Low‐energy green light alleviates senescence‐like phenotypes in a cell model of photoaging

Abstract

Background

Ultraviolet B (UVB) affects diverse pathways in skin cells, resulting in skin photoaging. Skin fibroblasts internalize and degrade elastin and collagen, playing prominent roles in photoaging. Green light is used in many fields of dermatology, but few studies have examined its role in photoaging. The present work aimed to assess low‐energy green light for its effects in a previously proposed cell model of photoaging and to explore the possible anti‐photoaging mechanism.

Methods

The stress‐induced premature senescence (SIPS) model was constructed via repeated treatment of MDFs with UVB. Senescence‐like phenotypes were compared among normal, low‐energy green light pretreatment and UVB groups, for example, cell morphological properties, senescence‐associated β‐galactosidase (SA‐β‐gal) amounts, extracellular matrix (ECM) biosynthesis and degradation, and autophagy.

Results

In comparison with the UVB group, the green light pretreatment group showed significantly decreased number of senescent mast cells and markedly declined signal intensity and amounts of SA‐β‐gal‐positive cells. Furthermore, green light pretreatment directly affected ECM by increasing type I and type III collagen production and decreasing MMP‐1 amounts. Moreover, changes in autophagy levels induced by green light pretreatment provided a potential mechanism underlying its anti‐aging property.

Conclusions

Low‐energy green light pretreatment improves senescence‐like phenotypes in vitro, indicating a possible application for anti‐aging in clinic after future research has uncovered the potential mechanism.

INTRODUCTION

Ultraviolet light, especially UVB and UVC, represents the top cause of extrinsic aging, also referred to as photoaging, which is characterized by rhytids, decreased skin elasticity, lentigines, and mottled pigmentation. Autophagic death represents an important and dynamic cellular event in which unwanted and impaired organelles/proteins are degraded. Dermal fibroblasts biosynthesize collagen and perform extracellular matrix (ECM) remodeling. The latter cells have critical functions in photoaging and anti-aging, particularly in case of skin exposure to cumulative damage by ultraviolet exposure. The major aspects of photoaging comprise decreased ability to secrete collagen and increased matrix metalloproteinase (MMP) amounts resulting from ultraviolet exposure in fibroblasts. Therefore, many targets of photoaging therapy are hypothesized to play a role by acting on dermal fibroblasts, including intense pulsed light. Although the specific mechanism of action is unclear, the change of cellular autophagy may be one of the mechanisms by which light plays its biological role.

Autophagy is an important biological process to maintain cell homeostasis. Studies have shown autophagy contributes to UV-induced skin light injury. Autophagy induction protects cells against UV-associated apoptosis and promotes the purge of oxidized phospholipids and protein aggregates. Autophagy impairment was recently reported in chronologically aged skin fibroblasts, deteriorating skin integrity, and inducing skin fragility.

Green light is a visible light wave with a wavelength of 532 nm, whose effect on cell photoaging has not been reported. The current work aimed to examine the effect of green light treatment on photoaging in vitro and to explore the underpinning mechanism. A reproducible photodamage model of mouse dermal fibroblasts (MDFs) was recently established by our team via multiple UVB exposures, demonstrating diverse senescence-like phenotypes such as flattened morphology, upregulation of MMPs, enhanced ECM degradation, and increased SA-β-gal staining signals.

MATERIALS AND METHODS

Cells and treatments

The Committee on the Ethics of Animal Experiments of Tongji University reviewed the experimental protocols, and the experiments were carried out in accordance with the Animal Experiment Guidelines of Tongji University. MDFs were obtained from neonate C57BL/6 mice following a recently described protocol. Cell culture was carried out in DMEM containing 10% fetal bovine serum (FBS), glutamine, penicillin, and streptomycin at 37°C in a humid environment with 5% CO₂. Subculture was performed at 80% confluence, and passage 1–2 cells were utilized in various arrays.

At 12 h post seeding, the medium in the green light group was changed with phosphate buffered saline (PBS). Cells underwent exposure to four doses of green light of 532 nm at 1.93 mJ/cm² (Shanghai Intelligent Equipment Co, China) for 60 s before treatment with another four doses of UVB at 120 mJ/cm² (Narrowband TL 20 W/01 RS lamp, Philips, The Netherlands) for 120 s as described in a previous study. Immediately following irradiation, DMEM containing 1% FBS was added to replace PBS in the treatment group. The control group was handled similarly but with no UVB and green light exposure. Post-exposure, cells were further incubated in DMEM containing 10% FBS for recovery.

Morphological analysis

At 24 h following the final UVB exposure, cells underwent seeding in 35-mm plates and a 48-h culture in DMEM containing 10% FBS. Then, fixation (4% paraformaldehyde) and permeabilization (0.3% Triton X-100) were carried out for 15 min at ambient, before incubation with FITC-Phalloidin (Sigma, USA) for 30 min. Finally, 4, 6-diamidino-2-phenylindole (DAPI) counterstaining was performed, and data analysis utilized an Olympus IX70-S1F2 fluorescence microscope (Olympus, Japan) at 200×.

SA-β-Gal detection

SA-β-Gal staining followed a previous protocol. Briefly, following the final UVB irradiation, cells of each group were further cultured for 48 h in complete medium. Upon fixation, staining was carried out with a kit provided by CST (#9860, USA) as directed by the manufacturer. SA-β-Gal-positive cells were counted for totally 400 cells/dish with Image-Pro Plus (IPP) 6.0 (Media Cybernetics, USA). Data were provided as a percent of all cells counted.

Protein quantification and Western blotting

Cell collection was carried out 48 h following the final UVB irradiation to quantify soluble proteins. The number of cells was obtained for normalization, and cell lysates were prepared. Total protein was quantitated with the BCA kit (Pierce, USA).

Immunoblot was carried out as recently described. Briefly, 12% SDS-PAGE separation was followed by transfer onto PVDF membranes and incubation with primary antibodies targeting collagen I, collagen III, MMP-1, and Beclin 1 (Abcam, UK). Detection was performed with horseradish peroxidase (HRP)-linked secondary antibodies. Enhanced chemiluminescence was used for development, with a kit from Pierce (USA).

Transfection of GFP-RFP-LC3

Totally 48 h following the final UVB exposure, cells were transferred into 35-mm plates for another 24-h culture. Next, transfection was carried out with GFP-RFP-LC3 (Hanbio, China) at a multiplicity of infection (MOI) of 100 for 24 h. For assessment, GFP-LC3 puncta in each cell were counted by microscopy. Thirty random cells were analysed, and data were provided as mean quantity of puncta per cell.

Statistical analysis

SPSS13.0 (SPSS, USA) was utilized for data analysis by paired Student's t-test and one-way ANOVA. Data are mean ± SD, and p < 0.05 indicated statistical significance.

RESULTS

Pretreatment with 532 nm green light improves the morphology of photoaging cells

Enlargement and flattening (senescence-like morphology) were detected in UVB-irradiated cells (Figure ) under an optical microscope. This result was more intuitive and straightforward after phalloidin staining (Figure ). Cells pretreated with 532 nm green light showed reduced lengths and widths compared with the UVB model group. Soluble protein content was assessed in each group per cell for quantifying the degree of cell hypertrophy. Protein amounts per cell were higher in the UVB group in comparison with control cells, while green light led-pretreated cells markedly declined soluble protein content in each cell (Figure ).

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Green light pretreatment reduces SA-β-Gal amounts

SA-β-Gal staining was carried out 72 h following the final exposure to UVB to examine if green light pretreatment prevents MDF senescence. The results demonstrated intense SA-β-Gal signals in UVB-induced MDFs, which were reduced in green light-pretreated cells (Figure ). Furthermore, the amounts of positive cells were effectively reduced by green light pretreatment (Figure ).

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Green light pretreatment counteracts UVB-induced alteration of cell secretory functions in MDFs

Reduced type I and III collagen amounts and MMP-1 upregulation are characteristic changes occurring in photoaging. Therefore, the possible effects of green light intervention on collagen and MMP-1 amounts were examined. Totally, 48 h following the final exposure, markedly increased MMP-1 release and starkly reduced type I and III collagen protein amounts were found in the UVB group. Pre-incubation with 532 nm green light resulted in decreased release of MMP-1 and partially reduced UVB-associated downregulation of type I and III collagen proteins (Figure ).

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Green light pretreatment stimulates autophagy in photoaging MDFs

Following UVB exposure, both autophagosome and autolysosome (yellow and red puncta, respectively), amounts were reduced in photoaged cells, and these effects were alleviated by pre-incubation with 532 nm green light (Figure ). Immunoblot revealed UVB downregulated the autophagy-associated protein Beclin-1, suggesting autophagy suppression in this photodamage model. Meanwhile, pre-incubation with green light (532 nm) increased Beclin-1 expression (Figure ). Therefore, pretreatment with green light markedly decreased the suppressive effects of UVB on autophagy.

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DISCUSSION

Photoaging represents a skin degenerative disorder different from endogenous aging, regulated by genes. It is mostly caused by solar ultraviolet (UV) that induces MMPs and reduces collagen expression in skin fibroblasts, which have a critical function in ECM remodeling. In addition, most physical blockers and sunscreens are widely applied to prevent UV penetration of skin cells, reducing its deleterious dermatological impact. The low-energy green laser is broadly utilized clinically, for example, for treating erythematous skin lesions, tattoo pigment remonval, and managing retinopathy of prematurity. Nevertheless, few studies have examined the effect of green light on photoaging.

This study found that pretreatment with 532 nm green light could counteract some UVB-related senescence characteristics in MDFs. UVB-exposed MDFs pretreated with low energy green light retained an elongated cell shape with markedly reduced signal intensity and rate of SA-β-Gal-expressing cells. The aging skin commonly shows altered pigmentation, sallowness, and serious wrinkling. The potential change increases collagen degradation by MMPs. Multiple reports indicate the skin amounts of several MMPs in humans or mice, including MMP-1, MMP-2, MMP-3, and MMP-9, are increased upon UV irradiation. The present study detected MMP-1 (collagenase), collagen-1, and collagen-3. We here demonstrated that low-energy green light (532 nm) suppressed collagen-1 and collagen-3 breakdown by UVB via MMP-1 downregulation.

Next, the potential mechanism behind the anti-photoaging effects of green light in MDFs after UVB irradiation was explored. Studies have shown autophagy contributes to UV-induced skin light injury.

Autophagy is an essential biological process that maintains cell homeostasis by clearing damaged proteins. It contributes to multiple pathologies such as neurodegenerative disorders, malignancies, infectious diseases, and particularly aging. Other scholars have reported a complex role for autophagy in response to UV-triggered oxidative stress injury, removing oxidized protein and lipid molecules, and reducing antioxidation in diverse cell types. Based on the above findings, we explored whether low-energy green light improves the basic autophagy level of cells and then showed its ability to resist cell photoaging. This work revealed starkly decreased cell autophagy after photoaging. However, pretreatment with low-energy green light could increase the basic autophagy level of MDFs, which may alleviate the initial mitochondrial damage, protein aggregation, and ROS accumulation. Instead of dysregulating autophagy, which may result in exacerbated autophagic cell death as well as fibroblast dysfunction, low-energy green light seems to enhance the ability of cells to respond to UV light damage by enhancing their basic autophagy level. However, this study only verified the changes in the number of autophagosomes and the content of Bcl-2 protein in each group. A more comprehensive exploration of the relevant indicators of autophagy to explain the mechanism of green light will be a further research direction, which is also the deficiency of this study.

Overall, this study showed pretreatment with green light ameliorates senescence-like phenotypes such as flattened morphology and SA-β-gal biosynthesis. Besides, ECM degradation was prevented by low-energy green light via MMP-1 downregulation and collagen upregulation. Few studies have examined the anti-aging property of green light in vitro, providing insights into its potential clinical use for rejuvenation. Naturally, further investigation is warranted to further examine the molecular pathways involved in this anti-aging effect.

ACKNOWLEDGMENTS

This work was supported by grants from National Natural Science Foundation of China (No. 81773347).

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are openly available in [repository name] at [DOI].

ETHICAL APPROVAL

The Committee on the Ethics of Animal Experiments of Tongji University reviewed the experimental protocols, and the experiments were carried out in accordance with the Animal Experiment Guidelines of Tongji University.

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