research article photobiomodulation therapy decreases ...rua vergueiro, / liberdade, - s ao paulo,...

Research ArticlePhotobiomodulation Therapy Decreases OxidativeStress in the Lung Tissue after Formaldehyde ExposureRole of OxidantAntioxidant Enzymes

Rodrigo Silva Macedo1 Mayara Peres Leal1 Tarcio Teodoro Braga2 Eacuteric Diego Barioni3

Stephanie de Oliveira Duro3 Anna Carolina Ratto Tempestini Horliana1

Niels Olsen Saraiva Cacircmara2 Tacircnia Marcourakis3 Sandra Helena Poliselli Farsky3

and Adriana Lino-dos-Santos-Franco1

1Postgraduate Program in Biophotonics Applied to Health Sciences University Nove de Julho (UNINOVE)Rua Vergueiro 235249 Liberdade 01504-001 Sao Paulo SP Brazil2Department of Immunology University of Sao Paulo Sao Paulo Brazil3Department of Clinical and Toxicological Analyses Faculty of Pharmaceutical Sciences University of Sao PauloSao Paulo Brazil

Correspondence should be addressed to Adriana Lino-dos-Santos-Franco alsantosfrancogmailcom

Received 18 February 2016 Revised 28 April 2016 Accepted 4 May 2016

Academic Editor Helen C Steel

Copyright copy 2016 Rodrigo Silva Macedo et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Formaldehyde is ubiquitous pollutant that induces oxidative stress in the lung Several lung diseases have been associated withoxidative stress and their control is necessary Photobiomodulation therapy (PBMT) has been highlighted as a promissorytreatment but its mechanisms need to be better investigated Our objective was to evaluate the effects of PBMT on the oxidativestress generated by FA exposure Male Wistar rats were submitted to FA exposure of 1 or vehicle (3 days) and treated or notwith PBMT (1 and 5 h after each FA exposure) Rats treated only with laser were used as control Twenty-four hours after the lastFA exposure we analyzed the effects of PBMT on the generation of nitrites and hydrogen peroxide oxidative burst glutathionereductase peroxidase S-transferase enzyme activities the gene expression of nitric oxide cyclooxygenase superoxide dismutasethe catalase enzyme and heme oxygenase-1 PBMT reduced the generation of nitrites and hydrogen peroxide and increasedoxidative burst in the lung cells A decreased level of oxidant enzymes was observed which were concomitantly related to anincreased level of antioxidants This study provides new information about the antioxidant mechanisms of PBMT in the lung andmight constitute an important tool for lung disease treatment

1 Introduction

Oxidative stress is characterized by the imbalance between anincreased generation of reactive oxygen and nitrogen species(RONS) and a reduced antioxidant capacity RONS are pro-duced in high quantities by endogenous metabolisms whichare represented primarily by the activation of neutrophilsthe products of cyclooxygenase (COX) lipoxygenase (LOX)and nitric oxide synthases (NOS) [1ndash3] At the same time

RONS are also produced in the body through exogenous fac-tors including formaldehyde (FA) particulate matter (PM)metals and quinones [4ndash9]

We have identified oxidative stress as an importantpathway in which FA exerts its toxic effects with a disrup-tion of the lung physiological balance between the oxidantand antioxidant systems modulating positively with a neu-trophilic lung inflammation [9 10] FA is ubiquitous pollutantfound in many industries offices and laboratories and it is

Hindawi Publishing CorporationMediators of InflammationVolume 2016 Article ID 9303126 9 pageshttpdxdoiorg10115520169303126

2 Mediators of Inflammation

also emitted in the domestic ambient of homes in thingssuch as furniture building materials and chipboards and inheating and cooking fumes [11]

Neutrophils that are activated by different biochemicalmechanisms produce high quantities of RONS and inflam-matory cytokines leading to a severe destruction of the lungtissues [12 13] However the deleterious effects of RONSare neutralized naturally by the lung through the defensiveantioxidant system Among the main antioxidant sources inthe lung are glutathione reductase peroxidase s-transferaseenzymes superoxide dismutase the catalase enzyme andheme oxygenase-1 [14ndash16]

Several lung diseases such as asthma chronic obstructivepulmonary diseases and lung fibrosis have been associatedwith oxidative stress [17ndash19] Lung diseases constitute animportant public health problem and the control of oxidativestress into the lung is necessary In this context photo-biomodulation has been highlighted as a promissory treat-ment because of the absence of side effects displaying lowcosts and a noninvasiveness However its mechanisms needto be better investigated and understood

Some studies have evaluated the antioxidant effects oflaser therapy by using different models in vivo and in vitro[20ndash23] When considering the antioxidant effects of PBMTin the lung tissues studies have reported that in an animalexperimental model laser treatment restored the balancebetween the oxidant and the antioxidant mediators raisingthe PPAR expression and consequently the production ofHSP70 [22]

Based upon the oxidative stress that is induced by FAexposure in the lung tissues and the antioxidant effects ofPBMT we evaluated the generation of nitrites and hydrogenperoxide oxidative burst glutathione reductase peroxidases-transferase enzyme activities the gene expression of nitricoxide cyclooxygenase superoxide dismutase the catalaseenzyme and heme oxygenase-1Thus this studymay provideinformation about the antioxidant mechanisms of PBMT inlung diseases

2 Materials and Methods

The experiments were approved by the Committee on theEthics of Animal Experiments of the University of Nove deJulho (CoEP-UNINOVE Permit Number AN00292014)

21 Animals Male 2-month-old Wistar rats (40) wereobtained from the University Nove de Julho and maintainedin a light and temperature-controlled room (1212-hour light-dark cycle 21 plusmn 2∘C) with free access to food and water

22 Formaldehyde (FA) Exposure Group of rats (5chamber)were exposed to FA inhalation (1 90minday) or vehicle(distilled water) for 3 consecutive days Thus we utilized astandard glass chamber (20 L) coupled to an ultrasonic neb-uliser device (Icelreg Brazil) which produces an aerosol withparticles between 05 and 1 micron to generate a constantairstream in an aqueous solution of formalin [9 10 24]

23 Photobiomodulation Therapy According to Miranda daSilva et al [25] rats received infrared laser (CWDiode Laser-MMOptics Sao Paulo Brazil) 1 and 5 h after each FA orvehicle inhalation The irradiation was performed directly inthe skin in nine points of respiratory tract (3 points in the tra-chea and 3 points in the right and left lung lobes) After 24 hof last FA exposure the analyses were performed We usedthe following parameters output power of 30mW 660 nmwavelength 60 spoint and spot size of 014 cm2 resultingin an irradiance of 210mWcm2 and radiant exposure of1286 Jcm2 The optical power was calibrated using a New-port 1835 C multifunction optical power meter (EquiplandOklahoma Road Sao Jose CA USA) The laser power wasmonitored during laser irradiation by collecting laser lightwith a partial reflection (4) mirror The laser irradiationdose was set at 18 J for 1min [24ndash28]

24 Experimental Groups The rats were assigned into 4experimental groups N nonmanipulated rats FA identifiedas rats submitted to FA inhalation L identified as rats treatedonly with laser and FA + L identified as rats subjected toFA inhalation and treated with laser The rats were killed bysectioning the abdominal aorta under deep anaesthesia withketamine-xylazine by intraperitoneal route (100mgkg and20mgkg resp) 24 h after the last FA inhalation

25 Quantification of Nitrites in the Supernatant of Bron-choalveolar Lavage (BAL) The concentration of nitrites(NO2) was determined in the BAL supernatants samples

Nitrites levels were quantified according to the Griessmethod The optical density (540 nm) was recorded using amicroplate reader (Bio-Tek Instr USA) and the nitrites levelswere obtained using a standard curve of NaNO

2(5ndash60120583M)

26 Quantification of Hydrogen Peroxide in the Bronchoalve-olar Lavage (BAL) Cell Suspension The hydrogen peroxidelevels were quantified in sample of BAL cells The BAL cellsuspension (1times 105 cellsmL in phenol red solution) was stim-ulate with PMA (10 ngwell) and incubated at 37∘C 5 CO

2

for 1 hour After this time the reaction was stopped by theaddition of 10 120583L of NaOH 1N The optical density (620 nm)was recorded using a microplate reader (Bio-Tek Instr USA)and the hydrogen peroxide levels were obtained using astandard curve of H

2O2(0ndash200 nM) and expressed in

H2O21 times 104 cells

27 Determination of Gene Expression of Oxidants andAntioxidants Enzymes in the Lung Tissue Lung sampleswere snap-frozen in liquid nitrogen The total RNA wasisolated from lung tissue using Trizol Reagent (InvitrogenCarlsbad CA) according to Invitrogen RNA concentrationswere determined by spectrophotometer absorbance readingsat 260 nm First-strand cDNAs were synthesized using theMML-V reverse transcriptase (Promega Madison WI) RT-PCR was performed using the SYBR Green real-time PCRassay (Applied Biosystem USA) for the following moleculeshypoxanthine guanine phosphoribosyl transferase (HPRT)(sense) 51015840-CTC ATG GAC TGA TTA TGG ACA GGA C-31015840

Mediators of Inflammation 3

and (antisense) 51015840-GCA GGT CAG CAA AGA ACT TATAGC C-31015840 iNOS (sense) 51015840-AGT GAG GAG CAG GTTGAG GA-31015840 and (antisense) 51015840-GCT GTA ACT CTT CTGGGT GT-31015840 RT-PCR was performed using the TaqManreal-time PCR assay (Applied Biosystem USA) for thefollowing molecules COX-1 (Rn00566881 m1lowast) COX-2(Rn01483828 m1lowast) SOD-1 (Rn00566938 m1lowast) SOD-2 (Rn00690587 g1lowast) and catalase (Rn00560930 m1lowast)Cycling conditions were as follows 10min at 95∘C followedby 45 cycles at 20 s each at 95∘C 20 s at 58∘C and 20 s at 72∘CAnalysis was performed using Sequence Detection Software19 (SDS) and mRNA expression was normalized to HPRTexpression

28 Quantification of Enzymatic Activities of GlutathionePeroxidase Reductase and S-Transferase Glutathione perox-idase (GPx) activity was determined using tert-butylhydrop-eroxide as the substrate and the formation of oxidized glu-tathione (GSSG) was indirectly monitored through NAPDHconsumption (5min wavelength of 340 nm) Glutathionereductase (GR) activity was determined by the reductionof GSSG to GSH measured through NADPH consump-tion which was monitored spectrophotometrically (10minwavelength of 340 nm 37∘C) Glutathione S-transferase(GST) activity was determined by measuring the conjugationof 1-chloro-24-dinitrobenzene (CDNB) with reduced glu-tathione The formation of the complex was monitored spec-trophotometrically (5min wavelength of 340 nm 25∘C)TheGPx GR and GST assays were performed in a Power Wavetimes340 spectrophotometer (Bio-Tek Instruments INC softwareKC4 v30)

29 Evaluation of Oxidative Burst in the BronchoalveolarLavage (BAL) Cells Quantification of oxidative burst wasperformed using BAL total cells (2 times 105 cellswell) estimatedby means of 2101584071015840-dichlorofluorescin diacetate (DCFH-DA)fluorescence cells Direct measurement of the mean fluores-cence recorded on green channel was recorded as oxidativeburst A flow cytometer (FACS Calibur Becton DickinsonImmunocytometry Systems San Jose CA USA) interfacedwith a Macintosh G4 computer was used Data from 10000events were collected in list mode and analyzed in CellQuest (Becton Dickinson Immunocytometry Systems) Flu-orescence data were plotted on log scale Green fluorescencefrom DCFH was measured at 530 plusmn 30 nm (FL1 detector)

210 Statistical Analysis The statistical analysis was per-formed using the GraphPad Prism software (GraphPadSoftware Inc) The normality test was performed usingKolmogorov-Smirnov test Since the data were parametricwe used one-way ANOVA followed by Studentrsquos Newman-Keuls Differences were considered significant when 119875 lt005

3 Results

31 Photobiomodulation Therapy Reduced the Generation ofHydrogen Peroxide (H2O2) and Nitrites (NO2) Induced by FAExposure in the Lung To investigate the effects of PBMT on

generation of RONS in the lung tissue we quantified theH2O2andNO

2in the bronchoalveolar lavageData of Figure 1

showed that treatment with laser reduced the levels of H2O2

(a) as well as NO2(b) when compared to nontreated group

(FA group) and did not differ from nonmanipulated and lasergroups (N and L groups) We can also observe that FA expo-sure increased the generation of NO

2and H

2O2in relation to

basal N and L groups

32 Photobiomodulation Therapy Increased the OxidativeBurst in the Lung In order to investigate the effects of PBMTon the functional state of BAL cells we evaluated the oxidativeburst Figures 2(a) and 2(b) showed that the treatmentwith laser increased the oxidative burst in BAL cells whencompared to the FA N and L groups

33 Photobiomodulation Therapy Reduced the Gene Expres-sion of Nitric Oxide Synthase (iNOS and cNOS) and Cyclooxy-genase (COX-2) Induced by FA Exposure in the Lung Inorder to understand the possible mechanism involved inreactive oxygen and nitrogen species (RONS) after PBMTwe investigated important enzymes that generate RONSFigure 3(a) showed that PBMT reduced the gene expressionof COX-2 when compared to the N L and FA groups Nodifferences were observed between FA L and N groups

In Figures 3(b) and 3(c) we can observe that PBMTdecreased the gene expression of cNOS and iNOS respec-tively when compared to the FAgroup anddid not differ fromL and N groups We also showed that FA exposure increasedthe gene expression of both enzymes (cNOS and iNOS) inrelation to nonmanipulated rats (N group) and rats treatedonly with laser (L group)

34 Photobiomodulation Therapy Increased the Gene Expres-sion of Superoxide Dismutase (SOD-1 and SOD-2) and HemeOxygenase-1 (HO-1) without Changing Catalase after FAExposure in the Lung We investigated the involvement ofPBMT in the gene expression of antioxidant enzymes Fig-ure 4(a) showed that PBMT increased the gene expression ofSOD-1 when compared to the FA L and N groups On theother hand in Figure 4(b) we showed that no differences wereobserved in the gene expression of catalase when rats weretreated with laser (FA + L group) We also showed that FAexposure increased the gene expression of catalase in relationto control groups (N and L)

In Figure 4(c) we can observe that PBMT increased thegene expression of HO-1 when compared to the FA L and Ngroups We also observed that FA exposure did not inducealteration in the expression of HO-1 in relation to controlgroups (N and L)

35 PhotobiomodulationTherapy Increased theActivity of Glu-tathione S-Transferase (GST) and Peroxidase (GPX) withoutChanging Glutathione Reductase (GR) after FA Exposure inthe Lung We also investigated the involvement of PBMT inthe activity of glutathione enzymes that exert an importantprotection of oxidant species in the lung tissue Figures 5(a)and 5(c) showed that PBMT increased the activity of GSTand GPX respectively when compared to the FA L and N


L FAN

H2O2

(1times104

cells

)

0

50

100

150

120579

lowast

lowast

FA + L(a)

00

05

10

15

20

L FAN

120579

lowast

NO2minus

(mM

)

FA + L(b)

Figure 1 Photobiomodulation therapy reduces generation of H2

O2

and NO2

after FA exposure in the lung Group of rats was exposed or notto FA inhalation (1 90minday 3 days) and treated or not with laser (30mW 18 J 60 spoint total 540 s 1 and 5 h after each FA inhalation)In parallel group of rats were treated only with laser and nonmanipulated rats were used to obtain basal parameters The quantification ofH2

O2

and NO2

(a b) was determined 24 h after the last FA inhalation Data mean plusmn SEM of 6 animals per group lowast119875 lt 005 in relation to Nand L groups 120579119875 lt 005 in relation to FA group

N L FA

120579

lowast

DCF

H-D

A

FA + L0

100

200

300

400

oxid

ativ

e bur

st (I

FM)

(a)

0

500 FITC pos

Cou

nt

15K

10K

0minus103

104

105

103

FITC-A

FITC pos

0 105

minus103

103

104

FITC-A

0

100

200

300

400

500

Cou

nt

FITC pos

0 103

104

105

minus103

FITC-A

0

500

10K

15K

Cou

nt

L FA FA + L

(b)

Figure 2 Photobiomodulation therapy increases oxidative burst in the lung Group of rats was exposed to FA inhalation (1 90minday3 days) and treated or not with LLLT (30mW 18 J 60 spoint total 540 s 1 and 5 h after each FA inhalation) In parallel group of rats weretreated only with laser and nonmanipulated rats were used to obtain basal parameters The oxidative burst was determined 24 h after the lastFA inhalation Data mean plusmn SEM of 6 animals per group lowast119875 lt 005 in relation to N and L groups 120579119875 lt 005 in relation to FA group


L FAN

COX-2

(2minus

DD

CT)

00

05

10

15

20

25

120579

lowast

FA + L(a)

0

1

2

3

cNO

S (2minus

DD

CT)

120579

lowast

L FAN FA + L(b)

00

05

10

15

20

iNO

S (2minus

DD

CT)

L FAN

120579

lowast

FA + L(c)

Figure 3 Photobiomodulation therapy decreases oxidant enzymes after FA exposure in the lung Group of rats was exposed to FA inhalation(1 90minday 3 days) and treated or not with LLLT (30mW 18 J 60 spoint total 540 s 1 and 5 h after each FA inhalation) In parallel groupof rats were treated only with laser and nonmanipulated rats were used to obtain basal parameters The oxidant enzymes were determined24 h after the last FA inhalation Data mean plusmn SEM of 6 animals per group lowast119875 lt 005 in relation to N and L groups 120579119875 lt 005 in relation toFA group

groups On the other hand PBMT did not interfere in the GRactivity (Figure 5(b))

4 Discussion

Photobiomodulation therapy was an effective treatment foroxidative stress that was induced by an FA exposure in thelung tissue as it reduced the generation of H

2O2 NO2 and

the gene expression of oxidant enzymes that were concomi-tantly related to the increased gene expression of antioxidantenzymes Photobiomodulation therapy also increased theactivity of glutathione enzymes (GST and GPX) which arehighly important for the protection of the lung against oxygenand nitrogen reactive species (RONS)

When considering the fact that oxidative stress is animportant pathway by which FA induces a lung inflamma-tion we have shown here the protective effects of PBMT Aswe expected FA exposure evoked an increased generationof RONS in the cells recruited into the lung with respect toH2O2and NO

2 which was reversed by PBMT These results

can explain the previous results published by our group thatshowed a reduced lung inflammation after PBMT [25]This is

since RONS has been implicated in initiating inflammatoryresponses in the lungs through the activation of transcriptionfactors such as the nuclear factor NF-120581B leading to anenhanced gene expression of proinflammatory mediators

The beneficial effects of PBMT were corroborated by adetermination of the oxidative burst in the BAL cells thatwere predominantly neutrophils The oxidative burst exertsan important role in the killing activity by the generation ofoxygen reactive species by neutrophils An elevated oxidativeburst was found after PBMT showing that this treatmentimproves the capacity of neutrophils to defend the bodyagainst microorganisms although this process generatesreactive species Thus we must consider that reactive speciesexert a dual role in the organism protecting andor prejudic-ing

PBMT reduced the generation of NO2and H

2O2and

simultaneously increased the oxidative burst These datacould be considered controversial if it is not taking intoaccount that several pathways on phagocytes can generatereactive species which are simultaneously inactivated byantioxidant enzymes In this context phagocytes such asmacrophages and neutrophils generate reactive species via


L FAN

SOD

-1(2minus

DD

CT)

0

2

4

6

8 120579

lowast

lowast

FA + L(a)

lowast

00

05

10

15

20

25

CAT

(2minus

DD

CT)

L FAN FA + L(b)

120579

lowast

0

2

4

6

8

10

HO

-1(2minus

DD

CT)

L FAN FA + L(c)

Figure 4 Photobiomodulation therapy increases antioxidant enzymes after FA exposure in the lung Group of rats was exposed to FAinhalation (1 90minday 3 days) and treated or not with LLLT (30mW 18 J 60 spoint total 540 s 1 and 5 h after each FA inhalation) Inparallel group of rats were treated only with laser and nonmanipulated rats were used to obtain basal parameters The antioxidant enzymeswere determined 24 h after the last FA inhalation Datameanplusmn SEMof 6 animals per group lowast119875 lt 005 in relation toN and L groups 120579119875 lt 005in relation to FA group

oxidative burst regulated by NADPH-oxidase This latterenzyme generates superoxide radical that is subsequentlyconverted into hypochlorous acid a potent bactericidal agentThus increased oxidative burst may be observed in reducedlevels of H

2O2 In addition different mechanisms of defense

against H2O2production are available such as glutathione

peroxidase that was increased by PBMT which contributeto reduced levels of H

2O2 It is important to mention that

our results corroborate those obtained by Dolgushin et al[29] which showed that PBMT increased killing activity byneutrophils

Since in previous studies we have shown that FAexposure causes a disruption of the physiological balancebetween the oxidant and antioxidant enzymes in the lungtissue most likely by favoring the oxidant pathways and thuspositively modulating the lungrsquos inflammation [11 20] wehave investigated the effects of PBMT on the gene expressionof these oxidant and antioxidant enzymes in the lung tissue

Based on previous studies we suppose that RONS pro-duced during FA inhalation generated by oxidantantioxi-dant enzymes imbalance in the lung tissue might alter the

metabolism of lung phagocytes which in turn could increasethe release of inflammatory mediators as well as reactivespecies amplifying the lung inflammatory response [30] Inaddition it is reasonable to admit that we analyzed enzymesin the whole lung tissue including parenchyma muscle cellsstructural cells and phagocytes

Our data has shown that PBMT reversed the increasedgene expression of cNOS and iNOS in the lung tissue afterFA exposureThese results corroborate with the reducedNO

2

released by the BAL cells Similarly PBMT also reducedthe gene expression of COX-2 that is an important oxidantenzyme and one that generates potent inflammatory media-tors including eicosanoids We can infer this reduction in theCOX as well as in the NOS and where this is caused by aPBMT these results might be responsible at least in part forthe decreased generation of oxidative species released duringan FA exposure and culminating in a reduced lung inflamma-tion as noted previously [25]

In the literature few studies showed the PBMT effects inmodel of lung diseases [22 25ndash28] Almost the works showedthe anti-inflammatory and antioxidants effects of PBMT in


L FAN0

2

4

6

8G

ST ac

tivity

(Ug

)120579

lowast

FA + L(a)

L FAN00

05

10

15

20

GR

activ

ity (U

g)

FA + L(b)

0

2

4

6

8

GPX

activ

ity (U

g)

L FAN

120579

lowast

FA + L(c)

Figure 5 Photobiomodulation therapy increases activity of glutathione s-transferase and peroxidase enzymes after FA exposure in the lungGroup of rats was exposed to FA inhalation (1 90minday 3 days) and treated or not with LLLT (30mW 18 J 60 spoint total 540 s 1 and 5 hafter each FA inhalation) In parallel group of rats were treated only with laser and nonmanipulated rats were used to obtain basal parametersThe activities of glutathione s-transferase reductase and peroxidase were determined 24 h after the last FA inhalation Data mean plusmn SEM of6 animals per group lowast119875 lt 005 in relation to N and L groups 120579119875 lt 005 in relation to FA group

experimental models of arthritis Thus studies that inves-tigate the effects of PBMT in lung diseases are importantshowing the alternative therapy without side effects

As mentioned above we also evaluated the antioxidantenzymes including SOD CATHO-1 GPX GR andGSTTheincreased gene expression of SOD and HO-1 demonstratedthe protective antioxidant mechanisms that are induced byPBMT Thus we can infer that the SOD and HO-1 geneexpressions were augmented after PBMT as a compensatorymechanism that prevented a pulmonary tissue disease fromthe oxidative damage induced by FA It is known that HO-1 is a rate-limiting enzyme in heme catabolism degradingheme to free iron biliverdin and carbon monoxide SOD inturn promotes the degradation of the superoxide anion inhydrogen peroxide and it is less reactiveThese products exerta cytoprotective mechanism against oxidative stress [31] Onthe other hand no differences were observed in CAT levels

Another important antioxidant source in the lung isglutathione which is essential for a defensive response tooxidants and inflammatory agents by repairing the oxidized

and damaged molecules and helping to regulate a variety ofcellular functions [32] Our study has found that the activitiesof GST and GPX were increased by PBMT As previouslymentioned these data can explain the reduced levels ofH

2O2

since GPX is a potent antioxidant against H2O2

Here we have utilized an interestingmodel of lung diseaseinduced by pollutionMoreover several lung diseases such asasthma a chronic obstructive pulmonary disease and lungfibrosis have all been associated with oxidative stress [18 1933] and in addition the pollution might aggravate these dis-eases In this context taking everything into account our datahas shown that PBMT might be as a promissory treatmentfor lung diseases mediated by oxidative stress Additionallythis treatment is without side effects it presents low costs andit demonstrates a noninvasive therapy

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper


Acknowledgments

This study was sponsored by Fundacao de Amparo a Pesquisado Estado de Sao Paulo (FAPESP) Adriana Lino-dos-Santos-Franco is a Research Fellow from FAPESP (201500830-9)

References

[1] A Manda-Handzlik and U Demkow ldquoNeutrophils the role ofoxidative and nitrosative stress in health and diseaserdquo Advancesin Experimental Medicine and Biology vol 857 pp 51ndash60 2015

[2] H RGriffiths I H KDias R SWilletts andADevitt ldquoRedoxregulation of protein damage in plasmardquo Redox Biology vol 2no 1 pp 430ndash435 2014

[3] H LHsieh andCM Yang ldquoRole of redox signaling in neuroin-flammation and neurodegenerative diseasesrdquo BioMed ResearchInternational vol 2013 Article ID 484613 18 pages 2013

[4] N D Betancourt-Martınez J Jimenez-Villarreal P Carranza-Rosales et al ldquoSperm chromatin dispersion by formaldehydein wistar ratsrdquo Genetics and Molecular Research vol 14 no 3pp 10816ndash10826 2015

[5] B Silva Ibrahim C Miranda da Silva E D Barioni et alldquoFormaldehyde inhalation during pregnancy abolishes thedevelopment of acute innate inflammation in offspringrdquo Toxi-cology Letters vol 235 no 2 pp 147ndash154 2015

[6] H Fukui S Endoh M Shichiri et al ldquoThe induction of lipidperoxidation during the acute oxidative stress response inducedby intratracheal instillation of fine crystalline silica particles inratsrdquo Toxicology and Industrial Health 2014

[7] M Maiellaro M Correa-Costa L B Vitoretti et al ldquoExposureto low doses of formaldehyde during pregnancy suppressesthe development of allergic lung inflammation in offspringrdquoToxicology and Applied Pharmacology vol 278 no 3 pp 266ndash274 2014

[8] L Forchhammer S Loft M Roursgaard et al ldquoExpression ofadhesionmolecules monocyte interactions and oxidative stressin human endothelial cells exposed to wood smoke and dieselexhaust particulate matterrdquo Toxicology Letters vol 209 no 2pp 121ndash128 2012

[9] A Lino-dos-Santos-FrancoMCorrea-Costa ACCDos San-tos Durao et al ldquoFormaldehyde induces lung inflammation byan oxidant and antioxidant enzymes mediated mechanism inthe lung tissuerdquo Toxicology Letters vol 207 no 3 pp 278ndash2852011

[10] A Lino-dos-Santos-FrancoHVDomingos A P L DOliveiraet al ldquoDifferential effects of formaldehyde exposure on the cellinflux and vascular permeability in a rat model of allergic lunginflammationrdquo Toxicology Letters vol 197 no 3 pp 211ndash2182010

[11] T Salthammer ldquoFormaldehyde in the ambient atmospherefrom an indoor pollutant to an outdoor pollutantrdquoAngewandteChemiemdashInternational Edition vol 52 no 12 pp 3320ndash33272013

[12] B Amulic C Cazalet G L Hayes K D Metzler and AZychlinsky ldquoNeutrophil function frommechanisms to diseaserdquoAnnual Review of Immunology vol 30 pp 459ndash489 2012

[13] J A Marwick D A Dorward C D Lucas et al ldquoOxygen levelsdetermine the ability of glucocorticoids to influence neutrophilsurvival in inflammatory environmentsrdquo Journal of LeukocyteBiology vol 94 no 6 pp 1285ndash1292 2013

[14] S K Biswas and I Rahman ldquoEnvironmental toxicity redoxsignaling and lung inflammation the role of glutathionerdquoMolecular Aspects of Medicine vol 30 no 1-2 pp 60ndash76 2009

[15] V L Kinnula and J D Crapo ldquoSuperoxide dismutases in thelung and human lung diseasesrdquoAmerican Journal of Respiratoryand Critical Care Medicine vol 167 no 12 pp 1600ndash1619 2003

[16] R Stocker Y Yamamoto A F McDonagh A N Glazer and BN Ames ldquoBilirubin is an antioxidant of possible physiologicalimportancerdquo Science vol 235 no 4792 pp 1043ndash1046 1987

[17] H Lee J R Park E J Kim et al ldquoCigarette smoke-mediatedoxidative stress induces apoptosis via the MAPKsSTAT1 path-way in mouse lung fibroblastsrdquo Toxicology letters vol 240 no 1pp 140ndash148 2016

[18] T A Moore K K Schmid A Anderson-Berry and A MBerger ldquoLung disease oxidative stress and oxygen require-ments in preterm infantsrdquo Biological Research For Nursing 2015

[19] R-M Liu and L P Desai ldquoReciprocal regulation of TGF-120573 andreactive oxygen species a perverse cycle for fibrosisrdquo RedoxBiology vol 6 pp 565ndash577 2015

[20] A C-H Chen P R Arany Y-Y Huang et al ldquoLow-Levellaser therapy activates NF-120581B via generation of reactive oxygenspecies in mouse embryonic fibroblastsrdquo PLoS ONE vol 6 no7 Article ID e22453 2011

[21] F K Ibuki A Simoes J Nicolau and F N Nogueira ldquoLaserirradiation affects enzymatic antioxidant system of streptozo-tocin-induced diabetic ratsrdquo Lasers in Medical Science vol 28no 3 pp 911ndash918 2013

[22] F M de Lima R Albertini Y Dantas et al ldquoLow-level lasertherapy restores the oxidative stress balance in acute lung injuryinduced by gut ischemia and reperfusionrdquo Photochemistry andPhotobiology vol 89 no 1 pp 179ndash188 2013

[23] M Migliario P Pittarella M Fanuli M Rizzi and F RenoldquoLaser-induced osteoblast proliferation is mediated by ROSproductionrdquo Lasers in Medical Science vol 29 no 4 pp 1463ndash1467 2014

[24] A Lino dos Santos Franco H V Domingos A S Damazoet al ldquoReduced allergic lung inflammation in rats followingformaldehyde exposure long-term effects on multiple effectorsystemsrdquo Toxicology vol 256 no 3 pp 157ndash163 2009

[25] C Miranda da Silva M Peres Leal R A Brochetti et al ldquoLowlevel laser therapy reduces the development of lung inflamma-tion induced by formaldehyde exposurerdquo PLoS ONE vol 10 no11 Article ID e0142816 2015

[26] J P S Peron A A de Brito M Pelatti et al ldquoHuman tubal-derived mesenchymal stromal cells associated with low levellaser therapy significantly reduces cigarette smoke-inducedCOPD in C57BL6 micerdquo PLoS ONE vol 10 no 8 Article IDe0136942 2015

[27] V R Silva PMarcondesM Silva et al ldquoLow-level laser therapyinhibits bronchoconstriction Th2 inflammation and airwayremodeling in allergic asthmardquo Respiratory Physiology andNeurobiology vol 194 no 1 pp 37ndash48 2014

[28] F M de Lima F Aimbire H Miranda R D P Vieira A Pde Oliveira and R Albertini ldquoLow-level laser therapy attenu-ates the myeloperoxidase activity and inflammatory mediatorgeneration in lung inflammation induced by gut ischemia andreperfusion a dose-response studyrdquo Journal of Lasers inMedicalSciences vol 5 pp 63ndash70 2014

[29] I I Dolgushin V A Markova and O A Gizinger ldquoMonitoringof the effect of low-intensity laser radiation with constant pulsegeneration on neutrophil granulocytes in vitrordquo Bulletin of


Experimental Biology andMedicine vol 150 no 2 pp 222ndash2242010

[30] A Lino-dos-Santos-Franco H V Domingos A P Oliveira etal ldquoDifferential effects of formaldehyde exposure on the cellinflux and vascular permeability in a rat model of allergic lunginflammationrdquo Toxicology Letters vol 197 no 3 pp 211ndash2182010

[31] N G Abraham and A Kappas ldquoPharmacological and clinicalaspects of heme oxygenaserdquo Pharmacological Reviews vol 60no 1 pp 79ndash127 2008

[32] K Doreswamy B Shrilatha T Rajeshkumar and MuralidharaldquoNickel-induced oxidative stress in testis of mice evidence ofDNA damage and genotoxic effectsrdquo Journal of Andrology vol25 no 6 pp 996ndash1003 2004

[33] H Lee J R Park Kim E J Kim et al ldquoCigarette smoke-mediated oxidative stress induces apoptosis via the MAPKsSTAT1 pathway in mouse lung fibroblastsrdquo Toxicology Lettersvol 240 no 1 pp 140ndash148 2015

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


also emitted in the domestic ambient of homes in thingssuch as furniture building materials and chipboards and inheating and cooking fumes [11]

Neutrophils that are activated by different biochemicalmechanisms produce high quantities of RONS and inflam-matory cytokines leading to a severe destruction of the lungtissues [12 13] However the deleterious effects of RONSare neutralized naturally by the lung through the defensiveantioxidant system Among the main antioxidant sources inthe lung are glutathione reductase peroxidase s-transferaseenzymes superoxide dismutase the catalase enzyme andheme oxygenase-1 [14ndash16]

Several lung diseases such as asthma chronic obstructivepulmonary diseases and lung fibrosis have been associatedwith oxidative stress [17ndash19] Lung diseases constitute animportant public health problem and the control of oxidativestress into the lung is necessary In this context photo-biomodulation has been highlighted as a promissory treat-ment because of the absence of side effects displaying lowcosts and a noninvasiveness However its mechanisms needto be better investigated and understood

Some studies have evaluated the antioxidant effects oflaser therapy by using different models in vivo and in vitro[20ndash23] When considering the antioxidant effects of PBMTin the lung tissues studies have reported that in an animalexperimental model laser treatment restored the balancebetween the oxidant and the antioxidant mediators raisingthe PPAR expression and consequently the production ofHSP70 [22]

Based upon the oxidative stress that is induced by FAexposure in the lung tissues and the antioxidant effects ofPBMT we evaluated the generation of nitrites and hydrogenperoxide oxidative burst glutathione reductase peroxidases-transferase enzyme activities the gene expression of nitricoxide cyclooxygenase superoxide dismutase the catalaseenzyme and heme oxygenase-1Thus this studymay provideinformation about the antioxidant mechanisms of PBMT inlung diseases

2 Materials and Methods

The experiments were approved by the Committee on theEthics of Animal Experiments of the University of Nove deJulho (CoEP-UNINOVE Permit Number AN00292014)

21 Animals Male 2-month-old Wistar rats (40) wereobtained from the University Nove de Julho and maintainedin a light and temperature-controlled room (1212-hour light-dark cycle 21 plusmn 2∘C) with free access to food and water

22 Formaldehyde (FA) Exposure Group of rats (5chamber)were exposed to FA inhalation (1 90minday) or vehicle(distilled water) for 3 consecutive days Thus we utilized astandard glass chamber (20 L) coupled to an ultrasonic neb-uliser device (Icelreg Brazil) which produces an aerosol withparticles between 05 and 1 micron to generate a constantairstream in an aqueous solution of formalin [9 10 24]

23 Photobiomodulation Therapy According to Miranda daSilva et al [25] rats received infrared laser (CWDiode Laser-MMOptics Sao Paulo Brazil) 1 and 5 h after each FA orvehicle inhalation The irradiation was performed directly inthe skin in nine points of respiratory tract (3 points in the tra-chea and 3 points in the right and left lung lobes) After 24 hof last FA exposure the analyses were performed We usedthe following parameters output power of 30mW 660 nmwavelength 60 spoint and spot size of 014 cm2 resultingin an irradiance of 210mWcm2 and radiant exposure of1286 Jcm2 The optical power was calibrated using a New-port 1835 C multifunction optical power meter (EquiplandOklahoma Road Sao Jose CA USA) The laser power wasmonitored during laser irradiation by collecting laser lightwith a partial reflection (4) mirror The laser irradiationdose was set at 18 J for 1min [24ndash28]

24 Experimental Groups The rats were assigned into 4experimental groups N nonmanipulated rats FA identifiedas rats submitted to FA inhalation L identified as rats treatedonly with laser and FA + L identified as rats subjected toFA inhalation and treated with laser The rats were killed bysectioning the abdominal aorta under deep anaesthesia withketamine-xylazine by intraperitoneal route (100mgkg and20mgkg resp) 24 h after the last FA inhalation

25 Quantification of Nitrites in the Supernatant of Bron-choalveolar Lavage (BAL) The concentration of nitrites(NO2) was determined in the BAL supernatants samples

Nitrites levels were quantified according to the Griessmethod The optical density (540 nm) was recorded using amicroplate reader (Bio-Tek Instr USA) and the nitrites levelswere obtained using a standard curve of NaNO

2(5ndash60120583M)

26 Quantification of Hydrogen Peroxide in the Bronchoalve-olar Lavage (BAL) Cell Suspension The hydrogen peroxidelevels were quantified in sample of BAL cells The BAL cellsuspension (1times 105 cellsmL in phenol red solution) was stim-ulate with PMA (10 ngwell) and incubated at 37∘C 5 CO

2

for 1 hour After this time the reaction was stopped by theaddition of 10 120583L of NaOH 1N The optical density (620 nm)was recorded using a microplate reader (Bio-Tek Instr USA)and the hydrogen peroxide levels were obtained using astandard curve of H

2O2(0ndash200 nM) and expressed in

H2O21 times 104 cells

27 Determination of Gene Expression of Oxidants andAntioxidants Enzymes in the Lung Tissue Lung sampleswere snap-frozen in liquid nitrogen The total RNA wasisolated from lung tissue using Trizol Reagent (InvitrogenCarlsbad CA) according to Invitrogen RNA concentrationswere determined by spectrophotometer absorbance readingsat 260 nm First-strand cDNAs were synthesized using theMML-V reverse transcriptase (Promega Madison WI) RT-PCR was performed using the SYBR Green real-time PCRassay (Applied Biosystem USA) for the following moleculeshypoxanthine guanine phosphoribosyl transferase (HPRT)(sense) 51015840-CTC ATG GAC TGA TTA TGG ACA GGA C-31015840






3 Results







2and H

2O2in relation to









L FAN

H2O2

(1times104

cells

)

0

50

100

150

120579

lowast

lowast

FA + L(a)

00

05

10

15

20

L FAN

120579

lowast

NO2minus

(mM

)

FA + L(b)


O2

and NO2


O2

and NO2


N L FA

120579

lowast

DCF

H-D

A

FA + L0

100

200

300

400

oxid

ativ

e bur

st (I

FM)

(a)

0

500 FITC pos

Cou

nt

15K

10K

0minus103

104

105

103

FITC-A

FITC pos

0 105

minus103

103

104

FITC-A

0

100

200

300

400

500

Cou

nt

FITC pos

0 103

104

105

minus103

FITC-A

0

500

10K

15K

Cou

nt

L FA FA + L

(b)



L FAN

COX-2

(2minus

DD

CT)

00

05

10

15

20

25

120579

lowast

FA + L(a)

0

1

2

3

cNO

S (2minus

DD

CT)

120579

lowast

L FAN FA + L(b)

00

05

10

15

20

iNO

S (2minus

DD

CT)

L FAN

120579

lowast

FA + L(c)



4 Discussion


2O2 NO2 and








2O2and



L FAN

SOD

-1(2minus

DD

CT)

0

2

4

6

8 120579

lowast

lowast

FA + L(a)

lowast

00

05

10

15

20

25

CAT

(2minus

DD

CT)

L FAN FA + L(b)

120579

lowast

0

2

4

6

8

10

HO

-1(2minus

DD

CT)

L FAN FA + L(c)












2




L FAN0

2

4

6

8G

ST ac

tivity

(Ug

)120579

lowast

FA + L(a)

L FAN00

05

10

15

20

GR

activ

ity (U

g)

FA + L(b)

0

2

4

6

8

GPX

activ

ity (U

g)

L FAN

120579

lowast

FA + L(c)






2O2



Competing Interests



Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results







2and H

2O2in relation to









L FAN

H2O2

(1times104

cells

)

0

50

100

150

120579

lowast

lowast

FA + L(a)

00

05

10

15

20

L FAN

120579

lowast

NO2minus

(mM

)

FA + L(b)


O2

and NO2


O2

and NO2


N L FA

120579

lowast

DCF

H-D

A

FA + L0

100

200

300

400

oxid

ativ

e bur

st (I

FM)

(a)

0

500 FITC pos

Cou

nt

15K

10K

0minus103

104

105

103

FITC-A

FITC pos

0 105

minus103

103

104

FITC-A

0

100

200

300

400

500

Cou

nt

FITC pos

0 103

104

105

minus103

FITC-A

0

500

10K

15K

Cou

nt

L FA FA + L

(b)



L FAN

COX-2

(2minus

DD

CT)

00

05

10

15

20

25

120579

lowast

FA + L(a)

0

1

2

3

cNO

S (2minus

DD

CT)

120579

lowast

L FAN FA + L(b)

00

05

10

15

20

iNO

S (2minus

DD

CT)

L FAN

120579

lowast

FA + L(c)



4 Discussion


2O2 NO2 and








2O2and



L FAN

SOD

-1(2minus

DD

CT)

0

2

4

6

8 120579

lowast

lowast

FA + L(a)

lowast

00

05

10

15

20

25

CAT

(2minus

DD

CT)

L FAN FA + L(b)

120579

lowast

0

2

4

6

8

10

HO

-1(2minus

DD

CT)

L FAN FA + L(c)












2




L FAN0

2

4

6

8G

ST ac

tivity

(Ug

)120579

lowast

FA + L(a)

L FAN00

05

10

15

20

GR

activ

ity (U

g)

FA + L(b)

0

2

4

6

8

GPX

activ

ity (U

g)

L FAN

120579

lowast

FA + L(c)






2O2



Competing Interests



Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















L FAN

H2O2

(1times104

cells

)

0

50

100

150

120579

lowast

lowast

FA + L(a)

00

05

10

15

20

L FAN

120579

lowast

NO2minus

(mM

)

FA + L(b)


O2

and NO2


O2

and NO2


N L FA

120579

lowast

DCF

H-D

A

FA + L0

100

200

300

400

oxid

ativ

e bur

st (I

FM)

(a)

0

500 FITC pos

Cou

nt

15K

10K

0minus103

104

105

103

FITC-A

FITC pos

0 105

minus103

103

104

FITC-A

0

100

200

300

400

500

Cou

nt

FITC pos

0 103

104

105

minus103

FITC-A

0

500

10K

15K

Cou

nt

L FA FA + L

(b)



L FAN

COX-2

(2minus

DD

CT)

00

05

10

15

20

25

120579

lowast

FA + L(a)

0

1

2

3

cNO

S (2minus

DD

CT)

120579

lowast

L FAN FA + L(b)

00

05

10

15

20

iNO

S (2minus

DD

CT)

L FAN

120579

lowast

FA + L(c)



4 Discussion


2O2 NO2 and








2O2and



L FAN

SOD

-1(2minus

DD

CT)

0

2

4

6

8 120579

lowast

lowast

FA + L(a)

lowast

00

05

10

15

20

25

CAT

(2minus

DD

CT)

L FAN FA + L(b)

120579

lowast

0

2

4

6

8

10

HO

-1(2minus

DD

CT)

L FAN FA + L(c)












2




L FAN0

2

4

6

8G

ST ac

tivity

(Ug

)120579

lowast

FA + L(a)

L FAN00

05

10

15

20

GR

activ

ity (U

g)

FA + L(b)

0

2

4

6

8

GPX

activ

ity (U

g)

L FAN

120579

lowast

FA + L(c)






2O2



Competing Interests



Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















L FAN

COX-2

(2minus

DD

CT)

00

05

10

15

20

25

120579

lowast

FA + L(a)

0

1

2

3

cNO

S (2minus

DD

CT)

120579

lowast

L FAN FA + L(b)

00

05

10

15

20

iNO

S (2minus

DD

CT)

L FAN

120579

lowast

FA + L(c)



4 Discussion


2O2 NO2 and








2O2and



L FAN

SOD

-1(2minus

DD

CT)

0

2

4

6

8 120579

lowast

lowast

FA + L(a)

lowast

00

05

10

15

20

25

CAT

(2minus

DD

CT)

L FAN FA + L(b)

120579

lowast

0

2

4

6

8

10

HO

-1(2minus

DD

CT)

L FAN FA + L(c)












2




L FAN0

2

4

6

8G

ST ac

tivity

(Ug

)120579

lowast

FA + L(a)

L FAN00

05

10

15

20

GR

activ

ity (U

g)

FA + L(b)

0

2

4

6

8

GPX

activ

ity (U

g)

L FAN

120579

lowast

FA + L(c)






2O2



Competing Interests



Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















L FAN

SOD

-1(2minus

DD

CT)

0

2

4

6

8 120579

lowast

lowast

FA + L(a)

lowast

00

05

10

15

20

25

CAT

(2minus

DD

CT)

L FAN FA + L(b)

120579

lowast

0

2

4

6

8

10

HO

-1(2minus

DD

CT)

L FAN FA + L(c)












2




L FAN0

2

4

6

8G

ST ac

tivity

(Ug

)120579

lowast

FA + L(a)

L FAN00

05

10

15

20

GR

activ

ity (U

g)

FA + L(b)

0

2

4

6

8

GPX

activ

ity (U

g)

L FAN

120579

lowast

FA + L(c)






2O2



Competing Interests



Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















L FAN0

2

4

6

8G

ST ac

tivity

(Ug

)120579

lowast

FA + L(a)

L FAN00

05

10

15

20

GR

activ

ity (U

g)

FA + L(b)

0

2

4

6

8

GPX

activ

ity (U

g)

L FAN

120579

lowast

FA + L(c)






2O2



Competing Interests



Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article photobiomodulation therapy decreases ...rua vergueiro, / liberdade, - s ao paulo,...

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