EFFECT OF PHOTOBIOMODULATION THERAPY IN AN EXPERIMENTAL MODEL OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE: A DOSIMETRIC STUDY

The aim was to evaluate the effects of different dosimetric parameters in an experimental COPD model treated with photobiomodulation therapy (PBMT). C57BL/6 mice were divided into groups: Basal, COPD, and COPD treated with PBMT at doses of 1 J, 3 J, 5 J, and 7.5 J. Treated groups received diode laser (660 nm, 100 mW) for 10s, 30s, 50s, and 120s over 15 consecutive days. COPD was induced by orotracheal instillation of cigarette extract twice a week for 45 days. Analyses included cell counts, immune cell profiling by flow cytometry, pulmonary infiltration of inflammatory markers, necrosis, apoptosis, and ROS production. Data were analyzed by one-way ANOVA followed by the Newman-Keuls test, with statistical significance set at 5% (p < 0.05). PBMT effectively reduced inflammatory cell infiltration, with the most significant anti-inflammatory effects observed at 1 J and 3 J. [1]¿p#1 EFFECT OF PHOTOBIOMODULATION THERAPY IN AN EXPERIMENTAL MODEL OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE: A DOSIMETRIC STUDY Cintia Estefano Alves*1 Tawany Gonçalves Santos*1 Luana Beatriz Vitoretti*1 Stella Zamuner*1 Rodrigo Labat*1 José Antonio Silva Junior*1 Flavio Aimbire*2 Renata Kelly da Palma*3,4 Ana Paula Ligeiro de Oliveira*1 1. Programa em Medicina-Biofotônica, Universidade Nove de Julho, SP, Brazil; 2. Instituto de Ciências e Tecnologia,Universidade Federal de São Paulo, Unifesp, São José dos Campos, Brazil, 3. Faculty of Health Sciences at Manresa, University of Vic-Central University of Catalonia (UVic-UCC), Manresa, Spain. 4. University Center of Anápolis, Anápolis, Brazil.

The aim was to evaluate the effects of different dosimetric parameters in an experimental COPD model treated with photobiomodulation therapy (PBMT). C57BL/6 mice were divided into groups: Basal, COPD, and COPD treated with PBMT at doses of 1 J, 3 J, 5 J, and 7.5 J. Treated groups received diode laser (660 nm, 100 mW) for 10s, 30s, 50s, and 120s over 15 consecutive days. COPD was induced by orotracheal instillation of cigarette extract twice a week for 45 days. Analyses included cell counts, immune cell profiling by flow cytometry, pulmonary infiltration of inflammatory markers, necrosis, apoptosis, and ROS production. Data were analyzed by one-way ANOVA followed by the Newman-Keuls test, with statistical significance set at 5% (p < 0.05). PBMT effectively reduced inflammatory cell infiltration, with the most significant anti-inflammatory effects observed at 1 J and 3 J.

COPD, Laser, photobiomodulation, lung, inflammation, cytokines

Chronic obstructive pulmonary disease (COPD) affects 210 million people and is the fourth leading cause of death, accounting for 4.8% of global deaths. 1 Although it has gained medical attention, COPD remains poorly understood by the public and health authorities. The primary risk factor for the disease is exposure to toxic particles and gases, such as cigarette smoke. 2 However, cases also occur among individuals who have never smoked, due to exposure to biomass burning, hookahs, pipes, electronic cigarettes, and others. In 1-2% of cases, COPD is caused by a genetic factor, alpha-1 antitrypsin deficiency. 3 COPD is a chronic, progressive, and partially reversible disease characterized by chronic bronchitis (airway obstruction) and pulmonary emphysema (destruction of the lung wall due to exposure to harmful substances). 4 Oxidative stress plays a crucial role in the development of COPD, causing direct damage to the respiratory tract and amplifying other pathological mechanisms. 5,6 Its effects include apoptosis, extracellular matrix remodeling, alveolar epithelial injury, impaired mitochondrial respiration, lipid peroxidation, mucus hypersecretion, and inactivation of surfactants and antiproteases. COPD is a neutrophilic pathology, with neutrophils being the main inflammatory cells in the lungs, responsible for producing collagenase and elastase, which destroy collagen and elastin, leading to pulmonary emphysema. 7 Cells such as eosinophils and CD8 T lymphocytes may also be elevated. 8 In the lung parenchyma, there is an increase in pro-inflammatory cytokines such as IL-6, IL-8, TNF-α, and IL-1β. 9 Smoke containing toxic particles releases inflammatory mediators, including macrophages, neutrophils, and Tc1 cells, which contribute to lung destruction and increased mucus secretion, characterizing chronic bronchitis. Both pathologies can occur simultaneously. The primary goal of COPD treatment is to stabilize the disease and prevent acute exacerbations. The diagnosis of COPD involves a medical evaluation of symptoms, an analysis of personal and family history, followed by clinical tests such as spirometry, cardiopulmonary exercise testing, and imaging exams (chest X-ray and computed tomography). The main symptoms include dry cough, expectoration, shortness of breath, frequent respiratory infections, fatigue, and wheezing. COPD is also associated with systemic effects such as inflammation and skeletal muscle dysfunction. Although treatable, the disease can cause extrapulmonary effects such as weight loss and nutritional deficiencies, worsening the patient’s overall condition and compromising their autonomy. 10 COPD is associated with periodic respiratory exacerbations, which are highly heterogeneous events linked to increased systemic and airway inflammation and physiological changes. 11-13 These exacerbations are characterized by worsening lung function and increased dyspnea, cough, and sputum production. 14 They have a significant impact on patients’ quality of life, as they frequently experience shortness of breath and acute worsening of symptoms during pulmonary crises. The high frequency of hospitalizations due to these crises contributes to the high cost of the disease for healthcare systems. As COPD progresses, symptoms such as rapid weight loss, pneumonia, osteoporosis, and lung cancer become more common. COPD treatment generally includes oxygen therapy and medications such as corticosteroids, bronchodilators, and anticholinergics. However, these treatments have adverse effects. 15,16 Corticosteroids, although effective in treating asthma, are less effective in COPD as they do not reverse pulmonary inflammation, making them of limited efficacy for treating the disease. Photobiomodulation Therapy (PBMT) involves exposing cells or tissues to low levels of red and infrared light, with ”low level” referring to the use of light with lower energy densities compared to other therapies. Photobiomodulation stimulates healing, relieves pain, and reduces inflammation by modulating metabolic events through photochemical and photophysical processes, such as increased cellular metabolism and stimulation of mitochondrial activity. 17,18 The effects of PBMT include enhanced phagocytosis by macrophages, increased secretion of fibroblast growth factors, and intensified fibrin and collagen resorption. Although the exact mechanisms of how laser therapy affects cells are not fully understood, it is believed that the laser is absorbed by chromophores in cell membranes, interfering with the respiratory chain and increasing cellular ATP levels. 19 This allows stressed cells to return to homeostasis, promoting cell division and increased fatty acid production. Scientific literature on the effects of photobiomodulation therapy (PBMT) in COPD is limited, but recent studies indicate that 660 nm light can reduce both inflammation and alveolar enlargement in cigarette smoke-induced COPD. 20 A laser dosimetry study in chronic Asthma demonstrated that the laser dose has a direct influence on treatment outcomes, with the best efficacy observed at a dose of 3 J of 660 nm laser, significantly reducing key disease parameters in the asthma group compared to the control group. 21 Based on this study, a similar pilot protocol was developed, applying the same experiment in a COPD model. Similar to asthma, the 3 J radiant energy was found to be effective, substantially reducing inflammatory cytokines in lung infiltrates, blood, and the lungs of treated animals. Considering the positive effects of photobiomodulation therapy in reducing inflammatory cells in pulmonary diseases, this study aims to evaluate the impact of different radiant laser energy levels on inflammatory parameters such as cell migration, mediator release, necrosis, and apoptosis. The goal is to determine the optimal radiant energy for treating COPD patients. 2.0 Objectives 2.1 Primary objective: To evaluate the effects of photobiomodulation therapy with different levels of radiant energy in an experimental model of COPD. 2.2. Secondary objectives: ● To evaluate the levels of cytokines secreted by BAL cells; [1]¿p#1 ● To quantify the total and differential cells present in BAL and in the blood of animals; ● To analyze airway remodeling by histology; ● To evaluate pulmonary mechanics; 3.0 Materials and methods 3.1 Study design The experiment was previously approved by CEUA (animal use ethics committee) under number AN006.2013. Thirty-six male mice of the C57BL/6 lineage were used. The animals were obtained from the vivarium of Universidade Nove de Julho and raised in a controlled environment, with humidity (50% - 60%), luminosity (12h light/12h dark) to simulate day and night, and temperature between (22° - 25°). Figure 1. Flowchart with experimental groups. 3.2. Sample size Six animals were used in each experimental group, based on the literature that proved to be a sufficient number of animals for the induction of COPD. 22 3.3. Randomization For this experiment we used random randomization as a criterion. 3.4. Cegamento All experiments and analyses were carried out by the researcher himself. All experiments and analyses were carried out by the researcher himself. 3.5. Statistical methods Data were analyzed using GraphPad Prism 3.1 software (California, USA). The normal distribution of data was assessed using the Kolmogorov-Smirnov test. Data with parametric distribution were subjected to One-way ANOVA followed by the Newman-Keuls test for comparison between groups. Data with nonparametric distribution were subjected to One-way ANOVA on Ranks followed by the Dunn test for comparison between groups. Graphs were created using GraphPad Prism 3.1 software (California, USA). 3.6. Experimental animals The animals used, from the C57BL/6 lineage, males, were on average 6 months old at the beginning of the experiments, weighing between 20g and 26g. 3.7. Experimental procedures Figure 2. Illustrative diagram of the experiment. 3.7. Cigarette extract For the preparation of the cigarette extract, Marlboro brand cigarettes (tar: 13.0 mg, nicotine: 1.10 mg, carbon monoxide: 10 mg) were used. The cigarettes were burned, and the smoke was passed through a hose, with one end connected to a conical tube containing 1X PBS buffer solution, using a ratio of 1 cigarette per 4 ml of buffer solution (1 cigarette in 4 ml). At the other end of the hose, a vacuum pump with constant pressure was attached, where the cigarette was introduced. To induce the disease using cigarette extract, the animals were anesthetized intramuscularly injection of 2% xylazine (0.06 ml/100g) 10% ketamine (0.08 ml/100g), 4 µl per animal. The animals were positioned vertically in a way that allowed proper access to the administration route. 30 µl of the cigarette extract was administered via orotracheal route, three times per week for 7 weeks (49 days). 3.8. Irradiation After 35 days of disease induction, the COPD+LASER group received a punctual application of a diode laser, wavelength of 660nm, irradiated area of 0.785 cm2, in three regions: one below the trachea, and the other two in each lung lobe (right and left). The time and power varied for each group as shown in the following diagram. The MMO Optics laser/Model: TF Premier PLUS was used. The animals were immobilized and irradiated at the points illustrated in the following Table 1. Dosimetric parameters. [1]¿p#1 | Radiant energy (J) | 1 | 3 | 5 | 7,5 | | Wavelength (nm) | 660 | 660 | 660 | 660 | | Spectral width (nm) | ±20 | ±20 | ±20 | ±20 | | Operating mode | Continuous | Continuous | Continuous | Continuous | | Average radiant power ( mW) | 100 | 100 | 100 | 100 | | Radiant exposure (J/cm²) | 22 | 66 | 111 | 116 | | Beam area on target (cm²) | 0,045 | 0,045 | 0,045 | 0,045 | | Exposure time (s) | 10 | 30 | 50 | 75 | | Frequency (Hz) | Unique | Unique | Unique | Unique | | Application points | 3 points | 3 points | 3 points | 3 points | | Application technique | Contact | Contact | Contact | Contact | 3.9. Euthanasia After 45 days of experiment, the animals were euthanized with a high dosage of anesthesia, with intramuscular (i.m.) injection of xylazine 2% (0.06 ml/100 g) ketamine 10% (0.08 ml/100 g). The substance was administered minutes before exsanguination, using a 1 ml syringe and a 25 x 5 mm hypodermic needle. After blood collection, for smear slides and differential cell count in circulation, the animals were tracheostomized and cannulated, and the lungs were lavaged with 3 x 0.5 ml of phosphate buffered saline (PBS). 3.10. Assessment of lung inflammation in bronchoalveolar lavage (BAL) The recovered lavage volume was centrifuged at 1600 rpm at 4°C for 5 minutes. The supernatant was stored at -70°C for cytokine analysis by ELISA. The cell button was resuspended in 1 ml of phosphate-buffered saline (PBS) and used to determine the total number of cells in the BAL performed by counting in the Neubauer Chamber. The remaining resuspended material was used to prepare cytospin slides. A 200 µl sample from each animal was used for differential cell counting, centrifuged for 10 minutes, 450 rpm in the Cytospin-2 model equipment, Shandon Instruments Sewickley, PA. The slides were stained using the Instant Prov staining technique: 1) place the slides in the Instant Prov I staining tank, leave for 10 seconds, remove and let drain for 5 seconds. (2). Place the slides in the Instant Prov II staining tank, leave for 10 seconds, remove and let drain for 5 seconds. (3). Place the slides in the Instant Prov III staining tank, leave for 20 seconds, remove, let drain for 5 seconds and wash the slides in running water. After staining, 100 cells were counted to determine the differential count. 3.11 Flow Cytometry After lung extractions, the tissue was fragmented and incubated for 30 min at 37°C with constant agitation in 2 mg/ml collagenase IV and 1 mg/ml deoxyribonuclease I (DNAse) (Sigma). After this period, we introduced Hank’s balanced solution (HBSS) together with EDTA to slow down the digestion of the material. After crushing and filtering the lung fragments through a 40 mm sieve, the contents were centrifuged for 10 min at 1,500 rpm, and the pellets were then resuspended in PBS buffer. After the incubation period, the samples were resuspended in 200 μl of the same buffer after being cleaned with PBS containing 0.01% BSA and sodium azide. Samples were acquired on a BD Accuri flow cytometer and analyzed using CSampler software (Becton Dickinson—BD ®, East Rutherford, NJ, USA). 4. Results 4.1 Effects of PBMT on the number of cells present in BAL in a COPD model The data on the effects of PBMT on the quantification of cells present in bronchoalveolar lavage (BAL) are presented in (Fig. 3). We found a significant increase in the total number of cells (A), macrophages (B), neutrophils (C) and lymphocytes (D) in the COPD group induced by cigarette smoke extract when compared to basal group (Fig. 3A, 3B, 3C and 3D). On the other hand, regarding the total number of cells and neutrophils, we observed a decrease in all groups submitted to PBMT when compared to the COPD group (Fig. 3A and 3C). In addition, we verified a reduction in the number of macrophages in the COPD+PBMT groups (3 J and 5 J) in relation to the COPD group (Fig. 3B). In figure 3D, we observed a reduction in the COPD+PBMT groups (5 J and 7.5 J) when compared to the COPD group. [1]¿p#1 Figure 3. Effect of photobiomodulation (PBMT) on the influx of inflammatory cells into the lungs in an experimental model of COPD. (A) Total cell count, (B) macrophages, (C) neutrophils, and (D) lymphocytes, recovered from the bronchoalveolar lavage (BAL) 24 hours after the last orotracheal application. Data are presented as mean ± standard error of the mean (S.E.M.). *P < 0.05; ***P<0.001 compared to the basal group; ∆P<0.05; ϕ P<0.001 compared to the COPD group. 4.2 Effects of PBMT on the quantification of cells present in the lung in a COPD model Data on the effects of PBMT on the quantification of cells present in the lung are presented in (Fig. 4) We observed an increase in the number of neutrophils (Ly6G+), macrophages (CD11b+), dendritic cells (CD11c+), and total lymphocytes (CD3+) in the lungs of animals in the COPD group compared to the baseline group (Fig. 4A, 4B, 4C, and 4D). Regarding PBMT, we observed that all radiant energy levels used were able to reduce the number of macrophages and lymphocytes compared to the COPD group (Fig. 4B and 4D). Additionally, only the COPD+PBMT groups (3 J, 5 J, and 7.5 J) reduced the number of neutrophils (Fig. 4A) when compared to the COPD group. Furthermore, we observed a reduction in the number of dendritic cells in the COPD+PBMT groups (3 J and 5 J) compared to the COPD group (Fig. 4C). Figure 4. Effect of photobiomodulation therapy (PBMT) on the number of inflammatory cells in the lung in an experimental model of COPD . In (A) the number of neutrophils, (B) macrophages, (C) dendritic cells, (D) total lymphocytes, present in the lung. Data represent the mean ± standard error of the mean (S.E.M.). P < 0.05. *P < 0.05; ***P<0.001 compared to the basal group; ∆P<0.05; ϕ P<0.001 compared to the COPD group. 4.3 Effects of PBMT on necrosis, apoptosis and reactive oxygen species production in leukocytes in the lung [1]¿p#1 We observed that the application of cigarette extract in the COPD group led to an increase in leukocyte necrosis, apoptosis, and the production of reactive oxygen species (ROS) compared to the baseline group (Fig. 5A, 5B, 5C, and 5D). On the other hand, all COPD+PBMT groups reduced leukocyte necrosis compared to the COPD group (Fig. 5A). Furthermore, as observed in Fig. 5B and 5C, there was a reduction in apoptosis and ROS production in the COPD+PBMT (7.5 J) group compared to the COPD group. Figure 5. Effect of photobiomodulation (PBMT) on necrosis, apoptosis and production of reactive oxygen species in pulmonary leukocytes in an experimental model of COPD. Results represented in CD 45 + cell gate, being (A) necrosis values, (B) apoptosis and (C) production of reactive oxygen species. Data represent the mean ± standard error of the mean (S.E.M.). P < 0.05. *P < 0.05 compared to the basal group; ∆P<0.05; ϕ P<0.001 compared to the COPD group. 4.4 Effect of PBMT therapy on the percentage of CD3 + TGF- β + (A) and CD3 + IL-1 β + (B) lymphocytes in BAL in an experimental model of COPD We observed that the induction of chronic obstructive pulmonary disease (COPD) using cigarette extract resulted in a significant increase in the percentage of TGF-β-producing lymphocytes (Fig. 6A) and IL-1β (Fig. 6B) in the bronchoalveolar lavage (BAL) of animals in the COPD group compared to the baseline group. While the levels of TGF-β-producing lymphocytes showed no significant differences in any of the COPD+PBMT groups, the COPD+PBMT (7.5 J) group presented a significantly reduced percentage of IL-1β-producing lymphocytes compared to the COPD group (Fig. 6B). Figure 6. Effect of PBMT therapy on the percentage of CD3 + TGF- β + (A) and CD3 + IL-1 β + (B) lymphocytes in BAL in an experimental model of COPD. In (A) the values of CD3⁺TGF-β⁺ lymphocytes and in (B), CD3⁺IL-1β⁺ lymphocytes in the bronchoalveolar lavage. Values expressed as mean and standard deviation. * p<0.05 when compared to the basal group; ∆ p<0.01 when compared to the COPD group. [1]¿p#1 4.5 Effect of PBMT therapy on the quantification of CD11b + TGF- β + (A) and CD11b + IL-1 β + (B) macrophages in BAL in an experimental model COPD We observed that the induction of chronic obstructive pulmonary disease (COPD) by cigarette extract resulted in a significant increase in the percentage of TGF-β-producing macrophages and CD11b+ cells in the bronchoalveolar lavage (BAL) of animals in the COPD group compared to the baseline group (Fig. 7A and 7B). While a reduction in TGF-β-producing macrophages was observed in the COPD+PBMT (5 J and 7.5 J) groups compared to the COPD group (Fig. 7B). Figure 7. Effect of PBMT therapy on the quantification of CD11b + TGF- β + (A) and CD11b + IL-1 β + (B) macrophages in BAL in an experimental model of chronic obstructive pulmonary disease. In (A) values of CD11b⁺TGF-β⁺ macrophages in (B) CD11b⁺IL-1β⁺ macrophages in bronchoalveolar lavage. Values expressed as mean and standard deviation. *P<0.05; ** P<0.01 when compared to the basal group; ∆ P<0.05 when compared to the COPD group.

Diseases associated with smoking, such as chronic obstructive pulmonary disease (COPD), reduce patients’ quality of life and pose a significant social and economic burden. Current treatments have limited efficacy and high costs for public health, highlighting the need for alternative therapies. Inhaled glucocorticoids are frequently prescribed for COPD without concrete evidence of their efficacy. However, many patients show little or no response to these medications. Thus, there is an urgent need to develop new anti-inflammatory therapies with immunomodulatory activity, providing alternative strategies for COPD treatment. Photobiomodulation (PBMT) has been considered a promising therapy. Previous studies have confirmed that PBMT acts on COPD by inhibiting the production of the purinergic receptor P2X7, which plays an important role in the inflammatory response and cell death regulation, a prominent phenomenon in the disease. However, research indicates that insufficient radiant energy may not produce the desired effects, while excessive doses may lead to adverse outcomes. This has motivated a study focused on the effect of different laser radiant energies on COPD, aiming to select the energy that provides the best anti-inflammatory and lung repair effect. After COPD induction, different doses of radiant energy (1, 3, 5, and 7.5 Joules) were applied. The results showed that PBMT significantly reduced the number of inflammatory cells involved in COPD exacerbation, such as macrophages and neutrophils, which release reactive oxygen species (ROS) responsible for membrane rupture and receptor, transcription factor, and enzyme inactivation In a previous study, we found that low-level laser therapy (LLLT) in the red range (660 nm) in an experimental COPD model promotes the attenuation of major disease outcomes. This effect includes the reduction of P2X7 purinergic receptor expression, suggesting that PBMT also plays a modulatory role in purinergic signaling, a molecular mechanism implicated in COPD pathogenesis. Another previous study explored red laser dosimetry in asthma, demonstrating that this approach reduced inflammatory cytokine levels and airway remodeling, including mucus overproduction and collagen accumulation, both relevant disease markers. COPD is associated with systemic inflammation, which can affect various organs and systems, contributing to comorbidities such as osteoporosis, anemia, diabetes, metabolic syndrome, and depression. Inflammatory mediators are considered the main culprits for these changes Cytometric analysis revealed a significant increase in neutrophils, macrophages, dendritic cells, and total lymphocytes in the lungs of the COPD group, highlighting an exacerbated inflammatory response due to cigarette extract exposure. The exacerbated inflammation triggered by smoke exposure is one of the main pathological factors in COPD, and cell infiltration is a reflection of this. On the other hand, the literature shows that the application of adequate PBMT doses can reduce inflammatory cell infiltration in the airways in experimental COPD models. This may be due to PBMT’s ability to modulate inflammatory cell function and reduce the production of inflammatory mediators. We observed that PBMT reduced these cellular populations in a dose-dependent manner, with 3 J and 5 J doses showing the best results. This effect is consistent with another dosimetry study, which showed a significant reduction in these cells in a house dust mite (HDM)-induced asthma experimental model treated with PBMT. In COPD, there is a predominance of pro-inflammatory Th17 cells and reduced regulatory T cell function, contributing to chronic inflammation and lung tissue destruction. New studies suggest that PBMT significantly reduces cell migration to the lung, as well as cytokine and chemokine levels. This effect may be related to the increase in IL-10-producing regulatory T cells, which possibly suppress effector T cells, promoting immune homeostasis PBMT considerably reduced leukocyte necrosis compared to the COPD group, indicating a protective effect against cell damage caused by inflammation. This was confirmed by another study investigating PBMT’s effects on extrapulmonary changes caused by experimental emphysema in rats. The results indicated a decrease in the number of inflammatory cells in the bronchoalveolar lavage (BAL) and a significant reduction in CKMB levels (a myocardial injury marker) in the PBMT-treated group compared to the untreated emphysema group. These findings suggest that PBMT has a protective effect against cell damage caused by inflammation, not only in the lungs but also in extrapulmonary organs However, no significant differences in cell apoptosis were observed between groups. On the other hand, the relationship between apoptosis and inflammation in COPD is complex, and the absence of significant differences may be influenced by experimental variables. Another study using osteosarcoma cells subjected to PBMT showed that the laser does not induce cell apoptosis but increases the expression of the pro-apoptotic gene (BAX). Although COPD induction did not alter ROS levels, PBMT with 7.5 J led to an increase in ROS production. This increase may be related to an immunomodulatory response, as the same dose reduced IL-1β expression in lymphocytes, suggesting a possible anti-inflammatory effect. However, excessive ROS production is associated with oxidative stress. Another PBMT study in COPD demonstrated that 1 J/cm² and 5 J/cm² doses in different circumstances decreased pulmonary ROS and ATP production in BAL, suggesting that PBMT modulates lung inflammation by regulating ROS. The impact of this increase on COPD progression still requires further investigation. Another finding indicating immune response modulation was the intracellular cytokine analysis of BAL cells, which showed alterations in lymphocyte and macrophage subpopulations. This technique allows for the identification of changes in these subpopulations, providing insights into the immune mechanisms involved. It corroborates a previous study that investigated the anti-inflammatory effect of low-power laser therapy (660 nm) on pleurisy in rats. The findings showed that laser treatment significantly reduced pro-inflammatory cytokine concentration and increased the anti-inflammatory cytokine IL-10 in pleural fluid, indicating PBMT’s anti-inflammatory action COPD induction led to a significant increase in the percentage of CD3+ lymphocytes producing TGF-β and IL-1β in the BAL of COPD animals. This increase reflects the exacerbated inflammatory response characteristic of COPD pathogenesis, where TGF-β contributes to tissue remodeling and pulmonary fibrosis, while IL-1β is strongly associated with acute and chronic inflammatory responses. No significant changes were observed in the percentage of TGF-β-producing lymphocytes in any of the treated groups. This may indicate that the laser does not directly influence the production of this factor or may be related to specific disease indicators, as results from another study on laser therapy in an experimental model of bleomycin-induced idiopathic pulmonary fibrosis treated with PBMT showed a significant reduction in total pulmonary TGF-β. However, the COPD+PBMT group treated with 7.5 J showed a significant reduction in IL-1β production, indicating a modulatory effect on the lymphocytic inflammatory response. Another study in an experimental COPD model also demonstrated PBMT’s efficacy in reducing IL-1β production. Additionally, COPD induction increased the percentage of CD11b+ macrophages producing TGF-β and IL-1β in the BAL, reflecting these cells’ role in chronic inflammation and disease progression. The data suggest that PBMT can modulate COPD’s inflammatory response, reducing IL-1β- and TGF-β-producing macrophages in a dose-dependent manner. Although there are no specific studies on PBMT’s influence on CD11b+ macrophages producing TGF-β and IL-1β, available evidence suggests that PBMT may modulate macrophage inflammatory responses, promoting a shift to an anti-inflammatory phenotype and reducing pro-inflammatory cytokine production. The findings indicate that PBMT exerts significant anti-inflammatory effects, reducing inflammatory cell recruitment and protecting against cell damage in the experimental COPD model. The mechanism appears to be related to cellular response modulation, particularly in macrophages and lymphocytes, without inducing apoptosis but potentially influencing ROS levels. The effectiveness of PBMT varied depending on the radiant energy used, with 3 J and 5 J being the most effective in reducing inflammatory parameters in COPD. This suggests that selecting laser parameters is critical to maximizing therapeutic benefits. Thus, after determining the optimal radiant energy with anti-inflammatory and lung tissue repair effects, this therapy may be an important complementary tool in treating pulmonary diseases, including COPD.

The present study demonstrates that photobiomodulation therapy (PBMT) holds significant potential as a therapeutic strategy for chronic obstructive pulmonary disease (COPD) by modulating the inflammatory response and protecting against cellular damage. PBMT effectively reduced the infiltration of inflammatory cells, particularly macrophages and neutrophils, which play a key role in COPD exacerbation. Among the energy levels tested, 3 J and 5 J were the most effective for COPD treatment. Additionally, PBMT exhibited a protective effect by reducing inflammation-induced cell necrosis without significantly altering apoptosis levels. Although ROS induction was not modified in COPD, PBMT at 7.5 J moderately increased ROS production, suggesting a potential immunomodulatory role, as controlled ROS levels can influence cell signaling. Based on these findings, PBMT emerges as a promising approach for COPD treatment, primarily due to its ability to attenuate inflammation and regulate immune responses. Further studies are warranted to optimize PBMT parameters and elucidate its long-term effects on disease progression. [1]¿p#1 Compliance with Ethical Standards Conflict of interest statement: There is no conflict of interests Role of funding source: To finance scientific research in the state of São Paulo Ethical Approval: The clinical study protocol was submitted and approved by the Ethics and Research Committee of University Nove de Julho - nº AN006.2013 Referências 1. Boers E, Barrett M, Su JG, Benjafield AV, Sinha S, Kaye L, Zar HJ, Vuong V, Tellez D, Gondalia R, Rice MB, Nunez CM, Wedzicha JA, Malhotra A. Global Burden of Chronic Obstructive Pulmonary Disease Through 2050. JAMA Netw Open. 2023 Dec 1;6(12):e2346598. doi: 10.1001/jamanetworkopen.2023.46598. 2. Devereux G. ABC of chronic obstructive pulmonary disease. 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Authors Metrics & Citations Metrics Article Usage 445views 249downloads Citations Download citation Cintia Estefano Alves, Tawany Gonçalves Santos, Luana Beatriz Vitoretti*1, et al. EFFECT OF PHOTOBIOMODULATION THERAPY IN AN EXPERIMENTAL MODEL OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE: A DOSIMETRIC STUDY. Authorea. 24 April 2025. DOI: https://doi.org/10.22541/au.174548193.38539134/v1 DOI: https://doi.org/10.22541/au.174548193.38539134/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.