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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

It remains unknown if the oxidative stress can be regulated by low-level laser therapy (LLLT) in lung inflammation induced by intestinal reperfusion (i-I/R). A study was developed in which rats were irradiated (660 nm, 30 mW, 5.4 J) on the skin over the bronchus and euthanized 2 h after the initial of intestinal reperfusion. Lung edema and bronchoalveolar lavage fluid neutrophils were measured by the Evans blue extravasation and myeloperoxidase (MPO) activity respectively. Lung histology was used for analyzing the injury score. Reactive oxygen species (ROS) was measured by fluorescence. Both expression intercellular adhesion molecule 1 (ICAM-1) and peroxisome proliferator-activated receptor-y (PPARy) were measured by RT-PCR. The lung immunohistochemical localization of ICAM-1 was visualized as a brown stain. Both lung HSP70 and glutathione protein were evaluated by ELISA. LLLT reduced neatly the edema, neutrophils influx, MPO activity and ICAM-1 mRNA expression. LLLT also reduced the ROS formation and oppositely increased GSH concentration in lung from i-I/R groups. Both HSP70 and PPARy expression also were elevated after laser irradiation. Results indicate that laser effect in attenuating the acute lung inflammation is driven to restore the balance between the pro- and antioxidants mediators rising of PPARy expression and consequently the HSP70 production.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

The pathophysiologic mechanisms of acute lung injury (ALI) after intestinal ischemia-reperfusion (i-I/R) are complex. Intestinal I/R increases vascular permeability following shock, severe trauma or cardiac and aortic surgery. Several investigators have deliberated upon mechanisms of this injury, postulating that the postischemic gut serves as a priming bed for circulating leukocytes [1].

Previous studies have established the ability of neutrophils to mediate remote organ injury after intestinal I/R by rendering animals neutrophic or by blocking neutrophil adhesion to the vascular endothelium [2]. ICAM-1 (CD54) is a gene encoding for an intercellular adhesion molecule 1 (ICAM-1) found at low concentrations in leukocyte and endothelial cell membranes and this has been implicated in the inflammatory process. The interaction between leukocytes and endothelium, which is regulated by adhesion molecules, is the most critical step for leukocyte-mediated organ injury [3].

Oxidant stress due to free radicals and/or reactive oxygen species (ROS) is known to cause organ injury. A growing body of evidence indicates that oxidative stress plays an important role in the pathogenesis of many clinical conditions [4] involving cardiovascular diseases [5], liver diseases [6], diabetes [7] and lung diseases [8, 9]. The important assumption that oxygen free radicals play a crucial role in the pathogenesis of I/R has been supported by many evidences that the intestinal reperfusion-induced oxidant stress in the lung is supported by glutathione (GSH) consumption and concomitant formation of oxidized GSH in the gastrointestinal mucosa subjected to I/R.

Peroxisome proliferator-activated receptors (PPAR) are members of the superfamily of nuclear receptors containing transcription factors that regulate gene expression [10]. In lung tissues, PPARy is most abundant in airway epithelial cells [11]. In addition, PPARy is also expressed in smooth muscle cells, myofibroblasts, endothelial cells of the pulmonary vasculature and inflammatory cells, such as alveolar macrophages, neutrophils, eosinophils, lymphocytes and mast cells [12, 13]. PPARy expression is increased in bronchial submucosa, airway epithelium and smooth muscle cells of septic as compared with healthy subjects [14]. It has been hypothesized that the upregulation of PPARy in acute lung inflammation represents a self-regulatory mechanism for preventing airway inflammation and remodeling. Recent studies have shown that activation of PPARy reduces expression of various cytokines and activation of neutrophils, which are increased in lung inflammation, suggesting a therapeutic potential of PPARy agonists [15, 16]. Some authors have attributed this protective effect to be related to activation of the heat shock proteins (HSPs) response [17, 18].

HSP70, commonly referred to as stress protein, is one of the main stress proteins induced by heat shock in mammals, which help to protect cells from stress. The expression of HSP70 after sublethal insults can induce stress tolerance and protect against subsequent potentially lethal injury [19]. The heat shock response is one of the more commonly described examples of stress adaptation and is characterized by the rapid expression of a unique group of proteins known as HSP. The expression of HSP is well described in both whole lungs and in specific lung cells from a variety of species and in response to a variety of stressors [20]. More important, data from various animal models of ALI and in vitro experiments demonstrate that HSP, especially HSP70, have an important cytoprotective role during lung inflammation and injury [21]. Based on the studies above mentioned it remains clear that the strategies, which favor the reduction in oxidative stress and increase the HSP70 synthesis production are effective to prevent i-I/R-induced lung injury.

Therapeutic advantages of low-level laser therapy (LLLT) for inflammatory pathologies have been suggested by several authors [22-25]. Some reports have referred that laser therapy can interfere positively to relieve the clinical signals and the late and early symptoms of lung inflammation [26-28]. Studies were performed to understand which cellular signaling is responsible by LLLT anti-inflammatory action in the acute lung inflammation [29-31]. It is reasonable to suggest that LLLT would bring beneficial results on nonallergic lung inflammation such as that induced by i-I/R. It is important to empathize that all studies above mentioned were done in a noninvasive way.

Considering the LLLT effects on the unbalance between the actions of tumor necrosis factor (TNF) and IL-10 in acute lung inflammation [32], the present work aims to investigate if LLLT could modulate the acute lung inflammation by upregulating the anti-inflammatory protein HSP70 in a model of i-I/R in rat.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Animals

Thirty-five Wistar male rats were randomly allocated into five groups as shown in Table 1. The experiments were carried out on male Wistar rats weighting 200–220 g each, maintained under standard conditions of temperature (22–25°C), relative humidity (40–60%) and a 12 h light/dark cycle, with access to food and water ad libitum. The extruded type food for animal feeding had the following composition: protein (22%), ether extract (4%), fibrous matter (8%), mineral matter (10%), calcium (1.40%) and phosphorus (0.80%). The animal housing and use were in accordance with the guidelines of the Committees on Care and Use of Laboratory Animal Resources of the University of Vale do Paraíba and the University of São Paulo, Institute of Biomedical Sciences, which are similar to the guidelines of the Canadian Council of Animal Care. All rats were placed in a common box and divided randomly into groups of seven animals (n = 7) each.

Table 1. Experimental groups
GroupDescription
NaïveAnimals nonmanipulated
ShamAnimals submitted to the same surgical procedures, but not submitted neither to the ischemia nor reperfusion
LaserAnimals nonmanipulated and irradiated with laser
i-I/RAnimals subjected to i-I/R
i-I/R + laserAnimals subjected to i-I/R and treated with laser
i-I/R rat model

Under ketamine-xylazine anesthesia (100 and 20 mg kg−1 respectively), rats were subjected to laparotomy and occlusion of the superior mesentery artery for 60 min using a microsurgical vascular clip (Vascu-statt, Scanlan International, Saint Paul, MN). The intestinal reperfusion was reestablished by the clip release. During the ischemic period (60 min), the abdominal cavity was maintained wrapped with a plastic film to avoid excessive heat loss. Then, the abdominal cavity was sutured after the clip removal. The animals were killed 2 h later by exsanguination via the abdominal aorta under an excess of anesthesia.

Laser diode and strategy of treatment

A 660 nm laser diode (CW diode laser; MM Optics, São Carlos, SP) with an output power of 30 mW and 0.08 cm2 of spot size was employed. The diode laser was adjusted to an energy density of 6.9 J cm−2, corresponding to a power density of 38.4 mW cm−2. The optical power was calibrated utilizing a Newport Multifunction Optical Meter (model 1835C; Newport Corp., Irvine, CA). The laser stability during irradiation was monitored by collecting light from a partially reflecting surface (4%). Laser was applied with a unique dose of 5.4 J for 180 s, over a unique point on the skin in direction of the trachea distal. Two series of laser irradiation protocols were undergone: firstly, the animals received laser irradiation 5 min after the initial or 5 min before the end of the intestinal reperfusion; secondly: animals were irradiated at 30 min after the beginning of the reperfusion.

Experimental groups

Rats were divided into five groups (n = 7 animals for each group): Group naïve: consisted of nonmanipulated rats. Group sham: rats submitted to surgical procedures including mesenteric artery dissection but not submitted neither to arterial occlusion (ischemia) nor reperfusion. Group i-I/R: rats with occlusion of the superior mesenteric artery and also reperfusion. Group laser: nonmanipulated rats, but irradiated with laser. Group i-I/R + laser: consisted of rats submitted to i-I/R and irradiated with laser. The experimental groups are summarized in Table 1.

Lung MPO activity

Myeloperoxidase (MPO) was measured as an index of the presence of neutrophils. Lung tissue samples were obtained from rats killed 2 h after intestinal reperfusion. The lungs were perfused via the pulmonary artery with pH 7.0 phosphate-buffered saline (PBS) containing 5 IU mL−1 heparin. Briefly, to normalize the pulmonary MPO activity among all animals of the group, whole lung was homogenized with 3 mL g−1 PBS containing 0.5% of hexadecyltrimethylammonium bromide and 5 mm EDTA, pH 6.0. The homogenized samples were sonicated (Vibra Cell; Sonics Materials, Newtown, CT) for 1 min and then centrifuged at 37 000 g for 15 min. Samples of lung homogenates (20 μL) were incubated for 15 min with H2O2 and ortodianisidine; the reaction was stopped by the addition of 1% NaNO3. Absorbance was determined at 460 nm using a microplate reader (Synergy H4; Bio-Tek Instruments, Winooski, VT).

Lung microvascular leakage

Lung vascular permeability was assessed by Evans blue dye extravasation. In brief, Evans blue dye (25 mg kg−1) was given intravenously to rats 5 min before animals were killed. Two hours after intestinal reperfusion the rats were killed, the lungs perfused as described above and then two samples of lung parenchyma were removed. Both samples were weighted, one placed in formamide (4 mg mL−1 wet weight) at 20°C for 24 h, whereas the other was put to dry in oven (60°C) till constant weight. The concentration of Evans blue dye in formamide was determined by spectrophotometry at a wavelength of 620 nm (Synergy 2; Bio-Tek Instruments) using the standard dilution of Evans blue in formamide (0.3–100 μg mL−1). The dry/wet ratio of each lung sample was determined (index of edema) and used for the final calculation of Evans blue extravasation, which was expressed as μg Evans blue/g of dry weight.

Lung histology

Lungs were removed 2 h after intestinal reperfusion and fixed in 4% buffered formaldehyde overnight. Then, the fragments were washed with PBS, dehydrated through a graded series of ethanol, diaphonized with Xylol and embedded with Paraplast. Thereafter, the samples were cut into sections of 3 μm thick and stained with hematoxylin and eosin. A single pathologist, who was blinded to all groups, examined the pathological specimens. At least two different sections of each specimen were examined to determine the degree of injury. Lung polymorphonuclear neutrophil (PMN) sequestration was quantified by counting alveolar septal wall PMNs. Only peripheral lung parenchyma was examined. Microscopic fields containing other structures such as airways, large vessels and pleura were excluded. The inflammatory cells infiltration was examined with a Leica microscope (Leica Microsystems Inc., Buffalo Grove, IL) (magnification: 100×).

Bronchoalveolar lavage fluid (BALF): Elapsed 2 h after intestinal reperfusion, animals were anesthetized and the trachea cannulated. BALF was collected from the airway lumen by flushing the airways through the tracheal cannula with 10 mg kg−1 of Roswell Park Memorial Institute medium (RPMI) 1640. This procedure was repeated and then made a pool of samples for each animal, which were refrigerated for later use. For ROS measurement, BALF was centrifuged at 400 g during 10 min at 20°C, the supernatant was discarded and the pellet was resuspended (1 mL) in phosphate buffer solution (PBS). This last solution was preferred over the saline because of its pH buffering capacity.

ROS and PPAR- ץmeasurements: Cells from BALF were adjusted to 1 × 106 cells mL−1 in PBS. To prove that the LLLT modulates the ROS generation, the 2′,7′-dichlorofluorescin diacetate (DCFH-DA; MoBiTec, Göttingen, Germany) was used for ROS detection. DCFH-DA diffuses into the cell and it is hydrolyzed by intracellular esterases to polar 2′,7′-dichlorofluorescin. This nonfluorescent fluorescin can be oxidized to the highly fluorescent 2′,7′-dichlorofluorescein by intracellular oxidants. Cells were cultured to adhere on the plates and incubated with 10 μm DCFH-DA for 30 min. Next, the cultures were washed twice with RPMI 1640 and subsequently treated as described before. Fluorescence baseline was measured with a fluorimeter (FLUOstar; BMG LabTechnologies, Offenburg, Germany) immediately after wood dusts were added. The results are given as percentage change over baseline values. The assessment of PPAR-y activity was determined in nuclear extracts from BALF cell with the TransAM enzyme-linked immunosorbent assay (ELISA) Kit (R&D System, Minneapolis, MN) according to the manufacturer's instructions. Briefly, BALF cells lysates were centrifuged (15 000 g, 30 min, 4°C), and the supernatant (nuclear extract) was collected for evaluation of PPAR-y activity.

Gene expression of ICAM-1 and PPAR-ץ by RT-PCR: For mRNA analysis, the thoracic cavity of rats was exposed and the heart and lung were removed in bloc 2 h after intestinal reperfusion. The pulmonar artery was cannulated and then the vasculature pulmonar was perfused with ice-cold sterile phosphate buffer solution (PBS) using a peristaltic pump (Thermo Fisher Scientific, Suwannee, GA) to remove the intravascular blood. Lung fragments were cut into 5 mm pieces using a tissue chopper, flash frozen in liquid nitrogen and stored at −80°C for real time-polymerase chain reaction (RT-PCR) analysis of genes expression. For that assay, total RNA was isolated from lung by TRIzol reagent (Gibco BRL, Gaithersburg, MD) according to the manufacturer's protocol. RNA was subjected to DNase I digestion, followed by reverse transcription to cDNA. PCR was performed in a 7000 sequence detection system (ABI Prism; Applied Biosystems, Foster City, CA) using the SYBRGreen core reaction kit (Applied Biosystems). Primers used for PPARy mRNA quantification were forward primer 5′-ATGCCATTCTGGCCCACCAACTT-3′ and reverse primer 5′-CC CTTGCATCCTTCACAAGCATG-3′. For ICAM-1 mRNA the primers used for quantification were forward primer 5′-CACCTCTCAAGCAGAGCACAG-3′ and reverse primer 5′-GGGTTCCATGGTGAAGTCAAC-3′. Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5′-CTCTACCCACGGCAAGTTCAA-3′ and reverse 5′-GGGATG ACCTTGCCCACAGC-3′, were used as control. Quantitative values for PPARy, ICAM-1 and GAPDH mRNA transcription were obtained from the threshold cycle number, where the increase in the signal associated with an exponential growth of PCR products begins to be detected. Melting curves were generated at the end of every run to ensure product uniformity. The relative target gene expression level was normalized on the basis of GAPDH expression as endogenous RNA control. ΔCt values of the samples were determined by subtracting the average Ct value of PPARy or ICAM-1 mRNA from the average Ct value of the internal control GAPDH. As it is uncommon to use ΔCt as a relative data due to this logarithmic characteristic, the 2−ΔCt parameter was used to express the relative expression data. Results are expressed as a ratio relative to the sum of GAPDH transcript level as internal control.

Immunohistochemical of ICAM-1 in lung

Paraffin sections of lung tissue were processed for standard immunohistologic staining by the labeled streptavidin-biotin method using polyclonal rabbit antirat ICAM-1 antibody diluted at 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA). The secondary antibody consisted of biotinylated sheep antirabbit immunoglobulins (Santa Cruz Biotechnology) containing 10% normal rat serum. The tertiary antibody was streptavidin-horseradish peroxidases conjugate (Santa Cruz Biotechnology). The positive reaction was visualized as a brown stain following treatment with 3,3-diaminobenzidine. Sections were counterstained with Mayer's hematoxylin solution. Immunohistochemical photographs (five photographs from each sample collected from all rats in each experimental group) were assessed using Imaging Densitometer (AxioVision, Zeiss, Gottingen, Germany) and a computer program (AxioVision).

Quantification of HSP70 and GSH in lung homogenate

Lung fragments were homogenized in 1 mL (0.4 m of NaCl and 10 mm of NaPO4) containing antiproteases (0.1 mm phenylmethylsulfonyl fluoride, 0.1 mm benzethonium chloride, 10 mm EDTA and 20 KI aprotinin A) and 0.05% Tween 20. HSP70 levels in lung homogenates were quantified by commercially available ELISA kits according to the manufacturer's instructions (R&D Systems; BioRad, Protein Assay, Mississauga, ON, Canada, respectively). The detection limit of assays was found to be in the range 1–5 pg mL−1. The data were expressed as HSP70/protein content (Lowry method) and the levels of lung protein were expressed as pg mg−1. For GSH measurements, the lung fragments were equally homogenized as described above and then posteriorly the GSH measure was performed using a colorimetric method at 412 nm (QuantiChrom Glutathione Assay Kit; BioAssay Systems, Hayward, CA).

Reagents

Ketamine and Xylazine were purchased from Cristalia (São Paulo, Brazil). The reagents for MPO activity and lung microvascular leakage were obtained from Sigma–Aldrich (St. Louis, MN). Xylol and Paraplast were purchased from Merck (São Paulo, SP, Brazil) and Sigma–Aldrich. ELISA kits for ICAM-1, TNF, HSP70 and GSH, as well as the primer for PPARץ were obtained from R&D Systems.

Statistical analysis

Statistical differences were evaluated by analysis of variance (ANOVA) and Tukey–Kramer Multiple Comparisons Test to determine differences between groups. Results were considered significant when P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

LLLT on acute lung inflammation at different times of irradiation during the intestinal reperfusion

To investigate if the laser irradiation is efficient in controlling the acute lung inflammation at different times during the intestinal reperfusion, the laser irradiation was performed 5 min after initial of intestinal reperfusion or 5 min before the end (corresponding to 115 min from the initial). The Fig. 1 illustrates the inefficiency of LLLT on acute lung inflammation in both conditions (5 or 115 min after initial of intestinal reperfusion). The inflammatory markers chosen were MPO activity, ROS production, GSH generation and HSP.

image

Figure 1. LLLT on acute lung inflammation in different moments of irradiation during the intestinal reperfusion. The laser effect on the inflammatory markers when the animals were irradiated with laser at 5 or 115 min after initial of intestinal reperfusion. Each inflammatory marker is represented as follow: (A and B)—MPO activity; (C and D)—ROS production; (E and F)—glutathione level; (G and H)—HSP70 protein concentration. LLLT was fixed at a dose of 5.4 J for 180 s, applied punctually on the skin in direction of the trachea distal. Results were considered significant when P < 0.05.

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LLLT on ALI

MPO activity and neutrophils migration

Figure 2 represents the MPO content (2A) and the neutrophils influx (2B) into lung. The Fig. 2A shows that i-I/R induces a large increase in MPO content in lung homogenates in comparison with animals from naïve group. This increase was significantly attenuated by the laser when compared with animals subjected to intestinal reperfusion but not irradiated. The laser irradiation on animals nonreperfused produced a discrete rises in neutrophil influx in comparison to naïve group. The Fig. 2B illustrates a marked increase in PMN influx into alveoli after intestinal I/R. In this case, the LLLT also reduced significantly the PMN influx when compared with i-I/R group. The MPO activity was not influenced by laser irradiation in animals nonreperfused. Results demonstrate that there was no difference for both MPO activity and PMN influx between sham and naïve groups.

image

Figure 2. LLLT on acute lung injury. (A) The effect of LLLT on the level of myeloperoxidase (MPO) measured by the biochemistry method. (B) The laser effect on polymorphonuclear (PMN) number in rat lung from three groups studied. (C) The laser irradiation was tested by the extravasation content of Evans blue dye that represents the pulmonary microvascular leakage. (D) The scores of lung injury after intestinal reperfusion. (E) The inflammatory cellular infiltrate in lung parenchyma after intestinal I/R from animals treated or not treated with laser irradiation. LLLT was fixed at 5.4 J for 180 s, applied punctually on the skin in direction of the trachea distal, at half an hour after the beginning of intestinal reperfusion. Results were considered significant when P < 0.05.

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Lung microvascular leakage

Figure 2C represents the pulmonary edema in response to i-I/R. In this assay, we determined vascular permeability in lung 120 min after reperfusion using the Evans blue extravasation technique. The Evans blue extravasation into the lung was significantly higher in comparison with naïve group. It can be observed that such increase in the pulmonary microvascular leakage after i-I/R was significantly reduced by the LLLT. On the contrary, LLLT was not efficient on pulmonary microvascular leakage in animals nonreperfused. Otherwise, the pulmonary microvasculature leakage was not different between the sham and naïve groups.

Lung injury score

The Fig. 2D revealed normal lung parenchyma in the naïve group, but severe lung injury in the i-I/R group. Furthermore, animals reperfused and irradiated had significantly less severe lung injury than those from the i-I/R group, as indicated by the lower total lung injury scores.

Neutrophil infiltration

The Fig. 2E illustrates the neutrophil infiltration into lung after i-I/R and treated with low-level laser. There is a marked increase in the number of inflammatory cells in lung from animals of i-I/R group when compared with naïve group. On the other hand, such increase was reduced when animals were treated with laser. This is in agreement with the reduction in lung injury score and MPO activity observed after laser therapy. The laser irradiation on animals nonreperfused had no effect on neutrophil influx.

LLLT on lung ICAM-1

The Fig. 3 shows the LLLT effect on ICAM-1 mRNA expression as well as the lung localization of ICAM-1 marked with immunohistochemical staining for ICAM-1. ICAM-1 mRNA expression in the lung was significantly higher in the i-I/R group compared with the naïve group; however, there was a significant decrease in ICAM-1 expression for lungs harvested from animals treated with laser irradiation (Fig. 3A). The Fig. 3B confirms the presence of ICAM-1 in lungs from challenged animals with intestinal reperfusion and it is also has evidenced the effect of the LLLT directly on ICAM-1. Laser irradiation was not efficient on ICAM-1 expression in animals nonreperfused.

image

Figure 3. LLLT on ICAM-1. The changes in ICAM-1 mRNA expression (A) and the immunohistochemical localization of ICAM-1 in lung (B) are represented. For measurement of ICAM-1 mRNA expression, the lung tissue was submitted to analysis of real time-PCR, whereas that for immunohistochemical localization of ICAM-1 in lung, the positive reaction was visualized as a brown stain (B). LLLT was fixed at 5.4 J for 180 s, applied punctually on the skin in direction of the trachea distal, at half an hour after the beginning of intestinal reperfusion. Results were considered significant when P < 0.05.

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Influence of LLLT on ROS generation in BALF

As shown in Fig. 4, the ROS concentration in BALF after the intestinal reperfusion period was considerably increased in comparison with the naïve group, increase that was partially reverted by the laser treatment. Otherwise, the laser irradiation induced a discrete rise on ROS concentration when compared with naïve group.

image

Figure 4. LLLT on ROS. The ROS production in BALF cells incubated with DCFH-DA to permit quantification of the ROS production that was measured using the DCF fluorescence indicator. The results are given as percentage change from baseline values. LLLT was fixed at 5.4 J for 180 s, applied punctually on the skin in direction of the trachea distal, at half an hour after the beginning of intestinal reperfusion. Results were considered significant when P < 0.05.

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LLLT on GSH in lung

The Fig. 5 evidences the significant effect of the low-level laser on GSH concentration in BALF fluid. The level of GSH was detected by a colorimetric method (412 nm wavelength) in animals subjected to intestinal reperfusion or not and irradiated with laser or not. The GSH level was severely reduced after intestinal reperfusion when compared with naïve group. On the contrary, the GSH level was increased significantly after laser irradiation at 5.4 J applied in the direction of the trachea distal, 30 min after initial of intestinal reperfusion. Laser irradiation had no effect on ICAM-1 expression in animals nonreperfused.

image

Figure 5. LLLT on GSH. The concentration of GSH in lung tissue is represented in this figure. Lung fragments were equally homogenized and the GSH concentration measure was performed using a colorimetric method at 412 nm. LLLT was fixed at 5.4 J for 180 s, applied punctually on the skin in direction of the trachea distal, at half an hour after the beginning of intestinal reperfusion. Results were considered significant when P < 0.05.

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Influence of LLLT on HSP70 generation in lung

The effect of phototherapy on anti-inflammatory HSP70 in lung tissue after i-I/R is disclosed in Fig. 6. There is no upregulation of HSP70 in lung after intestinal reperfusion in comparison with respective naïve group; however, the level for HSP70 in lung from inflamed animals was more pronounced when it received laser irradiation. Laser irradiation of animals from i-I/R group produced a discrete rise of HSP70 anti-inflammatory protein when compared with the naïve group.

image

Figure 6. LLLT on HSP 70. The HSP 70 levels in lung homogenates. The HSP 70 concentration was quantified by ELISA commercially available kits, according to the manufacturer's instructions. The detection limit of assays was found to be in the range: 1–5 pg mL−1. Levels of lung protein were expressed as pg mg−1. LLLT was fixed at 5.4 J for 180 s, applied punctually on the skin in direction of the trachea distal, at half an hour after the beginning of intestinal reperfusion. Results were considered significant when P < 0.05.

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LLLT on peroxisome proliferator activating receptor-ץ

In Fig. 7 are displayed the measurements on the PPARy activity in BALF cells (7A) and mRNA expression in lung tissue (7B). There was a significant upregulation of this nuclear factor in lung from animals subjected to i-I/R in comparison with the naïve group. When animals subjected to i-I/R received laser irradiation, the PPARץ mRNA expression in lung tissue was reduced significantly when compared with animals inflamed but not treated. Laser irradiation discretely interfered on both PPARy activity and mRNA expression in animals nonreperfused.

image

Figure 7. LLLT on PPAR-y. The PPAR-y mRNA expression in lung as well as the activity in BALF cells are displayed in this figure. Activity of PPAR-y (A) in nuclear extract from BALF cells was measured by ELISA kit. PPAR-y mRNA expression (B) in lung tissue was evaluated by real time-PCR. LLLT was fixed at 5.4 J for 180 s, applied punctually on the skin in direction of the trachea distal, at half an hour after the beginning of intestinal reperfusion. Results were considered significant when P < 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Since 1967, over 100 phase III, randomized, double-blind, placebo-controlled, clinical trials (RCTs) with low-level laser irradiation have been published and supported by more than 1000 laboratory studies that investigated the primary mechanism and the cascade of secondary effects that contribute to the arrangement of the local tissue, as well as systemic effects. RCTs with positive outcomes have been published on pathologies as diverse as osteoarthritis [33], tendinopaties [34], wounds [35], back pain [36], muscle fatigue [37], peripheral nerve injuries [38], strokes [39] and pulmonary disorders such as asthma and chronic obstructive bronchitis [26, 27]; nevertheless, results have not been always positive. That failure can be attributed to several factors: dosimetry (inadequate or too much energy delivered, inadequate or too much irradiance, inappropriate pulse structure, irradiation of insufficient area of the pathology), inappropriate anatomical treatment location and concurrent patient medication, such as steroidal and nonsteroidal anti-inflammatory drugs. Several experimental studies have been performed in the last 6 years to clarify which inflammatory mediator is the target for LLLT in pulmonary disorders, initiating in this way the understanding of which cellular signaling is responsible by the laser anti-inflammatory effect in inflammatory conditions that compromised the lung and airway.

The aim of this study was to investigate if the LLLT can control the oxidative stress in lung, attenuating the features of the acute lung inflammation induced by intestinal ischemia and reperfusion through a mechanism that upregulates the generation/release of anti-inflammatory proteins from lung tissue. Diverse authors have showed that laser irradiation can restore the homeostatic equilibrium of cells activated by different stimuli [40], i.e. LLLT can modulate the cellular response to stimulate or inhibit the cellular functions to obtain the homeostatic equilibrium.

It is well known that laser irradiation reduces markedly the release of proinflammatory mediators in lung tissue from animals subjected to acute lung inflammation induced by endotoxin, TNF or immune-complex reaction [41-43]. Moreover, we recently demonstrated that LLLT decreased the pulmonary microvascular leakage and MPO activity in a model of acute lung inflammation induced by intestinal I/R through a mechanism that involves the downregulation of proinflammatory mediators, such as TNF, and a significant rise of IL-10 anti-inflammatory protein. In addition, pharmacological studies evidenced that laser effect on TNF and IL-10 occurs through independent pathways for each cytokine. That means that the laser induced increase in IL-10 concentration in lung tissue is not an indirect response to the decreasing in TNF protein concentration after intestinal reperfusion.

Considering the results exposed above, we pursued in this study to investigate if LLLT effect in controlling the balance between Th1/Th2 cytokines after i-I/R can also be applied to oxidative stress mediators generated during i-I/R.

Some authors have analyzed the temporal profile of acute lung inflammation induced by 45 min of ischemia followed by 30 min, 2 and 4 h of intestinal reperfusion [44]. It was observed that there is a time-dependent increase in pulmonary microvascular leakage as well as MPO activity, reaching a maximum of 2 h after intestinal I/R. Our results are in agreement with those reported by these authors. In this study, we demonstrated that rats treated with laser irradiation at 30 min after initial of intestinal reperfusion presented a reduction in Evan blue content as well as MPO activity in lung tissue. At the same time, we also showed that the number of inflammatory cells in BALF from rats inflamed was markedly reduced by the laser treatment. This suggests that LLLT beneficial effect is linked to mechanisms of endothelium–leukocyte interaction. We have previously reported that LLLT reduced the pulmonary microvascular leakage and MPO activity in a model of acute lung inflammation induced by 45 min of ischemia followed by 4 h of reperfusion [32]. In this study, we observed that LLLT is also efficient in reducing the features of acute lung inflammation induced by intestinal I/R after 2 h of the intestinal reperfusion. An important point to be emphasized is the capacity of the laser therapy to provide beneficial effect in lung at early as well as lately times post the intestinal reperfusion.

The effect of the low-level laser on inflammatory infiltrate in BALF as well as in lung tissue can be explained by its action on ICAM-1 adhesion molecules that play an important role in the endothelium–leukocyte interaction. It was reported in the literature that animals submitted to 60 min of intestinal ischemia followed by 90 min of reperfusion synthesized an elevated level of ICAM-1 in lung tissue [21]. Our results in the present study show that the ICAM-1 protein in lung was markedly reduced in animals subjected to intestinal reperfusion and laser treatment. Furthermore, the effect on ICAM-1 mRNA expression shows that the laser is able to interfere in one of the steps involved in the ICAM-1 synthesis. This last effect evidences the important role of laser irradiation in cellular signaling that controls the neutrophils influx and consequently the exacerbation of inflammatory response.

Concerning the lung edema induced by intestinal reperfusion, Marcus et al. [3] showed that the loss of the endothelial barrier function requires neutrophil adhesion. In this sense, the results of the current study suggest that LLLT beneficial effect in attenuating the pulmonary microvascular leakage can be because it restores the endothelial cytoskeleton allowing the vascular permeability to be reduced. This hypothesis is based on the results from Mafra de Lima et al. [45], reporting that the lung inflammation and endothelial cell damage in rats subjected to lipopolysaccharide from E. coli was significantly attenuated after low-level laser treatment. It have been demonstrated that the lung inflammation features are more critical between 90 and 120 min after intestinal reperfusion [46]. Our results corroborated with these findings.

Therefore, our results indicated that the beneficial effect of LLLT on lung inflammation induced by i-I/R is directly related to the reduction in both vascular permeability and inflammatory cells migration. Moreover, it can be seen from the current study that laser can also interfere in the generation/release of others cellular mediators that can be considered to be important for tissue reperfusion reactions, like the ROS and the GSH antioxidant. In fact, the balance between the oxidant/antioxidant responses has a key role in the lung inflammation caused by intestinal reperfusion [47]. We have also demonstrated that phototherapy can reduce the ROS production and modulates the macrophage inflammatory protein-2 expression generated by alveolar macrophages activated with lipopolysaccharide or H2O2 [30]. This suggests that LLLT can influence the toxic products of the oxidative stress of the pulmonary system.

I/R injury is a potent inflammatory trigger that increases cytokine release, ROS generation and endothelial activation, with consequent nitric oxide production and expression of adhesion molecules [48]. Studies reported in the literature have shown that neutrophils are activated following tissue reperfusion and that lung injury is associated with an increasing accumulation of neutrophils that once activated are able to release mediators of oxidative stress into lung [49]. Our results showed that laser therapy besides to reduce the lung inflammation also downregulates the production of ROS into lung after 2 h of the intestinal reperfusion. On the contrary, we found in the current study that the GSH level into lung from animals inflamed by i-I/R and treated with laser was higher than from animals subjected to i-I/R but not laser treated. These findings highlights the laser treatment ability in controlling the balance between pro- and antioxidative mediators as the LLLT acts dually, i.e. decreasing the ROS levels at the same time that increasing the GSH level through two independent mechanism in lung for animals submitted to i-I/R. Results strengthen the idea that low-level laser can control the acute lung inflammation by biomodulating some oxidative stress mediators. Assuming that LLLT increases the GSH levels in lung after intestinal reperfusion, we investigated if the laser therapy could increase the synthesis of anti-inflammatory proteins that are released during the intestinal reperfusion, such as the HSP.

The HSP70 is one of the main stress proteins induced by heat shock in mammals, which helps to protect cells from stress [19]. The heat shock response is a highly conservative defense mechanism that provides cytoprotection from oxidative stress and ischemia-reperfusion injury. Similar to that reported by Guiqi [21], we found in this study that the HSP70 concentration in lung tissue was not altered after intestinal reperfusion. Although the i-I/R does not influence the HSP70 levels in lung tissue, we observed that LLLT markedly increases the HSP70 concentration into lung tissue from rats submitted to 2 h of intestinal reperfusion. This shows that the laser irradiation at low levels increase the generation of anti-inflammatory proteins that helps to control the acute lung inflammation. Although we do not discard the possibility that the laser may act also on others anti-inflammatory proteins, our results reinforce the idea that the anti-inflammatory effect of the laser is based on the modulation of the immune response to achieve and maintain the cellular homeostatic equilibrium, because of that the laser therapy may be considered as a facilitator of homeostasis. The majority of drugs available for treating the acute pulmonary inflammation reduces or effectively inhibits the proinflammatory proteins; nevertheless, that is not always enough to restore the pro- and anti-inflammatory balance. On the other hand, the laser therapy controlled pulmonary inflammation reestablishing the balance between pro-and anti-inflammatory mediators.

The peroxisome proliferator-activated receptor-y (PPAR-y) nuclear transcription factor is involved in generation of HSP70 anti-inflammatory protein in diverse organs, including the pulmonary system. Although the PPARץ expression and synthesis play an important role as an endogenous regulator in gut inflammation induced by oxidative stress [49], some authors have evidenced that PPARγ expression is a function of the inflammatory response, as PPARγ is downregulated in the bronchial epithelium and in the endothelium of thoracic aortas in rats subjected to polymicrobial sepsis [50]. PPARγ expression and DNA binding are also markedly reduced in lungs of mice subjected to endotoxic shock and they are associated with massive lung injury and neutrophil infiltration [51]. With this regard, PPARγ ligands have been shown to ameliorate lung injury during sepsis or endotoxin challenge in mice and rats [52]. This protective effect appears to be related to activation of the heat shock response [7].

Regarding to LLLT effect on PPARץ expression in lung from animals challenged with intestinal reperfusion, we found a marked rise of PPARץ mRNA expression after laser treatment. This rise was also observed for PPARy activity in BALF cells from animals subjected to i-I/R and laser treatment. We have previously demonstrated in vitro as well as in vivo studies that LLLT can interfere with nuclear transcription factors, such as NF-κB, for reducing the acute lung inflammation induced by endotoxin [53, 54]. However, in this study, the low-level laser acted oppositely on PPARץ expression in lung from animals subjected to i-I/R. This is curious because PPARץ is a transcription factor that participates directly as a physiologic endogenous regulator that preserves the lung integrity. Thus, on the basis of our results we can infer that LLLT works as a homeostatic facilitator increasing the expression of a transcription factor that is signaling the synthesis of anti-inflammatory proteins, such as HSP70.

The beneficial effect to use concomitant laser-pharmacologic drugs therapy for treatment of patients with airway and lung disorders is very questionable. One of the main drawbacks of such therapy is the fact that the laser light effect could be masked by other drugs or even active principle of anesthesia. It was demonstrated in our article published in the year of 2005 [41], on the use of laser therapy for treatment of experimental acute lung inflammation, that the laser has no efficacy on systemic inflammation because circulating inflammatory mediator's concentration is not altered after LLLT. That means that laser therapy have limitations for controlling the systemic inflammatory response. On the contrary, the laser therapy works very well locally in controlling the lung inflammation and at the same time does not present side effects.

Regard to the possible interference of the anesthesia with the laser effects on airway and lung tissues, it is important to point out that all animals in our experiment were anesthetized and it was observed noninterference with the inflammatory response of the animals from any group studied. The response of the lung tissue to i-I/R was not altered by anesthesia, meaning that it has no anti-inflammatory effect on this experimental model. This fact reinforces the idea that the laser is responsible for attenuating the lung inflammation after i-I/R.

Data obtained in this study showed that the laser does not have good efficacy to control the lung inflammation when it is applied either shortly after the initial or at a time very proximal to the end of reperfusion. These results lead us to suppose that laser does not work well in the first case because at that time there is still no expressive migration of inflammatory cells to lung and therefore the concentration of inflammatory mediators is low. On the other hand, laser has no anti-inflammatory effect when it is applied close to the end of intestinal reperfusion because pulmonary inflammation has already worsened a lot at that time. This means that the effect of the laser treatment depends on the time of irradiation.

Therefore, we chose 30 min after intestinal reperfusion to irradiate the animals because at this time the inflammatory stimulus produced in the mesenteric artery has already reached the lung, stimulating immediately the infiltration of inflammatory cells and consequently, the presence of their mediators. Moreover, the laser needs certain time to interact with the cellular mechanisms that are responsible for the beneficial effect of the laser therapy.

The biological compounds present in the animal body may have also a non-negligible influence on the interaction of the laser with the animal tissues. In fact, it is well established that the physiological composition of both tissue and corporal fluids determine the laser irradiation parameters that should be used, like absorption and scattering that directly influence the quantity of light that attain the target tissue. Admitting that low-level laser could be widely used as a coadjuvant therapy in clinical treatment of lung disorders, the presence of the physiological composition of both tissue and the corporal fluid will be always a reality.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

In conclusion, our study evidences that LLLT attenuated ALI induced by intestinal I/R in rats and that its protective mechanism seems to inhibit proinflammatory response and oppositely to enhance HSP70 protein level by increasing the PPARץ expression.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

We acknowledge the financial support by Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (FAPESP; grant no. 2008/08838-5).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
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