Low-level red light inhibits human retinal pigment epithelial cell fibrosis via UBE2C in a myopia-simulating hypoxic microenvironment

Abstract

Background

Low-level red light (LLRL) irradiation may inhibit myopia occurrence and progression. Understanding how LLRL inhibits fibrosis in human retinal pigment epithelial (hRPE) cells is critical to inhibiting myopia progression and developing novel therapeutic strategies. Here, we explored the effects of LLRL on hRPE cells in a myopia-simulating hypoxic microenvironment and elucidated the mechanisms through which it inhibits scleral remodeling.

Methods

We first used the MTT assay to analyze hRPE cell proliferation under hypoxic conditions after LLRL irradiation at varying frequencies over different durations. RNA sequencing was used to screen for key signaling molecules leading to hRPE cell fibrosis. Western blotting, reverse transcription quantitative polymerase chain reaction, and immunofluorescence assay were used to detect the role of ubiquitin binding enzyme E2 C (UBE2C) in hRPE cell fibrosis under LLRL irradiation.

Results

LLRL was noted to regulate the extracellular matrix, inhibiting fibrosis in hypoxic hRPE cells. Moreover, supernatant of LLRL-treated hypoxic hRPE cells inhibited scleral remodeling in human scleral fibroblasts. Mechanistically, LLRL inhibited cell fibrosis by regulating UBE2C activation of the AKT/mTOR pathway.

Conclusion

In a hypoxic environment, LLRL irradiation can prevent fibroblast transformation in hRPE cells, indicating its potential in scleral remodeling inhibition. Our results revealed the molecular mechanism through which red light controls myopia and provide evidence for further basic and clinical research.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s40001-025-02774-2.

Introduction

Myopia incidence is increasing globally. The global prevalence of myopia and high myopia is expected to reach 49.8% and 9.8% by 2050 [1]. High myopia considerably increases the risks of complications, such as myopic macular degeneration and retinal detachment, which can cause irreversible visual impairment [2]. At present, the primary treatment methods for myopia control include defocus glasses, orthokeratology lenses, and atropine eye drops. Each of these methods has limitations, such as inefficient myopia control, refractive regression after discontinuation, and various complications (e.g., glaucoma). Therefore, development of newer, safer, and effective methods for the prevention and control of myopia, particularly high myopia, is warranted.

The main pathological manifestation of myopia is external environment changes, which lead to changes in the retina and choroid, secretion of various cytokines, thinning of scleral collagen fibers, and an increase in the scleral stretch rate; these effects make scleral collagen fibers more susceptible to increased eye load. Recent studies have reported that hypoxia is a major risk factor for myopia. Various myopia animal models have been noted to have strong HIF-1α expression in their retinas, choroids, and scleral tissues [3, 4]. Under hypoxic conditions, the expression of α-smooth muscle actin (SMA; a scleral remodeling marker protein) and matrix metalloproteinase (MMP) 2 increases, but that of collagen type I alpha 1 chain (COL1A1) decreases; this leads to an imbalance between scleral ECM synthesis and metabolism, leading to scleral remodeling [5, 6].

Patients with high myopia and myopia animal models exhibit retinal neuronal apoptosis. In a myopia model with 3 weeks of form deprivation, severe swelling of retinal cell mitochondria, downregulation of BCL2, and upregulation of apoptotic signaling pathway proteins (i.e., Bax and caspase 3) occurred before pathological changes occurred in retinal morphology. BCL2 is associated with retinal pigment epithelial (RPE) cell apoptosis; it inhibits apoptosis by interfering with factors such as Bax [7].

Complex signaling pathways in human RPE (hRPE) cells are closely associated with myopia occurrence and progression. hRPE cells contain or express factors that can mediate retinal–scleral signals [8]. In particular, mTOR controls myopia occurrence and progression by regulating metabolism in hRPE cells [9]. hRPE cells increase dopamine(DA) synthesis and secretion, regulating scleral chondrocyte proliferation and preventing myopia progression [10]. Acetylcholine can promote transforming growth factor (TGF) β production in hRPE cells, leading to scleral remodeling and ultimately regulating myopia progression [11]. Gamma-aminobutyric acid and all-trans-retinoic acid from hRPE cells can stimulate scleral fibroblasts to worsen myopia progression [12, 13]. Increased α-SMA expression promotes hRPE cell fibrosis, an effect similar to that observed in scleral fibroblasts during myopia [14]. TGF-β1 is a critical factor promoting the transformation of RPE cells to myofibroblasts via epithelial–mesenchymal transition (EMT). Recent studies have reported that the YAP/TAZ, Nrf2, and AKT/GSK-3β pathways are associated with age-related macular disease-related EMT in RPE cells [15, 16]. In summary, hRPE cells are crucial in the development of scleral remodeling due to myopia.

In recent years, low-level red light (LLRL) therapy affords favorable clinical control in individuals with myopia. In a 12-month multicenter randomized clinical trial involving 264 children aged 8–13 years, LLRL therapy demonstrated more effective myopia control than single-vision spectacles: at the 6-month follow-up, 16.1% and 23.2% of the children with myopia demonstrate a reduction in equivalent diopter of 0.25D and eye axis shortening of > 0.05 mm, respectively, without side effects [17]. Clinical studies have reported that LLRL stimulation increases macular choroidal thickness and leads to transient shortening of the eye axis, possibly relieving retinal tissue hypoxia [18]. Retinal epithelium has a barrier effect, and LLRL must cross the choroid to exert its effect. Therefore, RPE cells may demonstrate changes in corresponding pathways and secrete corresponding signaling molecules to regulate myopia-related pathological changes in the choroid and sclera [19]. However, the biological function of red light in preventing myopia progression and the underlying mechanisms remain unclear.

Herein, we determined whether LLRL irradiation of hRPE cells in a myopia-simulating hypoxic microenvironment triggers the activation of myopia-inhibiting signaling pathways, involving proteins such as α-SMA, MMP2, and BCL2. We also assessed the scleral remodeling-inhibiting effects of LLRL irradiation not only within hypoxic hRPE cells, but also in human scleral fibroblasts (HSFs) treated with LLRL-treated hypoxic hRPE cell supernatant. High-throughput sequencing and gene enrichment analyses were also used to elucidate and verify key genes affecting scleral remodeling.

Material and methods

Cell culture

An hRPE cell line and HSFs were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured according to the manufacturer's instructions. hRPE cells and HSFs were maintained in Dulbecco’s modified Eagle medium (DMEM)/F12 (Gibco, Grand Island, NY, USA) and DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), respectively, at 37 °C under 5% CO₂. Hypoxic cells were cultured at 0.1% oxygen concentration. All cells were subjected to mycoplasma testing and short tandem repeat fingerprinting.

LED light illumination

In our experiments, the control groups were exposed to white LED lights (450–460 nm; 2954 K; Honyar Electrical, Hangzhou, China), whereas the experimental groups were exposed to red LED lights (630 nm; 600 lx; 2134 K; Honyar Electrical). All cells were placed in a dark room until irradiation. For irradiation, the cells were moved out of the dark room and irradiated for appropriate durations at appropriate frequencies. For cells receiving irradiation multiple times, the irradiation timepoints were distributed evenly every 12 h. We employed conditions for LLRL irradiation based on previous animal and clinical studies on myopia [17, 20].

Plasmid, reagents, and transfection

The coding sequence of the ubiquitin binding enzyme E2 C gene (UBE2C) was subcloned into the lentivirus vector pSin-EFα-puro, and empty pSin-EFα-puro was employed as the negative control (NC).

An RNA interference (RNAi) sequence was subcloned into the lentivirus vector PLKO.1 to knock down UBE2C expression, and a scramble sequence was used as the NC. The target sequence was shUBE2C: 5′-CCTGCAAGAAACCTACTCAAATTCAAGAGATTTGAGTAGGTTTCTTGCAGG-3′. In brief, the lentivirus was co-transfected with the packaging plasmids into 293 T cells using Lipofectamine 3000 (Thermo Scientific, Waltham, MA, USA), and the resulting lentivirus was collected at 48 and 72 h after transfection. Next, hRPE cells were infected with this lentivirus over 7 days, and puromycin (TargetMol, Boston, MA, USA) was used to screen for stable cells. Finally, all cells were maintained in 1 µg/mL puromycin until further experimentation.

MTT assay for LLRL irradiation parameter optimization

To determine the optimal parameters of LLRL irradiation, hypoxic hRPE cells were exposed to LLRL for 20, 40, 60, 120, or 180 s. Another batch of hypoxic hRPE cells was exposed to LLRL for 20 s once, twice, or three times every 12 h, or for 40 s once, twice, or three times every 12 h.

The viability of hRPE cells, plated in 96-well plates (Corning, NY, USA) at 2.0 × 10³ cells per well, was assessed using an MTT assay kit (Solarbio, Beijing, China) under hypoxic and normoxic conditions. The cell survival rate (%) was calculated based on the resulting absorbance at 500 nm on a microplate reader (Synergy LX; BioTek Instruments).

Experimental hypoxic model with hRPE and transfection

hRPE cells were seeded into six-well plates and cultured under 0.1% oxygen for 24 h. The cells were then transfected with plasmid and RNAi vectors using Lipofectamine 3000, followed by incubation for 36 h. Finally, the cells were harvested for further analysis.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted using a Total RNA Extraction Reference RNA Extraction Kit (Invitrogen, Waltham, MA, USA), according to the manufacturer’s instructions. Then, it was subjected to reverse transcription (RT) into cDNA by using Hiscript Reverse Transcriptase (Vazyme, Nanjing, China). Next, quantitative polymerase chain reaction (qPCR) was performed using the cDNA as a template, as described previously [21], with glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) expression as the housekeeping control. The cycle threshold (Ct) value was determined and used to quantify relative expression levels based on the 2^−ΔΔCt method.

We used the following RT-qPCR primers: ASMA, 3′-AAAAGACAGCTACGTGGGTGA-5′ (forward), 3′-GCCATGTTCTATCGGGTACTTC-5′ (reverse); MMP2, 3′-TACAGGATCATTGGCTACACACC-5′ (forward), 3′-GGTCACATCGCTCCAGACT-5′ (reverse); COL1A1, 3′-GAGGGCCAAGACGAAGACATC-5′ (forward), 3′-CAGATCACGTCATCGCACAAC-5′ (reverse); UBE2C, 3′-GACCTGAGGTATAAGCTCTCGC-5′ (forward), 3′-TTACCCTGGGTGTCCACGTT-5′ (reverse); BCL2, 3′-GGTGGGGTCATGTGTGTGG-5′ (forward), 3′-CGGTTCAGGTACTCAGTCATCC-5′ (reverse); and GAPDH, 3′-GGAGCGAGATCCCTCCAAAAT-5′ (forward), 3′-GGCTGTTGTCATACTTCTCATGG-5′ (reverse).

Western blotting

Western blotting was performed, as described previously [22]. The following primary antibodies (Abcam, Cambridge, MA, USA) were used: anti-α-SMA, anti-COL1A1, anti-BCL2, anti-MMP2, anti-UBE2C, anti-AKT, anti-p-AKT, anti-mTOR, anti-p-mTOR, anti-Smad2/3, anti-p-Smad2/3, anti-HIF-1α, and anti-GAPDH.

Immunofluorescence staining

Cells were plated on glass coverslips and incubated overnight for attachment. Next, the cells were fixed with 4% formaldehyde for 30 min, followed by 0.5% Triton X-100 for 30 min. The cells were then blocked with 3% bovine serum albumin for 2 h and incubated with anti-α-SMA (Abcam) overnight at 4 °C. Finally, the cells were incubated with the secondary antibody (Goat anti-Rabbit IgG (H + L) Cross-Absorbed Secondary Antibody, Alexa Fluor 488;1:1000; A-11008; Thermo Scientific) and 4′,6-diamidino-2-phenylindole at room temperature for 2 h and imaged under a Zeiss LSM800 with Airyscan.

Supernatant intervention

hRPE cells were passaged in six-well plates in DMEM/F12 without FBS for 24 h and divided into three groups: normoxia, hypoxia, and LLRL + hypoxia. After irradiation treatments, the supernatants of the treated cells were collected and used to culture HSFs. The cultured HSFs were subjected to Western blotting and RT-qPCR to determine α-SMA, COL1A1, BCL2, and MMP2 expression.

RNA sequencing

We prepared a sequencing library using a VAHTS Stranded mRNA-seq Library Prep Kit for Illumina V2 (Vazyme Biotech, NR612-02), according to the instructions. Reads were aligned to the human Ensemble genome GRCh38 (mouse Ensemble genome GRCm38) by using Hisat2 aligner (version 2.1.0) under the parameter “–rna-strandness RF.” The reads mapped to the genome were analyzed using featureCounts (version 1.6.3). Differential gene expression analysis was performed using the R package DESeq2. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the R package clusterProfiler (version 3.6.0).

Cell treatment

hRPE cells were treated with the mTOR pathway inhibitor rapamycin (Selleck, Shanghai, China) and cultured in growth medium under 0.1% oxygen for 48 h. Next, 10 µM rapamycin was added to UBE2C-overexpressing and control cells. We collected the cells for further analysis after their density reached 100%.

Statistical analysis

All results are presented as means ± standard deviations (SD) of at least three repeated individual experiments. To analyze between-group differences, two-tailed Student’s t test and one-way analysis of variance were performed using SPSS (version 21.0; IBM, Armonk, NY, USA). The significance threshold was set at P < 0.05. Gene set enrichment analysis (GSEA) was performed using the software program GSEA (https://www.gsea-msigdb.org/gsea/index.jsp).

Results

LLRL inhibits fibroblast metaplasia in hRPE cells under hypoxic conditions

First, we examined differences in the expression of key fibroblast metaplasia indicators in hypoxic hRPE cells. Hypoxic hRPE cells demonstrated significantly higher ASMA and MMP2 expression but considerably lower COL1A1 and BCL2 expression than normoxic hRPE cells (P < 0.05; Fig. 1A, B). Moreover, immunofluorescence staining revealed that α-SMA expression was significantly higher in hypoxic hRPE cells than in normoxic hRPE cells (Fig. 1C).

Fig. 1.

Hypoxia induces hRPE cell fibrosis. A Western blotting and B RT-qPCR for ECM-related gene expression in hypoxic and normoxic hRPE cells. C Immunofluorescence staining showing α-SMA upregulation in hypoxic hRPE cells. Data are presented as means ± SDs. *P < 0.05

We subsequently screened for optimal LLRL irradiation parameters with protective effects in hRPE cells. In particular, we detected the effects of 630-nm, 600-lx red light on hRPE cell proliferation under normoxic and hypoxic conditions by using the MTT assay. After exposing the cells to LLRL irradiation at varying durations and frequencies and detecting cell viability 24 h later, we found that 20-s irradiation led to the highest proliferation activity in hypoxic hRPE cells (P < 0.05); in contrast, the proliferation activity decreased after irradiation for longer durations. In normoxic hRPE cells, the proliferation activity decreased significantly after 40 s of irradiation (Fig. 2A). Moreover, we determined the optimal daily frequency for 20-s LLRL irradiation to protect hRPE cells. After 24 h of treatment, one and two 20-s LLRL exposures every 12 h had protective effects in hypoxic hRPE cells, with two 20-s exposures having the most significant effects (P < 0.05; Fig. 2B).

Fig. 2.

LLRL protects hRPE cells under hypoxic conditions. A MTT assay for hRPE cell viability after LLRL irradiation (wavelength, 630 nm; intensity 600 lx) for 0, 10, 20, 40, 60, 120, or 180 s under normoxic and hypoxic conditions. B MTT assay for hRPE cell viability after LLRL irradiation (wavelength, 630 nm; intensity, 600 lx) for 20 s once, twice, or three times, or for 40 s once, twice, or three times, under hypoxic conditions. Data are presented as means ± SDs. *P < 0.05

We also noted that LLRL irradiation inhibited the mRNA and protein expression of ASMA and MMP2 and increased that of COL1A1 and BCL2 in hypoxic hRPE cells (Fig. 3A, B). Moreover, immunofluorescence staining revealed enhanced α-SMA expression in LLRL-irradiated hypoxic hRPE cells (Fig. 3C). These results indicated that LLRL protects retinal pigment epithelium and prevents fibroblast metaplasia.

Fig. 3.

LLRL protects against hRPE cell fibrosis. A Western blotting and B RT-qPCR for ECM-related gene expression in hypoxic hRPE cells exposed to LLRL for 20 s twice every 12 h. C Immunofluorescence staining showing α-SMA upregulation in LLRL-treated hypoxic hRPE cells compared with untreated hypoxic hRPE cells. Data are presented as means ± SDs. *P < 0.05

To determine whether LLRL irradiation prevents hypoxic hRPE cells from promoting fibrosis in HSFs, we exposed HSFs to the supernatant of LLRL-treated hypoxic hRPE cells. The results demonstrated significant differences in the mRNA and protein expression of ASMA, MMP2, COL1A1, and BCL2 (P < 0.05; Supplemental Fig. 1A, B), confirming the potential of LLRL irradiation in preventing hRPE cells from secreting cytokines that promote scleral remodeling.

UBE2C is a potential gene leading to hRPE cell fibrosis

High-throughput RNA sequencing demonstrated that UBE2C expression was upregulated in hypoxic hRPE cells but downregulated in LLRL-irradiated hypoxic hRPE cells (Fig. 4A). Moreover, UBE2C mRNA and protein expression was significantly higher in hypoxic hRPE cells than in normoxic hRPE cells, whereas it was lower in LLRL-irradiated hypoxic hRPE cells than in hypoxic hRPE cells (Fig. 4B, C).

Fig. 4.

UBE2C is upregulated in hypoxic hRPE cells. A mRNA sequencing, B Western blotting, and C RT-qPCR assay for differentially expressed genes in hRPE cells. UBE2C was upregulated in hypoxic hRPE cells compared with LLRL-treated hypoxic hRPE cells and normoxic hRPE cells. Data are presented as means ± SDs. *P < 0.05

LLRL inhibits hRPE cell fibrosis via UBE2C

Gene Ontology enrichment analyses revealed that the differentially expressed genes were mainly enriched in ECM receptor function (Fig. 5A). The key signaling molecules considered in this study, namely α-SMA, MMP2, COL1A1, and BCL2, are all related to ECM secretion. After UBE2C overexpression, hypoxic hRPE cells demonstrated upregulation of UBE2C, ASMA, and MMP2 mRNA and protein expression (Fig. 5B) and downregulation of COL1A1 mRNA and protein expression (Fig. 5C). These results indicated that UBE2C is a potential key gene leading to fibroblast metaplasia via hRPE cells and that LLRL may exert its effects by inhibiting its expression.

Fig. 5.

UBE2C promotes hRPE cell fibrosis. A KEGG pathway enrichment analysis of differentially expressed genes. Hypoxia exposure and LLRL irradiation mainly enriched expression of molecules related to ECM regulation. B RT-qPCR for the relation between UBE2C overexpression and ECM-related genes. C Western blotting for UBE2C, α-SMA, MMP2, and COL1A1 expression in UBE2C-overexpressing hypoxic hRPE cells. Data are presented as means ± SDs. *P < 0.05

Next, we used UBE2C RNAi to detect key protein expression in hRPE cells in the hypoxia, hypoxia + LLRL, and hypoxia + RNAi groups and found that UBE2C RNAi and LLRL irradiation had similar effects in the cells. In particular, UBE2C RNAi led to downregulation of UBE2C, ASMA, and MMP2 mRNA and protein expression but upregulation of that of COL1A1 (Fig. 6A, B). These results further confirmed that LLRL irradiation exerts its effects via UBE2C.

Fig. 6.

LLRL achieves cell protection by regulating UBE2C expression. A Western blotting and B RT-qPCR for the relationship of ECM-related gene expression with UBE2C knockdown and LLRL irradiation. Data are presented as means ± SDs. *P < 0.05

LLRL exerts its effect through the UBE2C-mediated AKT/mTOR pathway

We further assessed the mechanisms of action of UBE2C in hRPE cells. We performed KEGG pathway enrichment analysis and observed that in hypoxic hRPE cells, upregulated differentially expressed genes were mainly enriched in the mTOR pathway (Fig. 7A). Western blotting revealed the upregulated expression of the mTOR pathway proteins AKT, p-SMAD2/3, SMAD2/3, and HIF1-α in hypoxic hRPE cells. For subsequent analysis, we divided hypoxic hRPE cells into four groups based on the treatment strategy: NC, UBE2C overexpression, blank control, and LLRL + UBE2C overexpression groups. The results demonstrated that p-mTOR, p-AKT, and p-SMAD2/3 expression was the lowest in the LLRL + UBE2C overexpression group but the highest in the UBE2C overexpression group. In contrast, HIF1-α expression was the highest in the LLRL + UBE2C overexpression group but the lowest in the UBE2C overexpression group (Fig. 7B). These results confirmed that LLRL irradiation affects mTOR pathway protein expression via UBE2C, thereby altering hRPE cell activity.

Fig. 7.

UBE2C promotes hRPE cell fibrosis via mTOR pathway activation. A Gene enrichment analysis showing that genes differentially expressed between cells treated and not treated with LLRL were mainly concentrated in the mTOR pathway. B Western blotting for mTOR pathway protein expression in UBE2C-overexpressing LLRL-treated hRPE cells. Data are presented as means ± SDs. *P < 0.05

We subsequently treated UBE2C-overexpressing hRPE cells with an mTOR pathway inhibitor and detected changes in mTOR pathway proteins through Western blotting. The results demonstrated that a reduction in AKT, mTOR, SMAD2/3, and HIF-1α phosphorylation, as well as mTOR pathway inhibition (Fig. 8A). RT-qPCR further revealed that mTOR pathway inhibition led to downregulation of ASMA and MMP2 mRNA expression but upregulation of COL1A1 mRNA expression (Fig. 8B). Taken together, these results indicated that UBE2C promotes hRPE cell fibrosis via mTOR pathway regulation.

Fig. 8.

mTOR pathway inhibition reverses hRPE cell fibrosis. A Western blotting for mTOR pathway-related protein expression and B RT-qPCR for ECM-related gene expression in rapamycin-treated UBE2C-overexpressing hRPE cells. Data are presented as means ± SDs. *P < 0.05

Discussion

In this study, we first observed that LLRL irradiation inhibited EMT-related fibrogenesis via RPE cells induced under hypoxic conditions and suppressed cytokine secretion, stimulating HSF remodeling. These results suggested that LLRL prevents myopia progression via RPE cells. Notably, high-throughput RNA sequencing revealed that UBE2C, which is typically involved in cancer progression, was a crucial factor regulating RPE cell fibrosis, and its expression could be regulated using appropriate LLRL. In UBE2C overexpression and inhibition experiments, LLRL was further confirmed to downregulate UBE2C expression, thus inhibiting RPE cell fibrosis. In general, our results indicated that LLRL irradiation and UBE2C prevent scleral remolding via EMT inhibition in RPE cells.

Red light is beneficial to the human body at low doses; however, at excessive doses, it can lead to impaired physiological functions. Highly proliferating hRPE cells may demonstrate optimal myopia-related biological functions under varied experimental conditions; therefore, in the present study, we used the MTT assay to determine the optimal LLRL irradiation frequency and duration for increasing hRPE cell proliferation. At low doses (0.001–0.10 J/cm²), red light irradiation can cause photostimulation or photoinhibition [23]. Most related studies have used 620–670-nm, 300–1200-lx red light in their irradiation experiments. Low-dose red light has been noted to repair spinal cord injuries, promote skin wound healing, and accelerate nerve regeneration [24]. In ophthalmic research, LLRL has been reported to protect photoreceptor cells and reduce neuronal apoptosis [25]. In general, LLRL may exert protective effects by reducing tissue inflammation, increasing mitochondrial metabolism, and alleviating oxidative stress damage.

Norton et al. reported that tree shrews living in a low-energy, long-wavelength red light environment had shorter eye axes and that their refractive errors shifted toward hyperopia [26]. In monkeys, cooler, long-wavelength light was noted to significantly inhibit eye axis elongation than warmer, short-wavelength light [27]. The myopia-inhibiting effects of low-energy red light depend on the local signal transduction mechanisms in the retina. hRPE cells are crucial components in regulating myopia occurrence and progression. Low-energy, short-term light exposure can activate self-protection mechanisms in RPE cells. Moreover, low-energy light can induce apoptosis, whereas high-energy light can cause necrosis [28]. Based on these results, we exposed hRPE cells to LLRL irradiation in a myopia-simulating hypoxic microenvironment to elucidate possible molecular mechanisms underlying the myopia progression-inhibiting effects of LLRL. We observed that exposing the cells to 630-nm, 600-lx red light for 20 s twice every 12 h resulted in the highest proliferation activity—consistent with the previous results. Moreover, exposure to hypoxia alone downregulated the expression of the apoptosis-related gene BCL2, whereas LLRL irradiation with hypoxia exposure upregulated it. Taken together, these results indicated that at the molecular level, LLRL irradiation for optimal duration and frequency promotes apoptosis and inhibits necrosis.

In a hypoxic microenvironment, RPE cells undergo EMT, and TGF-β is an important factor causing RPE cells to transform into fibroblasts [29, 30]. In the current study, hypoxic hRPE cells demonstrated upregulated expression of extracellular matrix proteins α-SMA and MMP2 and downregulated expression of COL1A1. Similarly, studies on fundus diseases have reported that RPE cells secrete various cytokines, such as three TGF-β isoforms, HIF-1, VEGF, and extracellular matrix [31].

During RPE cell fibrosis in a myopic microenvironment, cytokines such as α-SMA, TGF-β, and MMP2 secreted may promote scleral remodeling [32]. In the current study, we treated HSFs with hypoxic hRPE cell supernatant and noted an upregulation in α-SMA and MMP2 expression in HSFs, promoting scleral remodeling; in contrast, the supernatant of LLRL-irradiated hypoxic hRPE cells downregulated the expression of these proteins, inhibiting scleral remodeling. Therefore, LLRL may inhibit scleral remodeling promoted by hypoxic hRPE cells.

We subsequently used high-throughput RNA sequencing to screen for genes through which LLRL inhibits hRPE cell fibrosis. The results demonstrated that hypoxia significantly upregulated UBE2C expression, whereas LLRL irradiation significantly downregulated it even in the hypoxic microenvironment. UBE2C, encoding an E2 ubiquitin-conjugating enzyme family protein [33], is located on human chromosome 20q13.12. UBE2C is 179 amino acids long with a molecular weight of 19.6 kDa. It regulates the ubiquitination process by forming a thioester bond with ubiquitin molecules through the cysteine at position 114 in the E2 nuclear domain. The enzyme is essential for cell-cycle regulation; it is involved in separase inhibitor degradation, enabling normal sister chromatid separation and metaphase-to-anaphase transition in cells [34, 35]. Okamoto et al. noted that UBE2C expression is low in many normal tissues but high in most cancer cell lines. UBE2C expression is significantly higher in primary tumors of the lung, stomach, bladder, and uterus than in the adjacent normal tissues. The enzyme can promote malignant tumor invasion and proliferation, enabling cancer progression [36, 37]. In recent years, UBE2C has also been associated with skeletal muscle development. For instance, Chen et al. reported that UBE2C can promote embryonic mouse skeletal muscle development: UBE2C knockout significantly reduced expression of the skeletal muscle marker proteins MYOG and MYHC and restricted skeletal muscle development and regeneration [38]. UBE2C has also been linked to diseases such as pulmonary fibrosis and ovarian dysfunction [39, 40]. In the current study, LLRL irradiation downregulated UBE2C expression in hRPE cells for one major reason: LLRL activated ubiquitin throughout the retina, including RPE cells, and thus increased degradation of excess rhodopsin, which is controlled by the ubiquitin–proteasome system [41, 42].

In the present study, UBE2C was noted to upregulate HIF-1α, α-SMA, and MMP2 expression in hypoxic hRPE cells, indicating that UBE2C promotes hRPE cell fibrosis [43]. Studies on idiopathic pulmonary fibrosis have reported that upregulated UBE2C expression is related to tissue fibrosis, which is often deleterious to the tissues. In addition, studies on heart diseases have found that UBE2C may promote calcific aortic valve disease progression through HIF-1α pathway upregulation [44]. UBE2C also promotes MMP2 expression in lung cancer cells [45]. Direct evidence indicating the relationship between UBE2C and α-SMA remains unavailable. Nevertheless, UBE2C is associated with tissue fibrosis, and cells of fibrotic tissues often demonstrate upregulated α-SMA expression, similar to that observed in the current study. Thus, UEB2C may promote α-SMA expression. In the present study, LLRL intervention downregulated UBE2C expression in hypoxic hRPE cells, representing the major protective effect of moderate red light irradiation in tissues: inhibition of hypoxia-induced scleral remodeling signal transduction, a deleterious biological signaling pathway. Our subsequent experiments using overexpression and RNAi techniques corroborated the aforementioned results.

The mTOR pathway is strongly associated with myopia occurrence and progression [46]. In a study on a unilateral form deprivation myopia mouse model, single-cell RNA sequencing revealed that the differentially expressed genes were significantly enriched in the mTOR pathway and the hypoxic signaling pathway in the cardiovascular system [3]. In the present study, high-throughput RNA sequencing results indicated that the mTOR pathway proteins were strongly expressed in hypoxic hRPE cells, confirming that the mTOR pathway-mediated damage in hRPE cells leads to myopia occurrence and progression. In a study, insulin-treated hRPE cells were noted to demonstrate significant activation of the mTOR pathway and upregulation of growth factor and MMP2 expression. Moreover, electron microscopy revealed dilated and foamed endoplasmic reticulum in the treated hRPE cells, indicating significant degeneration [9].

A study reported that mTOR pathway activation can mediate TGF-β1-induced pulmonary fibrosis, indicating the role of mTOR in tissue fibrosis development [47]. In the present study, UBE2C overexpression led to upregulation of p-mTOR and p-AKT expression, whereas UBE2C inhibition resulted in its downregulation. Therefore, UBE2C regulates the mTOR pathway in hRPE cells. Similar to the current results, studies on cancers in multiple tissues have found that UBE2C can regulate the PI3K/AKT/mTOR pathway [48, 49]. RPE cells initiate the antioxidant effects via PI3K/Akt/mTOR pathway inhibition [50]. Moreover, UV-induced photodamage leads to PI3K/Akt/ERK pathway activation, exacerbating RPE cell damage [51]. Therefore, we speculate that LLRL irradiation also inhibits RPE cell fibrosis via UBE2C-mediated PI3K/Akt/mTOR pathway inhibition. In the current study, we also assessed the expression of SMAD2/3 and HIF1-α, which are crucial signaling molecules that also lead to tissue fibrosis [52, 53]. We noted that UBE2C inhibition resulted in downregulation of p-SMAD2/3 and HIF1-α expression. Thus, UBE2C can affect hRPE cell fibrosis through the SMAD2/3 pathway as well.

Conclusion

The current results indicated that in a hypoxic microenvironment, hRPE cells’ EMT may be closely associated with scleral fibroblast remodeling during myopia development and progression. LLRL was noted to protect hRPE cells by inhibiting UBE2C-initiated fibrosis under hypoxic conditions and regulating proteins involved in pathways closely related to fibrosis, such as mTOR and SMAD2/3. However, additional studies investigating the mechanism through which hRPE cells stimulate fibrosis via scleral fibroblasts, as well as the potential roles of UBE2C in other fundus diseases (e.g., age-related macular degeneration), are warranted. In the future, we aim to verify the function of UBE2C in myopia animal models.

Author contributions

H.J.Q. conceived and supervised the project, and was responsible for all experiments. X.W.Z. wrote the manuscript. Y.P.G. and X.W.Z. performed all in vitro experiments. Y.L.L., X.F.W., and L.T. were responsible for data analysis and interpretation. All authors read and approved the final manuscript.

Funding

This work was supported by a grant from the Nansha District Science and Technology Plan Project in Guangzhou City [grant number 2021MS008].

Data availability

No datasets were generated or analysed during the current study.

Declarations

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.