Protocol for Studying Honokiol Regulation of Transforming Growth Factor Beta 1/Smad Signaling in Ultraviolet A–induced Skin Photoaging Models

Samenvatting

This protocol investigates whether honokiol ameliorates UVA-induced photo-aging in human dermal fibroblasts and mouse skin by activating the TGF-β1/Smad pathway and reducing MMP expression.

Samenvatting

Honokiol (HNK), a key component derived from Magnolia officinalis, has the potential to treat skin diseases. At present, the impact and mechanism of action of HNK on Ultraviolet A (UVA)-induced photodamage in the skin are unclear. A photo-aging damage model of HDF cells and hairless mouse skin was constructed by UVA induction. The impact of HNK on the viability of normal HDF cells and UVA-induced HDF cells was examined through the Cell viability assay. SOD, GSH, CAT, and MDA levels in HDF cells were measured using different kits, and ROS levels were measured using DCFH-DA and Dihydroethidium. SA-β-galactosidase and TUNEL staining detected senescence and apoptosis in HDF cells, and flow cytometry assessed cell cycle progression. Histopathological damage to the skin was evaluated through pathological staining. In addition, immunofluorescence and Western blot were used to assess Ki67, collagen, matrix metalloproteinase (MMP), and TGF-β1/Smad pathway-associated protein levels. HNK (2.5-10 µM) treatment had no adverse impact on normal HDF cell viability but attenuated UVA-induced HDF cell damage, reducing oxidative stress and apoptosis. UVA induced senescence of HDF cells, induced S-phase arrest, downregulated Ki67, decreased collagen content, and increased MMP expression, while HNK effectively attenuated these UVA-induced abnormalities. In addition, UVA caused deeper wrinkles, decreased water content and collagen content, and increased epithelial thickness in mouse skin, whereas subcutaneous injection of HNK attenuated pathological damage to skin tissue and increased water and collagen content. Furthermore, HNK activated the TGF-β1/Smad pathway, and the TGF-β1 inhibitor SB431542 impaired the protective effect of HNK against photo-aging damage in HDF cells. In conclusion, HNK has the potential to be a drug for ameliorating photo-aging damage by reducing MMP expression through activation of the TGF-β1/Smad pathway, thereby inhibiting UVA-induced photo-aging in HDF cells and mouse skin.

Inleiding

The process of skin aging is intricate and divided into two main forms: natural aging and photo-aging¹. Studies indicate that as much as 80% to 90% of skin aging is caused by exogenous factors in the environment (e.g., ultraviolet rays, air pollution), of which long-term ultraviolet radiation-induced photo-aging has the most serious effects^2,3. Ultraviolet radiation is divided into UVC, UVB, and UVA. Since UVC cannot penetrate the ozone layer, current research on photo-aging focuses on UVA (320-400 nm) and UVB (280-320 nm)^4,5,6. Compared with UVB, UVA has lower energy, but it is more penetrating, passing through the epidermis and reaching deep into the dermis, which is an important cause of serious skin aging⁷. Previous studies have shown that the damage caused by 57 mJ/cm² UVB and 20 J/cm² UVA irradiation to HaCaT cells is comparable⁸. Skin photo-aging causes changes in the function and structure of the skin, specifically manifested as skin surface tissue sagging, deepening wrinkles, loss of collagen, and the appearance of gray skin color visible to the naked eye; in severe cases, a variety of benign, precancerous lesions and tumors can occur^9,10. Along with the increase in research on aging and anti-aging, the health risks of photoaging have become one of the most important health concerns worldwide. Therefore, it is crucial to investigate the pathophysiological processes underlying UVA-induced photo-aging damage, with a view to developing new therapeutic options for its treatment.

Extracted from Magnolia officinalis, a traditional Chinese medicinal herb, Honokiol (HNK) is a significant bioactive compound with the chemical formula of C₁₈H₁₈O₂¹¹. HNK exhibits various pharmacological activities, and its phenolic hydroxyl group is highly susceptible to oxidation and has strong antioxidant and free radical scavenging effects¹². In addition, HNK has antibacterial, anti-inflammatory, antiviral, antitumor, antidepressant, neuroprotective, and anti-premature ovarian failure pharmacological effects^13,14,15,16. Oral HNK has been determined to be essentially safe in animal studies and clinical studies, and no significant adverse effects have been identified^17,18,19. Recent studies have found that HNK prevents UVB-induced skin cancer²⁰, and inhibits UVB-induced skin inflammatory response and DNA hypermethylation²¹, suggesting its potential use in treating skin conditions. Compared with widely used anti-photo-aging agents, including vitamin C and resveratrol, HNK has distinct and superior properties: its lipophilic structure confers better transdermal permeability than hydrophilic vitamin C²²; and it exhibits higher photostability and in vivo bioavailability than resveratrol²³. These preclinical findings support the notion that HNK has promising translational potential as a safe and effective candidate for skincare products and clinical interventions against ultraviolet-induced skin photodamage. However, the impact of HNK treatment on skin photo-aging damage caused by UVA has not yet been reported.

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine crucial for cell growth and development, tissue repair and regeneration, and the growth and development of cells, tissues, and organs^24,25. It has been shown that TGF-β1 signals to the cell primarily through members of the Smad protein family, leading to the specific regulation of transcription of downstream target genes. When TGF-β1 signaling is activated, it results in phosphorylation of Smad2 and Smad3^26,27. Recent studies have shown a strong connection between the TGF-β1/Smad pathway and skin fibrosis and photo-aging^28,29. It has been reported that the mammalian target of rapamycin activates the TGF-β1/Smad pathway in rat skin tissues, which in turn promotes the synthesis of ceramides, which are important for retaining skin moisture and maintaining the skin barrier³⁰. Notably, it has been reported that HNK modulated the TGF-β1/Smad pathway in hepatic stellate cells, which, in turn, ameliorated liver fibrosis³¹. Therefore, cellular and mouse models of UVA-induced photo-aging injury were established, with 2.5–10 µM HNK treatment in HDF cells and 5 mg/kg or 10 mg/kg HNK intervention in hairless mice, to explore the effects of HNK on photo-aging damage and its potential role in modulating the TGF-β1/Smad pathway. This study aimed to develop new therapeutic strategies for UVA-induced skin photodamage.

Protocol

All cell culture experiments were performed in accordance with institutional biosafety guidelines and conducted under Biosafety Level 2 (BSL-2) laboratory conditions. Animal experiments were performed after approval from the Animal Ethics Committee of the 988th Hospital of the Joint Logistic Support Force of the Chinese People’s Liberation Army (approval number: 2023-089).

All experimental procedures were conducted in strict accordance with institutional and national guidelines for the care and use of laboratory animals. At the end of the experiment, mice were euthanized via an overdose of sodium pentobarbital (100 mg/kg, intraperitoneal injection). All efforts were made to minimize animal suffering during housing, treatment, and sampling procedures. The reagents, chemicals, equipment, and software used in the protocol are listed in the Table of Materials.

1. Cell culture and processing

1. Culture human dermal fibroblasts (HDF) in fibroblast culture medium. Maintain cultures at 37 °C in a saturated-humidity environment with 5% CO₂.
2. Replace the culture medium every 3 days, and passage HDF at a ratio of 1:3 when confluent.
3. Irradiate HDF with UVA lamps (emission spectrum: 320-400 nm) at a dose of 5 J/cm².
4. Replenish with fresh medium immediately after 30 min of UVA irradiation. Repeat UVA irradiation once daily for 8 consecutive days³².
5. Treat HDF with 2.5, 5, 10, 20, or 40 µM HNK for 24 h or 48 h in the dose-screening experiments³³. Treat HDF with 2.5, 5, or 10 µM HNK for 24 h followed by UVA irradiation (5 J/cm²) in subsequent experiments.
6. Expose HDF to 10 µM HNK for 4 h, then incubate with 10 µM TGF-β1 inhibitor SB431542 for 24 h, and finally irradiate with UVA (5 J/cm²) in the HNK+SB431542 group.

2. Cell viability assessment

1. Inoculate HDF into 96-well cell culture plates at a density of 1×10⁴ cells/well.
2. After different treatments, add 10 µL of 10% water-soluble tetrazolium salt reagent to each well to a final volume of 100 µL per well, and incubate the cells at 37°C for 1.5 h in the dark.
3. Measure the OD450 values of the cells using a microplate reader.

3. Determination of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), and glutathione (GSH) levels

1. Wash HDF with 1 mL pre-cooled PBS (0.01 M, pH 7.4), add 200 µL SOD sample preparation solution (from SOD Assay Kit) to lyse cells on ice for 15 min.
2. Centrifuge the lysate at 8000 × g for 5 min at 4 °C, and collect the supernatant as the assay sample.
3. Use a BCA (Bicinchoninic Acid) protein assay kit to determine the protein concentration of the supernatant, and adjust the sample to 50 µg total protein in a 50 µL volume.
4. Mix the 50 µg protein sample with the SOD assay buffer to a final reaction volume of 200 µL, and incubate the mixture at 37 °C for 30 min in the dark, and then measure the OD450 value.
5. Take the same cell lysate supernatant used for the SOD assay, measure CAT, GSH, and MDA levels using the respective assay kits.

4. Measurement of cellular ROS levels

1. Treat HDF cells with different conditions, rinse twice with 500 µL PBS, and mix well with 500 µL of 10 µM reactive oxygen species fluorescent probe and incubate at 37 °C for 20 min away from light.
2. Centrifuge at 1000 × g for 5 min at room temperature to collect the cells, and then rinse two times with 500 µL PBS.
3. Resuspend the cells in 500 µL cell culture medium, and then analyze the staining by flow cytometry.
4. Record fluorescence intensity using flow cytometry analysis software, and then calculate ROS levels based on the fluorescence intensity higher ROS corresponds to stronger green fluorescence).

5. Senescence-associated β-galactosidase (SA-β-galactosidase) staining

1. Seed HDF into 6-well cell culture plates at a density of 1×10⁶ cells/well and culture to adherence. After different treatments, wash HDF once with 2 mL PBS.
2. Add 1 mL β-galactosidase staining fixative to each well, and incubate the cells at room temperature with fixative for 15 min.
3. Wash the cells 3 times with 2 mL PBS, add 1 mL staining working solution to each well, and then incubate at 37°C overnight in the dark.
4. Observe and photograph the cells using a light microscope (senescent cells appear blue).

6. Cell cycle assay

1. Seed HDF into 6-well plates at 1×10⁶ cells/well; after different treatments, rinse cells twice with 2 mL PBS.
2. Centrifuge at 1000 × g for 5 min at room temperature to collect cells, add 1 mL of 70% ethanol (cell fixative), and mix gently by pipetting. Place the fixed cells in a 4°C refrigerator for 12 h.
3. Prepare 500 µL per sample of cell staining solution by mixing propidium iodide (PI, final concentration 50 µg/mL) and RNaseA (final concentration 200 µg/mL) in PBS. Expose cells to 500 µL of staining solution, incubate in the dark at 37°C for 30 min.
4. Assess fluorescence intensity using flow cytometry. Perform cell-cycle analysis using a flow cytometer.

7. TUNEL staining

1. Expose HDF cells from different treatment groups to 1 mL of 4% paraformaldehyde and incubate at room temperature for 30 min.
2. Add 1 mL of PBS containing 0.3% Triton X-100 to the cells and incubate at room temperature for 5 min.
3. Wash the cells twice with PBS, then add 500 µL TUNEL assay solution dropwise to evenly cover the cells. Incubate at 37°C in the dark for 1.5 h.
4. Add 200 µL of DAPI staining solution to cover the cells, and incubate away from light for 10 min at 25 °C.
5. Seal the cells with a fluorescence signal-preservation mounting medium, then observe and photograph them using a fluorescence microscope (TUNEL-positive cells show green fluorescence).

8. Immunofluorescence staining

1. Seed HDF cells from different treatment groups into 12-well plates. Once the cell density reaches 50%-60%, expose the cells to 1 mL of 4% paraformaldehyde and incubate at room temperature for 25 min.
2. Add 1 mL of 5% bovine serum albumin (BSA) dropwise to block the cell surface, and incubate at room temperature for 30 min.
3. Add 500 µL of primary antibodies Ki67 (1:300), collagen I (1:100), matrix metalloproteinase (MMP)-1 (1:50), or MMP-3 (1:100) dropwise, and incubate at 4°C overnight.
4. On the following day, incubate the cells with 500 µL of fluorescein isothiocyanate (FITC)-labeled secondary antibody (1:100) at 37°C away from light for 1 h.
5. Add 200 µL of DAPI staining solution to cover the cells, incubate away from light for 10 min, observe using a fluorescence microscope, and analyze with image analysis software (collagen I and MMP-1 positive cells show red fluorescence, Ki67 and MMP-3 positive cells show green fluorescence).

9. ELISA

1. Add 100 µL of HDF cell culture supernatant to wells of MMP-1 or MMP-3 ELISA kit plates, and incubate at 37°C for 2 h.
2. Wash the wells 3 times with 200 µL of PBS, then add 100 µL of the corresponding antibody to each well and incubate at 37 °C in the dark for 1 h.
3. Rinse the wells 3 times with 200 µL of washing buffer (provided in the kit), shake off excess solution, and incubate the samples with 100 µL of streptavidin-HRP for 30 min at 37°C in the dark.
4. After rinsing with PBS, add 100 µL of substrates A and B according to the kit instructions and incubate at 37°C in the dark for 10 min.
5. Add 50 µL of termination solution, mix well, assess the OD₄₅₀ value using a microplate reader, and calculate the concentration.

10. Photo-aging damage mouse model construction

1. House SPF-grade female SKH1 hairless mice (15-20 g, 4-5 weeks old) at 22 °C with 55%-60% humidity and allow autonomous access to food and water. Disinfect facilities regularly, including food containers, water bottles, and drinking spouts.
2. Randomly assign mice to the control group (n=6) and the model group (n=18). Irradiate model group mice with UVA at 0.35 J/cm² for 8 weeks³⁴; house control group mice in the same environment without UVA irradiation.
3. Divide the model group mice equally into 3 subgroups (n=6 each).
4. Administer subcutaneous injections of HNK at 5 mg/kg or 10 mg/kg in 100 µL volume twice a week to two subgroups during UVA irradiation (record as the 5 mg/kg HNK group and 10 mg/kg HNK group, respectively). Administer subcutaneous injections of 100 µL saline twice a week to the third subgroup during UVA irradiation (record as UVA group).
5. Evaluate skin wrinkle formation and water content after successful modeling³⁵.
6. Euthanize mice with sodium pentobarbital (100 mg/kg) intraperitoneally, and then collect dorsal skin tissues for further experiments.

11. Measurement of skin collagen I content

1. Collect 50 mg of mouse dorsal skin tissues, grind them thoroughly in liquid nitrogen, and lyse them using 500 µL of pre-cooled RIPA lysis buffer for 30 min on ice.
2. Centrifuge the lysate at 8000 × g for 5 min at 4 °C, collect the supernatant, and measure the collagen content using a pro-collagen I C peptide assay kit according to the manufacturer’s instructions.

12. Hematoxylin and eosin (HE) staining

1. Fix mouse skin tissues in 10 mL 4% paraformaldehyde at room temperature for 24 h, dehydrate in gradient ethanol (50%, 75%, 95%, 100%, each for 5 min), embed in paraffin, and section into 4 µm slices.
2. Bake the slices at 65°C for 8 h, deparaffinize in xylene twice for 10 min each, and rehydrate in gradient ethanol at room temperature.
3. Stain the slices with hematoxylin solution for 8 min, rinse with running tap water for 1 min, differentiate with differentiation solution for 30 s, and stain with eosin for 1 min at room temperature.
4. Dehydrate the stained slices in gradient ethanol, clear in xylene, seal with mounting medium, and observe under a microscope (The cell nucleus appears blue-purple, while the cytoplasm, collagen fibers, and erythrocytes appear pink to varying degrees).

13. Masson staining

1. Stain paraffin sections of mouse skin tissues using Masson trichrome staining kit³⁶. Deparaffinize paraffin sections in xylene twice for 10 min each, add Weigert iron hematoxylin staining solution, and stain for 10 min.
2. Differentiate with acid ethanol differentiation solution for 30 s, rinse with running tap water for 5 min, then stain with Ponceau-fuchsin staining solution dropwise for 10 min at room temperature. Perform reverse bluing with Masson's blue solution and then rinse with distilled water for 1 min.
3. Prepare a weak acid working solution at a ratio of distilled water: weak acid solution (provided in the kit) = 2:1; add 1 mL weak acid working solution dropwise and wash for 30 s at room temperature.
4. Pour off excess liquid, add 1 mL phosphomolybdic acid solution dropwise, and treat for 2 min at room temperature. Add 1 mL of the weak acid working solution dropwise, then wash for 30 s.
5. Pour off excess liquid, add 1 mL aniline blue staining solution dropwise and stain for 1 min. Add 1 mL weak acid working solution dropwise and wash for 30 s.
6. Dehydrate quickly in 95% ethanol for 2 s. Dehydrate in absolute ethanol twice, 10 s each time.
7. After drying, clear the sections in xylene, and mount with neutral gum. Observe the distribution of collagen fibers in each group under a microscope (Collagen fibers appear blue, while muscle fibers, cytoplasm, and erythrocytes are bright red, and cell nuclei appear distinctly blue-black).

14. Measurement of skin tissue ROS levels

1. Take frozen sections of mouse skin tissue (prepared within 1 h of excision), spread 1 mL cleaning solution dropwise over the entire section surface, and let stand at room temperature for 10 min.
2. Shake off the washing solution, then add 1 mL of 10 µM Reactive oxygen species probe dropwise to evenly cover the sections; incubate at 37 °C away from light for 30 min.
3. Rinse the sections twice with PBS, add cover slips, and observe using a fluorescence microscope (ROS-positive areas exhibit red fluorescence; the lower the ROS level, the weaker the red fluorescence signal).

15. Biochemical indicators testing

1. Collect mouse serum and prepare detection reagents according to the instructions of the corresponding assay kits.
2. Measure the activities of aspartate transaminase (AST) and alanine transaminase (ALT) in mouse serum using the respective kits.
3. Determine the levels of blood urea nitrogen (BUN) and creatinine (CRE) in mouse serum using the respective kits.

16. Western blot

1. Collect 50 mg of tissue or 1×10⁶ cells, lyse the samples on ice for 30 min using 200 µL RIPA lysis buffer to obtain proteins.
2. Centrifuge at 8000 × g for 5 min at 4 °C and collect the supernatant. Assess protein content in cells and tissues using the BCA protein concentration assay kit.
3. Perform electrophoresis of sample proteins on 12% SDS-PAGE gel, and transfer separated proteins to PVDF membranes at 4 °C.
4. Block PVDF membranes with 5% BSA for 3 h at room temperature. Rinse the membranes with TBST (contains 20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween-20) 3 times, then incubate overnight at 4°C with primary antibodies: P53 (1:1000), P21 (1:1000), P16 (1:100), collagen I (1:1000), Cyclin A2 (1:500), Cyclin-dependent kinase 2 (CDK2, 1:1000), MMP-1 (1:500), MMP-3 (1:1000), TGF-β1 (1:1000), Smad2 (1:100), Smad3 (1:1000), p-Smad2 (1:1000), or p-Smad3 (1:1000).
5. On the second day, wash the membranes with TBST 3 times, then incubate with 1 mL of goat anti-rabbit IgG (1:10000) at room temperature for 2 h. Apply 500 µL of the enhanced chemiluminescence (ECL) reagent evenly to the membranes, then scan them using a gel imaging system.
6. Perform gray value analysis using image analysis software. Express the relative expression level as the ratio of its gray value to that of GAPDH.

17. Statistical analysis

1. For in vitro cell-based assays, all experiments are independently repeated thrice (n=3, biological replicates).
2. For in vivo animal experiments, 6 mice are included per group (n=6), which complies with the 3R principles for laboratory animal welfare and ethics.
3. All results are expressed as mean ± standard deviation. Perform statistical analysis using statistical analysis software. Data normality was tested using the Shapiro-Wilk test.
4. For normally distributed data, use one-way ANOVA for multi-group comparison, followed by Tukey’s post-hoc test for pairwise comparisons between groups. For non-normally distributed data, apply the nonparametric Kruskal-Wallis test. P< 0.05 indicates a significant difference.
5. Generate graphs using graphing software.

Resultaten

HNK alleviates oxidative stress damage in HDF cells caused by UVA
The impacts of various concentrations of HNK treatment for 24 h or 48 h on the viability of HDF cells were examined by Cell viability assay to screen for the appropriate concentration and treatment duration. The results showed that 2.5, 5, and 10 µM of HNK did not significantly affect the viability of normal HDF cells after 24 h or 48 h of treatment, demonstrating that HNK is non-toxic to normal cells at these doses. Notably, HNK at co...

Discussie

Ultraviolet light is one environmental factor that harms cells and organisms. Previous studies have reported that UVA, with its high penetrating power, is the main type of ultraviolet light that reaches the Earth's surface³⁸. Fibroblasts are important functional cells in the dermis, and their aging can directly or indirectly cause skin aging³⁹. UVA-induced photo-aging damage in the HDF cell model and the hairless mouse model is widely used in the study of skin photo-aging

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
ALT activity assay kit	Beyotime Biotechnology, Shanghai, China	P2711M	alanine transaminase activity detection kit
Amplex Red CRE assay kit	Beyotime Biotechnology, Shanghai, China	S0291S	creatinine detection kit
AntiFade mounting medium	Beyotime Biotechnology, Shanghai, China	P0126	fluorescence signal preservation mounting medium
AST activity assay kit	Beyotime Biotechnology, Shanghai, China	P2715S	aspartate transaminase activity detection kit
BCA protein assay kit	Beyotime Biotechnology, Shanghai, China	P0012	protein concentration determination kit
BSA	Sigma-Aldrich, St. Louis, MO, USA	V900933	blocking reagent for non-specific binding
BUN Assay kit	Beyotime Biotechnology, Shanghai, China	S0574S	blood urea nitrogen detection kit
CAT Assay kit	Beyotime Biotechnology, Shanghai, China	S0051	catalase activity detection kit
CCK-8 reagent	Sigma-Aldrich, St. Louis, MO, USA	96992	cell viability assay solution which is a water-soluble tetrazolium salt
CDK2 antibody	Invitrogen, Carlsbad, CA, USA	PA5-79024	primary antibody for CDK2 detection
Chemiluminescent agent ECL	MedChemExpress, Monmouth Junction, NJ, USA	HY-K1005	enhanced chemiluminescence detection reagent
Collagen I antibody	Invitrogen, Carlsbad, CA, USA	PA1-26204	primary antibody for collagen I detection
Cyclin A2 antibody	Invitrogen, Carlsbad, CA, USA	PA5-34682	primary antibody for Cyclin A2 detection
DAPI staining solution	Sigma-Aldrich, St. Louis, MO, USA	D9542	nuclear staining dye
DCFH-DA fluorescent probe	MedChemExpress, Monmouth Junction, NJ, USA	HY-D0940	reactive oxygen species fluorescent probe
Differentiation solution	Beyotime Biotechnology, Shanghai, China	C0161s	reagent for hematoxylin differentiation step
Dihydroethidium probe	Beyotime Biotechnology, Shanghai, China	S0063	reactive oxygen species probe
Fibroblast culture medium	Sunncell Biotechnology Co., Ltd., Wuhan, Hubei, China	SNPM-H342	complete culture medium for fibroblast growth
FITC-labeled goat anti-rabbit IgG	Invitrogen, Carlsbad, CA, USA	F-2765	fluorescein isothiocyanate (FITC)-labeled secondary antibody
Flow cytometry	BD biosciences, San Jose, CA, USA	BD FACSCaliburTM	flow cytometer
FlowJo software	BD biosciences, San Jose, CA, USA	v10.8	flow cytometry analysis software
Fluorescence microscope	Leica, Heidelberg, Germany	DM IL LED	microscope for fluorescence imaging
GAPDH antibody	Invitrogen, Carlsbad, CA, USA	PA1-987	internal control antibody for protein normalization
gel imaging system	Invitrogen, Carlsbad, CA, USA	iBright CL1500	imaging system for protein detection
goat anti-rabbit IgG	Invitrogen, Carlsbad, CA, USA	31460	secondary antibody for immunodetection
Graphpad Prism 9.0 software	GraphPad Software, San Diego, CA, USA	Prism 9.0	graphing software
GSH Assay kit	Beyotime Biotechnology, Shanghai, China	S0053	glutathione content detection kit
HE staining kit	Beyotime Biotechnology, Shanghai, China	C0105S	histological staining kit for tissue morphology analysis
HNK	MedChemExpress, Monmouth Junction, NJ, USA	HY-N0003	honokiol, bioactive compound
Human dermal fibroblasts	Sunncell Biotechnology Co., Ltd., Wuhan, Hubei, China	SNP-H342	primary human skin fibroblast cells
IBM SPSS Statistics 26.0 software	IBM Corporation, Armonk, NY, USA	IBM SPSS Statistics 26.0	statistical analysis software
ImageJ software	Wayne Rasband, National Institute of Mental Health, USA	version 1.54h	image analysis software
Ki67 antibody	Invitrogen, Carlsbad, CA, USA	MA5-14520	primary antibody for cell proliferation marker detection
Light microscope	Leica, Heidelberg, Germany	DM3000	optical microscope for brightfield imaging
Masson trichrome staining kit	Solarbio, Beijing, China	G1340	collagen and connective tissue staining kit
MDA Assay kit	Beyotime Biotechnology, Shanghai, China	S0131S	malondialdehyde level detection kit (lipid peroxidation marker)
Microplate Reader	Thermo Fisher Scientific, Waltham, MA, USA	1410101	absorbance measurement instrument for cell-based assays
MMP-1 antibody	Invitrogen, Carlsbad, CA, USA	PA5-27210	primary antibody for matrix metalloproteinase-1 detection
MMP-1 ELISA kit	Enzyme-linked Biotechnology, Shanghai, China	ml038199	quantitative detection of MMP-1 in biological samples
MMP-3 antibody	Abcam, Cambridge, MA, USA	ab52915	primary antibody for matrix metalloproteinase-3 detection
MMP-3 ELISA kit	Enzyme-linked Biotechnology, Shanghai, China	ml105322	quantitative detection of MMP-3 in biological samples
Neutral gum	Beyotime Biotechnology, Shanghai, China	C0173	mounting medium
P16 antibody	Invitrogen, Carlsbad, CA, USA	PA5-20379	primary antibody for p16 protein detection
P21 antibody	Invitrogen, Carlsbad, CA, USA	MA5-14949	primary antibody for p21 protein detection
P53 antibody	Abcam, Cambridge, MA, USA	ab131442	primary antibody for p53 protein detection
paraformaldehyde	Solarbio, Beijing, China	P1110	cell and tissue fixation reagent
PI	Beyotime Biotechnology, Shanghai, China	ST512	propidium iodide for DNA staining in cell cycle analysis
Pro-collagen I C peptide assay kit	TAKARA, Tokyo, Japan	MK101	collagen synthesis detection kit
p-Smad2 antibody	Invitrogen, Carlsbad, CA, USA	44-244G	primary antibody for phosphorylated Smad2 detection
p-Smad3 antibody	Invitrogen, Carlsbad, CA, USA	44-246G	primary antibody for phosphorylated Smad3 detection
PVDF membranes	Invitrogen, Carlsbad, CA, USA	88520	protein transfer membrane for western blotting
RIPA lysis buffer	Beyotime Biotechnology, Shanghai, China	P0013B	protein extraction buffer for cell and tissue lysates
RNaseA	Beyotime Biotechnology, Shanghai, China	ST579	ribonuclease for RNA removal during DNA staining
SA-β-galactosidase staining kit	Beyotime Biotechnology, Shanghai, China	C0602	cellular senescence detection kit
SB431542	MedChemExpress, Monmouth Junction, NJ, USA	HY-10431	TGF-β1 signaling pathway inhibitor
SDS-PAGE gels	Invitrogen, Carlsbad, CA, USA	WG1403BX10	polyacrylamide gels for protein electrophoresis
SKH1 hairless mice	Vitalriver, Beijing, China		animal model for skin photo-aging studies
Smad2 antibody	Invitrogen, Carlsbad, CA, USA	51-1300	primary antibody for Smad2 detection
Smad3 antibody	Invitrogen, Carlsbad, CA, USA	51-1500	primary antibody for Smad3 detection
SOD Assay kit	Beyotime Biotechnology, Shanghai, China	S0101S	superoxide dismutase activity detection kit
TGF-β1 antibody	Abcam, Cambridge, MA, USA	ab215715	primary antibody for TGF-β1 detection
Triton X-100	Sigma-Aldrich, St. Louis, MO, USA	X-100	non-ionic detergent for cell membrane permeabilization
TUNEL assay solution	Beyotime Biotechnology, Shanghai, China	C1086	apoptosis detection reagent for DNA fragmentation
Xylene	Sigma-Aldrich, St. Louis, MO, USA	247642	organic solvent for tissue deparaffinization