Protective Effect of Photobiomodulation against Hydrogen Peroxide-Induced Oxidative Damage by Promoting Autophagy through Inhibition of PI3K/AKT/mTOR Pathway in MC3T3-E1 Cells

Photobiomodulation (PBM) has been repeatedly reported to play a major role in the regulation of osteoblast proliferation and mineralization. Autophagy is closely associated with various pathophysiological processes in osteoblasts, while its role in oxidative stress is even more critical. However, there is still no clear understanding of the mechanism of the role of autophagy in the regulation of osteoblast mineralization and apoptosis under oxidative stress by PBM. It was designed to investigate the impact of 808 nm PBM on autophagy and apoptosis in mouse preosteoblast MC3T3-E1 treated with hydrogen peroxide (H2O2) through PI3K/AKT/mTOR pathway. PBM could inhibit MC3T3-E1 cell apoptosis under oxidative stress and promote the expression of osteogenic proteins, while enhancing the level of autophagy. In contrast, 3-methyladenine (3-MA) inhibited the expression of osteoblast autophagy under oxidative stress conditions, increased apoptosis, and plus counteracted the effect of PBM on osteoblasts. We also found that PBM suppressed the activated PI3K/AKT/mTOR pathway during oxidative stress and induced autophagy in osteoblasts. PBM promoted autophagy of MC3T3 cells and was further blocked by 740 Y-P, which reversed the effect of PBM on MC3T3 cells with H2O2. In conclusion, PBM promotes autophagy and improves the level of osteogenesis under oxidative stress by inhibiting the PI3K/AKT/mTOR pathway. Our results can lay the foundation for the clinical usage of PBM in the treatment of osteoporosis.


Introduction
Osteoporosis, as a common systemic bone metabolism disease, is characterized by bone mass reduction and deterioration of bone structure resulting in enhanced bone fragility and increased fracture risk [1]. Osteoporosis adversely affects human health, especially in the aging populations. Globally, osteoporotic fractures occur approximately every 3 second, and nearly half of women and one-quarter of men over 50 years of age will suffer an osteoporotic fracture [2]. Hence, osteoporosis is a major threat to people's health and living standards. Therefore, effective treatment options for osteoporosis need to be established.
Oxidative stress is an important pathological process involved in osteoporosis [3]. During the development of oxidative stress, excessive reactive oxygen species (ROS) production destroys the balance between oxidation and antioxidant defense system, leading to osteoblast metabolic function disorder and apoptotic pathway activation [4,5]. ROS, produced by oxidative stress, are the main intracellular signal transducers that maintain autophagy [6]. Moreover, a key role of autophagy is to regulate the levels of ROS [7,8]. A few studies have indicated that regulating autophagy in cells under oxidative stress can effectively reduce osteoblast apoptosis and promote osteoblast differentiation, thus reducing bone loss [9,10].
Photobiomodulation (PBM), a traditional physical therapy technique, has been extensively used in the treatment of skin disease, nerve injury, and other diseases owing to its anti-inflammatory and tissue repair-promoting effects and fewer side effects. Previous studies have shown that the main pathways of PBM include antiapoptosis, oxidative stress regulation, and autophagy [11][12][13]. However, Chang et al. revealed that PBM also enhance the proliferation and differentiation of osteoblasts and provided a new method to the regulation of osteoporosis and osteogenesis [14].
Thus, to explore the potential mechanism by which PBM regulates osteoporosis, an in vitro oxidative stress model was established, and the influence of PBM on H 2 O 2 induced apoptosis and autophagy in MC3T3-E1 cells and bone formation was examined. We found that PBM regulated autophagy by affecting the PI3K/AKT/mTOR pathway, thus regulating osteoblast differentiation. The aim of this study was to investigate the potential of PBM in the regulation of osteoporosis. In addition to providing new treatment options for the prevention and cure of osteoporosis, these findings will also provide new avenues for research.

Irradiation Tools and
Photobiomodulation. 808 nm fiber-coupled laser device (MW-GX-808/1000 mW, near IR spectroscopy) for PBM treatment of MC3T3-E1 cells, manufactured by Changchun Leishi Optoelectronic Technology Co., Ltd. The parameters of photobiomodulation were as follows: 808 nm sequential wavelength, 6 mW/cm 2 power density, 4.5 cm 2 spot size, 17.01 J irradiated energy, 3.78 J/cm 2 power density, and 630 s irradiation for one time per 24 h. The distance between the optical fiber and the bottom of the petri dish was about 21 cm. Equation of energy calculation formula is as follows: energy density ðJ/cm 2 Þ × spot size ðcm 2 Þ = power ðWÞ × time ðsÞ (Table 1)        2.12. Alizarin Bordeaux Staining. Extracellular matrix mineralization was assayed with an alizarin red S (C0148S, Beyotime Biotechnology, China). Seed 5 × 10 4 cells/well in 6-well culture dishes. After cell apposition and dissemination for 24 h, H 2 O 2 treatment was performed by 200 μm H 2 O 2 treatment for 4 h; then, PBM irradiation was performed for 630 s. After 48 h, cells were cultured for 21 d using osteogenic induction medium; and the medium was changed every 3 d. The cells were rinsed in PBS without calcium and magnesium after medium removal. The cells were fixed in fixative for 20 min and washed in PBS three times. The cells were stained with alizarin red stain solution for 30 min. The last step was to wash the cells using deionized water; next, all cells were photographed with microscope.
2.13. Statistical Analysis. All the experimental results in this experiment are data obtained in at least 3 independent repetitions. ImageJ (V1.47, National Institutes of Health, USA) software helps to analyze immunofluorescence data of fluorescence intensity and the gray scale of WB bands. LC3 immunofluorescence results were performed using ImageJ Pro Plus software (V6.0, Media Cybernetics, USA). The statistical analyses and line graph and histogram in the results were conducted and drawn using GraphPad Prism (V9.0 GraphPad Software, La Jolla, CA) software. All data are expressed as mean ± standard deviation ðSDÞ. Unpaired t -test was used for comparison between the two groups. One-way analysis of variance was used to determine differences between three or more groups, and Tukey's multiple comparison test was performed if statistically significant. p value < 0.05 was considered as statistically significant difference.

PBM Enhanced the Activity of MC3T3 Cells under
Oxidative Stress. Oxidative stress has been shown to induce apoptosis in a variety of cells [15][16][17]. Initially, using different concentrations of H 2 O 2 to treat MC3T3 cells (200, 300, 400, 500, and 600 μmol/L) for 4 h [18]. Cell Counting Kit was used to detect absorbance values, calculate cell viability, and observe the effect of different concentrations of hydrogen peroxide on the proliferation of MC3T3 cells. According to Figure 1(a), the viability of MC3T3 cells gradually diminished with the enhancement in H 2 O 2 concentration vs. the control group. Our results showed a dose-dependent inhibitory effect (p < 0:001). With the exception of the group of 200 μmol/L H 2 O 2 , there was a significant decrease in cell viability in all groups of H 2 O 2 compared to the control group. After PBM irradiation, the survival rate of the PBM+H 2 O 2 group was notably lower vs. control group (p < 0:001), where the cell activity of the 200 μmol/L H 2 O 2 group was close to 60%-70% than that of the control group. Furthermore, all PBM+H 2 O 2 groups was increased vigorously compared with H 2 O 2 group, with the 200 μmol/L H 2 O 2 group showing a significant improvement in cell activity after PBM irradiation (p < 0:05), and PBM can promote cell survival with H 2 O 2 condition. At the same time, the degree of apoptosis osteoblasts exposed to different concentrations of hydrogen peroxide was using the TUNEL assay (Figures 1(b) and 1(c)). Within the concentration range we selected, the apoptosis of osteoblasts was positively proportional to the concentration of hydrogen peroxide. Based on the above results, 200 μmol/L H 2 O 2 was selected to establish an osteoblast oxidative damage model in MC3T3 cells. MC3T3 cells treated with 200 μmol/L hydrogen peroxide were irradiated by PBM.

RUNX2
Osx   The multiplication of MC3T3 cells was detected by CCK-8 at different time points (0, 12, 24, 36, and 48 h) after irradiation. The results revealed that MC3T3 cells were significantly enhanced after 24 h of PBM (p < 0:05). The activity of MC3T3 cells treated with 200 μmol/L hydrogen peroxide was time-dependently reduced, and the activities of MC3T3 cells were restored after PBM and significantly increased after 24 h (p < 0:01), suggesting that PBM can inhibit the oxidative damage caused by hydrogen peroxide to MC3T3 cells and improve the survival rate of MC3T3 cells (Figure 1(d)). To examine whether the influence of PBM on cells correlates with its antioxidant properties, we used the fluorescent probe DCFH-DA to generate ROS. The data from Figures 1(e) and 1(f) shows that ROS expression was clearly increased after treatment of MC3T3 cells with H 2 O 2 . However, when the cells were irradiated with PBM, the expression of ROS was reduced, and the outcomes revealed that PBM attenuated the level of oxidative stress in MC3T3 cells. After irradiation with PBM, enhanced Bcl-2 expression and decreased expression were observed. Besides, the apoptosis of cells irradiated by PBM has been significantly inhibited (Figures 2(a)-2(c)). Similarly, flow cytometry results confirmed an increased apoptosis rate in MC3T3 cells treated with 200 μmol/L hydrogen peroxide. The apoptosis rate of MC3T3 cells irradiated with PBM did not show appreciable changes. However, the apoptotic rate of H 2 O 2 -stimulated MC3T3 cells was significantly reduced with PBM treatment (Figures 2(d) and 2(e)). As a positive control, the ROS scavenger N-acetylcysteine (NAC) significantly reduced oxidative stress levels after 2 h of cell pretreatment (Figures 2(f)-2(h)). These achievements suggest that PBM attenuates H 2 O 2 -induced apoptosis in MC3T3 cells.
The expression of osteogenic-related proteins RUNX2, Osterix, OPN and OCN was detected by Weatern blotting method. Also, the degree of cell differentiation and mineralization was observed using ALP and alizarin red staining to investigate the influence of PBM on H 2 O 2 -induced osteoblast dysfunction. The presence of H 2 O 2 significantly reduced the expression of osteogenic-related proteins in cells compared to controls, and PBM significantly restored the expression of these proteins inhibited by H 2 O 2 (Figures 3(a) and 3(b)). The RT-PCR outcomes are presented in Figure 3(c); the mRNA levels of RUNX2, osterix, OPN, and OCN were clearly reduced in the H 2 O 2 group as compared to the control group. The mRNA expression levels of RUNX2, osterix, OPN, and OCN were increased in the H 2 O 2 -PBM group with respect to the H 2 O 2 group. To confirm the level of osteoblast differentiation and mineralization in the presence of PBM with H 2 O 2 , ALP staining and alizarin red staining were performed. ALP is an indicator of osteoblast differentiation cells irradiated by PBM and subsequently cultured with osteogenic induction solution. At 14 days, ALP expression was reduced in MC3T3 cells from H 2 O 2 -treated cells compared to the control group. The ALP staining intensity of PBM-irradiated MC3T3 cells slightly increased. Compared to that of the H 2 O 2 group, the ALP staining intensity of the H 2 O 2 group after PBM irradiation was significantly increased (Figure 3(d)). Alizarin red staining was used to measure osteoblast differentiation twenty-one days after induction of cells with osteogenic induction solution, what the results showed is that calcium deposition decreased in the H 2 O 2 group compared with that in the control group; the level of calcium deposition was slightly increased in the PBM-irradiated MC3T3 cells. After PBM, calcification was significantly greater in the H 2 O 2 group than that in the control set (Figure 3(e)). According to these results, PBM promoted osteogenic differentiation of MC3T3 preosteoblasts.  For further studies on osteoblast differentiation influenced by PBM with oxidative stress, MC3T3 cells were pretreated with NAC (2 mmol/L) and then cultured with H 2 O 2 . In the western blotting experiment, we also observed an elevated expression of osteogenic proteins in cells exposed to the antioxidant NAC, and this effect was more pronounced after PBM (Figures 3(f) and 3(g)). Taken together, these results proved that PBM can reduce apoptosis triggered by oxidative stress and promote osteogenic differentiation in MC3T3 cells, and the combined effect of PBM with antioxidant NAC is more significant.    (Figure 4(a)). LC3B is an essential marker of macroautophagy. We detected autophagy in MC3T3 cells by western blotting, and based on the literature, it is also clear from our results that the autophagy level of normal MC3T3 cells increased after PBM [19]; however, the difference was not found to be statistically meaningful (p > 0:05).
The autophagy level of MC3T3 cells induced by H 2 O 2 is shown in Figure 4(b); in comparison with the controlling group, LC3B-II/LC3B-I ratio was enhanced and p62 protein level was reduced in the H 2 O 2 group (p < 0:05), LC3B-II/ LC3B-I ratio was further increased, and p62 expression was significantly inhibited after PBM (p < 0:05) (Figure 4(c)). The cellular ultrastructure was viewed by transmission electron microscopy (TEM), and this approach is considered to be the standard for detecting autophagy. Ultrastructure and number of autophagic vesicles in osteoblasts under oxidative stress were observed by TEM (Figure 4(d)). As anticipated, autophagosomes were enclosed by a double-membrane structure in the cells. The autophagic vesicles of MC3T3 cells were slightly increased after PBM irradiation comparing with the control group, but it was not statistically significant. The number of H 2 O 2 -induced autophagosomes in MC3T3 cells was significantly increased after PBM compared with the H 2 O 2 group. Besides, H 2 O 2 enhanced the accumulation of LC3B puncta in the nucleus; however, this phenomenon was further enhanced after PBM (p < 0:05) (Figures 4(e) and 4(f)). Such

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Oxidative Medicine and Cellular Longevity results suggested that H 2 O 2 can induce cellular autophagy and that PBM may further promote autophagy by activating the self-repair of cells under oxidative stress to scavenge excessive ROS in cells, thus promoting osteoblast differentiation. 13 Oxidative Medicine and Cellular Longevity related proteins were significantly increased; ALP and alizarin red staining results also showed more clearly that PBMirradiated MC3T3 cells induced by 3-MA and H 2 O 2 ; the calcification level of MC3T3 cells was lower than that of H 2 O 2 +PBM group ( Figure 5(i)). Collectively, these results suggest that 3-MA counteracts the influence of PBM on H 2 O 2induced cells and confirm that PBM inhibited apoptosis of MC3T3 cells and enhanced osteogenic differentiation ability by promoting autophagy.

Autophagy Inhibitors Can
3.5. PI3K/AKT/mTOR Pathway Is Engaged in the Promotion of H 2 O 2 -Induced Autophagy in MC3T3 Cells by PBM. The PI3K/AKT/mTOR pathway is essential to activation of autophagy. To investigate the effects of PI3K/AKT/mTOR pathway in H 2 O 2 -induced regulation of PBM in MC3T3 cells, western blotting was used to test the activation of this pathway (Figure 6(a)). As shown in Figure 6(b), the expression levels of p-PI3K/PI3K, p-Akt/AKT, and p-mTOR/ mTOR were slightly reduced in the H 2 O 2 group compared with the controlling group. But without statistical significance (p > 0:05), there are significantly lower expression levels of p-PI3K, p-Akt, and p-mTOR in the H 2 O 2 +PBM group than in the H 2 O 2 group (p < 0:05). In conclusion, these results suggest that PBM may enhance autophagy through inhibiting the PI3K/Akt/mTOR pathway.
To validate whether PBM promotes autophagy in MC3T3 cells through the PI3K/AKT/mTOR pathway, we examined changes in the pathway and autophagy after treatment with the PI3K agonist 740 Y-P and PBM. As illustrated in Figures 6(c) and 6(d), western blotting results showed the protein expression levels of p-PI3K, p-Akt, and p-mTOR in the different groups. The expression levels of p-PI3K/PI3K, p-Akt/AKT, and p-mTOR/mTOR proteins were significantly increased in the cells after PI3K agonist treatment compared with the H2O2 group. The expression levels of p-PI3K/PI3K, p-Akt/AKT, and p-mTOR/mTOR were significantly decreased after PBM irradiation compared with the PI3K agonist group. As shown in Figures 6(e) and 6(f), the immunofluorescence results showed that the green fluorescent scattered spots around the nucleus were dramatically reduced, and the autophagy protein LC3B was reduced, which verified that the PI3K receptor agonist 740 Y-P inhibited autophagy by promoting PI3K. After PBM, the green fluorescent spots around the nucleus increased significantly, which could significantly promote the autophagy level of MC3T3 cells under H 2 O 2 conditions. These outcomes illustrated that 740Y-P decreased LC3B expression and inhibited autophagy in MC3T3 cells under H 2 O 2 condition through activating the PI3K/AKT/mTOR pathway. PBM then enhanced cellular autophagy by inhibiting activation of the PI3K/AKT/mTOR pathway in MC3T3 cells with H 2 O 2 .

Discussion
Oxidative stress occupies the core site in the pathological process of osteoporosis, and osteoporosis caused by excessive oxidative stress may be a key factor affecting its prognosis [20,21]. Oxidative stress can cause a series of alterations in cells and activate the apoptotic signaling pathway, leading to cellular dysfunction [22]. The excessive production of ROS is part of the major pathophysiological basis of oxidative stress. Being a product of normal redox reactions in vivo, ROS can participate in the modulation of bactericidal, detoxification, and multiple metabolic pathways under normal physiological conditions [23]. However, oxidative stress caused by the excessive production of ROS, which damages multiple components of osteoblasts and inhibits osteoblast proliferation, differentiation, and bone formation, has been shown to be a vital factor affecting the development of osteoporosis [24][25][26]. In the previous study, we choose H 2 O 2 as an effective oxidant to induce oxidative stress, which could induce excess ROS produced by the organism to penetrate the cell membrane and diffuse rapidly intracellularly, thereby mimicking the pathophysiological process of oxidative stress [23,27].
Photobiomodulation therapy, also known as weak lowlaser-level therapy [28], which is noninvasive, anti-inflammatory, and prorepair, has been widely used in clinical practice. With the gradual exploration of PBM, many researchers have investigated the role of PBM in osteoblasts and found that PBM can significantly facilitate multiplication of MC3T3-E1 cells [29]. De Marco et al. suggested that PBM could improve fracture healing in rats and promote bone regeneration in bone defects [30]. Other studies have suggested that different wavelengths of PBM have significant regulatory effects on osteoblast differentiation [14]. We had previously applied PBM to the therapy of spinal cord injury and found that PBM could reduce the inflammatory response, reduce oxidative stress, inhibit neuronal apoptosis, and promote repair of spinal cord injury functions [31][32][33]. Although oxidative stress is one of the main inhibitors of osteoblast differentiation, PBM regulates osteoblast proliferation. In particular, the mechanisms of that regulate osteoblast differentiation during oxidative stress are not clear. Therefore, we investigated the regulatory mechanism of PBM in osteoblast differentiation under oxidative stress.
In this study, we constructed an osteoblast oxidative damage model using MC3T3-E1 cells cultured with the appropriate concentration of H 2 O 2 (200 μmol/L) in vitro.
To confirm the effect of hydrogen peroxide on the oxidative damage of osteoblasts, we evaluated the activity of osteoblasts using CCK-8. The apoptosis rate of osteoblasts was measured using flow cytometry, and the oxidative stress level was measured using an ROS probe. With PBM treatment, H 2 O 2 -induced MC3T3-E1 cell activity increased and the intensity of ROS florescence became weak, indicating that PBM has an antiapoptotic effect on the survival of oxidized osteoblasts. The findings supported that PBM may act as an antioxidant to regulate the oxidative stress levels of osteoblasts.
Autophagy is one of major mechanisms that promotes cell viability. It functions as a regulator in intracellular homeostasis mainly through the degradation and recycling of intracellular metabolites. In stressful microenvironments such as oxidative stress, hypoxia, and nutrient deprivation conditions, autophagy is activated, which in turn inhibits the accumulation of ROS, thus exerting an inhibitory effect on apoptosis and reducing oxidative stress [34,35]. At the same time, researchers discovered that autophagy has a major role in maintaining bone homeostasis and osteoblast differentiation [36]. Intracellular mineralized crystals can be transported by autophagosomes to promote extracellular mineralization [37]. In contrast, after the inhibition of autophagy, in vitro studies showed that the degree of cell mineralization was reduced, and bone mass and volume in vivo were also reduced, ultimately leading to oxidative stress [38]. Bostanciklioglu et al. used different laser irradiation wavelengths in a rat model of mucositis and found that autophagy was activated [39]. These results demonstrate not only that autophagy has a critical and central role in osteogenesis and differentiation but also that PBM plays a regulatory role in autophagy.
TEM of autophagic vesicles and other subcellular structures in a double-layer membrane-like structure is the gold standard for detecting autophagy [40]. However, it can only reflect the presence or absence of autophagy and not the level of autophagic activity. Among the numerous studies on autophagy, the ratio of LC3-II/LC3-I is a classical marker of autophagy, which reflects the strength of autophagic activity, whereas the degradation of p62 is an important marker of autophagy [41,42]. Accordingly, we detected the expression of autophagy marker proteins LC3-I, LC3-II, and p62 using western blotting to reflect the impact of H 2 O 2 on osteoblast autophagy and the therapeutic effect of PBM.
In the present study, LC3 expression increased after H 2 O 2 induction in MC3T3 cells, suggesting that oxidative damage to MC3T3 cells by H 2 O 2 stimulated osteoblast autophagy. This discovery is in line with the role indicated by She et al. in their study on enhanced autophagy of osteoblastoma MG63 cells under oxidative stress conditions [43]. We considered that after the oxidative stress occurs, large amounts of ROS were generated, and apoptosis was promoted. Nevertheless, apoptosis is accompanied by the activation of autophagy to remove damaged organelles in a timely manner. This phenomenon is a self-help behavior of cells, but their ability is limited. After PBM treatment, we found a further rise in LC3 expression, which indicates a further enhancement of autophagy levels in MC3T3 and a decrease in intracellular oxidative stress. We found that PBM can effectively remove damaged fragments from osteoblasts by inducing autophagy, reducing oxidative stress, and inhibiting apoptosis. Therefore, we speculate that a further increase in autophagy after PBM irradiation of osteoblasts under oxidative stress conditions could be an important way to protect cells from the antioxidant stress response.
Investigating the role of autophagy on osteoblasts under oxidative stress conditions and in protecting osteoblasts from PBM-induced oxidative damage, we used 3-MA to inhibit autophagy in osteoblasts in our research, and our results suggested that suppression of autophagy after H 2 O 2 -induced oxidative damage in osteoblasts resulted in increased levels of oxidative stress and reduced osteogenic differentiation of osteoblasts. The protection of PBM on osteoblasts is invalid after the inhibition of autophagy, which is consistent with the protective effect of autophagy on osteoblasts damaged be oxidation reported by Shi et al. and Yang et al. [44,45]. Hence, PBM may defend osteoblasts from oxidative damage by regulating the level of autophagy in damaged osteoblasts. In summary, we demonstrated that PBM reduced oxidative stress, promoted osteoblast survival and differentiation, and attenuated the effects of H 2 O 2 on osteoblast apoptosis. We also found that during this process, PBM promoted the level of autophagy in osteoblasts under oxidative stress, increased the expression of autophagy-related proteins LC3-II and LC3-I, and diminished the expression of p62, while 3-MA reversed the regulation of oxidative stress levels by PBM. This indicated that the antioxidant protective effect of PBM on osteoblasts damaged by oxidative stress is closely related to autophagy. Therefore, we propose that PBM may reduce oxidative stress levels through inducing autophagy to inhibit the influence of H 2 O 2 in osteoblast apoptosis and promote osteoblast survival and differentiation. However, the mechanism by which PBM induces autophagy and the pathways by which PBM activates to promote autophagy remain unclear.
It is a common knowledge that the PI3K/AKT/mTOR pathway is engaged in various cellular physiological and pathological processes, including cell growth, apoptosis, and autophagy [46,47]. Suppression of PI3K/AKT/mTOR pathway may activate autophagy [48,49]. Thus, we examined the impact of PBM on PI3K/AKT/mTOR osteoblasts under oxidative stress. As an intracellular phosphatidylinositol kinase, PI3K has a serine/threonine kinase activity. Phosphorylated PI3K can produce a second messenger phosphatidylinositol-3,4,5-triphospholipid (PIP3) on the cell membrane, which binds to the intracellular signaling proteins AKT and phosphoinositol-dependent enzymes (PDK1). AKT activation is caused by phosphorylation of the threonine and serine sites of AKT [50,51]. As a negative regulator of autophagy initiation, mTOR blocks ATG1-activated kinase [52]. H 2 O 2 induced oxidative stress in osteoblasts after PBM, and PI3K/AKT/mTOR protein phosphorylation levels were detected. The findings revealed that the expression of p-PI3K, p-AKT, and p-mTOR was slightly decreased under oxidative stress conditions. PI3K/ AKT phosphorylation levels were significantly reduced and LC3 expression levels were enhanced after PBM irradiation, suggesting that PBM inhibits the activation of PI3K/AKT/ mTOR pathway by downregulating PI3K/AKT/mTOR phosphorylation, thereby promoting autophagy. To investigate further the inhibitory role of PBM on this pathway, we employed 740 Y-P, PI3K agonist, and revealed that 740Y-P markedly upregulated the expression of PI3K/ AKT/mTOR phosphorylated protein in MC3T3 cells and facilitated PI3K/AKT/mTOR pathway activation. When 740 Y-P was used in combination with PBM, the promotion of autophagy by PBM was no longer significant, indicating that the inhibitory action of PBM on the PI3K/AKT/mTOR pathway was counteracted by 740 Y-P.
Our present study has some limitations. Because our existing optical fiber used for in vivo research can only be used for local irradiation and cannot meet the requirements of whole-body irradiation of animals, we are improving the optical fiber to be more suitable for PBM irradiation for 15 Oxidative Medicine and Cellular Longevity osteoporosis treatment in vivo. Therefore, we have not yet carried out verification in vivo, but this research will be conducted in the future. The regulation of autophagy is complex, and we only confirmed the impact of PBM on this pathway. However, the direct target of PBM on osteoblasts and how PBM inhibits phosphorylation need to be further studied.

Conclusion
In brief, we cultured MC3T3-E1 osteoblast precursor cell with an appropriate concentration of H 2 O 2 , constructed an osteoblast oxidative stress model, and irradiated the cells with near infrared light at 808 nm. We observed that photobiomodulation attenuated H 2 O 2 -induced oxidative stress in MC3T3-E1 cells and activated autophagic osteoblasts in response to oxidative stress. In addition, we found that PBM enhanced autophagy by inhibiting PI3K/AKT/mTOR pathway-related signaling proteins, thereby promoting osteoblast differentiation (Figure 7). These findings suggested that photobiomodulation has a beneficial impact on H 2 O 2induced osteogenic differentiation of MC3T3-E1 cells.

Data Availability
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.  Figure S1: the effect of PBM on H 2 O 2 -induced Bcl-xl and bad expression in MC3T3 cells was detected by western blot. Bad expression level was significantly upregulated, and Bclxl protein expression level was downregulated after H 2 O 2 treatment in MC3T3 cells. After irradiation with PBM, enhanced Bcl-xl expression and decreased expression were observed. Figure S2: PBM can promote the expression of autophagy protein Beclin-1 induced by H 2 O 2 in MC3T3 cells. Figure