Hyperoside may improve UVB skin aging by boosting cell cleanup through the miR-361-5p/PI3K/Akt/mTOR pathway

The human keratinocyte cell line HaCaT (SNL-163) was obtained from Wuhan SUNNCELL (Wuhan, China). Hyperoside (HY; purity ≥ 98%, CAS No.: 482-36-0) was purchased from Beijing Solarbio Science and Technology Co., Ltd. (Jiangsu, China). The following assay kits were acquired from Beyotime Biotechnology (Shanghai, China): BCA Protein Concentration Assay Kit (P0012), Methylthiazolyldiphenyl-tetrazolium Bromide (MTT) Cell Viability Kit (ST316), Autophagy Staining Kit (C3018S), Senescence-Associated β-galactosidase Staining Kit (C0602), Reactive Oxygen Species Assay Kit (CA1410), and EdU-555 Cell Proliferation Assay Kit (C0075S). The Cell Cycle Detection Kit (KGA511) was supplied by KeyGEN BioTECH (Nanjing, China). MitoSOX Red Mitochondrial Superoxide Indicator (HY-D1055) was procured from MedChemExpress (Shanghai, China).

Antibodies were sourced as follows: p21 (10355-1-AP), Beclin1 (11306-1-AP), GAPDH (60004-1-Ig), LC3 (14600-1-AP), PI3K (20584-1-AP), p-Akt (66444-1-Ig), LaminB1 (12987-1-AP), TOM20 (11802-1-AP), and Tubulin (11224-1-AP) were purchased from Proteintech Group (Wuhan, China); γ-H2AX (M63324S) was from Abmart (Shanghai, China); Akt (YP-mAb-14652), mTOR (YP-mAb-14867), p-mTOR (YP-mAb-14329), and p-PI3K (YP-Ab-17956) were obtained from Upingbio (Hangzhou, China); p16 (CY5316), PINK1 (BY0130), Parkin (CY6641), and p62 (CY9081) were from Abways Technology (Shanghai, China).

HaCaT cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a 5% CO₂ humidified atmosphere. Upon reaching 90% confluence, cells were detached using trypsin and subcultured. All experiments were conducted using cells between passages 2 and 7 during the exponential growth phase.

HaCaT cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well and treated with various concentrations of HY (1–1000 μM) for 24 h. For UVB irradiation experiments, a UVB lamp (280–315 nm spectrum) was positioned 5 cm above the culture dish, and cells were exposed to doses ranging from 20 to 140 mJ/cm². After irradiation, cells were incubated for an additional 24 h. Subsequently, 10 μL of MTT solution (5 mg/mL) was added to each well, and the plate was incubated for 4 h. The medium was then aspirated, and 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. Absorbance was measured at 490 nm using a microplate reader (Molecular Devices, LLC, San Jose, CA, USA).

To evaluate the protective effects of HY against UVB-induced damage, a series of functional assays was performed. Unless otherwise specified, cells were pretreated with the indicated concentrations of HY for 24 h, exposed to a specific dose of UVB (detailed in each assay below), and then further incubated for subsequent analysis. All experiments were independently repeated in triplicate.

Total RNA was extracted from HaCaT cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. cDNA was synthesized from 1000 ng of RNA using a reverse transcription kit (Takara Bio Inc., Kusatsu, Shiga, Japan). Quantitative PCR was performed using SYBR^® Premix Ex Taq™ II (Takara Bio Inc., Kusatsu, Shiga, Japan) in a 20 μL reaction mixture containing forward and reverse primers and cDNA template. Amplification was carried out for 40 cycles under the following conditions: initial denaturation at 95 °C for 30 s; amplification at 95 °C for 5 s and 60 °C for 1 min; melting curve analysis at 95 °C for 1 s, 55 °C for 55 s, and 97 °C for 1 s; and final cooling at 37 °C for 30 s. The LightCycler^® 96 System was used for detection. GAPDH served as the internal control. Primer sequences are listed in Table 1.

Proteins were extracted from skin tissues or HaCaT cells using high-efficiency RIPA lysis buffer according to the manufacturer's protocol. Protein concentration was quantified using a bicinchoninic acid (BCA) assay kit (KeyGEN BioTECH, Nanjing, China). Samples (4 μg/μL, 6 μL per lane) were separated by 10% sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Burlington, MA, USA). Membranes were blocked with 5% skim milk for 2.5 h, incubated with primary antibodies at 4 °C overnight, and then probed with appropriate secondary antibodies for 2 h at room temperature. Protein bands were visualized using an automated chemiluminescence imaging system and analyzed with ImageJ software (v1.8.0).

HaCaT cells were cultured on confocal dishes and treated as indicated. After treatment, cells were fixed with 4% formaldehyde for 20 min at 4 °C, washed three times with PBS, and permeabilized with 0.2% Triton X-100 for 30 min. Blocking was performed with 10% goat serum for 30 min at room temperature. Cells were incubated with primary antibodies overnight at 4 °C, followed by incubation with fluorescently labeled secondary antibodies for 1.5 h in the dark. The primary antibodies and dilutions used were as follows: anti-γ-H2AX antibody at 1:500; anti-LaminB1 antibody at 1:500; anti-Tubulin antibody at 1:1000; anti-TOM20 antibody at 1:200; anti-Beclin1 antibody at 1:300; anti-LC3B anti-body at 1:200; anti-p21 antibody at 1:500. All images were acquired using a Nikon A1R+ confocal microscope system with consistent acquisition parameters to ensure comparability [21].

HaCaT cells were seeded in 6-well plates at 1 × 10⁵ cells per well and pretreated with HY (1 or 5 μm) for 24 h prior to UVB irradiation. After UVB exposure, cells were incubated with 10 μm 2′,7′-dichlorofluorescin diacetate (DCFH-DA) or 5 μm MitoSOX reagent for 30 min at 37 °C in the dark. Following three washes with PBS, fluorescence was quantified by flow cytometry or visualized under a fluorescence microscope.

Samples were high-pressure frozen using a Leica EMPACT2 system and subjected to freeze substitution in anhydrous acetone containing 1% OsO₄ and 0.1% uranyl acetate using a Leica EM AFS2 unit (Leica Microsystems, Wetzlar, Germany). The temperature was raised stepwise from −85 °C to 0 °C over several days. Samples were embedded in Spurr resin, and ultrathin sections were cut with a Leica EM UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany). Sections were stained with uranyl acetate and lead citrate and examined under a Philips CM100 transmission electron microscope operating at 100 kV.

The 2D structure of HY was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/↗ accessed on 13 May 2025, Compound CID: 5,320,951). Potential protein targets of HY were predicted using the following online platforms: SwissTargetPrediction (http://www.swisstargetprediction.ch/↗ accessed on 13 May 2025) with the species set to "Homo sapiens"; and PharmMapper (http://www.lilab-ecust.cn/pharmmapper/↗ accessed on 13 May 2025) with the species set to "Human". All predicted targets were merged, and duplicates were removed to obtain the potential target set of HY. Targets related to "photoaging" were retrieved by searching the following databases: DisGeNET (https://www.disgenet.org/↗, version 7.0 accessed on 23 May 2025), GeneCards (https://www.genecards.org/↗, version 5.14, accessed on 23 May 2025), OMIM (https://www.omim.org/↗ accessed on 23 May 2025), and DrugBank (https://go.drugbank.com/↗, version 5.1.9 accessed on 23 May 2025). All database searches were conducted between 13 and 23 May 2025. Using keywords such as "photoaging", "skin aging", and "UV-induced skin aging", results from each database were combined and deduplicated to generate the disease target set. Common targets between HY and photoaging were identified by intersecting the respective target sets. A protein–protein interaction (PPI) network was constructed using the STRING database (https://string-db.org/↗, version 12.0 accessed on 27 May 2025). The key parameters were set as follows: organism limited to "Homo sapiens"; a minimum required interaction score of >0.90 (highest confidence); and disconnected nodes were hidden in the network. The resulting TSV data were imported into Cytoscape software (version 3.10.3) for network visualization and topological analysis. The CytoNCA plugin was used to identify hub targets in the network based on parameters including "Degree Centrality", "Betweenness Centrality", and "Closeness Centrality". The crystal structures of the core targets PI3K (PDB ID: 5JHB) or Akt (PDB ID: 1H10) were downloaded from the PDB database (https://www.rcsb.org/↗ accessed on 29 May 2025). Pre-docking preparations were performed using AutoDock Tools 1.5.7: water molecules and the original ligand were removed, polar hydrogen atoms were added, and Gasteiger charges were calculated. The 3D structure of HY was energy-minimized and set as a flexible ligand. The docking grid box was centered on and covered the active site of PI3K. Molecular docking was performed using AutoDock Vina 1.1.2, with the exhaustiveness parameter set to 32. The resulting binding conformations were ranked by binding affinity (unit: kcal/mol), and the binding modes were visualized and analyzed using PyMOL (version 2.5.0).

MiR-361-5p mimic and si-PI3K, along with their respective negative controls, were purchased from GenePharma (Shanghai, China). HaCaT cells in the logarithmic growth phase were seeded into 6-well plates at 5 × 10⁵ cells per well and transfected using GP-transfect-mate reagent (Genepharma Co., Ltd., Shanghai, China) according to the manufacturer's instructions.

Forty-eight male Institute of Cancer Research (ICR) mice (4–5 weeks old, 17 ± 1 g) were supplied by Jiangsu Qinglongshan Biotechnology Co., Ltd (Nanjing, China). Mice were housed under a 12 h light/dark cycle with ad libitum access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University (Approval No.: 2025-01-001. Animal Approval Date: 1 January 2025) and complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Mice were randomly divided into six groups (n = 8 per group): control, UVB model, low-dose HY (LH) (10 mg/mL), medium-dose HY (MH) (20 mg/mL), high-dose HY (HH) (40 mg/mL), and vitamin E (VE) positive control. Dorsal hair was removed prior to UVB irradiation. Mice were irradiated with 150 mJ/cm² UVB three times per week. Topical applications of HY or VE at the indicated concentrations were administered 30 min before each irradiation. The solutions were applied evenly to the shaved dorsal skin area (approximately 4 cm × 5 cm). A fixed volume of 100 μL was applied per mouse, delivering doses of 10, 20, and 40 mg/kg for the low-, medium-, and high-dose HY groups, respectively (based on an average mouse weight of 25 g, this equates to 0.5, 1.0, and 2.0 mg of HY per cm² of skin). At the endpoint, dorsal skin samples were collected for analysis [22].

Skin tissues were fixed, paraffin-embedded, sectioned, and dewaxed. Masson's trichrome staining was used to evaluate collagen content, and hematoxylin and eosin (H&E) staining was applied to assess histological changes. Slides were scanned using a NanoZoomer 2.0 RS digital slide scanner and analyzed with ImageJ.

Paraffin sections were dewaxed, rehydrated, and subjected to antigen retrieval. Endogenous peroxidase activity was blocked, and sections were incubated with primary and secondary antibodies. Signal was developed using 3,3-N-diaminobenzidine tertrahydrochloride (DAB), and nuclei were counterstained with hematoxylin. Slides were dehydrated, mounted, and imaged under a microscope, and analyzed with ImageJ software (version 1.54f) [23].

Data were analyzed using GraphPad Prism 10 and expressed as mean ± standard error of the mean (SEM). For normally distributed data with homogeneous variance, one-way analysis of variance (ANOVA) was applied; otherwise, non-parametric tests were used. A p-value < 0.05 was considered statistically significant.

Based on the pharmacological profile of HY, we hypothesized that it might exert protective effects against UVB radiation-induced skin aging. To test this hypothesis, we first evaluated the impact of varying concentrations of HY on the viability of HaCaT cells. Results indicated that HY at concentrations of 1 μm and 5 μm did not exhibit significant cytotoxicity (Figure 1A). Using the MTT assay, we assessed the influence of different UVB irradiation intensities on cell viability and established an irradiation dose of 60 mJ/cm² as a standardized parameter for subsequent experiments (Figure 1B). Cell cycle analysis revealed that UVB irradiation induced G2/M phase arrest, which was effectively reversed by HY treatment, with the most pronounced effect observed at 5 μm (Figure 1C,D). Furthermore, cell invasion assays demonstrated that HY significantly alleviated UVB-induced suppression of HaCaT cell migration (Figure 1E,F). Colony formation assays indicated that UVB exposure markedly reduced cell viability, whereas HY treatment notably enhanced proliferative capacity (Figure 1G,H), consistent with previously reported findings [21]. Immunofluorescence staining showed a substantial increase in apoptosis in UVB-irradiated cells, which was markedly attenuated by HY treatment (Figure 1I,J). In summary, HY effectively counteracts UVB-induced reduction in HaCaT cell viability and suppresses apoptosis, demonstrating significant protective activity.

To evaluate the effect of HY on UVB-induced cellular senescence, we performed SA-β-gal staining on HaCaT cells. The results indicated that the number of SA-β-gal-positive cells increased significantly after UVB irradiation, whereas HY treatment markedly reduced the proportion of positive cells (Figure 2A,B). Cellular senescence is often accompanied by the secretion of the senescence-associated secretory phenotype (SASP) [24], which comprises various pro-inflammatory cytokines, growth factors, chemokines, and matrix-remodeling enzymes [25]. Accordingly, we examined the mRNA expression levels of IL-1α, TNF-α, and VEGF. HY significantly suppressed the expression of TNF-α and VEGF while upregulating IL-1α (Figure 2C), with the most pronounced effects observed at a concentration of 5 μm. Furthermore, p16 and p21 are well-established and reliable markers of senescence [26]. As shown in Figure 2E–G, UVB irradiation notably increased the expression of p16 and p21, and this effect was reversed by HY treatment in a concentration-dependent manner. The phosphorylated form of histone H2AX (γH2AX) serves as a marker of the DNA damage response (DDR) [27]. Its phosphorylation level was significantly reduced following HY treatment (Figure 2D,I). Senescent cells often undergo morphological alterations accompanied by downregulation of LaminB1, establishing LaminB1 as an indicator of senescence [28]. Confocal microscopy revealed that the fluorescence intensity of LaminB1 was significantly weakened in the UVB group, whereas high concentrations of HY noticeably enhanced its expression (Figure 2H,J). These results demonstrate that HY effectively protects HaCaT cells from UVB-induced cellular senescence.

ROS have been demonstrated to play a critical role in skin photoaging [29]. Using flow cytometry and immunofluorescence assays, we observed a significant increase in DCFH-DA-positive cells following UVB irradiation, whereas HY treatment markedly reduced intracellular ROS levels (Figure 3A,B). Mitochondria represent a major target of ROS, and severe oxidative damage often leads to mitochondrial dysfunction [30]. Further analysis via flow cytometry and immunofluorescence confirmed that UVB irradiation elevated mitochondrial superoxide (MitoSOX) production in HaCaT cells, with the mean fluorescence intensity increasing approximately 1.4-fold compared to the control group. HY treatment effectively suppressed this UVB-induced rise in MitoSOX (Figure 3C,D).

Additionally, monodansylcadaverine (MDC), a fluorescent dye commonly used to label autophagic vesicle structures, was employed to evaluate the formation of autophagic vesicles. The results revealed that HY treatment significantly increased the number of MDC-labeled vesicles in HaCaT cells—approximately 1.8-fold higher than that in the UVB group (Figure 3E). Meanwhile, TMRE staining was used to assess mitochondrial membrane potential as an indicator of membrane integrity. UVB irradiation resulted in a pronounced loss of mitochondrial membrane potential, which was notably reversed by HY treatment (Figure 3F).

Western blot analysis indicated altered autophagic processes in UVB-exposed cells, characterized by decreased p62 degradation and downregulated Beclin1 expression. In contrast, HY treatment led to a significant reduction in p62 and upregulation of Beclin1. Notably, HY administration also increased the protein levels of Parkin and TOM20. The increase in Parkin is consistent with early events in the initiation of mitophagy. However, the concurrent increase in TOM20, a mitochondrial outer membrane protein, is incongruent with its typical degradation during the completion of mitophagy. This pattern may suggest a complex dynamic balance between mitophagic initiation and mitochondrial biogenesis under HY intervention, or a potential blockage in the later stages of the mitophagic flux. Further investigation is required to elucidate the precise mechanism underlying this observation (Figure 3G–K).

HaCaT cells were subjected to immunofluorescence staining using antibodies against LC3 and TOM20. The results demonstrated that HY treatment increased the co-localization of LC3 and TOM20 compared to the UVB-treated group (Figure 4A,B). To further validate the induction of mitophagy, the mitophagy agonist Adezmapimod (Ade) was employed [31]. Western blot analysis revealed that Ade reversed the UVB-induced alterations in the expression of Beclin1, p62, LC3, Parkin, PINK1, and TOM20. Moreover, the combination of Ade and HY resulted in a more pronounced activation of mitophagy (Figure 4C–H). Immunofluorescence staining further confirmed that Ade enhanced the co-localization of Beclin1 and TOM20 in HaCaT cells, an effect that was significantly strengthened when Ade was used in combination with HY (Figure 4I–K). Additionally, transmission electron microscopy (TEM) was used to examine mitochondrial ultrastructure. The UVB group exhibited evident mitochondrial membrane damage, while autophagosomes were clearly observed in the HY-treated group. In conclusion, these results collectively indicate that HY promotes mitophagy, with enhanced efficacy observed when co-administered with Ade.

Figure 5A illustrates the molecular structure of HY, a bioactive flavonoid compound. Potential protein targets of HY were systematically identified through database mining using SwissTargetPrediction (yielding 100 targets) and PharmMapper (yielding 35 targets). After removing duplicates, 127 unique potential target proteins were retained. Concurrently, photoaging-related targets were retrieved from the DisGeNET, GeneCards, OMIM, and DrugBank databases, resulting in 2921 non-redundant disease targets following deduplication. Venn diagram analysis revealed 80 common targets between HY and photoaging (Figure 5B). These overlapping targets were subsequently subjected to PPI network analysis. The PPI network was constructed using the STRING database (version 12.0) under strict parameters: organism limited to Homo sapiens, minimum interaction confidence score > 0.9, and exclusion of disconnected nodes (Figure 5C). The resulting network data in TSV format were imported into Cytoscape 3.10.3 for topological analysis and visualization (Figure 5D). Integrated network pharmacological analysis systematically identified Akt, SRC, and EGFR as key targets of HY (Figure 5E). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that the differentially expressed genes are potentially involved in the PI3K/AKT signaling pathway (Figure 5F). Enrichment analysis of potential targets suggested that the anti-photoaging mechanism of HY primarily involves protein kinase activity and cell migration processes (Figure 5G). Given that PI3K is the most common upstream regulator of Akt and that Akt closely interacts with mTOR, the PI3K/Akt/mTOR signaling pathway—which plays crucial roles in cell survival, metastasis, metabolism, angiogenesis, and inflammatory recruitment—is likely central to HY's activity [32]. Molecular docking results further demonstrated a strong binding affinity between HY and PI3K or Akt (Figure 5H). In summary, our integrated analysis predicts that HY exerts anti-photoaging effects primarily through modulation of the PI3K/Akt signaling pathway.

To further investigate whether HY regulates photoaging via the PI3K/Akt/mTOR signaling pathway, we examined the expression of phosphorylated PI3K (p-PI3K), Akt (p-Akt), and mTOR (p-mTOR) by Western blot. The results indicated that HY dose-dependently suppressed UVB-induced activation of the PI3K/Akt/mTOR pathway, and a synergistic effect was observed when HY was combined with Ade (Figure 6A–D). However, whether HY exerts its anti-photoaging effects by inhibiting the PI3K/Akt/mTOR pathway to activate mitophagy remained unclear. Therefore, we constructed PI3K-knockdown (si-PI3K) HaCaT cells using validated siRNA protocols (Figure 6E). Western blot analysis confirmed successful knockdown of PI3K protein expression (Figure 6F,G). Subsequent evaluation of mitophagy-related protein expression in si-PI3K cells revealed that Parkin, Beclin1, and PINK1 levels increased with rising HY concentration, exhibiting a clear concentration-dependent response. In contrast, TOM20 and LC3 levels did not show concentration dependence, and no significant synergistic effect was observed with Ade co-treatment (Figure 6H–P). These findings suggest the potential involvement of a negative feedback mechanism, which warrants further investigation. Immunofluorescence detection of p21 protein expression yielded consistent results (Figure 6Q,R). Taken together, these data indicate that HY likely activates mitophagy through inhibition of the PI3K/Akt/mTOR cascade to mitigate photoaging, though excessive suppression of this pathway may induce compensatory feedback responses.

In human skin, miRNAs serve critical regulatory functions in development, morphogenesis, and tissue homeostasis by modulating processes such as cell proliferation, differentiation, immune regulation, and wound healing [33]. By fine-tuning the expression of protein-coding genes, miRNAs act as subtle yet potent genetic regulators, making them promising candidates for maintaining skin health and counteracting premature aging [34,35]. Considering the pivotal role of the PI3K/Akt/mTOR signaling pathway in controlling cell proliferation, survival, and senescence [36], we hypothesized that its dysregulation represents a key mechanism underlying UVB-induced photoaging and that its activity may be modulated by upstream miRNAs. Therefore, this study aimed to identify specific miRNAs that regulate photoaging through the PI3K/Akt/mTOR pathway.

Based on our previous research, we identified 28 differentially expressed miRNAs (9 upregulated and 19 downregulated) in UVB-irradiated skin, with expression changes ranging from +1.7 to +8.4 (upregulation) and −1.9 to −25.0 (downregulation) [37]. Among these, miR-501-5p (+6.8) and miR-361-5p (+8.4) were the most significantly upregulated, while miR-23a (−25.0) and miR-323-3p (−7.7) were the most downregulated [37]. To identify miRNAs most likely to regulate the PI3K/Akt/mTOR pathway, we performed bioinformatic analysis using the miRDB (141 targets) and TargetScan (1190 targets) databases. Cross-referencing yielded four candidate miRNAs targeting PI3K with significant differential expression: miR-501-5p, miR-361-5p, miR-532-5p, and miR-139-5p (Figure 7A). RT-qPCR validation confirmed that miR-361-5p exhibited the most pronounced differential expression (Figure 7B), consistent with findings reported by Kraemer et al., further supporting the involvement of miRNAs in photoaging.

To investigate whether HY mediates its anti-photoaging effects via miR-361-5p in HaCaT cells, we constructed miR-361-5p mimics and confirmed their transfection efficiency (Figure 7C). Western blot analysis showed that miR-361-5p overexpression activated the PI3K/Akt pathway, whereas HY treatment inhibited its activation. Additionally, miR-361-5p mimic transfection suppressed mitophagy, an effect that was reversed by HY (Figure 7D–N). Confocal microscopy revealed reduced Beclin1 and TOM20 colocalization in miR-361-5p-overexpressing cells, which was significantly restored upon HY treatment (Figure 7O,P). We further evaluated the effect of HY on miR-361-5p-induced senescence using immunofluorescence and SA-β-gal staining. Compared to the UVB group, miR-361-5p-transfected cells showed elevated p21 expression, increased β-galactosidase activity, and a higher proportion of senescent cells. HY treatment counteracted these effects, reducing p21 levels, β-galactosidase activity, and senescence (Figure 7Q,R). Taken together, these results demonstrate that HY mitigates UVB-induced cellular senescence by downregulating miR-361-5p, thereby inhibiting the PI3K/Akt/mTOR pathway and promoting mitophagy.

Photoaging leads to significant deterioration of skin appearance, commonly manifested as wrinkles, laxity, erythema, and hyperpigmentation. The experimental design of the mouse photoaging model in this study is illustrated in Figure 8A. Treatment groups included LH, MH, HH, and a positive control VE group. Weekly photographic records were taken throughout the study. After 28 days, dorsal skin samples were collected to evaluate the effects of each treatment group on UVB-induced aging.

As shown in Figure 8B, the model group exhibited significant skin thickening, redness, and deep wrinkle formation, while all treatment groups showed varying degrees of improvement. The LH group still had slight redness and numerous wrinkles, with limited improvement; the MH group displayed mild sunburn with few wrinkles; both the HH and VE groups showed no obvious signs of photoaging, though fine wrinkles remained in the VE group. In terms of body weight, the model group showed suppressed weight gain, while the treatment groups exhibited a steady increasing trend (Figure 8C).

Histological analysis further revealed the protective effects of the treatments at the cellular level. Masson's trichrome staining indicated a substantial loss of dermal collagen in the model group—approximately 45.12% reduction compared to the control. All treatment groups increased dermal collagen content to varying degrees, with the HH group showing a 51.31% increase compared to the model group (Figure 8D,E). H&E staining demonstrated hyperkeratosis, acanthosis, and inflammatory cell infiltration in the model group, which were alleviated to some extent in the LH, MH, and HH groups. The HH group showed the most significant effect, with an epidermal thickness of only 26.58 ± 2.13 μm, significantly lower than that of the model group (53.44 ± 6.73 μm) (Figure 8F,G).

Western blot analysis indicated decreased expression of COL-1 and increased expression of MMP-9, p21, p53, and p16 in the model group. These changes were markedly reversed in the HH group (Figure 8J–N). Immunohistochemical findings further confirmed these results, showing the highest MMP-1 expression in the UVB-induced model group and the lowest in the HH group (Figure 8H,I). In summary, UVB significantly degrades skin collagen and elastic fibers, leading to structural alterations and accelerated cellular senescence, while high-dose HY treatment effectively mitigates these changes, preserving skin structural and functional integrity.