs (passage ≤ 15) to hydrogen peroxide (H₂O₂, 300 μM) for 5 days, and HDFs at the same passage without H₂O₂ exposure were used as controls. Replicative senescent fibroblasts were obtained by serial subculture until passage ≥ 40, at which point cells exhibited typical morphological and proliferative features of senescence. All experiments were performed immediately after completion of the 5 day H₂O₂ exposure or upon reaching the designated passage number.

Cell viability was evaluated using an MTT assay. Human dermal fibroblasts were seeded in 6-well plates at a density of 1 × 10⁵ cells per well and treated with MAPE (0.002–0.02%) for 5 days under the same replicative senescence conditions used for the SA-β-gal assay. After treatment, MTT solution was added and incubated for 3 h at 37 °C. The resulting formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm using a Epoch microplate reader (BioTek Instruments, Winooski, VT, USA).

Total RNA was isolated with the MiniBEST Universal RNA Extraction Kit (9767A; Takara Bio, Shiga, Japan) according to the supplier’s protocol. First-strand cDNA was generated from 1 µg total RNA using the Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Quantitative real-time PCR was carried out on a CronoSTAR™ 96 Real-Time PCR system (Takara Bio, Kusatsu, Japan). Gene-specific primer sequences are listed in Table 1.

Human dermal fibroblasts (HDFs) were seeded at 1 × 10⁵ cells per well in 6-well plates and incubated with test materials for 5 days. Senescence-associated β-galactosidase (SA-β-gal) activity was visualized using a Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, Danvers, MA, USA) following the manufacturer’s instructions [23]; cells positive for SA-β-gal appeared blue after overnight incubation with the staining solution. After overnight staining, blue-stained cells were imaged at ×100 magnification, and staining intensity was quantified as integrated density (IntDen) using ImageJ (Fiji, version 1.54p; NIH, Bethesda, MD, USA). For fluorescence-based detection of β-galactosidase activity, cells were labeled with SPiDER-βGal (Dojindo, Kumamoto, Japan) for 15 min at 37 °C, rinsed, and imaged by fluorescence microscopy under identical acquisition settings; the proportion of fluorescence-positive cells was determined from the acquired images.

Human dermal fibroblasts (HDFs) were seeded at 1 × 10⁵ cells per well in 6-well plates and incubated with test materials for 5 days. Cells were fixed in 4% formaldehyde for 15 min at room temperature, then blocked in 5% bovine serum albumin with 0.3% Tween-20 in PBS for 1 h. Primary antibodies against p21CIP1 and β-catenin (typical 1:500 dilution; CST, Danvers, MA, USA) were applied overnight at 4 °C, followed by species-appropriate Alexa Fluor-conjugated secondary antibodies (typical 1:1000 dilution; Invitrogen, Carlsbad, CA, USA). Nuclei were counterstained with Hoechst 33342 (1:10,000; 10 min, room temperature), and fluorescence was acquired using a K1-Fluo confocal microscope (Nanoscope Systems, Daejeon, Republic of Korea). Acquisition parameters were kept constant for all groups.

Intracellular ROS were assessed using 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) [24]. Cells were incubated with 5 µM H₂DCFDA for 30 min at 37 °C, rinsed with PBS, and imaged under identical acquisition settings using a confocal microscope. Fluorescence was quantified in ImageJ/Fiji and expressed as relative fluorescence intensity.

Interleukin-6 (IL-6) levels in culture supernatants were quantified using a Human IL-6 Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Human dermal fibroblasts (HDFs) were subjected to oxidative stress by continuous exposure to H₂O₂ (300 µM) for 5 days and co-treated with MAPE at 0.005%, 0.01%, or 0.02%. At the end of the treatment period, culture supernatants were collected and analyzed for IL-6.

Normal human dermal fibroblasts (HDFs; passages ≤10) were cultured to confluence (≈95–100%) in 6-well plates and switched to DMEM containing 1% FBS on the assay day. A linear wound was created across the cell monolayer using a sterile 200 µL pipette tip, and detached cells were removed by two PBS rinses. After scratching, cells were incubated with assay medium (DMEM with 1% FBS) containing MAPE (0.005, 0.01, or 0.02% w/v), or EGCG (10 µM) for 30 min, followed by the addition of H₂O₂ (300 µM) to induce oxidative impairment of cell migration. Corresponding vehicle-only wells (without H₂O₂) served as untreated controls. Wound recovery was quantified by measuring the cell-covered area within a fixed region of interest (ROI) at 72 h using ImageJ software. The cell-covered area was segmented from background using the threshold function, and the pixel area occupied by cells was recorded.

The relative recovery (%) was calculated according to the following formula:where Relative recovery ( % ) = × A sample A H 2 O 2 − A control A H 2 O 2 − 100

Acontrol = cell-covered area in the vehicle (no H₂O₂) group;

AH2O2 = cell-covered area in the H₂O₂-only group;

= cell-covered area in the test sample group. A sample

Normal human dermal fibroblasts (HDFs) were cultured in DMEM containing 10% FBS and 1% penicillin–streptomycin at 37 °C in a humidified 5% CO₂ incubator. At designated time points (3 or 7 days after seeding), cells were detached and viable cell numbers were quantified using an automated cell counter (LUNA-II™, Logos Biosystems, Anyang-si, Republic of Korea) with disposable counting slides according to the manufacturer’s instructions. Viable cell numbers reported by the instrument were used for calculations.

Population Doubling Level (PDL) and Population Doubling Time (PDT) were calculated according to the following equations:whereanddenote the initial and harvested cell numbers, respectively, andrepresents the incubation period in days. P D L P D T d a y = , ( ) = log 2 N N 0 t d a y ( ) P D L N 0 N t

All data are expressed as the mean ± SD, as indicated. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test, and differences were considered statistically significant at p < 0.05.

BG (1,3-butylene glycol) is commonly used as an extraction solvent in cosmetic manufacturing because of its good water compatibility and lower irritancy compared with ethanol [25]. In the present study, we applied a BG-based extraction process to propolis and compared a conventional BG-based extraction with a microwave-assisted BG-based extraction and other solvent systems.

To establish a comprehensive chemical fingerprint for MAPE, we initially performed a screening of 20 common phenolic compounds (listed in). None of these standard phenolic compounds were detected in MAPE under the present analytical conditions, suggesting that the extract possesses a phytochemical profile distinct from the screened phenolic standards (). Instead, quantitative analysis focused on three representative propolis flavonoids—chrysin, pinocembrin, and galangin—which were used as marker compounds for extract standardization. Section 2.1 Supplementary Table S1; Supplementary Figure S1

As shown in Figure 1A–D, HPLC analysis of three representative propolis flavonoids—chrysin, pinocembrin and galangin—showed that the microwave-assisted BG-based propolis extract (MAPE) contained slightly higher overall levels of these markers than the propolis extract obtained by BG extraction at 80 °C for 3 h, while both BG-based extracts yielded markedly higher levels than the propolis extract obtained by BG extraction at room temperature (24 h). In addition, when the total phenolic content was compared among six different extraction conditions (EtOH, DPG, water, microwaved water, BG without microwave and BG with microwave), the microwave-assisted BG process produced the highest value (1676.6 µg GAE/g), corresponding to a 2.3-fold increase over ethanol extraction and a 9.7-fold increase compared with BG extraction without microwave.

The superiority of the microwave-assisted BG process is likely attributable to rapid dielectric heating and increased internal pressure within the propolis matrix, which facilitate the release of phenolic compounds. Although the levels of the three quantified flavonoids were similar between the microwave-assisted and 80 °C BG extracts, the markedly higher total phenolic content suggests that microwave-assisted extraction improves the recovery of a broader range of phenolic constituents beyond these flavonoid markers.

On this basis, MAPE was used in the subsequent experiments to investigate its antioxidant and anti-aging effects in human dermal fibroblasts.

To determine whether MAPE could suppress oxidative stress-induced cellular senescence, normal human dermal fibroblasts (HDFs) were treated with H₂O₂ in the presence or absence of increasing concentrations of MAPE (0.005–0.02%) or the reference antioxidant EGCG (10 µM), a well-established anti-aging compound. As shown in Figure 2A, H₂O₂ treatment markedly increased nuclear p21CIP1 staining and the proportion of p21CIP1-positive cells, indicating the onset of cell cycle arrest. MAPE significantly reduced p21CIP1 immunoreactivity in a dose-dependent manner, with the 0.02% treatment restoring the signal to nearly basal levels. Quantitative RT-PCR further confirmed a marked down-regulation of CDKN2A and CDKN1A expression (Figure 2B,C), both of which are canonical markers of cellular senescence.

In parallel, IL6 mRNA expression and IL-6 protein secretion were markedly upregulated by oxidative stress but attenuated by MAPE (Figure 2D,E). IL-6 is a well-established SASP-associated cytokine [26,27]. ELISA analysis revealed that IL-6 secretion decreased to approximately 80% of the H₂O₂ control group following treatment with 0.02% MAPE, indicating potent anti-inflammatory and anti-SASP effects. Collectively, these findings indicate that MAPE effectively mitigates oxidative stress-induced fibroblast senescence, which is associated with suppression of CDKN2A and CDKN1A expression and reduced SASP cytokine production.

Consistent with the reduction in senescence-associated gene expression, MAPE also suppressed cellular senescence at the functional level. SA-β-galactosidase staining revealed a pronounced increase in senescence-positive fibroblasts after H₂O₂ exposure, characterized by flattened morphology and intense blue staining (Figure 2F). In contrast, cells treated with MAPE (0.02%) displayed fewer SA-β-gal-positive areas and retained a more elongated, spindle-like morphology similar to non-stressed controls. Quantitative image analysis confirmed that H₂O₂ treatment elevated β-gal activity intensity more than twofold compared to untreated controls, whereas MAPE reduced it by approximately 40% (p < 0.01 vs. H₂O₂ group). The magnitude of reduction was comparable to that observed with EGCG (10 µM), a well-established antioxidant reference.

These findings indicate that MAPE effectively alleviates oxidative stress-induced fibroblast senescence, restoring cellular morphology and reducing lysosomal β-gal activity associated with the senescent phenotype.

After confirming that MAPE suppressed senescence-associated markers and SASP factors (Figure 2), we next examined whether these protective effects were accompanied by improvements in redox balance. To investigate whether the anti-senescent effect of MAPE was associated with attenuation of oxidative stress, intracellular ROS levels were quantified using the DCFH-DA probe (Figure 3A).

Fluorescence microscopy revealed that exposure to H₂O₂ triggered intense green fluorescence indicative of ROS generation, which was visibly attenuated in cells co-treated with MAPE. Quantitative analysis confirmed that H₂O₂ markedly increased ROS fluorescence intensity, indicating strong oxidative insult. However, treatment with MAPE significantly reduced ROS accumulation in a concentration-dependent manner, lowering fluorescence intensity by approximately 35–45% compared with H₂O₂-only groups. The antioxidant reference EGCG produced a comparable reduction.

These findings indicate that MAPE alleviates intracellular oxidative stress. This reduction in ROS prompted us to investigate whether MAPE acts merely as a direct radical scavenger or if it functions by boosting endogenous antioxidant defense systems.

To elucidate the molecular mechanism underlying the observed ROS reduction, we evaluated the expression of canonical NRF2-regulated antioxidant genes. As shown in Figure 3B–D, MAPE treatment resulted in a significant upregulation of NQO1 and GCLM mRNA levels. Interestingly, GCLC expression showed no significant change, suggesting differential regulation of the glutathione synthesis subunits by MAPE. While H₂O₂ exposure alone induced a slight increase in GCLM expression consistent with a compensatory stress response, MAPE markedly potentiated this induction, elevating NQO1 and GCLM levels significantly beyond the stress-induced baseline.

Collectively, these data indicate that the restoration of redox balance observed in Figure 3A is associated with activation of NRF2-dependent antioxidant pathways. By boosting the transcription of key cytoprotective enzymes, MAPE reinforces intracellular redox homeostasis, thereby equipping fibroblasts with enhanced resistance against oxidative insults.

Since oxidative stress and fibroblast senescence are closely associated with ECM degradation, we next assessed the effect of MAPE on matrix remodeling genes. Exposure to H₂O₂ markedly increased MMP1 mRNA levels to approximately 3.08-fold of the untreated control, while reducing COL1A1 expression to about 0.48-fold, a pattern consistent with increased collagen breakdown and impaired dermal structure. MAPE treatment reversed both trends in a dose-related manner (Figure 4). At 0.02%, MAPE significantly suppressed the H₂O₂-induced MMP1 upregulation, inhibiting approximately 46.2% of the increase, and partially restored COL1A1 expression, recovering about 26.9% of the H₂O₂-induced loss relative to the basal control level, with an efficacy comparable to the positive control EGCG (10 µM).

These results suggest that MAPE not only limits oxidative stress-induced ECM degradation but also helps restore collagen synthesis capacity, which may contribute to the preservation of dermal structural integrity under aging- or stress-related conditions.

To determine whether the molecular effects of MAPE translate into functional recovery of fibroblasts, we performed a scratch-wound assay under oxidative conditions (Figure 5). In H₂O₂-treated fibroblasts, migratory capacity was markedly impaired, resulting in negligible wound closure compared to the near-complete closure observed in untreated controls. However, co-treatment with MAPE significantly enhanced fibroblast migration in a dose-dependent manner. Notably, treatment with 0.02% MAPE achieved a wound closure rate of approximately 65.3%, calculated relative to the H₂O₂-treated group, representing a marked improvement compared to H₂O₂ alone, while EGCG (10 μM) produced only a modest effect. These findings indicate that MAPE effectively rescues fibroblast motility and wound-healing capacity compromised by oxidative stress.

Taken together, our data suggest that MAPE protects dermal fibroblasts from oxidative injury through complementary mechanisms: (i) attenuation of intracellular ROS accumulation via activation of the NRF2-dependent antioxidant response and (ii) preservation of ECM integrity and fibroblast functionality. This dual protection of redox balance and structural homeostasis may underlie the anti-senescent phenotype and improved dermal homeostasis observed in our in vitro skin-aging model.

To further elucidate the molecular mechanism underlying the anti-senescent effects of MAPE, we examined genes involved in the Wnt/β-catenin pathway, which is essential for fibroblast proliferation, collagen synthesis, and tissue repair [6,7,8,9]. In H₂O₂-treated fibroblasts, the expression of WNT3A and LEF1 was markedly reduced, indicating suppression of canonical Wnt signaling under oxidative stress conditions. Treatment with MAPE significantly restored the expression of these genes in a concentration-dependent manner (Figure 6A,B). At the highest concentration (0.02%), WNT3A and LEF1 recovered to approximately 92.5% and 75.9% of the untreated control, respectively, suggesting restoration of Wnt-mediated transcriptional activity. The magnitude of this restoration was comparable to that observed with the well-known antioxidant EGCG (10 µM), supporting the ability of MAPE to modulate this pathway.

In contrast, the Wnt inhibitor DKK1, which was strongly upregulated in H₂O₂-treated fibroblasts, was significantly downregulated following MAPE treatment (Figure 6C). Since elevated DKK1 expression is known to inhibit β-catenin signaling and impair dermal regeneration in aged fibroblasts, its suppression implies a release of autocrine inhibition within the Wnt axis. Collectively, these results indicate that MAPE effectively counteracts the oxidative suppression of Wnt signaling by simultaneously upregulating activators (WNT3A, LEF1) and downregulating the inhibitor (DKK1).

To examine whether the anti-senescent effects of MAPE are associated with modulation of Wnt signaling, we compared its transcriptional responses with those of CHIR99021 (CHIR, 10 µM), a well-established canonical Wnt pathway activator. As expected, CHIR treatment markedly enhanced the expression of WNT3A and LEF1, two core components of the Wnt/β-catenin signaling cascade (Figure 7A,B). MAPE treatment produced a highly similar pattern, significantly upregulating both genes in H₂O₂-stressed fibroblasts. These results indicate that MAPE promotes activation of the canonical Wnt signaling pathway in a manner comparable to the potent Wnt agonist CHIR.

Downstream of Wnt signaling, both CHIR and MAPE suppressed the senescence and inflammatory markers IL6, CDKN1A, and MMP1 (Figure 7C–E), which are typically elevated in oxidative stress-induced fibroblast senescence. The reduction in IL6 and CDKN1A suggests inhibition of SASP propagation and cell-cycle arrest, while decreased MMP1 reflects recovery of ECM integrity.

In parallel, MAPE and CHIR both significantly increased the expression of COL1A1 and TERT (Figure 7F,G), indicating restoration of ECM synthesis and possible reactivation of telomerase-related regenerative capacity. While the effects were broadly comparable, the induction magnitude for COL1A1 was visibly higher in the CHIR group. These observations support the hypothesis that the anti-senescent effects of MAPE are associated with modulation of the Wnt/β-catenin signaling pathway. This is consistent with previous reports linking Wnt/β-catenin activity to the upregulation of TERT and attenuation of cellular senescence [28,29,30].

Collectively, these findings suggest that MAPE may function as a natural modulator of the Wnt signaling pathway, mimicking the regenerative transcriptional profile induced by chemical Wnt activation and linking redox modulation to reactivation of fibroblast regenerative signaling.

To examine whether MAPE exerts anti-senescent effects under intrinsic, replication-driven aging conditions, we evaluated its activity in late-passage human dermal fibroblasts (passage ≥ 40), a well-established model of replicative senescence. Compared with early-passage fibroblasts (p8–15), aged cells exhibited characteristic senescent morphology—enlarged, flattened cytoplasm with reduced proliferative capacity—and showed strong SA-β-galactosidase positivity along with a 3.87-fold prolongation in population doubling time, confirming a senescent state (Figure 8A,B).

At the molecular level, replicatively senescent fibroblasts demonstrated multiple hallmarks of intrinsic aging. Intracellular ROS levels, quantified by DCFH-DA fluorescence, were markedly elevated, indicating a high oxidative burden characteristic of replication-induced cellular stress (Figure 8C, left panels). In parallel, senescent fibroblasts showed strong upregulation of IL6 and MMP1, accompanied by a notable reduction in COL1A1 expression, reflecting a SASP-associated pro-inflammatory and ECM-degrading phenotype that contributes to dermal matrix deterioration during intrinsic skin aging (Figure 8D–F).

To exclude the possibility that the observed anti-senescent effects were due to altered cell survival, we evaluated the cytotoxicity of MAPE in replicatively senescent fibroblasts. MAPE did not induce detectable cytotoxicity within the tested concentration range after 5 days of treatment (). Supplementary Figure S2

Treatment with MAPE (0.005–0.02%) for 5 days significantly alleviated these senescent alterations. MAPE reduced intracellular ROS accumulation to levels comparable to the antioxidant reference EGCG (10 µM), indicating effective mitigation of oxidative stress (Figure 8C). Consistent with this reduction in oxidative stress, cellular senescence, assessed by SPiDER-GAL fluorescence, was significantly reduced in a dose-dependent manner following MAPE exposure (Figure 9A). In parallel, MAPE effectively reversed the core senescent molecular hallmarks (Figure 9B–E). IL6 and MMP1 expression decreased significantly (Figure 9B,C), while COL1A1 expression was restored (Figure 9D). Notably, TERT mRNA—barely detectable in vehicle senescent controls—was re-induced by MAPE (Figure 9E), suggesting a potential involvement of telomerase-associated mechanisms. The antioxidant comparator EGCG (10 µM) produced similar trends but generally did not exceed the highest MAPE dose.

Taken together, these data indicate that MAPE suppresses SASP (IL6/MMP1) and reinstates ECM synthesis (COL1A1) while reactivating TERT in replicatively aged fibroblasts, thereby shifting the cellular state toward a less senescent and more regenerative phenotype.

We investigated whether MAPE could activate Wnt/β-catenin signaling in replicative senescent fibroblasts. qPCR analysis showed that MAPE increased the expression of LEF1 and WNT3A in a dose-dependent manner (Figure 10A,B), while significantly reducing the expression of the Wnt inhibitor DKK1 (Figure 10C). A significant increase in CTNNB1(β-catenin) mRNA expression was also observed at 0.02% MAPE (Figure 10D). These results indicate that MAPE effectively reactivates the canonical Wnt pathway in senescent fibroblasts.

Immunofluorescence staining further supported this activation. MAPE enhanced β-catenin protein levels, and notably promoted its nuclear translocation (indicated by white arrowheads), particularly at 0.02%. This nuclear accumulation was comparable to that induced by CHIR99021, a well-established canonical Wnt activator used as a positive control (Figure 10E). Nuclear accumulation of β-catenin is a hallmark of canonical Wnt pathway activation, consistent with the transcriptional upregulation observed.

Collectively, these findings indicate that MAPE promotes activation of the Wnt/β-catenin signaling pathway in replicative senescent fibroblasts and suggest its potential to mitigate intrinsic cellular aging by enhancing Wnt-driven regenerative pathways.