Several studies have shown that LLL therapy at various wavelengths may be beneficial for viral infection. For example, green light lasers enhance tissue oxygenation, while blue light lasers are utilized in photobiomodulation therapy (PBMT) [29]. Lugongolo et al. in 2020 showed that PBMT enhanced apoptosis in human immunodeficiency virus (HIV-1)-infected cells but had no inhibitory effects on HIV-1 uninfected cells [30]. The goal of this study was to evaluate the in vitro growth behavior and hepatitis B subviral particle secretion in a biomodulated human hepatoma HepG2.2.15 cell line using (650 nm) low-level laser in doses effects. Our findings showed changes in cell viability and ultrastructure in both hepatoma cell line HepG2.215 and HepG2 cells, in addition to a reduction in HBVsvp production in response to LLLT irradiation, in a dosage- and time-dependent manner.

The efficacy of LLLT is regulated by numerous factors, such as wavelength, power output, energy density, and duration of radiation. However, considering the diversity of laser parameters, an accurate effective dose has not yet been established [31]. In our study, we utilized dose fluencies falling between 2 and 10 J/cm2 that has an inhibitory effect on cell lines and in the same line with the analysis of the current literature found that doses between 0.001 to 10 J/cm2 provide the ideal therapeutic window for photobiomodulation [32].

The human hepatoma cell line HepG2.2.15, containing an integrated HBV genome, was used as an expression system to produce HBVsvp [3]. In the present study, selection of the human hepatoma HepG2.2.15 cells transfected with HBV surface region as it promotes a stable HBV replication, production of HBVsvps as well as a very reach organ by cytochrome C oxidase enzyme containing mitochondria which is the specific chromophore of red laser light absorption [24, 33]. In vitro cell culture models, such as human hepatoma HepG2 cells, also constitute potent cell models for HBV and have been used in an increasing number of studies [24, 29]. The concentration of HBVsvps was measured in the cell culture supernatant of each laser-treated human hepatoma HepG2.2.15 cells groups at 2 J/cm2, 4 J/cm2, 8 J/cm2, 10 J/cm2 and the non-irradiated group (control), and the results of HBVsvp levels were compared in a dose- and time-dependent manner after laser irradiation in triplicate.

Before laser irradiation, morphological analysis of both human hepatoma HepG2 and human hepatoma HepG2.2.15 cell lines demonstrated no morphological changes with growth as flattened polygonal cells arranged in a monolayer, with certain cells having two nucleoli. Furthermore, human hepatoma HepG2.2.15 cells were grown in multiple adherent layers, and at late stages of growth, a circular form was observed (after 96 h) (Figs. 2 and 3). These findings are in line with those of Wang et al., who described the morphological characteristics of hepatocellular carcinoma cells (HepG2 and HepG2.2.15) [24].

Following laser irradiation (2–10 J/cm2), we examined the morphology of human hepatoma HepG2 cells and HepG2.2.15. Our findings showed that there were no morphological changes induced in all irradiated groups compared to the non-irradiated control group over incubation time intervals ranging from 24 to 96 h post-laser irradiation. This result may be due to the use of a one-shot low-output laser power (35 mW) that does not cause heating; even higher doses of 8 J/cm2 and 10 J/cm2 were obtained by increasing the exposure time, not by increasing the laser output power, which is in agreement with previous studies that applied variable laser power, 30 mW [34] and 100 mW LED, both of which showed a reduction in oral mucositis. In our study, a relative decrease in human hepatoma HepG2 cell condensation was observed in both irradiated groups, D (8 J/cm2) and E (10 J/cm2). In human hepatoma HepG2.2.15, all irradiation groups showed a relative decrease in cell condensation, with a relative increase in circular cells in the irradiated C (4 J/cm2), D (8 J/cm2), and E (10 J/cm2) groups (Fig. 3). This is in agreement with [30].

After laser irradiation (2–10 J/cm2), the cell proliferation rate evaluated by MTT assay in a dose- and time-dependent manner between the irradiated and control groups showed that after 24 h of incubation (short-term effect), human hepatoma HepG2.2.15 irradiated at 2 J/cm2, 8 J/cm2 and 10 J/cm2 showed a significant decrease in viability compared with the control group (P < 0.001) with a maximum inhibitory dose of 2 J/cm2 (Figs. 6 and 7). This effect, which, according to some previous studies, can be interpreted as a red laser promoting short-term activation of the respiratory chain, resulting in alterations in the redox state of both the mitochondria and cytoplasm [35]. Our results are inconsistent with those of Guimaraes et al., who found that applying 2 J/cm2 from a red diode laser with a wavelength of 660 nm and power of 100 mW reduced oral mucositis [36]. Our findings contradict those of other studies, which found no significant differences in cell proliferation of TZM-bl cells after 24-h incubation following red diode laser irradiation (660 nm), (100 mW) CW mode, with fluences of 2–10 J/cm2 [26], and in cell proliferation of human gingival fibroblasts after irradiating cells with increasing fluences of 0.5 J/cm2, 1.5 J/cm2, 3.5 J/cm2, and 7 J/cm2 [37]. This variation can be attributed to the nature of the cell line employed.

The lack of a direct correlation between dosage duplication and the effect produced is evident from our study’s cell proliferation examination where after 24-h incubation post-laser irradiation (short term effects) (Fig. 7), the cell proliferation of human hepatoma HepG2.2.15 cells irradiated with 2 J/cm2 dose was decreased by 15%, while 8 J/cm2 caused a decrease of 11%, finally, 10 J/cm2 induced decrease in cell viability by 9%. These fluctuations can be explained by the dose-dependence of the effect. This finding was reported in a previous study that used head and neck squamous cell carcinoma and applied single irradiation delivering 1 J/cm2 or 2 J/cm2 and observed that 1 J/cm2 promoted an increase in cell proliferation, while no effect was observed with 2 J/cm2 [38].

Our HBVsvps concentration results showed that the short-term (after 24 h incubation) effect of LLLT at all irradiation doses 2 J/cm2, 4 J/cm2, 8 J/cm2, and 10 J/cm2) on HBVsvps production was significantly decreased when compared to a non-irradiated control group, with a maximum inhibitory effect at 10 J/cm2 dose (P < 0.001) (Fig. 8). This agrees with a previous study in which the viral load was reduced by LLLT [39].

Regarding the moderate effect (after 48-h incubation) post-laser irradiation, the cell viability analysis of HepG2 cells showed a significant decrease with irradiated doses of 2 J/cm2, 4 J/cm2, 8 J/cm2, and 10 J/cm2 when compared to the control group (P < 0.001) (Fig. 4), and the maximum effect was recorded for both doses 4 J/cm2 and 8 J/cm2, which agrees with data from a previous study reporting reduced incidence of oral mucositis to 53% when compared to the untreated group (70–90%) by using 4 J/cm2 red laser therapy [40] and with the—in vitro—study of [41] they used LLLT doses (5 J/cm2 and 10 J/cm2) at 685 nm, 50 mW, on cancer (HeLa) cells and the cell viability by MTT was decreased 48 h after laser irradiation.

In contrast, after 48 h, when human hepatoma HepG2.2.15 cells were irradiated with doses ranging from 2 J/cm2 to 10 J/cm2, no significant variations in cellular viability or HBVsvps production were observed when compared to the control group (Figs. 6 and 8). This could be explained by the fact that different cells respond differently to light therapy depending on the nature of the cell line used. Our results are in agreement with those in [42]. They recorded that a 4.8 J/cm2 red laser (635 nm) did not stimulate HepG2 cell proliferation.

In this study, human hepatoma HepG2 and HepG2.2.15 cells were viable after 96 h of laser irradiation (late cellular response) and showed an insignificant decrease in HepG2 cells at all irradiation doses (2, 4, 8, and 10 J/cm2) (Fig. 5). The viability of human hepatoma HepG2.2.15 cells with irradiated doses (2 J/cm2, 4 J/cm2, 8 J/cm2, and 10 J/cm2) significantly decreased when compared to the control group (P < 0.001) (Figs. 6 and 7), and the maximum effect was recorded for the 4 J/cm2 dose, which is compatible with our morphological observation by inverted microscopy regarding the decrease in human hepatoma HepG2.2.15 cell condensation after 96 h (Fig. 3). While the inhibitory effect on HBVsvps concentration production compared to non-irradiated cells (control), all irradiated dose of 2 J/cm2, 4 J/cm2, 8 J/cm2, and 10 J/cm2 showed a decrease in HBVsvps production, with 10 J/cm2 being the maximum inhibitory dose (Fig. 8). Our late response findings agree with a study by Barasch and his team on HeNe 632.8 nm, applied energy density (4 J/cm2), single application, on leukemic human cells (HL60), cell viability was decreased at day 3, 4, and 6, analysis every 24 h for 6 days [43], but in contrast with Coombe et al. who found no significant early (24 h post-laser irradiation) or late (after10 days post-laser irradiation) effects of laser irradiation on protein expression and alkaline phosphatase activity [44], which could be attributed to utilizing different cell seeding density (20,000 cells per well) and LLLI irradiated fluencies of 0.27 J/cm2, 1 cm2, and 4 J/cm2 daily, while in our study, the seeding density of human hepatoma HepG2 and HepG2.2.15 was 10,000 cells irradiated only once.

The current study demonstrated that even when administered the same dose of LLLT, each cell line responded differently to it, where the effect of irradiation with the highest (10 J/cm2) dose on HepG2 cells after 96-h (long effect) recorded a maximum non-significant reduction of 45% compared to the control, whereas in human hepatoma HepG2.2.15 cells at 10 J/cm2, a minimum significant inhibitory effect of 18% was observed compared to the control. This is incompatible with the results of a previous study that used a diode laser at 636 nm and 5, 10, and 20 J/cm2 (exposure times = 534, 1068, and 2136 s). single application on lung cancer cells (A549), 20,000 cells were analyzed after 24 h, 48 h, and 72 h. Increases in all doses at 72 h [45] may be due to the number of cells 20,000 cells, while in our study 10,000, in addition, the exposure time for 10 J/cm2 was too long (1068 s). In our study, the laser power was 35 mW, and the exposure time was 36 s.

The results of HBVsvps production were partially compatible with the results of human hepatoma HepG2.2.15 cell viability by MTT, whereas the early effect of irradiation laser dose showed a significant decrease in both (human hepatoma HepG2.2.15 cell viability and HBVsvps production) except that group C (4 J/cm2) showed a non-significant decrease in viability (Figs. 7 and 8). In addition, the late effect (after 96 h), all irradiated human hepatoma HepG2.2.15 cell groups (B, C, D, and E) showed a significant decrease in cell viability, while a significant decrease in HBVsvps expression was recorded only in the supernatant of both irradiated groups B (2 J/cm2) and E (8 J/cm2) (Figs. 7 and 8). As a result, we believe that the mechanism of action that affects cell viability after laser irradiation is distinct from that affecting viral expression, and further research is needed. This agrees with [6], who recorded a bifacial effect of LLLT in chronic viral patients.

Our results showed that the maximum decrease in HBVsvps was recorded at 96 h in group E (10 J/cm2). Therefore, we studied ultrastructural changes in human hepatoma HepG2.2.15 cells and Hepg2 cells irradiated in group E (10 J/cm2).

Ultrastructural changes were compared between the irradiated HepG2 cells group E (10 J/cm2) and a non-irradiated control group A at 96 h. showed an increase in autophagy vacuoles, a decrease in the regularity of the nuclear membranes (Figs. 9 and 10), and a significant decrease (P < 0.05) in the nuclear surface area in group E (10 J/cm2) by approximately 53% compared to the control group (Fig. 11). This finding is consistent with that of Lynnyk et al. (2018), who investigated the mechanisms involved in the death process of the human hepatic cell line Huh7 under laser irradiation. The Lynnyk group decoupled distinct cell death pathways targeted by laser irradiation at different powers. Their data demonstrated that high-dose laser irradiation resulted in the highest levels of total reactive oxygen species production, leading to cyclophilin D-related necrosis via mitochondrial permeability transition. In contrast, low-dose laser irradiation results in the nuclear accumulation of superoxide and apoptosis execution [46, 47].

Electromicrographs of human hepatoma HepG2.2.15 cells from the irradiated group E (10 J/cm2) in comparison to the non-irradiated group A showed euchromatic nuclei with irregular nuclear membranes, but less than those in the control group A. Autophagy vacuoles were large, and some vacuoles were up to 1 μm in diameter (Figs. 12 and 13). Their nuclear surface area was not significantly decreased compared to that in the control group (P ˃ 0.05) (Fig. 14). Our findings agree with those of previous studies that evaluated the ultrastructural features of HepG2 cells, and the hepatocyte cell model induced viral replication and propagation of HBV for a long time 20 days and were compared with non-infected cells [48, 49].

An explanation for our morphometric findings in human hepatoma HepG2 and HepG2.2.15 from ultrastructure electrophotography evaluation leads us to believe that the decrease in cell condensation and viability is related to decreased proliferation ability, as evidenced by the decrease in the nuclear surface area induced by LLLI compared with the non-irradiated control group. This reduction in nuclear surface area might denote the arrested proliferating state of these cells because proliferating cells commonly show prominence in their nuclei, for example, malignant cells of human liver cell carcinoma [50]. Our findings agree with those of a previous study, suggesting that LLLI inhibits the proliferation of human hepatoma HepG2.2.15 cells by regulating cell cycle gene expression and inducing G1 phase arrest [24]. Moreover, Lynnyk et al. (2018) has been shown that red laser light may initiate apoptosis via the induction of reactive oxygen species-mediated mitochondrial permeability transition [27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51] they have also been reported that red light-induced cell damage is mainly caused by the production of reactive oxygen species (ROS) in the mitochondria.