Reactive oxygen species (ROS) such as dihydrogen dioxide (H₂O₂), superoxide anion radical (${\text{O}}_2^{-}$), and hydroxyl radicals (•OH) are byproducts generated during oxygen consumption in the human body; All these ROS act as intracellular signaling molecules in vivo. ROS also play essential roles in the control of cellular functions such as the electron transport chain in mitochondria and the activation of white blood cells [1, 2]. However, excessive ROS cause oxidative stress by disrupting the antioxidant balance in the body. Free radical toxicity causes non-selective and irreversible damage to cellular components such as fat, protein, sugar, and DNA, leading to conditions such as cancer, Alzheimer’s disease, heart disease, arteriosclerosis, inflammation, autoimmune diseases, and aging. A severe increase in the amount of ROS causes fatal damage to cells and eventually leads to cell death [3, 4].

Lasers and light-emitting diodes (LEDs), which are generally used as light sources of phototherapy, can show various effects depending on their intensity. Since the laser treats deep tissue lesions, it is much more applicable to invasive treatment and can be effective for surgery, deep treatment, or pain relief. On the other hand, LED is widely used for non-invasive (no cutting or burn) treatment, and has the advantage of effectively treating a very large area of lesions and diseased areas without invasive damage [5]. Photobiomodulation (PBM) refers to irradiating light of a specific wavelength to induce and change spontaneous bioactivity in damaged areas of human tissue. Irradiated light has beneficial effects at power densities (irradiances) of 1 mW/cm² to 5 W/cm², with a narrow spectral width in the red or near-infrared region. PBM has been widely recognized in medical practice for more than 50 years, and the recent use of PBM in an increasing range of pathologies has shown positive results [6, 7]. Several mechanisms have been proposed to explain the photobiological regulatory effects of PBM; The currently accepted hypothesis is that PBM of red/NIR light irradiated to cells or tissues is absorbed by mitochondria and promotes the production of adenosine triphosphate and nitric oxide; The generated adenosine triphosphate and nitric oxide promote the displacement of free radicals and the reduction of oxidative stress load on the organism by regulating the production of an appropriate amount of ROS when there is an imbalance between antioxidants and free radicals, and activates transcription factors such as NF-κB that induce expression [8, 9]. PBM also enhances cell survival by upregulating the expression of protective proteins that prevent cell death [10–13]. Although the mechanism underlying PBM has not yet been fully elucidated, low-energy PBM yields more positive results compared with irradiation using light of the same wavelength at a higher energy. Notably, PBM can inhibit apoptosis and promote cell proliferation, migration, and adhesion under low-energy red or near-infrared light [14]. However, it remains unclear whether PBM can prevent intracellular oxidative stress-induced apoptosis. Because ROS have important roles in cell death, we hypothesized that PBM could inhibit cell death by protecting cells from ROS-induced damage.

The purpose of this study is to identify the protective effect of PBM on H₂O₂-induced cell death, and then to reveal the underlying mechanisms of these effects and which stage of cell death is protected by PBM.

2.1. Cell Line and Cell Culture

Mouse macrophage RAW264.7 cells were purchased from ATCC (Rockville, MD, USA), Dulbecco’s modified Eagle medium supplemented with 10% (w/v) fetal bovine serum (35-015-CV; Corning, NY, USA) and 1% (v/v) penicillin/streptomycin (30-002-CI; Corning, NY, USA) was used as a culture medium and cultured under 5% CO₂ conditions at 37 ℃. When the cell culture plate was filled with about 70–80% of RAW 264.7 cells, the cells were washed once with phosphate-buffered saline (PBS) and subcultured every 2 days.

2.2. Cell Viability Assay

RAW264.7 cells were seeded in 96-well plates (30096; SPL Life Sciences Co., Ltd., Pocheon, Republic of Korea) at 5 × 10³ cells/well and cultured for 24 hours, then treated with 0.6, 0.7, and 0.8 mM H₂O₂ for an additional 24 hours. The cell culture medium was changed, 1 mg/mL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was added, and cells were cultured for 3 hours. After removal of the supernatant and addition of 150 μL dimethyl sulfoxide to melt the formed formazan crystals, the absorbance was evaluated at 570 nm using an ELISA reader (Tecan, Männedorf, Switzerland). Cell viability was indicated as a percentage of the untreated control group (i.e., not treated with H₂O₂).

2.3. LED Irradiation and Cytotoxicity

LEDs with maximum emission wavelengths of 630 and 850 nm were used by arranging a total of 126 LED chips (14 × 9) with 0.2 W intensity and PCB of 14.6 × 21.0 cm. Detailed information on the parameters of both LEDs is shown in Table 1. A 19.5 V power supply (GPS-2303; GW Instek, Taipei, Taiwan) was used to power the LED panel and the power of the light source was measured as previously described [15, 16]. A power of 10 mW/cm² was transferred to the cell at a position of about 5 cm from the bottom of the cell culture plate as shown in Fig. 1.

Figure 1. Photographs showing the lighting conditions during exposure of RAW264.7 cells to red (left) and near-infrared (right) LED lights. LED devices for a cell culture plate with an output irradiance of 10 mW/cm² light at a distance of 5 cm. To cool the LED board, a cooling fan is installed to control temperature rise.

TABLE 1

LED^a) parameter information.

Parameter	Value
Light Type	Light-emitting Diode
Number of Array	126
Mode	Continuous Wave (CW)
Wavelength (nm)	630 (broadband: 600–650 nm); 850 (broadband: 780–890 nm)
Aperture Diameter (mm)	2
Irradiance at Aperture (mW/cm2)	10
Beam Shape	Circular
Beam Profile	Gaussian

^a)LED manufacturer: Wontech Co., Ltd., Seongnam, Korea..

RAW264.7 cells (5 × 10³ cells/well) were seeded in 96-well plates and cultivated for 24 hours at 37 ℃ with 5% CO₂. Then, 630 and 850 nm LEDs were respectively irradiated to the cells using an intensity of 10 mW/cm² at 5-minute intervals for 20 minutes based on previous studies (total energy density: 3, 6, 9, and 12 J/cm²) [16]. After irradiation, cells were cultured for 24 hours and cell viability was measured as described above. To confirm that LED irradiation inhibited cytotoxicity in H₂O₂-treated cells, RAW264.7 cells were treated with 0.7 mM H₂O₂, and then 630 nm LED was irradiated at an intensity of 10 mW/cm² for up to 20 minutes at 5-minute intervals (total energy density: 3, 6, 9, and 12 J/cm²), and 850 nm LED was irradiated for up to 40 minutes at 10-minute intervals at the same intensity (total energy density: 6, 12, 18, and 24 J/cm²). Cell viability was measured after 24 hours as described above.

2.4. ROS Staining

Cover slips were placed in 6-well plates (SPL Life Sciences Co., Ltd.) and RAW264.7 cells were grown at a density of 3 × 10⁵ cells per well. After incubation for 24 hours, 0.7 mM H₂O₂ was added; cells were then irradiated with a 630 or 850 nm LED, respectively, as described above. After incubation for 2 hours, the culture medium was eliminated and cells were rinsed three times with 1 mL of 1× PBS for 5 minutes. In dark conditions, 150 μL of 10 μM dichlorofluorescein diacetate (DCFDA) was added and cells were reacted for 15 minutes at room temperature; They were then washed three times (5 minutes per wash) with 1 mL of 1× PBS. After removal of the cover slip and the surrounding PBS, Vectashield^® solution containing 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., CA, USA) was added as a mounting medium. The stained samples were visualized using a fluorescence microscope by randomly setting three fields per cover slip (BX53F2; Olympus, Tokyo, Japan). ROS intensity was quantified using ImageJ software (NIH, MD, USA).

2.5. TUNEL Assay

RAW264.7 (5 × 10⁵ cells/well) cells were seeded on 6-well plates covered with cover slips and cultured for 24 hours. After incubation, each well was treated with 0.7 mM H₂O₂, then irradiated with a 630 or 850 nm LED at 10 mW/cm² for 10 minutes or 40 minutes, respectively. After incubation for 24 hours, the culture medium was eliminated and the cells were rinsed twice with cold 1× PBS, then fixed with 4% (v/v) paraformaldehyde (pH 7.4) dissolved in PBS at 4 ℃ for 25 minutes. Subsequently, cells were washed thrice for 5 minutes at 25 ℃ with 1× PBS, permeabilized for 5 minutes at room temperature with 0.2% (w/v) Triton X-100 solution (9002-93-1; Sigma-Aldrich, Darmstadt, Germany) dissolved in PBS, and washed thrice for 5 minutes at room temperature with 1× PBS. After removing the cover slip and surrounding PBS, the cells were then covered with 100 μL of 1× reaction buffer, incubated at room temperature for 5–10 minutes, and washed with 1× PBS. Fifty microliters of staining solution prepared according to the manufacturer’s instructions (5 μL of dUTP conjugated dye, 10 μL of 5× reaction buffer, 1 μL of TdT recombinant enzyme, and 35 μL of deionized water; Bioacts, Incheon, Republic of Korea) were added to the cells, and then reacted in an incubator at 37 ℃ for 1 hours. Cells were rinsed thrice with PBS at room temperature for 5 minutes to remove unreacted dye-dUTP. Next, they were incubated with propidium iodide (PI) solution (1 μg/mL) for 15 minutes at room temperature in the dark. After the cells had been washed with PBS, Vectashield mounting solution (Vector Laboratories Inc.) was added; A sample of three fields per cover slip was visualized with a confocal microscope, and cells showing a positive signal in each stain were counted and quantified (FV3000; Olympus, Tokyo, Japan).

2.6. Flow Cytometry Analysis

After RAW264.7 cells had been cultured for 24 hours, treated with H₂O₂ and subjected to 630 or 850 nm LED irradiation, the medium was removed and cells were washed twice with 1× cold PBS. Cells were detached by treatment with 0.25% Trypsin-EDTA (25200-056; Gibco, MA, USA) and suspended in culture medium containing 10% FBS, then transferred to a new tube and centrifuged at 500 × g at 4 ℃ for 5 minutes to remove the supernatant. The cells were then rinsed with pre-chilled PBS and centrifuged at 500 × g for 5 minutes to remove the supernatant. Culture medium was added to resuspend the cells, and the number of cells was determined. Next, cells were incubated at room temperature for 30 minutes and centrifuged again at 500 × g for 5 minutes, and the supernatant was removed. Then, cell apoptosis was measured by flow cytometry using a dead cell apoptosis kit (V13242; Thermo Fisher Scientific, MA, USA) following the manufacturer’s instructions. In brief, 1× Annexin V binding solution (Thermo Fisher Scientific) was added to accomplish a final concentration of 1 × 10⁶ cells/mL, and 100 μL of the cell suspension was transferred to a new tube at a concentration of 1 × 10⁵ cells/100 μL. Then, 5 μL of fluorescein isothiocyanate (FITC) Annexin V (Thermo Fisher Scientific) and 1 μL of PI stock solution (100 μg/mL; Thermo Fisher Scientific) were added. Cells were incubated in the dark for 15 minutes at room temperature and then analyzed by flow cytometry by adding 100 μL of cold 1× Annexin V solution (Thermo Fisher Scientific).

2.7. Western Blotting Analysis

After adding H₂O₂, RAW264.7 cells irradiated with the 630 or 850 nm LED were collected, respectively, and homogenized using ice-cold modified RIPA buffer [Biosesang, Seongnam, Republic of Korea; 50 mM Tris-HCl, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1% (v/v) NP40, 1% (w/v) Triton X-100 (pH 7.4), and protease inhibitor cocktail]. Protein concentrations were determined using a protein assay kit (DC-protein assay kit; Bio-Rad, CA, USA). Proteins were mixed with 5× sample buffer (Bio-Rad). Proteins (40 μg per sample) were separated via 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions, then transferred to polyvinylidene difluoride membranes (88520; Bio-Rad). The membranes were blocked with 5% (w/v) bovine serum albumin (BSA; to detect Bax, Bcl-2, and cleaved PARP proteins) or 5% (w/v) skim milk (to detect procaspase-3 protein) for 2 hours. After they had been blocked, the membranes were incubated with primary antibody (Bcl-2, 1:1000, Cell Signaling Technology, 3498s; Bax, 1:1000, Cell Signaling Technology, 14796s; procaspase-3, 1:1000, Cell Signaling Technology, 9662s; cleaved PARP, 1:200, Santa Cruz, sc-56196; β-actin, 1:1000, Sigma-Aldrich, A1978) in 5% BSA or 5% skim milk for 12 hours at 4 ℃. The membranes were washed four times with 0.1% (v/v) Tween-20 in PBS, then incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody (LF8002; Ab Frontier, Seoul, Republic of Korea) for 1 hours at room temperature. Ponceau staining was performed to monitor loading; Bands were visualized using an enhanced electrogenerated chemiluminescence western blotting system (170-5061; Bio-Rad). Protein levels were analyzed and quantified using ImageJ (NIH).

2.8. Statistical Analysis

Independent experimental results are presented as mean ± standard deviation (SD). Comparisons of three groups or more than three groups of normally distributed data were accomplished by one-way ANOVA followed by multiple comparison tests to determine the least significant difference. Statistical analysis was employed GraphPad Prism software (ver. 7.0; GraphPad, CA, USA). All experiments have been conducted in triplets and recognized as statistically significant differences when the P-value was less than 0.05.

3.1. Cell Viability after H₂O₂ Treatment

To confirm the viability of RAW264.7 cells after treatment with different concentrations of H₂O₂, cells were incubated with 0.6, 0.7, and 0.8 mM H₂O₂ for 24 hours, then analyzed using MTT assays. As shown in Fig. 2(a), the number of cells decreased as the concentration of H₂O₂ increased; Compared with the cell viability of untreated cells, the cell viabilities after treatment with 0.6, 0.7, and 0.8 mM H₂O₂ were 76.7, 65.6, and 26.4%, respectively [Fig. 2(b); P < 0.05, P < 0.01, and P < 0.0001, respectively]. Therefore, 0.7 mM H₂O₂ with a cell viability of 65% was used for subsequent experiments.

Figure 2. Cell viability of H₂O₂-treated RAW264.7 cells. RAW264.7 cells were grown in 96-well culture plates (1 × 10⁵ cells/well) for 24 hours, then treated with 0.6, 0.7, or 0.8 mM H₂O₂ for 24 hours. (a) Cell morphology after treatment of RAW264.7 cells with 0.6, 0.7, or 0.8 mM H₂O₂ for 24 hours. Cells were observed using an inverted microscope (magnification, 100×). Scale bar, 200 μm. (b) After 24 hours, cell viability was determined by MTT assay. The percentage of cell viability was normalized to the untreated cells. Data are shown as means ± SD of values from three separate experiments (n = 3). ****P < 0.0001, **P < 0.01, and *P < 0.05 vs. untreated cells. One-way ANOVA and Sidak’s post hoc analyses were performed to compare all experimental conditions. MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.

3.2. Cell Viability after LED Irradiation

To assess the effects of 630 or 850 nm LED irradiation on cell viability, cells were irradiated with an intensity of 10 mW/cm² at 5-minute intervals for up to 20 minutes (total energy density: 3, 6, 9, and 12 J/cm²). After cultured RAW264.7 cells had been irradiated with each of the LEDs for different lengths of time, the cells were cultured for a day and viability was measured. As shown in Fig. 3, cell viability tended to increase as the duration of 630 nm LED irradiation increased. Notably, when irradiated with an intensity of 10 mW/cm² for 15 minutes (9 J/cm²) and 20 minutes (12 J/cm²), cell viability significantly increased by 106% and 109%, respectively, compared with the viability of non-irradiated cells [Fig. 3(a); P < 0.05 and P < 0.001, respectively]. Furthermore, when irradiated with an intensity of 10 mW/cm² for 20 minutes (12 J/cm²) at 850 nm, cell viability significantly increased by 110% compared with non-irradiated cells [Fig. 3(b); P < 0.05]. Thus, 630 nm or 850 nm LED irradiation did not affect RAW264.7 cell viability.

Figure 3. Effects of 630 and 850 nm LEDs on the cell viability of RAW264.7 cells. RAW264.7 cells were irradiated using a (a) 630 nm LED or (b) 850 nm LED with an intensity of 10 mW/cm² at 5-minute intervals for up to 20 minutes (total energy density: 3, 6, 9, and 12 J/cm²), then cultured for 48 hours. Cell viability was measured using colorimetric MTT metabolic activity assays. Each experiment was performed ≥3 times (n = 3). Data are shown as means ± SD. *P < 0.05 and ***P < 0.001 vs. untreated cells. One-way ANOVA and Dunnett’s post hoc analyses were performed to compare all experimental conditions. MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.

3.3. Antioxidant Effects of 630 nm and 850 nm LEDs

To investigate the antioxidant effects of 630 or 850 nm LED irradiation, H₂O₂-treated RAW264.7 cells were subjected to each LED irradiation, followed by assessment of cell viability. After treatment with 0.7 mM H₂O₂, cells were irradiated by an LED with an intensity of 10 mW/cm². The 630 nm LED irradiation was performed at 5-minute intervals for 20 minutes (total energy density: 3, 6, 9, and 12 J/cm²); the 850 nm LED irradiation was performed at 10-minute intervals for 40 minutes (total energy density: 6, 12, 18, and 24 J/cm²). As shown in the results of Fig. 4, the cell viability of RAW264.7 cells irradiated with 630 nm LED for 10 minutes (6 J/cm²); After H₂O₂ treatment was significantly increased by about 1.7-fold compared to non-irradiated cells (P < 0.001), but showed a tendency to decrease after irradiation for 15 minutes (9 J/cm²) and 20 minutes (12 J/cm²) [Fig. 4(a)]. In contrast, cell viability gradually increased as the duration of 850 nm LED irradiation increased [Fig. 4(b)]. In particular, cell viability was significantly increased by approximately 1.6-fold after 40 minutes (24 J/cm²) of irradiation, compared with the viability of non-irradiated cells (P < 0.0001). These results show the antioxidant effect of 630 nm and 850 nm LEDs on H₂O₂-induced oxidative stress. Therefore, subsequent experiments were performed using 10- and 40-minute (6 and 24 J/cm², respectively) durations of irradiation with the 630 nm and 850 nm LEDs, respectively, at an intensity of 10 mW/cm².

Figure 4. Effects of 630 and 850 nm LEDs on the cell viability of H₂O₂-treated RAW264.7 cells. Cell viability was analyzed by varying the duration of irradiation with 630 nm and 850 nm LEDs after RAW264.7 cells had been treated with 0.7 mM H₂O₂. (a) RAW264.7 cells were irradiated using a 630 nm LED with an intensity of 10 mW/cm² at 5-minute intervals for up to 20 minutes (total energy density: 3, 6, 9, and 12 J/cm²), then cultured to determine cell viability. (b) RAW264.7 cells were irradiated using an 850 nm LED with an intensity of 10 mW/cm² at 10-minute intervals for up to 40 minutes (total energy density: 6, 12, 18, and 24 J/cm²), then cultured for 48 hours to determine cell viability. Cell viability was measured using colorimetric MTT metabolic activity assays. Each experiment was performed ≥3 times (n = 3). Data are shown as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. untreated cells. One-way ANOVA and Tukey’s post hoc analyses were performed to compare all experimental conditions.

3.4. Inhibition of ROS Generation by 630 nm or 850 nm LED Irradiation

To determine whether the antioxidant effects of 630 or 850 nm LED irradiation could inhibit cell death, the inhibition of H₂O₂-induced ROS generation was assessed with DCFDA staining. As shown in Fig. 5(a), ROS were generated in non-irradiated cells upon treatment with H₂O₂. However, when RAW264.7 cells were treated with H₂O₂ and then irradiated with the 630 or 850 nm LED, ROS generation was significantly reduced. Quantification of ROS intensity in each group (using ImageJ software) revealed approximately 7.4- and 12.3-fold reductions of intensity in cells irradiated with the 630 nm and 850 nm LEDs, respectively, after treatment with H₂O₂ [P < 0.0001; Fig. 5(b)]. Therefore, it was assumed that the inhibition of apoptosis observed after irradiation with the 630 or 850 nm LED was not only due to inhibition of ROS generation but also by the promotion of the displacement of free radicals and reduction of oxidative stress load. There was no significant difference in ROS scavenging activity between the two LEDs.

Figure 5. Radical scavenging activity after 630 or 850 nm LED irradiation of H₂O₂-treated RAW264.7 cells. (a) Fluorescence image of RAW264.7 cells stained with DAPI and DCFDA. Cells were observed by fluorescence microscopy (magnification, 400×). Images are representative of three independent experiments (n = 12). Scale bar, 100 μm. (b) Histogram of the radical scavenging effects of 630 nm or 850 nm LED irradiation in H₂O₂-treated RAW264.7 cells. Data are shown as means ± SD (n = 12). ****P < 0.0001 vs. 0.7 mM H₂O₂-treated cells. One-way ANOVA and Dunnett’s post hoc analyses were performed to compare all experimental conditions. DAPI, 4,6-diamidino-2-phenylindole; DCFDA, dichlorofluorescein diacetate.

3.5. Inhibition of Apoptosis after Irradiation with 630 nm or 850 nm LED, as Determined by TUNEL Staining and Flow Cytometry

To investigate whether irradiation with both LEDs could inhibit apoptosis, H₂O₂-treated cells were irradiated with the 630 or 850 nm LED, cultured for 24 hours, stained with TUNEL, and observed by fluorescence microscopy to quantify TUNEL-positive cells/nuclei. Among cells that were not treated with H₂O₂, few exhibited green fluorescence; After treatment with 0.7 mM H₂O₂, most cells exhibited green fluorescence [Fig. 6(a)]. However, when H₂O₂-treated cells were irradiated with the 630 or 850 nm LED, the numbers of TUNEL-positive cells were significantly reduced by approximately 45.7% and 37.8%, respectively, compared with the number among cells that had been treated with 0.7 mM H₂O₂ alone [P < 0.01 and P < 0.001; Fig. 6(b)]. The statistical significance between the two LEDs through TUNEL staining could not be confirmed.

Figure 6. Effects of 630 or 850 nm LED irradiation on H₂O₂-induced apoptosis in RAW264.7 cells, as determined by TUNEL staining and flow cytometry. (a) Representative images of RAW264.7 cells doubly stained with TUNEL (top column) and PI (middle column) under different wavelength conditions (control, 630 nm LED, and 850 nm LED). Cells were observed by confocal microscopy (magnification, 400×). Images are representative of three independent experiments (n = 12). Scale bar, 100 μm. (b) Bar graph showing the percentage of TUNEL-positive cells relative to PI-positive cells after irradiation with each wavelength. (c) RAW264.7 cells were labeled with Annexin V-FITC and PI, then analyzed by flow cytometry. RAW264.7 cells were cultured for 24 hours, treated with H₂O₂, then irradiated with the 630 or 850 nm LED. Dot plots depict cell populations in quadrants. (d) H₂O₂-treated RAW264.7 cells were irradiated with the 630 or 850 nm LED, labeled with Annexin V-FITC and PI, and analyzed by flow cytometry. The histogram shows the percentage of RAW264.7 cells in late apoptosis. Data are shown as means ± SD (n = 12). ****P < 0.0001 and *P < 0.05 vs. 0.7 mM H₂O₂-treated cells. One-way ANOVA and Dunnett’s post hoc analyses were performed to compare all experimental conditions.

To further explore the relationship between cell death inhibition and the stage of apoptosis after LED irradiation of H₂O₂-treated RAW264.7 cells, cells were dyed with Annexin V-FITC and PI, then measured by flow cytometric analysis. As shown in Figs. 6(c) and 6(d), 26, 26.1% of H₂O₂-treated RAW264.7 cells entered late apoptosis. Among H₂O₂-treated cells that were irradiated with the 630- or 850-nm LED, the numbers of cells in late apoptosis were approximately 2.3- and 1.8-fold lower, respectively, compared with the number among cells that had been treated with H₂O₂ alone. The detailed FACS results are summarized in Table 2. Although it is regrettable that RAW264.7 cells without any treatment have already progressed to the early stage of apoptosis during the incubation process, H₂O₂ treatment caused RAW264.7 cells to rapidly enter the late stage of apoptosis, and LED irradiation inhibited this cell death progression. FACS results also could not confirm the statistical significance between the two LEDs [Fig. 6(d)].

TABLE 2

Flow cytometry results.

Sample	Viable (%)	Apoptotic Early (%)	Apoptotic Late (%)	Necrotic (%)
Control	25.74 ± 2.70***	73.28 ± 2.71	0.97± 0.72*	0
H2O2-treated	9.57 ± 3.79	62.80 ± 17.50	26.12 ± 11.81	1.5 ± 1.34
H2O2 + 630 nm	12.79 ± 0.56	75.35 ± 5.67	11.53 ± 4.72	0.31 ± 0.14
H2O2 + 850 nm	10.69 ± 4.03	73.91± 9.83	14.78 ± 6.85	0.60 ± 0.21

Data are the percentage of cells positively stained for Annexin V⁻/PI⁻ (viable cells), Annexin V⁺/PI⁻ (early apoptotic cells), Annexin V⁻/PI⁺ (necrotic cells), and Annexin V⁺/PI⁺ (late apoptotic cells). The experiment was repeated three times (n = 3). Data are expressed as the mean ± SD. ***P < 0.001 and *P < 0.05 vs. 0.7 mM H₂O₂-treated cells..

3.6. Western Blotting Analysis of the Apoptosis-inhibiting Effects of the 630 nm and 850 nm LEDs

The expression level of apoptosis-related proteins was assessed to determine whether the ability to inhibit ROS generation by LED irradiation inhibits apoptosis. After 630 nm LED irradiation of H₂O₂-treated cells, the expression level of Bax was reduced by a smaller amount compared with the expression in cells treated with H₂O₂ alone, although this difference was not statistically significant. Although the expression level of Bcl-2 also tended to increase slightly compared with the expression in cells treated with H₂O₂ alone, this difference also was not statistically significant. After 850 nm LED irradiation of H₂O₂-treated cells, the expression level of Bax was significantly decreased by approximately 1.4-fold compared with the expression in cells treated with H₂O₂ alone [P < 0.01; Figs. 7(a) and 7(b)]. Additionally, the expression level of Bcl-2 was significantly increased by approximately 1.1-fold compared with the expression in cells treated with H₂O₂ alone [P < 0.01; Figs. 7(a) and 7(b)]. The Bax/Bcl-2 expression ratio in cells irradiated with the 630 nm or 850 nm LED was significantly reduced by approximately 1.3- and 1.6-fold, respectively, compared with the ratio in cells treated with H₂O₂ alone (P < 0.01 and P < 0.0001), indicating that the expression level of Bcl-2 was increased relative to the expression level of Bax after LED irradiation [Fig. 7(b)].

Figure 7. Effects of 630 and 850 nm LED irradiation on the Bax/Bcl-2 ratio and the expression levels of procaspase-3 and cleaved PARP in H₂O₂-treated RAW264.7 cells. (a) The expression levels of Bcl-2 and Bax were analyzed by western blotting using specific antibodies. (b) The relative expression levels of Bcl-2 and Bax were quantified using β-actin as a loading control, and the data were used to calculate the Bax/Bcl-2 ratio. (c) The expression levels of procaspase-3 and cleaved PARP were analyzed by western blotting. (d) The relative expression levels of procaspase-3 and cleaved PARP-1 were quantified using β-actin as a loading control. All quantitative data were displayed as histograms. Data are shown as means ± SD. All experiments were performed in triplicate (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, compared with the control group.

To explore whether irradiation with each LED source inhibited the degradation of procaspase-3 and the cleavage of PARP, the expression levels of these proteins were assessed. As shown in Figs. 7(c) and 7(d), the respective expression levels of procaspase-3 were significantly 1.3- and 1.2-fold greater in 630 nm and 850 nm LED-irradiated cells than in cells treated with H₂O₂ alone (P < 0.01 and P < 0.05). Furthermore, PARP cleavage in cells irradiated with the 630 nm or 850 nm LED was significantly decreased by 2.6- and 1.6-fold, respectively, compared with the cleavage in cells that had been treated with H₂O₂ alone (P < 0.01 and P < 0.05). Although the expression of cleaved caspase 3 could not be confirmed, these results suggest that the inhibition of procaspase 3 degradation by LED irradiation reduced the activation of caspase 3 and inhibited the cleavage of the PARP protein, thereby proceeding with normal DNA replication and gene expression in cells, which inhibit apoptosis-related signaling. As a result of western blot analysis, the statistical significance between the two LEDs could not be confirmed.

In this study, RAW264.7 cell viability increased over time after irradiation with 630 and 850 nm LEDs. Although the mechanism underlying the effects of PBM remains unclear, our results are consistent with former studies that have shown the promotion of cell proliferation, differentiation, and maturation after irradiation with an LED or laser in the wavelength bands of 620 ± 20 nm or 825 ± 25 nm [17, 18]. On the other hand, some conflicting results have also been reported, possibly because different tissues and cells respond with different targets to specific light wavelengths [19, 20]. In Fig. 3, when H₂O₂-treated cells were irradiated with 630 and 850 nm LEDs, the optimal cell viability was shown at a 4-fold higher fluence at 850 nm than at 630 nm. These results are explained by the results of George et al. [21], who reported that 825 nm near-infrared irradiation at high fluence produced high production of beneficial ROS and harmful ROS and simultaneously started antioxidant action to produce twice as much ATP as 636 nm to maintain high cell viability. Although many in vitro reports have shown that ROS increase after PBM, we demonstrated that both 630 and 850 nm LEDs inhibit H₂O₂ oxidative stress-induced ROS generation and rescue RAW264.7 cells from oxidative stress-induced cell death [11, 22–24]. These results are consistent with the findings of Huang et al. [7], who induced oxidative stress in cortical neuron cells using hydrogen peroxide, cobalt chloride, and rotenone; They showed that irradiation with an 810 nm laser could reduce ROS levels in the cultured cortical neurons, preventing cell death. It is also consistent with the findings of Sun et al. [25], who demonstrated anti-inflammatory effects of 625 nm LED irradiation by scavenging of phorbol-12-myristate-13-acetate-induced ROS in HaCaT human keratinocytes. The same results were even found in previous studies by the authors using different light sources. After inducing oxidative stress in human dermal fibroblasts using H₂O₂, irradiation with organic light-emitting diodes was used to reduce the level of ROS formation induced, and this antioxidant effect was able to regulate the mRNA expression level of genes related to aging and tumor suppression [16]. This is an important result that shows that the physical difference of the light source is not important, but that irradiation at a specific wavelength suppresses ROS production induced by oxidative stress. Our results not only show that red and near-infrared LED illumination can help maintain cellular homeostasis, but also provide an accurate explanation of the paradox of the PBM mechanism proposed in many studies.

It was confirmed by TUNEL staining that 630 and 850 nm LED irradiation could inhibit cell death due to oxidative stress in H₂O₂-treated RAW264.7 cells. Indeed, when flow cytometry was used to further explore whether 630 and 850 nm LED irradiation inhibited oxidative stress-induced cell death, we found significant differences in the numbers of cells in late apoptosis between irradiated and non-irradiated H₂O₂-treated cells. Although it was difficult to suppress early apoptosis in the untreated control group in this experiment, the numbers of cells in late apoptosis were significantly reduced after irradiation with the 630 or 850 nm LEDs compared with cells that had been treated with H₂O₂ alone; These differences suggested a substantial reduction in the overall rate of cell death after irradiation. The two most important groups of proteins in apoptotic signaling are Bcl-2 family proteins and cysteine proteases known as caspases [26]. In many cells and tissues, the Bax/Bcl-2 ratio may be important in determining susceptibility to apoptosis. When this ratio is low, cells can resist apoptosis. Therefore, the Bax/Bcl-2 ratio can influence the progression of cell death. Previously, Li et al. [27], reported that irradiation with an 810 nm laser led to increases in Bax protein expression and the Bcl-2/Bax ratio in senescent rat skeletal muscle. This irradiation reduced the progression of myocyte apoptosis in sarcopenic muscles. Miracabad et al. [28], used 6-hydroxide dopamine to induce oxidative stress in PC-12 cells; Subsequent treatment with curcumin and a 630 nm LED resulted in a decrease in the Bax/Bcl-2 ratio. The reduction in the Bax/Bcl-2 ratio induced by irradiation with a 630 nm LED was able to protect neurons by reducing 6-hydroxide dopamine-induced neuronal cell death. Our results also showed suppression of H₂O₂-mediated oxidative stress-induced cell death with the reduction of the Bax/Bcl-2 ratio after irradiation with 630 and 850 nm LEDs, consistent with previous in vivo findings that PBM-induced suppression of apoptosis was achieved by inhibiting the expression of mitochondrial-derived apoptosis signaling pathway proteins [29–31].

Apoptosis is initiated by an imbalance in the expression levels of Bcl-2 and Bax in a non-normal cellular state, which leads to increased cytochrome C expression and the activation of caspase proteins [32, 33]. In particular, the cleavage of caspase-3 induced by various stimuli is an important priming event for apoptosis. Activated caspase-3 cleaves the DEVD site to activate PARP; This cleavage leads to a reduction in adenosine triphosphate and the initiation of apoptosis [34, 35]. Although the expression of activated caspase-3 could not be confirmed in this study, the expression level of procaspase-3 was not significantly reduced in H₂O₂-treated RAW264.7 cells irradiated with the 630 or 850 nm LED, compared with cells that had been treated with H₂O₂ alone, and it was similar to the level in the untreated control group. Notably, PARP cleavage was significantly reduced in H₂O₂-treated cells irradiated with the 630 or 850 nm LED compared with cells that had been treated with H₂O₂ alone. These results are consistent with the findings of Salehpour et al. [36], who reported that red and near-infrared laser treatment significantly reduced caspase-3 protein levels in an experimental animal model of brain oxidative stress induced by chronic administration of D-galactose.

The mechanism by which LLLT can increase ROS in unstressed normal cells, but decrease ROS in oxidatively stressed cells to have the opposite effect, could be a promising candidate for the treatment of chronic human diseases caused by excessive ROS induction. In particular, nerve cells, which contain a large amount of fat, are particularly vulnerable to oxidative stress due to their structural characteristics and show a close relationship with oxidative stress-induced neurological diseases and damage and neurodegenerative diseases such as Alzheimer’s dementia [37]. In addition, active oxygen increased by such oxidative stress breaks down the in vivo defense system and ultimately promotes skin aging as well as skin dryness, increased sensitivity, and pigmentation, and causes various skin diseases such as photosensitivity diseases or malignant tumors [38]. Therefore, in order to protect nerve cells from oxidative stress caused by these reactive oxygen species and delay skin aging, two LED lights that are non-invasive and have appropriate light output are thought to be able to effectively treat a wide range of parts and diseased areas.

Overall, the expression of Bax and Bcl-2 proteins involved in the mitochondrial apoptosis signaling pathway changed in the direction to inhibit apoptosis with the reduction of ROS level by 630 and 850 nm LED irradiation, which ultimately inhibits cell death by reducing procaspase degradation and PARP cleavage. In addition, the 850 nm near-infrared LED showed better results when the irradiation time was longer than that of the 630 nm red LED, but it was found that the cell death inhibitory effect did not change depending on the LED wavelength. These results suggest that LEDs with 630 and 850 nm wavelength light can be promising phototherapy candidates for the treatment of chronic human diseases caused by excessive ROS induction. Nevertheless, why the 850 nm near-infrared LED irradiation time should be longer than that of the 630 nm red LED for optimal effect should be further studied and determined in future studies.

In this study, we showed that 630 or 850 nm LED irradiation inhibited H₂O₂-induced oxidative stress-induced cell death in RAW264.7 cells. As a result of irradiating LEDs of two wavelengths to cells subjected to oxidative stress by H₂O₂, it was confirmed that the intracellular ROS level was significantly reduced. The ROS scavenging effect by LED irradiation suppressed double-stranded DNA breakage that occurred during apoptosis, and this result led to inhibition of the late apoptosis stage. In addition, the expression of apoptosis pathway proteins in a direction that inhibits apoptosis supported this result. In particular, the 630 nm LED is considered a positive light source in terms of energy efficiency, as it exhibits equivalent effects even with shorter exposure times compared to the 850 nm LED.

All authors would like to acknowledge the support of the Undergraduate Research Program (URP) of the Korea Foundation for the Advancement of Science & Creativity (KOFAC), which helped conduct the research.

This work was supported by the Korea Foundation for the Advancement of Science & Creativity (KOFAC), and funded by the Korean Government (MOE); Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Grant no. NRF-2020R1A6A1A03043283); National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science and ICT) (Grant no. 2022R1F1A1062944); National Research Facilities & Equipment Center (NFEC) grant funded by the Korea government (Ministry of Education) (Grant no. 2019R1A6C1010033).

The authors declare no conflict of interest.

Data are contained within the article.