Intense pulsed light rejuvenates UVB-induced photoaging in human keratinocytes and guinea pig skin by inhibition of ERK-AP-1-MMP pathway

Abstract

Skin photoaging is predominantly induced by ultraviolet (UV) irradiation. Intense pulsed light (IPL) is a commonly employed non-ablative treatment for photoaging. However, the effects and mechanisms of IPL on UV-induced skin photoaging remain insufficiently understood. In this study, we aimed to examine the anti-photoaging effects of IPL and elucidate the underlying mechanisms. This study revealed that UV triggered extracellular signal-regulated kinases (ERK) together with c-jun NH2-terminal kinase (JNK), while selectively suppressed UV-induced ERK phosphorylation while activating JNK in human skin keratinocytes. The different ERK/JNK expression patterns induced by UV and IPL resulted in distinct c-fos/c-jun (activator protein 1) phosphorylation, cyclin D1 expression, and matrix metalloproteinase (MMP) secretion. In vivo, IPL inhibited MMP expression in guinea pig skin and promoted c-fos/c-jun phosphorylation, epidermal proliferation, and collagen remodeling. These findings indicated that ERK was involved in IPL rejuvenation by regulating c-fos, c-jun, cyclin D1, and MMPs, providing a potential target for skin rejuvenation.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-50438-w.

Introduction

The skin serves as a protective barrier against radiation, chemical, and physical damage. The damaging factors, including ultraviolet (UV) radiation, climate change, sleep deprivation, and air pollution, induce skin aging. Among them, UV is a major cause of aging¹. The spectrum of solar radiation reaching Earth includes UV, visible light, and infrared radiation². Different wavelengths have varying effects, and UV (100–400 nm) can cause skin sunburn, inflammation, aging, and cancer^3,4. Intense Pulsed Light (IPL) is a type of filtered, high energy density pulsed light that emits polychromatic, broadband incoherent light with a wavelength range of 500–1200 nm. It was originally developed and introduced into clinical practice primarily based on the principle of selective photothermolysis, aiming to treat cutaneous vascular lesions (e.g., telangiectasias, port-wine stains), pigmented lesions (e.g., freckles, solar lentigines), and fine wrinkles. IPL has been demonstrated to have anti-aging efficacy in clinical practice; however, the mechanism remains unclear^5,6.

Mitogen-activated protein kinase (MAPK) primarily comprises extracellular signal-regulated kinases (ERK), p38 mitogen-activated protein kinase (p38), and c-jun NH2-terminal kinase (JNK), which are critical for cell proliferation, differentiation, and apoptosis^7–11. Numerous studies have demonstrated that MAPKs play an important role in UV-induced skin photoaging^12–14. However, research has indicated that IPL and lasers in the visible light and near-infrared range may activate the MAPK pathway and promote skin rejuvenation^15–18. It has been demonstrated that IPL can activate interleukin and transforming growth factor-β (TGF-β) signaling pathways, together with MAPK pathways^19,20. Activator protein 1 (AP-1), which is composed of c-jun and c-fos, serves as a key downstream target of the MAPK pathway. Activated MAPK modulates the c-fos/c-jun heterodimer (i.e., the AP-1 transcription complex), whose transcriptional activity is jointly determined by the phosphorylation status of its core subunits c-fos and c-jun, and thereby regulates a variety of cellular processes such as cell proliferation and collagen remodeling²¹. We further propose a mechanistic model underlying IPL-mediated MAPK modulation in skin rejuvenation. As a non-invasive photobiomodulatory modality, IPL induces mild, well-tolerated photothermal stress and low level ROS in keratinocytes without overt cytotoxicity. These sublethal stimuli activate membrane sensors and upstream kinase cascades, including MEKs and MKKs, leading to dose and time dependent, subtype-selective phosphorylation of MAPK members. Notably, optimal IPL treatment preferentially attenuates aberrant ERK hyperactivation while transiently and moderately stimulating JNK signaling. Such differential MAPK reprogramming remodels downstream AP-1 transcriptional activity, shifting cellular responses toward reparative collagen remodeling rather than pro-photoaging catabolic reactions, thereby contributing to the anti-photoaging effects of IPL.

We hypothesized that MAPKs play a critical role in UV-induced skin photoaging and IPL rejuvenation, and that IPL differentially modulates MAPK subtypes, suppressing ERK while activating JNK, thereby rebalancing AP-1 activity toward a rejuvenative profile. We investigated the effects of UV and IPL on the expression patterns of MAPKs, AP-1, cell proliferation, and matrix metalloproteinase (MMP) production in human skin keratinocytes (hKCs) and guinea pig skin. We speculate that IPL exerts anti-photoaging effects by selectively regulating the ERK/AP-1/MMP signaling pathway in epidermal keratinocytes. This study aims to verify the regulatory effects of IPL on MAPK/AP-1 pathway, cell proliferation and MMPs secretion in UVB-irradiated human keratinocytes in vitro, and to confirm the reparative effects of IPL on collagen remodeling and epidermal proliferation in photoaged guinea pig skin in vivo. The results revealed that 17 J/cm² of IPL (IPL17) could rejuvenate UV-irradiated human keratinocytes and guinea pig skin by regulating ERK/AP-1, thereby inhibiting MMP production and promoting cell proliferation.

Materials and methods

Source of hKCs

Primary hKCs were isolated from foreskin biopsies of adolescent donors (aged 12–16 years), procured from the Department of Dermatology at the Air Force Medical Center. This was followed by establishing consent protocols approved by the Ethics Committee (Approval 2023-13-S01).

Ethics statement

All procedures involving human tissues received approval from the Air Force Medical Center Ethics Committee (Approval 2023-13-S01) and were conducted in compliance with the principles of the Declaration of Helsinki and relevant institutional guidelines for human tissue research.

The animal experiments conducted in this study received approval from the Animal Ethics Committee of the Air Force Medical Center (Approval No. 2023-13-S01). These experiments were conducted in strict compliance with the Regulations for the Administration of Affairs Concerning Experimental Animals of China. Furthermore, the experiments were reported in compliance with the ARRIVE guidelines.

Materials

Human Keratinocyte Growth Supplement/fetal bovine serum was procured through Gibco™ (NY, USA). Trypsin-EDTA (0.25%), dimethyl sulfoxide, RIPA buffer, Dulbecco’s modified Eagle’s medium, BCA proteomic assay, penicillin-streptomycin solution, phosphate-buffered saline (PBS), and goat anti-murine IgG-HRP were obtained from Solarbio (Beijing, China). Goat anti-rabbit IgG-HRP was obtained by ZSGB-BIO (Beijing, China). EpiLife Medium, Cell Counting Kit-8 (CCK-8) Assay Kit was provided by Dojindo (Kumamoto, Japan). Additionally, PD98059 was obtained from MedChemExpress (USA). Phosphorylated c-fos (p-c-fos), phosphorylated p38 (p-p38), phosphorylated JNK (p-JNK), together with cyclin D1 (for Western blot), MMP-1, MMP-9 (for immunohistochemistry [IHC]), and phosphorylated c-jun (p-c-jun, for Western blot/IHC) were acquired through Cell Signaling Technology™ (USA). MMP-1, MMP-2, MMP-9, phosphorylated ERK (p-ERK), GAPDH (for Western blot), p-c-fos (for IHC), and MMP-3 (for Western blot and IHC) were obtained from Abcam™ (Cambridge, UK).

Cellular culturing

The hKCs were used in passages 1–4. Cell isolation and culture protocol were performed according to a prior study. Different intervention methods created three categories of hKCs: control group (without UV irradiation), UV-irradiated group, and UV + IPL-irradiated group. This study was approved by the Ethics Committee of the Air Force Medical Center’s (Beijing, China) Ethics Committee (2023-13-S01). Moreover, it was conducted with the written informed consent of the donors’ legal guardians.

Animals

A total of 30 white guinea pigs (males, 8 weeks old, weighing 300–400 g each) were obtained from Beijing Keyu Animal Breeding Center. The dorsal skin of each guinea pig was shaved before IPL/UV irradiation. About three groups of guinea pigs were randomly selected: The control group (not exposed to UV irradiation), the UV-irradiated group, and the UV + IPL-irradiated group (n = 10). The guinea pigs were anesthetized by intraperitoneal injection with sodium pentobarbital for skin biopsy. The guinea pigs were humanely euthanized under deep anesthesia induced by 1% sodium pentobarbital (100 mg/kg, intraperitoneal), followed by cervical dislocation to ensure death.

UV irradiation

A Philips ultraviolet-B (UVB) lamp (Netherlands) emitting in the 310–311 nm range was used. UVB intensity was measured by a UV radiometer. Before UVB irradiation, the medium was replaced with PBS. UVB doses were 50, 100, and 200 mJ/cm² for cells. A 100 mJ/cm² UVB dose was given to investigate the time effect of UVB irradiation²². The cells were collected 24 h following UVB irradiation. An improved irradiation method was used for animal experiments based on relevant reports²³. The guinea pigs were exposed daily to 100 mJ/cm² of UVB radiation for 10 weeks and the cumulative UVB exposure dose was 7.0 J/cm².

IPL irradiation

The guinea pigs were irradiated by an IPL device (M22, Lumenis Ltd., Israel). All treatments were performed at a wavelength of 590–1200 nm, a spot size of 15 × 35 mm, a pulse duration of 12 ms, a pulse delay of 12 ms, and two pulses. In the dosage-effect experiment, the dose of IPL irradiation on hKCs was 10, 17, and 23 J/cm². In the time-effect experiment on hKCs, IPL parameters were 17 J/cm². For animal experiments, the dorsal skin of guinea pigs was irradiated with 17 J/cm² of IPL once every three weeks for three consecutive times to emulate the clinical IPL rejuvenation process. Specimens were collected on the 1 st, 7th, and 14th day after the last IPL irradiation. The IPL wavelength range of 590–1200 nm was selected based on its widespread clinical use for photorejuvenation and photodamage repair. This broadband range combines visible light (acting on epidermal keratinocytes) and near-infrared light (penetrating into the dermis to affect collagen remodeling), enabling comprehensive photobiomodulation while avoiding excessive thermal injury to skin tissue.

Staining

Using staining kits, Masson’s Trichrome and Sirius Red Stain steps were performed according to routine protocols. Tissue sections were cut to 4 μm thickness, and photographs were captured using a Leica microscope from Germany. Each histological slide was examined by a pathology instructor blinded to the experimental group allocations, who randomly selected 5 fields of view per slide under a microscope to assess the pathological changes.

IHC

IHC was performed using an HRP-Peroxide-DAB protocol. Guinea pig skin tissues were fixed in 4% paraformaldehyde for 24 h to ensure optimal preservation of cellular and extracellular structures. After fixation, tissues were rinsed and dehydrated through a graded ethanol series (70%, 80%, 95%, and 100%). Subsequently, samples were cleared in xylene and embedded in paraffin wax using a standard protocol (60℃ for 2 h). Sections were cut at 4 μm thickness using a microtome (Leica RM2235, Germany) and mounted on glass slides. For antigen retrieval, slides were heated in citrate buffer (pH 6.0) at 95℃ for 15 min, followed by blocking with 5% bovine serum albumin for 1 h at room temperature. Primary antibodies (Table 1) were incubated overnight at 4℃ with the sections, including p-c-fos (1: 100), p-c-jun (1: 150), MMP-1 (1: 150), MMP-3 (1: 400), and MMP-9 (1: 400). Next, the biotinylated secondary antibodies were left to incubate at room temperature for 20 min. Lastly, the DAB (3,3-diaminobenzidine) color was developed, and nuclei were counterstained with hematoxylin for histological context. Stained sections were imaged using light microscopy (Leica, Germany). For IHC analysis, five fields of view per sample were randomly selected at 200× magnification, covering central and peripheral regions of the tissue section to ensure representative sampling. Fields were selected using a predefined grid pattern to avoid bias. Optical density measurements for p-c-fos, p-c-jun, and MMPs were performed blindly by two independent investigators, with the mean value derived from all fields used for statistical analysis. The methods used to score and quantify IHC and histological evaluations are based on established protocols from prior research²⁴.

Table 1.

Primary Antibodies for Western Blot and Immunohistochemistry.

Antibodies clone	Cat#	Source
Anti-ERK1 (phospho Y204) + ERK2 (phospho Y187)	ab47339	abcam
Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb	4668 S	CST
p38 MAPK (D13E1) XP® Rabbit mAb	8690T	CST
Phospho-c-fos (Ser32) (D82C12) XP Rabbit mAb	5348T	CST
Phospho-c-jun (Ser73) (D47G9) XP Rabbit mAb	3270T	CST
Anti-MMP-1 antibody	ab38929	abcam
Anti-MMP-2 antibody	ab86607	abcam
Anti-MMP-3 antibody	ab53015	abcam
Anti-MMP-9 antibody	ab38898	abcam
Cyclin D1 (E3P5S) XP® Rabbit mAb	55,506	CST
GAPDH	ab8245	abcam

Transmission electron microscope (TEM)

The tissues were fixed using a solution containing 2.5% glutaraldehyde and then postfixed with an osmium tetroxide solution (1%). The next step involved dehydrating tissue portions using a graded ethanol series and embedding them in epoxy resin. The copper grids were used to mount ultrathin slices, stained with lead citrate and uranyl acetate.

CCK-8 assay

Cell viability was measured through the CCK-8 assay. The hKCs were placed within a 96-well plate (5000 cells/well). The hKCs were incubated with a 10% CCK-8 solution (120 min, 37℃, 5% CO₂) on the first, third, and fifth day after UV or IPL irradiation. The microplate reader used to measure the absorbance value was Bio-Rad 680, USA, and the measurement was taken at 450 nm.

Western blot analysis

With the RIPA buffer, the hKCs were lysed, and the extracted proteins were separated by 10% SDS-PAGE. The resolved proteins were then transferred onto PVDF membranes. Primary antibodies were exposed to membranes (4℃,overnight). Antibodies (Table 1) were diluted 1: 1000 within 5% non-fat milk and included p-ERK, p-p38, p-JNK, p-c-jun, and p-c-fos, as well as MMP-1, MMP-2, MMP-3, MMP-9, cyclinD1 together with GAPDH. Secondary antibodies were applied to the membranes and incubated at ambient temperature for 1 h. Lastly, Blots were imaged using Gel imaging system (Micro, USA). The contrast and brightness of the images presented in the figures were linearly adjusted for the entire image using ImageJ to improve visibility.

MMP-2 and MMP-9 gel zymography

Gel zymography was performed according to the manufacturer’s instructions. Gelatin zymography gels comprise 10% polyacrylamide gels, a separating gel with 1 mg/mL gelatin. Protein extracts containing active MMPs were obtained from the supernatant of hKCs. The samples were denatured in SDS buffer, and 20 µg of protein was used for SDS-PAGE. After staining with Coomassie Brilliant Blue, the gel was decolorized and photographed with a gel imaging device. MMP activity was observed as stainless bands on the blue-stained gelatin backdrop.

Evaluation of images

Photographs of HE, TEM, Masson’s trichrome/Sirius red stain-steps, IHC, and Western blotting were assessed through Image J^® (version 1.50, NIH, USA). The quantifications of epidermal thickness, collagen fibers, MAPKs, MMPs, and AP-1 were performed blindly.

Statistical analyses

Statistical analyses were conducted using GraphPad Prism software (version 8.2.0) (San Diego, USA). Data are presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for cohort-comparative statistical analyses. Statistical significance was defined as p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***) compared with the control group.

Results

Different effects of UV and IPL on MAPKs/AP-1 expression in hKCs

The epidermis was the direct target of UV and IPL, so hKCs model was selected for further investigation. To investigate the effects of UV and IPL on the MAPK/AP-1 signaling pathway, we performed dose and time effect experiments in hKCs, detected the phosphorylation levels of key proteins by Western blot, and screened out the optimal irradiation doses and time points for each respectively. Dosage-influence experiments indicated that neither the control nor the UV-irradiated groups demonstrated a significant change in p-p38 levels. Additionally, p-ERK and p-JNK were notably elevated in a dose-dependent manner following UV irradiation at doses of 50, 100, and 200 mJ/cm² (Fig. 1A).

Fig. 1.

UV and IPL differentially regulate MAPK phosphorylation in hKCs. (A, C) Western blots indicating p-p38, p-ERK1/2, and p-JNK in hKCs treated with UVB (50, 100, and 200 mJ/cm², A) or IPL (10, 17, and 23 J/cm², C). Right panels: Quantification of phosphorylated MAPK levels normalized to GAPDH (n = 3 independent experiments). (B, D) Time-course analysis of MAPK phosphorylation after UVB (100 mJ/cm², B) or IPL17 (17 J/cm², D) at 0–120 min post-treatment. (E, F) Phosphorylation of AP-1 subunits (p-c-fos and p-c-jun) in hKCs treated with UVB (50, 100, and 200 mJ/cm², E) or IPL (10, 17, and 23 J/cm², F). Statistical significance: One-way ANOVA with Tukey’s post-hoc test (A, C, E, F) or two-way ANOVA with Bonferroni’s test (B, D). *p < 0.05, **p < 0.01, ***p < 0.001 versus control.

Time-effect experiments demonstrated that p-p38 levels showed no significant changes after 100 mJ/cm² UV irradiation. However, p-ERK and p-JNK peaked at 15 min, increased 2–3 fold, respectively, and then recovered to baseline at 120 min (Fig. 1B).

After different doses of IPL irradiation, the p-p38 level indicated no significant change, while p-JNK increased dose-dependently (17 and 23 J/cm²). It demonstrated a non-linear relationship between p-ERK and IPL irradiation. At a dose of 17 J/cm², IPL irradiation inhibited the ERK pathway. However, at a dose of 23 J/cm², it notably activated ERK phosphorylation (Fig. 1C). The ERK activation pattern following IPL irradiation differed from that of UV irradiation. In the time-effect experiment, IPL17 immediately inhibited p-ERK and gradually recovered to a normal level in 120 min. The p-JNK peaked at 15 min and restored to its normal level in 120 min (Fig. 1D).

The effect of UV and IPL irradiation on p-c-fos/p-c-jun was assessed through Western blot analysis. The findings indicate that p-c-fos was significantly inhibited at 50, 100, and 200 mJ/cm² doses of UV radiation. However, p-c-jun significantly increased to more than 7-fold following 200 mJ/cm² of UV irradiation (Fig. 1E). Additionally, p-c-fos indicated no significant changes after various doses of IPL irradiation. Notably, among different doses of IPL irradiation, the 17 J/cm² dose significantly increased the p-c-jun level 5-fold (Fig. 1F). These results demonstrated that UV and IPL resulted in different ERK/JNK and c-fos/c-jun expression patterns.

Different effects of UV and IPL on hKCs proliferation and MMPs expression

Next, we evaluated the effects of UV and IPL on cell proliferative capacity and the expression of MMPs, the downstream target genes of AP-1. We detected the protein expression levels of cyclin D1 and MMPs by Western blot, assessed cell proliferative capacity using the CCK-8 assay, and analyzed the enzymatic activity of MMPs and the changes in cellular ultrastructure via gelatin zymography and transmission electron microscopy (TEM), respectively. The activation of cyclin D1 by AP-1 is crucial for cell proliferation. The Western blot technique evaluated the proteomic expression level of cyclin D1. About 24 h after UV irradiation, cyclin D1 was significantly downregulated in a dose-dependent manner. However, cyclin D1 was increased after IPL irradiation (Fig. 2A). Cell proliferation assay by CCK-8 assay revealed that doses of 100 and 200 mJ/cm² notably decreased cell viability on the first and third day. Conversely, after IPL17 treatment, the cell viability was significantly increased on the first day (Fig. 2B). CCK-8 assay revealed consistent results with that of Western blot, in which UV suppressed hKCs proliferation. Simultaneously, IPL promoted hKCs proliferation, particularly at the dose of 17 J/cm².

Fig. 2.

IPL17 regulates cyclin D1 expression, cell viability, and MMP production in UVB-irradiated hKCs. (A) Western blot and quantification of cyclin D1 protein in hKCs treated with UVB (50, 100, and 200 mJ/cm², left) or IPL (10, 17, and 23 J/cm², right). The cyclin D1 levels were normalized to GAPDH (n = 3 independent experiments). (B) Cell viability (CCK-8 assay) of hKCs at 1, 3, and 5 days post-UVB (50, 100, and 200 mJ/cm², upper) or IPL (10, 17, and 23 J/cm², lower). Data expressed as % of control (non-irradiated cells). (C, D) MMP expression (MMP-1, −2, −3, −9) in hKCs treated with UVB (50–200 mJ/cm², C) or IPL (10–23 J/cm², D). Left: Western blots; right: Quantification normalized to GAPDH. (E) Gelatin zymography assay the MMP-2 and MMP-9 activity in UVB-irradiated hKCs (50–200 mJ/cm²). (F) Ultrastructure of hKCs detected by TEM (scale bar: 500 nm) in control, UVB (100 mJ/cm²), and IPL17 (17 J/cm²)-treated hKCs. *p < 0.05, **p < 0.01, ***p < 0.001 versus control. Data are represented as mean ± SEM.

MMP, a critical downstream target gene of AP-1, have been reported to participate in the degradation of the extracellular matrix (ECM) and in collagen remodeling. Consequently, we measured MMP levels in UV and IPL-irradiated hKCs. Western blot indicated that UV irradiation promoted significant dose-dependent increases in MMP-1, MMP-2, MMP-3, and MMP-9 (Fig. 2C). However, IPL irradiation resulted in a non-linear response in MMP levels. Additionally, IPL irradiation significantly reduced MMP-1 and MMP-3 levels at a dose of 17 J/cm². At a dose of 23 J/cm², there was significant upregulation of MMP-1 and MMP-3. Moreover, there were no significant changes in MMP-2 and MMP-9 expression levels among IPL-irradiated groups and the control (Fig. 2D). The gelatin zymography assay determined MMP-2 and MMP-9 expression profiles following UV/IPL exposure. About 24 h after UV irradiation, MMP-2 and MMP-9 functions were significantly enhanced. However, there was no significant variation in MMP-2 and MMP-9 expression after IPL irradiation (Fig. 2E). Previous studies indicate that the number of cellular microvilli is involved in the secretory activity of hKCs. It participates in ECM degradation, remodeling, and homeostasis²⁵. Additionally, we examined the subcellular structure of hKCs via TEM. Compared with the control, showed more microvilli after UV irradiation and reduced after IPL17 treatment. These results suggest that UV triggers cell injury, whereas IPL induces cell repair in hKCs (Fig. 2F).

IPL17 rejuvenated UV-irradiated hKCs in MMP production

To specifically evaluate the rejuvenating effects of IPL17 (17 J/cm²) on UVB-irradiated hKCs, we compared three groups: control (untreated), UVB-irradiated (100 mJ/cm²), and UVB + IPL17-treated (100 mJ/cm² UVB followed by 17 J/cm² IPL).We analyzed the phosphorylation states of ERK, c-fos, and c-jun by Western blot, and measured the expression of cyclin D1 and MMPs to elucidate the ERK/AP-1-mediated mechanism. IPL17 showed a distinct photobiomodulation effect on ERK compared with UV irradiation. Additionally, we investigated the effects and mechanism of IPL17 on ERK, c-fos/c-jun, and the downstream MMPs. The data indicated that IPL17 significantly suppressed p-ERK that stimulated by UV. Moreover, IPL17 significantly restored UV-induced p-c-fos downregulation and increased UV-induced p-c-jun upregulation (Figs. 3A-D). Compared with the UV-treated group, UV + IPL irradiation significantly upregulated cyclin D1 and downregulated MMP-1, MMP-2, and MMP-3 expression in hKCs. Furthermore, MMP-9 expression levels indicated no significant difference between UV + IPL and UV groups (Figs. 3E-G). From these findings, we speculate that IPL alleviated UV-induced photoaging via the ERK/AP-1 pathway.

Fig. 3.

IPL17 modulates ERK-AP1 signaling, cyclin D1, and MMP expression in UVB-irradiated hKCs. (A) Western blots indicating p-ERK1/2, p-c-fos, and p-c-jun in hKCs. GAPDH served as a loading control. (B-D) Quantification of p-ERK1/2 (B), p-c-fos (C), and p-c-jun (D) levels normalized to GAPDH. (E) Western blots for cyclin D1 and MMPs (MMP-1, −2, −3, −9) in hKCs. (F-G) Quantification of cyclin D1 (F) and MMPs (G) levels normalized to GAPDH. ^##p < 0.01, ^###p < 0.001 versus control; **p < 0.01, ***p < 0.001 versus UVB alone. ns, non-significance. Data are represented as mean ± SEM.

IPL17 on UV-irradiated guinea pig skin collagen fibers and epidermal thickness

To verify the in vivo anti-photoaging effect of IPL, we established a photoaging model by irradiating the dorsal skin of guinea pigs with ultraviolet (UV) radiation. We assessed the epidermal thickness and alterations in dermal collagen fibers via H&E staining, and Masson’s trichrome and Sirius Red staining, respectively, and observed the ultrastructure of collagen fibers using TEM. Epidermal thickness was significantly reduced in the UV-irradiated group compared with controls. However, UV + IPL irradiation significantly increased the epidermal thickness from the 7th day as compared with the control, as well as the UV-irradiated group (Fig. 4A). The UV + IPL-irradiated group demonstrated a significant reproduction of collagen fibers in the dermis (Figs. 4B-C). Additionally, TEM revealed that collagen fibers were rearranged into a dense distribution, and the average fiber diameter was larger than in the UV-irradiated group (Fig. 4D). Based on TEM observations, while individual fields may appear similar at first glance, quantitative analysis of multiple sections revealed that UVB irradiation resulted in significantly reduced collagen fiber diameter, looser packing, and irregular arrangement compared with the control group. IPL treatment markedly increased collagen fiber diameter and promoted dense, regular alignment, indicating obvious structural repair. These results demonstrated that IPL could repair UV-damaged skin from the epidermis to the dermis. Notably, the epidermal thickening induced by IPL was orderly and regenerative, distinct from the disordered, pathological epidermal hyperplasia associated with chronic UVB-induced photodamage.

Fig. 4.

Effects of UV and IPL on epidermal thickness, collagen content, and ultrastructure in guinea pig skin. (A) HE staining of the guinea pig skin and the epidermal thickness (200×). (B) Masson’s trichrome stain for collagen fiber density (200×). (C) Area density of collagen fibers by Sirius Red stain (scale bar = 100 μm). (D) Arrangement and average diameter of collagen fibers by TEM (scale bar = 500 nm). ^#p < 0.05 versus Control; *p < 0.05, **p < 0.01, ***p < 0.001 versus UVB alone. Data are represented as mean ± SEM.

Effects of IPL17 on AP-1/MMPs expression profiles within guinea pig skin

Finally, via IHC, we examined the effects of UV and IPL on the expression profiles of AP-1 subunits (p-c-fos, p-c-jun) and their downstream targets MMPs (MMP-1, MMP-3, MMP-9) in guinea pig skin, so as to investigate the underlying mechanism of the anti-aging and reparative effects of IPL in vivo. Compared with the control, p-c-fos significantly decreased following UV irradiation. Compared to the group exposed to UV radiation, p-c-fos increased significantly on the 1 st, 7th, and 14th day following IPL irradiation (Fig. 5A). Additionally, p-c-jun indicated a significant elevation following UV irradiation as compared with the control. IPL irradiation increased the p-c-jun expression level on the 1 st, 7th, and 14th day after UV irradiation (Fig. 5B). In comparison to the control, UV significantly boosted the MMP-1, MMP-3, and MMP-9 expression. In contrast, IPL downregulated MMP-1 and MMP-3 within guinea pig skin exposed to UV radiation. However, no discernible difference was observed in MMP-9 expression profiles across UV-irradiated and IPL groups (Figs. 5C-E). We have included a supplementary Table 1 that summarizes the key findings at each time point across all in vitro and in vivo experiments. These results indicated that IPL irradiation enhances collagen deposition by suppressing MMPs.

Fig. 5.

IHC examined the effect of UV and IPL on AP-1 and MMPs in guinea pig skin. (A-E) The samples received staining with p-c-fos, p-c-jun, together with MMPs antibodies, and photomicrographs were taken (200×). Optical density of the samples was assessed using ImageJ^®. Datasets are represented as mean ± the SEM. ^#p < 0.05, ## p < 0.01 versus Control; *p < 0.05, **p < 0.01, ***p < 0.001 versus UVB alone.

Discussion

Although IPL therapy has demonstrated clinical efficacy in reversing photoaging, the precise molecular mechanisms-particularly the dose-dependent regulation of ERK/AP-1-MMP pathways-remain inadequately understood. The selection of a broadband IPL wavelength range (590–1200 nm) is critical for the observed anti-photoaging effects in this study. Unlike narrowband light sources that target single chromophores, this broadband spectrum simultaneously interacts with multiple skin components: shorter wavelengths (590–900 nm) primarily act on epidermal keratinocytes to regulate cell proliferation and MAPK/AP-1 signaling, while longer near infrared wavelengths (900–1200 nm) penetrate deeper into the dermis to modulate fibroblast function and collagen remodeling. Such a broad range is necessary to achieve coordinated epidermal-dermal repair, which cannot be fully replicated by narrowband wavelengths alone. Although clinical practice uses various wavelength settings, the 590–1200 nm range provides balanced photobiomodulation featuring mild photothermal effects, making it suitable for exploring the molecular mechanisms of IPL-mediated rejuvenation without causing overt tissue damage. In this study, we demonstrated that IPL, particularly at a 17 J/cm² dose selectively inhibits UV-induced ERK phosphorylation while simultaneously activating JNK. This leads to a rebalances in AP-1 activity, suppressing MMP-1/MMP-3 expression and enhancing cyclin D1-mediated cellular proliferation.

MAPKs have been demonstrated to be involved in UV-induced skin aging^26,27. The activated MAPKs regulate the heterodimeric c-fos/c-jun (AP-1) complex to maintain skin homeostasis by targeting genes, including cyclin D1 and MMPs. Previous studies have revealed that UV and downstream MAPKs impair cell functions and induce tissue degradation^28–30. The results were also consistent with previous studies indicating that certain herbal extracts can prevent UV-induced skin photoaging via the ERK/AP-1 pathway, reducing MMP production and promoting collagen deposition^27,31,32.

The present study aimed to examine the impact and mechanism of photobiomodulation using IPL on hKCs and guinea pig skin. Our results revealed that UV and IPL activated the MAPK signaling pathway; however, they exhibited distinct activation patterns. UV exposure caused dose-dependent phosphorylation of ERK and JNK, without selectivity. This resulted in a decrease in p-c-fos combined with an increase in p-c-jun. Such imbalances in p-c-jun and p-c-fos directly affected downstream targets, including AP-1, cyclin D1, and MMPs. Conversely, IPL, particularly at a 17 J/cm² dose, significantly promoted p-JNK and suppressed p-ERK, leading to stable p-c-fos and increased p-c-jun levels. While JNK is classically associated with stress responses and apoptosis, its role is highly context-dependent, with emerging evidence supporting its involvement in tissue regeneration and proliferation^33,34. Consequently, IPL17 upregulated cyclin D1 and significantly downregulated MMP-1 and MMP-3.

Our research indicates that ERK phosphorylation levels exhibit a dose-dependent relationship with UV radiation. However, the level of ERK phosphorylation is associated with the dose of UV radiation and closely related to the duration of phosphorylation. While our study demonstrates that UV and IPL differentially regulate the phosphorylation of c-fos and c-jun (AP-1 components), we acknowledge that AP-1 functional activity-such as dimer composition specificity, DNA-binding capacity, or transcriptional output-was not directly measured. These aspects are critical for a complete understanding of AP-1’s role in photoaging and rejuvenation, as its biological effects depend on phosphorylation status and also on downstream transcriptional targets (MMPs and cyclin D1). Future studies incorporating electrophoretic mobility shift assays or AP-1 reporter systems could elucidate the functional consequences of the observed phosphorylation changes.

The therapeutic efficacy of IPL was also evident in vivo: treatment inhibited p-ERK while activating p-c-jun, enhancing epidermal proliferation and suppressing dermal MMP-1 and MMP-3, thereby attenuating collagen destruction. Special staining indicated that UV-caused collagen loss, fragmentation, and breakage of collagen fibers were significantly alleviated by IPL17 irradiation. Our study primarily elucidates the mechanism by which IPL17 suppresses UV-induced collagen degradation through the ERK/AP-1/MMPs pathway. However, the observed increase in collagen fiber density and diameter (Fig. 4) implies a potential involvement in collagen synthesis. This may be attributed to IPL-mediated stabilization of AP-1 and to the upregulation of cyclin D1, which enhances fibroblast proliferation and activity. Previous studies have demonstrated that the modulation of MAPK/AP-1 intersects with TGF-β/Smad signaling, a pivotal pathway in collagen production^35,36. To further understand IPL’s role in collagen homeostasis, future research should directly assess procollagen synthesis markers, such as PICP and TGF-β/Smad signaling activation. Furthermore, the mild decrease in collagen density from day 7 to day 14 reflects a dynamic remodeling process rather than therapeutic regression. Day 7 represents the peak of early collagen deposition and compaction, followed by gradual fiber maturation, alignment, and matrix homeostasis at day 14. This pattern is consistent with normal skin repair kinetics, where initial dense deposition transitions to stable, organized collagen structure.

The UV + IPL group demonstrated significantly greater collagen density and increased epidermal thickness compared with the UV-irradiated group at all corresponding time points (Figs. 4 and 5). This suggests that IPL actively facilitates recovery, rather than simply aligning with the natural repair processes. It is acknowledged that extending the observation period (4–8 weeks following the cessation of UVB exposure) could provide deeper insights into the long-term dynamics of natural recovery versus IPL-induced rejuvenation. This study primarily focuses on the impact of UVB irradiation on skin photoaging. While UVB is a key factor in solar elastosis and epidermal damage, we recognize that solar radiation also includes UVA and visible light. Consequently, the findings presented here cannot fully represent the complex interactions of signaling pathways activated by full-spectrum solar radiation in the skin, and there are certain limitations in achieving a comprehensive understanding of the skin photoaging process. This limitation represents an important avenue for future investigation.

While our study provides evidence that IPL17 regulates MMPs expression via the ERK/AP-1 pathway, we acknowledge that IHC-based quantification of MMPs reflects protein abundance rather than enzymatic activity. Although gelatin zymography confirmed changes in MMP-2/9 activity, future studies should incorporate additional functional assays (fluorogenic substrate assays or TIMP interaction studies) to fully characterize the post-translational regulation of MMPs in this context. Despite these limitations, our data collectively support the hypothesis that IPL17 mitigates UV-induced photoaging by suppressing MMP overexpression at transcriptional and translational levels. While our in vitro mechanistic studies focused on hKCs, we acknowledge that dermal fibroblasts primarily orchestrate collagen remodeling in skin. The observed in vivo effects of IPL17-including reduced MMP expression, increased collagen density, and epidermal proliferation-likely involve paracrine crosstalk between keratinocytes and fibroblasts.

Conclusion

Our study demonstrated that IPL inhibits the production of MMPs and promotes cell proliferation in human keratinocytes and guinea pig skin following ultraviolet irradiation via the ERK/AP-1 signaling pathway, thereby facilitating the regenerative repair of photoaged tissue. This study not only clarifies the specific effects of IPL’s photobiomodulatory action but also reveals its potential molecular targets and mechanisms of action, providing a theoretical basis for the application of IPL in the field of skin photodamage repair (Fig. 6).

Fig. 6.

Proposed mechanism of UV-induced photoaging and IPL-mediated rejuvenation via the ERK-AP-1-MMP axis.

UV irradiation (left, purple pathway) non-selectively activates both ERK and JNK, leading to diminished p-c-fos and elevated p-c-jun. This AP-1 imbalance drives MMPs overexpression and cyclin D1 downregulation, resulting in collagen degradation and proliferation impairment. IPL (right, red pathway) selectively inhibits UV-induced ERK phosphorylation while activating JNK, thereby increasing p-c-jun while preserving p-c-fos levels. This restoration of AP-1 equilibrium selectively suppresses MMP-1 and MMP-3 expression, upregulates cyclin D1, and ultimately contributes to the amelioration of photoaging phenotypes.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Congcong Liu and Wenzhi Hu wrote the main manuscript text. Mingmin Lu, Jiayi Xiang, Lina Tan and Lixia Zhang prepared the figures. Ye Tao and Kui Ma performed the statistical analysis and reviewed the manuscript. Xiaoyang Zhang, Congcong Liu and Zhaoting Yang made substantial contributions to the revised manuscript and editing relevant sections. Weijie Gu and Zhaoting Yang designed the study and supervised the experiments.

Funding

This work was supported by the National Natural Science Foundation of China (Grant Nos. 82373460 and 82003321).

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.