2.1. Clinical Observation of Rosacea Patients Treated With IPL
All 10 patients were treated with a Lumenis M22® (Lumenis Ltd., Israel) xenon-based IPL device, using a 590 nm filter with a sapphire-cooled 15 × 35 mm² cylindrical light guide (with a fluence of 11 J/cm2, single pulse, and pulse duration of 20 ms, two passes). Before and 1 month after initial IPL therapy, the severity of rosacea was evaluated by two independent senior dermatologists using the clinician’s erythema assessment (CEA) and investigator global assessment (IGA), two 5-point grading scales[7, 29]. Facial images were obtained and analyzed using the VISIA®6.0 complexion analysis system (Canfield Scientific Inc., Parsippany, NJ, USA). The facial images were saved, the red area was processed by ImageJ, and the percentage of erythema area was calculated and further analyzed using GraphPad Prism 6.
Twenty-eight female BALB/c mice (6–8 weeks old, weighing 18–22 g) were purchased from Puer BHQ Laboratory Animals, Inc. (certification no: SCXK HU 2018-0006; Shanghai, China). Five mice were housed per cage under pathogen-free conditions with soft bedding under controlled temperature (22 ± 2 ℃) and a 12 h light/dark cycle (lights on at 8:00 a.m.). Mice were acclimatized to the testing environment for 2 days before starting the experiments. All mice were matched for age and body weight in each experimental group.
2.3. Rosacea-like Inflammation Mouse Model
A well-established model was used based on the previous studies[5, 30]. Female BALB/c mice were shaved 24 h prior to the experiment. LL-37 (amino acid sequence: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTE) was synthesized by Sangon Biological Technology (Shanghai, China) at > 99% purity and was dissolved (320 µM) in phosphate-buffered saline (PBS) in advance and stored at 4 ℃ for further use. After weighing, mice were anesthetized through intraperitoneal injection of pentobarbital sodium (50 mg/kg), and then LL-37 (320 µM in PBS) was intradermally injected into a pre-drawn circle on the back to form a picumulus. Subsequently, the mice were returned to the animal room for regular feeding.
BALB/c mice were randomly divided into four groups (seven mice per group): control (PBS, 50 µL, i.d.), LL-37 (LL-37, 320 µM, 50 µL, i.d.), LL-37 + IPL (320 µM LL-37, 50 µL, i.d.+IPL treatment), and IPL group (PBS, 50 µL, i.d.+IPL treatment). For IPL therapy, the same Lumenis M22® device was used (with a fluence of 11 J/cm2, single pulse, and pulse duration of 20 ms) for a single pass on the treated sites of the mice at 12 h after the last injection.
For the LL-37 group, injections were administered four times every 12 h consecutively, while the control group was administered 50 µL PBS. IPL treatment was performed in the LL-37 + IPL and IPL groups 12 h after the last injection. The light guide was kept in contact with the treated sites during the treatment. Mice were photographed 1 h after the IPL irradiation. Erythema severity was assessed and quantified as a* value using an L*a*b colorimeter (CR-10 plus, Konica Minolta, Japan).
At 72 h after the last injection, samples of skin lesions were collected for further analysis.
2.4. Histopathologic Analysis of Human and Mouse Skin Samples
Mouse skin samples were fixed overnight in 4% buffered formalin for 24 h, embedded in paraffin, sliced into 4-mm sections, and stained with hematoxylin and eosin (H&E) or toluidine blue. Images were captured using an optical microscope (Leica, Germany) at 200× and 400× magnifications by a blinded observer. Degranulation of MCs was considered if four or more extruded granules could be seen neighboring a cell or the cell outline was disrupted. Conversely, intact cells demonstrated deep blue staining[30, 31].
2.5. Immunohistochemical Analysis of Human and Mouse Skin Samples
Paraffin sections of tissue were washed with PBS, dewaxed, treated with 3% H2O2 for 10 min, repaired with citrate buffer, blocked with serum, and then incubated with different antibodies. The antibodies against MMP-9 (R&D, Santa Clara, CA, USA; 2.0 mg/mL, diluted by 1:100), human-KLK5 (Abcam, Waltham, MA, USA; 1.295 mg/mL, diluted by 1:100), human-Cathelicidin (Abcam; 3.44 mg/mL, 1:150), human-IL-6 (Santa Cruz, USA; 200 µg/mL, diluted by 1:100), mouse-Cathelicidin (Biorbyt, St Louis, MO, USA; 1 mg/mL, 1:200), mouse-IL-6 (Santa Cruz, USA, 200 µg/mL, 1:200), TNF-α (Abcam; 0.2 mg/mL, 1:100), and IL-1β (Abcam; 2 mg/mL, 1:100), MC Tryptase (Hua Bio, China; 1 mg/mL, 1:150), Histamine (Novus biological, Englewood, CO, USA, 15 mmol/mL, 1:150) were incubated for 1 h at room temperature. Negative controls were incubated with the diluent instead of the primary antibody. The slides were incubated with HRP-labeled secondary antibody for 30 min, treated with diaminobenzidine, and counterstained with hematoxylin. Subsequently, the slides were differentiated, dehydrated, cleared, covered with neutral resin, and observed and imaged using a microscope (Leica, Germany) at 200× magnification. Light to dark brown staining indicates positive results. Immunohistochemical (IHC) staining was semi-quantitatively analyzed and assigned using ImageJ (v1.51, https://imagej.nih.gov/).
2.6. Immunofluorescent Examination of Mouse Skin Samples
The paraffin sections of mouse skin were washed, dewaxed, treated with 3% H2O2, repaired using antigen, blocked using donkey serum containing 0.3% TritonX-100 for 2 h at room temperature, and treated with primary antibodies including MMP-9, KLK5 at 4°C overnight. Next, the slices were incubated with the secondary antibody (Jackson, Louisiana, NO, USA; 15 mg/mL, 1:300) for 2 h at 37°C, and sections were subsequently washed and counterstained with DAPI for 10 min. Images were captured using a fluorescence microscope at 100× magnification (Leica, Germany).
2.7. Cell Culture and Grouping
The mouse MC tumor P815 cell line (RRID: CVCL_2154, ATCC TIB-64; American Type Culture Collection, Manassas, VA, USA) were provided by Nanjing University of Chinese Medicine and were cultured in RPMI-1640 medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin (Gibco) in a cell incubator, which was set at 5% CO2 at 37°C. The cells were equalized into Petri dishes (35 mm × 10 mm) and then stimulated using LL-37 (2.0 µM) for 6 h with or without IPL treatment (single pulse, 11 J/cm2 with a 20 ms pulse duration) immediately after LL-37 administration. Proteins and RNA were then extracted. Before treatment, the supernatant was discarded, and the cells were rinsed twice with PBS and then replaced with fresh RPMI-1640 medium (phenol red-free, serum-free) to exclude the influence of light. The levels of related proteins and RNAs were detected.
2.8. Cell Viability Assay
P815 cells were seeded in 96-well plates and processed for 6 h. CCK8 solution (supernatant; Yi Sheng Biotechnology, China) was added at 10 µL per well. After a 3-h incubation, the optical density (OD) at 450 nm was measured using a microplate reader (Thermo Fisher, Waltham, MA, USA).
2.9. Western Blot
Samples, including skin tissue segments, were collected, lysed in RIPA lysate with 1% protease and phosphatase inhibitor. Proteins were extracted using methanol and chloroform and quantified using the BCA Protein Assay (Thermo Fisher). Equivalent amounts of proteins (animal proteins, 30 µg; cell proteins, 10 µL) were loaded and separated by SDS-PAGE. MMP-9, and KLK5, cathelicidin, and GAPDH were detected using 8% and 10% SDS-PAGE gels, respectively. All proteins were transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA) and blocked for 2 h at room temperature. Ponceau S (Sangon Biological Technology, China) was used to stain and detect the total proteins in cell supernatant before being blocked. The membranes were then probed with primary antibodies overnight at 4°C. The dilutions of the antibodies against GAPDH (Santa Cruz; 100 µg/mL, 1:800), MMP-9 (Proteintech, Rosemont, IL, USA; 400 µg/mL, 1:1000), KLK5 (Abcam; 1 mg/mL, 1:1000), cathelicidin (Abcam; 3.89 mg/mL, 1:1000). Finally, the membranes were incubated with the corresponding secondary antibodies (Sigma, Burlington, MA, USA, 1:5000). The bands were developed using an enhanced chemiluminescence reagent (Beyotime, China). Data were obtained using the Molecular Imager and analyzed using ImageJ software (NIH, USA).
2.10. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from the mouse skin tissue and P815 cells using TRIzol reagent (Invitrogen, Waltham, MA, USA). According to standard protocols, 500 ng RNA was reverse-transcribed into 10 µL cDNA using HiScript® RT SuperMix (Vazyme, China). qRT-PCR was performed with SYBR Green Master Mix (Vazyme, China) in the QuantStudio 5 Real-Time PCR Detection System (Thermo Fisher). Relative expression levels were calculated using the 2−ΔΔCt method after normalization with GAPDH. We used the formulas ΔCt = Ctgene – CtGAPDH and ΔΔCt = ΔCt – mean value of ΔCt in the control group; all primers used are listed in Table S3.
2.11 Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA reagent kits (Yi Fei Xue Biotechnology, China) for histamine, β-Hex, TNF-α, IL-8, and IL-1β were used following the manufacturer’s protocols. Sample addition, incubation, washing, and developing were performed in sequence, and the OD of each well was measured at 450 nm.
2.12 Statistical Analysis
GraphPad Prism 6 (LaJolla, CA, USA) was used to conduct all statistical analyses. Student’s t-test was used to evaluate the differences between the two groups. Results between more than two groups were compared using one-way ANOVA. Data are presented as the mean ± SEM of independent experiments. All statistical tests described as significant were based on a criterion of P < 0.05.