Pterostilbene (PT) (purity≥99%) was provided by Proya Cosmetics Co., Ltd. (Hangzhou, China). The PT was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution of 400 mM. A PT cream (0.01%) and its corresponding vehicle cream were formulated by the same provider. The composition of the vehicle cream was as follows: Water, Glycerin, Caprylic/Capric Triglyceride, Cetearyl Alcohol, Glyceryl Stearate, Sodium Acrylate/Sodium Acryloyldimethyl Taurate Copolymer, 1,2-Hexanediol, Hydroxyacetophenone, Polyethylene glycol-100 Stearate, Isohexadecane, Polysorbate 80, Acrylates/C10-30 Alkyl Acrylate Crosspolymer, Tromethamine, ethylenediaminetetraacetic acid disodium, and Sorbitan Oleate.

Primary cultured HDFs were purchased from Guangdong BioCell Biotech Co., Ltd. (cat. no. PC 2031; lot no. 230131), which were derived from the skin of a patient between 8 and 10 years old. The cells were cultured in Dulbecco’s Modified Eagle Medium complete medium (DMEM; Gibco, 31600034) containing with 10% fetal bovine serum (FBS; Excell Bio, FSD500) and 1% penicillin-streptomycin (YEASEN, 60162ES76) in a 37 °C incubator with 5% CO₂.

HDFs were divided into the following groups: the Control group, the UVB model group, and the PT treatment groups. Cells in the PT treatment group were pretreated with PT for 24 h, whereas both the Control and UVB model groups received the DMSO at the same final concentration. For UVB irradiation, the 6-well plate with cells in phosphate-buffered saline (PBS) were placed in the ultraviolet illuminometer and the program is set (Power: 0.30 mW/cm²; Time: 10 min; Energy: 180 mJ/cm²). After the program finished, the cells were normally cultured for 24 h and then detected.

Young HDFs controls were defined as passages 5–10 (P5-10) cells from the primary cultured HDFs with minimal aging characteristics. Replicative senescence was achieved by serial passaging. Passages 35–40 (P35-40) HDFs were used as the senescent cells. During serial passage, PT was added to the culture medium to continuously treat the cells. The senescent group cells were maintained with the same concentration of DMSO throughout the passaging process.

HDFs were seeded at 8,000 cells/well in 96-well plates, treated for 24 h, and incubated with the CCK-8 reagent (APExBIO, K1018). The absorbance was measured at 450 nm.

SA-β-Gal staining was performed using an SA-β-Gal Staining Kit (Beyotime Biotechnology, C0602) according to the manufacturer instructions. Cells were fixed and incubated with the staining solution overnight at 37 °C overnight in a dry incubator without CO₂. The blue SA-β-Gal-positive cells were imaged and quantified using a bright field microscope.

Total RNA was extracted using FreeZol Reagent (Vazyme, R711) and reverse-transcribed into cDNA using HiScript Ⅳ All-in-One Ultra RT SuperMix for qPCR (Vazyme, R433) according to the manufacturer’s instructions. Real-time PCR was performed using Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Q712) on an Applied Biosystems™ QuantStudio™ 3 System (Thermo Fisher, ISO13485). Gene expression was normalized to GAPDH and the results were calculated using the 2−^ΔΔCt method. The primer sequences used for target gene expression identification were as follows: p16, sense 5′-TCG​CGA​TGT​CGC​ACG​GTA-3′ and anti-sense, 5′-CAT​CTA​TGC​GGG​CAT​GGT​TAC​TG-3’; GAPDH, sense 5′- GAA​AGC​CTG​CCG​GTG​ACT​AA-3′ and anti-sense 5′-GCA​TCA​CCC​GGA​GGA​GAA​AT-3’. All primers used for RT-qPCR were obtained from Shanghai GENEray Biotechnology Co., Ltd.

Total proteins were extracted from cells and tissues using RIPA lysis buffer (NCM, WB3100) supplemented with protease and phosphatase inhibitor cocktail (Beyotime, P1045). Protein samples normalized by BCA assay were separated by SDS-PAGE and transferred to PVDF membranes (Milipore, ISEQ00010-1EA). The membranes were blocked with 5% skim milk for 2 h at room temperature. After that, the membranes were incubated overnight with the primary antibodies at 4 °C. The primary antibodies used were Rabbit Anti-p21 (ABclonal, A19094; 1:1,000), Rabbit Anti-LC3 Ⅱ (Sigma-Aldrich, I7543; 1:1,000), Rabbit Anti-TOM20 (ABclonal, A19403; 1:1,000), Rabbit Anti-GAPDH (Proteintech, 10494-1-AP; 1:10,000). Next, the membranes were incubated with HRP-conjugated secondary antibodies and then visualized using an enhanced chemiluminescence (ECL) system. The intensity of the bands was quantified using ImageJ software.

15,000 cells per well grown on 24-well coverslips were fixed with 4% paraformaldehyde fix solution for 15 min, permeabilized with 0.2% Triton X-100 for 10 min, and blocked with 10% goat serum containing glycine for 30 min. Next, the cells were incubated overnight at 4 °C with the following primary antibodies: Rabbit Anti-Collagen Ⅰ (Proteintech, 14695-1-AP; 1:200), Rabbit Anti-Collagen Ⅲ (Abcam, ab7778; 1:200), Rabbit Anti-LC3Ⅱ (Proteintech, 14600-1-AP; 1:200), Mouse Anti-TOM20 (ABclonal, A27799; 1:200). Then the cells were washed with PBST and incubated with Alexa Fluor 488-conjugated goat anti-rabbit (ABclonal, AS053; 1:250) and Alexa Fluor 647-conjugated goat anti-mouse secondary antibodies (ABclonal, AS059; 1:250) for 1 h at room temperature. After a final wash with PBST, slides were mounted using Antifade Mounting Medium containing DAPI (Beyotime, P0131) for nuclear counterstaining. Images were captured using a fluorescence microscope.

Mitochondrial morphology: Cells seeded in confocal microdishes were staining coincubated with 200 nM MitoTracker (Thermo Fisher Scientific, M22426) and Hoechst (APExBIO, K2407) for 30 min at 37 °C. After staining is complete replace the staining solution with fresh prewarmed media. Fluorescent images were acquired using an Olympus FV3000 confocal microscope with a ×100 oil objective.

Mitochondrial respiration was analyzed using a Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies, 103,793–100), according to the manufacturer’s instructions. 15,000 HDFs per well were plated into the wells of an XF96 cell culture microplate and incubated at 37 °C in a CO₂ incubator overnight to ensure attachment. The Oxygen consumption rate (OCR) involved sequential injection of 1.5 μM oligomycin, 2 µM FCCP, 0.5 μM rotenone and 0.5 μM antimycin-A. Protein normalization was performed after the assay.

Six-week-old male C57BL/6 mice were maintained under a 12 h light/dark cycle with free access to water and food. As previous described (Budluang et al., 2025; Choi et al., 2020), mice received localized UVB irradiation on depilated dorsal skin three times weekly for 4 weeks. The exposure dose began at 1 minimal erythemal dose (MED) and was increased weekly by 1 MED, reaching a final dose of 4 MED. The cream containing 0.01% Pterostilbene was evenly applied daily to the exposed dorsal skin of the mice during the experiment. All experiments were approved by and conducted in accordance with the ethical guidelines of the Zhejiang University Animal Experimentation Committee and were completely compliant with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Mice dorsal skin samples were fixed in 4% paraformaldehyde in PBS, then embedded in paraffin. Subsequently, the samples were sectioned and stained using HE and Masson’s trichrome for histopathologic examination. Dermal thickness and collagen density were quantified using the ImageJ software.

All statistical analyses were conducted using GraphPad Prism version 9.0. Student’s t-test was used for comparisons between two groups, whereas one-way analysis of variance (ANOVA) or two-way ANOVA was applied for multiple-group comparisons. Data are represented as means ± SD, and statistical significance was set at p < 0.05.

We initially verified the anti-aging effects of PT in HDFs. In a UVB-induced acute oxidative stress model established in HDFs, and following confirmation of non-cytotoxic PT concentrations (Figures 1A,B), PT treatment significantly attenuated UVB-induced cellular senescence. This was manifested as a concentration-dependent decrease in senescence-associated markers, including SA-β-gal, p16, and p21 levels (Figures 1C–G). Furthermore, PT restored the expression of collagen I and III (Figures 1H–J). These results indicated that PT attenuated UVB-induced acute oxidative stress in HDFs.

To clarify whether PT protects against UVB-induced acute oxidative stress in HDFs by improving mitochondrial quality, both mitochondrial morphology and function were assessed. Mitochondria staining revealed that PT incubation restored the mitochondrial length and network size (Figures 2A–C), implying improved mitochondrial quality. Furthermore, flow cytometry analysis showed that PT prevented the loss of mitochondrial membrane potential by UVB exposure (Figure 2D). Consistently, MitoSOX fluorescence images revealed that PT suppressed UVB-induced mitochondrial ROS production, indicating improved respiratory efficiency (Figures 2E,F). To further quantify mitochondrial respiration, we assessed the oxygen consuming rate (OCR) in HFDs. The results demonstrated that 13 and 40 μM PT treatment significantly enhanced baseline respiration, ATP production, and maximal respiration compared to the UVB group (Figures 2G,H). Together, these results demonstrated that PT treatment ameliorated mitochondrial dysfunction in UVB-induced aged HDFs.

To further verify the anti-aging effect of PT on the dermis, a replicative senescence model was employed by serially passaging the human foreskin-derived HDFs. PT was administrated continuously during the aging passage of HDFs (Figure 3A). HFDs within P4-P10 were defined as young HDFs, whilst those from passage P35-P40 were considered senescent HFDs. PT treatment significantly reduced cellular senescence in replicative HDFs, as evidenced by a concentration-dependent decrease in senescence-associated markers, including SA-β-gal, p16, and p21 levels (Figures 3B–F). As shown in Figures 3G–I, immunofluorescence images revealed that PT restored the expressions of Collagen Ⅰ and Collagen Ⅲ in a concentration-dependent manner. Collectively, these findings demonstrate that PT treatment attenuates replicative senescent HDFs, supporting its potential anti-aging effect on the dermis.

To investigate the effects of PT on mitochondrial quality in senescent HDFs, mitochondrial morphology, mitochondrial membrane potential, ROS levels and mitochondrial respiratory function were evaluated. As shown in Figures 4A–C, senescent HDFs exhibited reduced mitochondrial length and network size, along with decreased MMP, all of which were reversed by PT treatment (Figure 4D). Furthermore, MitoSOX immunofluorescence images revealed that senescent HFDs displayed significantly increased mitochondrial ROS compared to young group, senescent HDFs exhibited significantly increased mitochondrial ROS, an effect significantly attenuated by 40 μM PT treatment (Figures 4E,F). Mitochondrial respiration was assessed via oxygen consumption rate measurements. Specifically, PT treatment at 13 and 40 μM significantly enhanced baseline mitochondrial respiration, ATP production, and maximal respiration compared to the senescent group (Figures 4G,H). Our result demonstrated that PT treatment reversed mitochondrial dysfunction in replicative senescent HDFs.

Our previous results proved that PT can improve mitochondrial function and enhance mitochondrial quality control, in which mitophagy plays a key regulatory role. We evaluated PT’s impact on mitophagy in replicative senescent HDFs. Co-localization fluorescence images displayed a significant reduction in the co-localization efficiency in senescent HDFs, whereas PT treatment enhanced the co-localization efficiency both in young and senescent HDFs (Figures 5A,B). Western blot results revealed that the expression levels of LC3 Ⅱ were significantly reduced and the expression levels of mitochondrial proteins TOM20 were significantly increased in senescent HDFs, which was reversed by PT treatment (Figures 5C,D). Thus, mitophagy was impaired and damaged mitochondrial accumulated in senescent HDFs, while PT promoted mitophagy to remove damaged mitochondrial and maintained mitochondrial mass homeostasis.

HE and Masson staining revealed that UVB irradiation significantly decreased dermal thickness and collagen density in mouse skin compared to the control group. Importantly, long-term continuous PT application significantly reversed these UVB-induced damages (Figures 6A–C). Western blot analysis showed that PT treatment mitigated the UVB-induced upregulation of p21 protein level in mouse skin (Figures 6D,E), suggesting its protective effect against photoaging. Furthermore, PT application restored the protein levels of autophagy marker LC3, which were reduced by UVB exposure (Figure 6F). This restoration of LC3 levels suggests that the PT promotes autophagy, which is a potential mechanism through which PT exerts its anti-photoaging effects.