β-glucan protects against burn-induced oxidative organ damage in rats

h -glucan protects against burn-induced oxidative organdamage in ratsHale Z.Toklu a ,Go ¨ksel S ¸ener a ,*,Nermina Jahovic b ,Bahar Uslu c ,Serap Arbak c ,Berrak C ¸.Yeg˘en b aMarmara University,School of Pharmacy,Department of Pharmacology,Haydarpas ¸a,Istanbul 34668,TurkeybMarmara University,School of Medicine,Department of Physiology,Istanbul,Turkey cMarmara University,Department of Histology and Embryology,Istanbul,TurkeyReceived 18April 2005;received in revised form 9May 2005;accepted 26July 2005AbstractThermal injury may lead to systemic inflammatory response,and multiple organ failure.Generation of reactive oxygen radicals and lipid peroxidation play important roles in burn-induced remote organ injury.In the present study,we investigated the putative protective effect of local or systemic h -glucan treatment on burn-induced remote organ injury.Wistar albino rats were exposed to 908C bath for 10s to induce thermal trauma.h -glucan (3.75mg/rat locally or 50mg/kg orally)or saline was administered immediately after the trauma and were repeated twice daily in 48h groups.Rats were decapitated either 6or 48h after burn injury and the skin,lung,liver,ileum and kidney tissues were taken for the measurement of malondialdehyde (MDA)—an index of lipid peroxidation —and glutathione (GSH)—a key antioxidant —levels.Neutrophil infiltration was evaluated by the measurement of tissue myeloperoxidase (MPO)activity,while the tumor necrosis factor-a (TNF-a )levels were measured in serum samples.Skin tissues were also examined microscopically.Severe skin scald injury (30%of total body surface area)caused significant decreases in GSH levels of the liver and intestinal tissues (p b 0.01–b 0.001),while MDA levels were significantly (p b 0.01–p b 0.001)increased at post-burn 6and 48h.Both local and systemic h -glucan treatments significantly reversed (p b 0.01–p b 0.001)the elevations in MDA levels,while reduced GSH levels were reversed back to control levels (p b 0.01–p b 0.001);and the raised MPO levels were significantly decreased (p b 0.05–p b 0.001).The results indicate that both systemic and local administration of h -glucan were effective against burn-induced oxidative tissue damage in the rat.h -glucans,besides their immunomodulatory effects,have additional antioxidant properties.Therefore,h -glucans merit consideration as therapeutic agents in the treatment of burn injuries.D 2005Elsevier B.V .All rights reserved.Keywords:Glucan;Burn;Thermal injury;Oxidative;Antioxidant1567-5769/$-see front matter D 2005Elsevier B.V .All rights reserved.doi:10.1016/j.intimp.2005.07.016*Corresponding author.Tel.:+902164142962;fax:+902163452952.E-mail address:gokselsener@ (G.S ¸ener).International Immunopharmacology 6(2006)156–169/locate/intimp1.IntroductionDespite recent advances,thermal trauma,which is one of the most common problems faced in the emer-gency room,may cause damage to multiple organs distant from the original burn wound and may lead to sepsis and multiple organ failure,and thereby morbidity and mortality in burn patients[1].Major thermal injury induces the activation of an inflammatory cascade, resulting in local tissue damage,which contributes to the development of subsequent immunosuppression, increased susceptibility to sepsis and deleterious sys-temic effects in all the organ systems distant from the original wound,yielding to multiple organ failure [2].The inflammatory response syndrome in shock-like states might frequently be accompanied by oxidative cell/tissue damage in one or more organ systems in the body[3,4].Several studies have demonstrated that burn injury is associated with oxygen radical-induced lipid peroxidation,which is an autocatalytic mechanism leading to oxidative destruction of cellular membranes, and their destruction can lead to the production of toxic, reactive metabolites and cell death[5,6].The role of free oxygen radicals in ischemic insult or organ failure is well documented[7,8].The inflammatory response, which leads to hyperactivation of tissue neutrophils also contributes to oxidative cell/tissue damage[3]. Macrophages are also major producers of pro-inflam-matory mediators and their productive capacity for these mediators is markedly enhanced following ther-mal injury[9].Production of pro-inflammatory media-tors such as prostaglandin E2,reactive nitrogen intermediates,interleukin(IL)-6and tumor necrosis factor(TNF)-a,is markedly enhanced following ther-mal injury[9,10].There have been several reports indicating that circulating levels of IL-1h,IL-6and TNF-a are increased in patients with burn injury[11]. Since it appears that tissue injury after thermal trauma is mediated by both reactive oxygen metabolites (ROM)and activated neutrophils and macrophages [12–14],antioxidants or free radical scavengers, given in the post-burn period,exert protective effects against thermal trauma-induced oxidative tissue damage and multiple organ failure[15,16].h-glucans are glucose polymers found in the cell walls of yeast,fungi and cereal plants.The beneficial effects on the immune system and the lack of toxic or adverse effects[17,18]had focused the studies on h-glucan molecule.Currently,h-glucan is accepted to be one of the most powerful immune response modifiers [19].Several studies conducted in the past decade have showed that it inhibits tumor development [20,21],enhances defense against bacterial,viral,fun-gal,parasitic challenge[22–26],activates macro-phages[27,28],induces production of cytokines [29–31],nitric oxide(NO),arachidonic acid metabo-lites[32,33],increases hematopoesis,exerts radiopro-tective effects,improves wound healing by inducing the macrophage release of wound growth factors [34,35]and lowers serum lipids[36,37].Among sev-eral mechanisms proposed for the protective effects of h-glucan,a major one is related to antioxidant capa-city of the molecule[38,39].Based on the free radical scavenging activity of h-glucan protection in different inflammatory models, we investigated the possible protective effects of h-glucan against burn-induced injury of the skin and the oxidative damage of the remote organs,by the histo-pathological examination of the skin tissue as well as by the measurement of serum TNF-a,malondialde-hyde(MDA)and glutathione(GSH)levels,and mye-loperoxidase(MPO)activity in the lung,liver,kidney and the intestine.2.Materials and methodsAll experimental protocols were approved by the Mar-mara University School of Medicine Animal Care and Use Committee.2.1.Animals and the induction of thermal injuryWistar albino rats of both sexes,weighing200to250g, were fasted for12h,but allowed free access to water before the experiments.The animals were kept in individual wire-bottom cages,in a room at a constant temperature(22F2 8C)with12-h light and dark cycles,and were fed a standard rat chow.The rats were randomly divided into the seven groups of eight rats(four males and four females)each: control group,vehicle-treated6and48h burn groups,local glucan-treated6and48h burn groups,and oral glucan-treated6and48h burn groups.Under brief ether anesthesia, dorsum of the rats was shaved,exposed to908C water bath for10s,which resulted in partial-thickness second-degree skin burn involving30%of the total body surface area.All the animals were then resuscitated with physiological salineH.Z.Toklu et al./International Immunopharmacology6(2006)156–169157solution(10ml/kg,subcutaneously,s.c.).Rats were decapi-tated either6or48h after burn injury.2.2.b-glucan treatmenth-glucan(1,3-1,6-h-d-glucan),which is in the micro-particulate form,was prepared from Saccharomyces cerevi-siae yeast(Mustafa Nevzat Drug Company,Turkey)and was suspended in saline.h-glucan was administered by an orogastric catheter at a dose of50mg/kg or was evenly pasted on the burn wound(3.75mg/rat)in its ointment form. The treatments were made immediately after the trauma and were repeated twice daily in48h groups.Vehicle-treated burn groups received either intragastric saline or ointment base(emulsifying wax 2.430g,carboxy polypropylene 0.060g,nipagin0.030g,nipazol0.015g,vaseline4.050 g,liquid paraffin1.620g,propylene glycol1.500g,sodium hydroxide0.0098g,distilled water qs30g)on their wounds.Since the results of all parameters were not differ-ent among both vehicle groups,the data of local and oral treatments were pooled.In order to rule out the effects of anesthesia,the same protocol was applied in the control group,except that the dorsums were dipped in a258C water bath for10s.The rats were decapitated either at6 or48h after the burn injury,and trunk blood was collected to measure serum TNF-a levels.The skin,lung,liver,ileum and kidney samples were immediately taken and stored at À708C.Malondialdehyde(MDA)and glutathione(GSH) levels,and myeloperoxidase(MPO)activity were measured later in all tissue samples.2.3.Biochemical assays2.3.1.Measurement of serum TNF-a levelsTNF-a was evaluated by a RIA-IRMA(radioimmunoas-say-immunoradiometric assay)method.All samples were assayed in duplicates using the commercial kit(Biosource Europe S.A.,Nivelles,Belgium).The activity of radioactive assays was measured by a gamma counter(LKB WALLAC 1270RACK Gamma Counter,Turku,Finland).TNF-a in the serum samples was expressed as ng/ml.2.3.2.Malondialdehyde(MDA)and glutathione(GSH) assaysTissue samples were homogenized with ice-cold150 mM KCl for the determination of MDA and GSH levels. The DMA levels were assayed for products of lipid perox-idation by monitoring thiobarbituric acid reactive substance formation as described previously[40].Lipid peroxidation was expressed in terms of MDA equivalents using an extinction coefficient of1.56Â105MÀ1cmÀ1and results are expressed as nmol MDA/g tissue.GSH measurements were performed using a modification of the Ellman proce-dure[41].Briefly,after centrifugation at3000r/min for10 min,0.5ml of supernatant was added to2ml of0.3mol/l Na2HPO4.2H2O solution.A0.2ml solution of dithiobisni-trobenzoate(0.4mg/ml1%sodium citrate)was added and the absorbance at412nm was measured immediately after mixing.GSH levels were calculated using an extinction coefficient of13600MÀ1cmÀ1.Results are expressed inA mol GSH/g tissue.2.3.3.Myeloperoxidase activityMPO activity in tissues was measured by a procedure similar to that described by Hillegass et al.[42].Samples of liver,lung or intestinal tissue were homogenized in50mM potassium phosphate buffer(PB),with pH6.0,and centri-fuged at41400g for10min.The pellets were then suspended in50mM PB containing0.5%hexadecyltrimethylammo-nium bromide(HETAB).After three freeze and thaw-cycles, with sonication between cycles,the samples were centrifuged at41400g for10min.Aliquots(0.3ml)were added to2.3ml of reaction mixture containing50mM PB,o-dianisidine,and 20mM H2O2solution.One unit of enzyme activity was defined as the amount of MPO present that caused a change in absorbance,measured at460nm for3min.MPO activity was expressed as U/g tissue.2.4.Histological preparation and analysisSkin tissue samples were fixed in10%formaldehyde and processed routinely for embedding in paraffin.Following dehydration in ascending series of ethyl alcohol,tissue samples were cleared in xylene.Paraffin sections of5–6 A m were stained with Masson’s trichrome in order to eval-uate collagen fibers.Tissue sections were also stained with Haematoxylin and Eosin(H&E)to indicate leukocyte infiltration.Activated mast cells were visualized by tolui-dine blue staining method.Microscopic scoring was done by experienced histologists,who were unaware of the treat-ments received by the animals.Epithelial degeneration, dermal edema,hair follicular degeneration,subcutaneous degeneration and vasocongestion were evaluated for the skin tissue.Scores for each criterion are given as0,none; 1,mild;2,moderate,3,severe.At least five microscopic areas were examined to score each specimen.2.5.Statistical analysisStatistical analysis was done using a GraphPad Prism3.0 (GraphPad Software,San Diego;CA;USA).All data are expressed as means F SEM.Groups of data were compared with an analysis of variance(ANOV A)followed by Tukey’s multiple comparison tests.Values of p b0.05were consid-ered as significant.H.Z.Toklu et al./International Immunopharmacology6(2006)156–169 1583.Results3.1.Serum NTF-a levelsIn the saline-treated burn groups,serum TNF-a levels in both early (6h)and later (48h)phases of the injury were significantly increased when compared to control group (p b 0.001).Although the TNF-a levels in the h -glucan-treated groups were still significantly higher than that of the control group,both oral and local h -glucan treatments significantly reduced the raised TNF-a levels (Fig.1).3.2.Malondialdehyde (MDA)levelsLipid peroxidation in the tissues was expressed as MDA levels.The MDA levels in the skin,liver,lung,kidney and ileum were significantly higher in the vehicle-treated burn groups than the levels in the control group (p b 0.01–p b 0.001).Oral or local treatments with h -glucan signifi-cantly reversed the elevations in MDA levels equally in all the tissues distant to burn injury (p b 0.05–p b 0.001;Figs.2a,3a,4a ).Although locally applied h -glucan reduced the MDA levels of the burned skin tissue significantly (p b 0.05–0.01),oral h -glucan-induced reductions in the skin MDA did not reach statistical significance.3.3.Glutathione (GSH)levelsBurn injury caused significant decreases (p b 0.001)in GSH levels of the skin and remote tissues,compared with the control group.In the skin,h -glucan treatment given either orally or locally,significantly (p b 0.001)reversed the GSH levels back to the control values in both early and late phases of burn injury (Fig.2b).However,in the early phase of injury,local treatment had no significanteffect on burn-depleted GSH contents of the tissues remote to burn (Figs.3b,4b,5b,6b ).Except the ileal tissue,topical treatment was found to be equally effective as the systemic administration when the post-burn application was repeated throughout the 48h follow-up period (p b 0.05–0.001).3.4.Myeloperoxidase (MPO)activityAs an indicator of tissue neutrophil infiltration,the MPO activities were significantly higher (p b 0.05–0.001)in all the tissues of the 6and 48h burn groups than those in the control group (Figs.2c,3c,4c,5c,6c ).In the 6th h of burn injury,tissue neutrophil infiltration in all the tissues was abolished by both local and systemic glucan treatments (p b 0.05–0.001).Similarly,renal and hepatic MPO activ-ities in the 48h burn group were also abolished by both treatment procedures.On the other hand,burn-induced neu-trophil infiltration in the skin and ileum at 48h was only blocked by topical glucan application,while oral h -glucan was effective in the late phase of pulmonary injury.3.5.Histopathological observationsBurn-induced cellular damage was observed in the skin tissues of 6and 48h burn groups (Fig.7a,d)demonstrating structural degeneration of epidermis and dermis with necro-sis,loosely arranged disintegrated collagen fibers,vasocon-gestion and edema,accompanied by inflammatory cell infiltration.These changes were reversed by h -glucan treat-ment (Fig.7b,c,e,f).Burn groups revealed extreme damage of epidermis hair follicles,increased activation of mast cells (Fig.8a,d)and extensive edema while mast cell activation seemed to be normal in h -glucan-treated groups (Fig.8b,c,e,f).Both topical and systemic administrations of h -glucan attenuated burn-induced dermal degeneration with densely arranged thick collagen fibers,while the totalC255075******+++++++++Burn6 h 48 h+++T N F (n g /m l )*********vehicle-treated oral glucan-treatedlocal glucan-treatedFig.1.The effect of oral or local h -glucan treatment on the serum tumor necrosis factor (TNF)-a levels.TNF-a was measured by a RIA-IRMA method 6or 48h following burn trauma (n =8in each group).Results were analysed in one-way ANOV A and expressed as mean F SEM.***p b 0.001:compared with the control (C)group.+++p b 0.001:compared with the respective vehicle-treated burn group.H.Z.Toklu et al./International Immunopharmacology 6(2006)156–169159damage scores were also reduced significantly (p b 0.001;Fig.9).4.DiscussionIn accordance with our previous observations [1,15,16],the results of the present study demonstratethat burn injury caused oxidative tissue damage not only in the skin but also in the liver,lung,kidney and ileum,as assessed by increased lipid peroxidation and decreased GSH levels.h -glucan treatment depressed lipid peroxidation and replenished GSH content in the tissues,verifying the protective effect of h -glucan against oxidative tissue injury.Since oxidative injury of these tissues was accompanied with neutrophilBurnC6 h 48 hBurnM D A (n m o l /g )skin0.00.51.01.5******+++++++G S H (µm o l /g )C1020306 h 48 hBurn**++M P O (U /g )*+*+vehicle-treated oral glucan-treated local glucan-treateda)b)c)Fig.2.The effect of oral or local h -glucan treatment on the malondialdehyde (MDA;an index of lipid peroxidation),glutathione (GSH;a key antioxidant)and myeloperoxidase (MPO;indirect measurement of neutrophil infiltration)levels in the skin.MDA,GSH and MPO levels were determined by spectrophotometric methods 6or 48h after burn trauma (n =8in each group).Results were analysed in one-way ANOV A and expressed as mean F SEM.*p b 0.05,**p b 0.01,***p b 0.001:compared with the control (C)group.+p b 0.05,++p b 0.01,+++p b 0.001:compared with the respective vehicle-treated burn group.H.Z.Toklu et al./International Immunopharmacology 6(2006)156–169160infiltration,while h -glucan treatment,along with its antioxidant activity,suppressed neutrophil accumula-tion,it may be suggested that burn-induced oxidative injuries in these tissues are neutrophil-dependent and are reversible by h -glucan treatment.Furthermore,increased serum TNF-a ,which plays a pivotal role in the inflammatory processes,was also reduced by h -glucan treatment.Local application of glucan was found to be more effective especially in the directly affected skin tissue,while systemic administration was more potent on the distant organs in both the early and later phases of injury.Among h -glucans,the major components of yeast,bacterial,and fungi cell walls,the h -glucans derived from S.cerevisiae have been the most extensively studied class with highest biological effects [18].Detailed analysis of the interaction of human cells with h -glucans has demonstrated that complementM D A (n m o l /g )lungG S H (µ m o l /g )6 h 48 hBurnM P O (U /g )a)b)c)Fig.3.The effect of oral or local h -glucan treatment on the malondialdehyde (MDA;an index of lipid peroxidation),glutathione (GSH;a key antioxidant)and myeloperoxidase (MPO;indirect measurement of neutrophil infiltration)levels in the lung.MDA,GSH and MPO levels were determined by spectrophotometric methods 6or 48h after burn trauma (n =8in each group).Results were analysed in one-way ANOV A and expressed as mean F SEM.**p b 0.01,***p b 0.001:compared with the control (C)group.+p b 0.05,++p b 0.01,+++p b 0.001:compared with the respective vehicle-treated burn group.H.Z.Toklu et al./International Immunopharmacology 6(2006)156–169161receptor type 3[43,44],and novel Dectin-1receptors are primarily responsible for both the binding and biological effects [45–47].It has been suggested that h -glucans bind to scavenger receptors and inhibit the interactions of monocyte membranes with classical scavenger ligands [19].h -glucan receptor activity has also been reported on a variety of leukocytes,including macrophages,neutrophils,eosinophils and natural killer cells,as well as on non-immune cells including endothelial cells,alveolar epithelial cells and fibroblasts [19].Recent studies have shown that h -glucan exerts the protective effect by the inhibition of early activation of tissue nuclear factor (NF)-n D and IL-6[48].h -glucans,especially in particulate form,were shown to induce pro-inflammatory and antimicrobial responses through the toll-like receptors (TLR),the signal transducing molecules critical for the induction of innate immunity,especially TLR-2C255075100******++++++++++++6 h 48BurnM D A (n m o l /g )liverC1234*******+++++++Burn6 h 48hG S H (µ m o l /g )C1020306 h 48hBurn++++++***++++++M P O (U /g )*vehicle-treated oral glucan-treated local glucan-treateda)b)c)hFig.4.The effect of oral or local h -glucan treatment on the malondialdehyde (MDA;an index of lipid peroxidation),glutathione (GSH;a key antioxidant)and myeloperoxidase (MPO;indirect measurement of neutrophil infiltration)levels in the hepatic tissues.MDA,GSH and MPO levels were determined by spectrophotometric methods 6or 48h after burn trauma (n =8in each group).Results were analysed in one-way ANOV A and expressed as mean F SEM.*p b 0.05,***p b 0.001:compared with the control (C)group.++p b 0.01,+++p b 0.001:compared with the respective vehicle-treated burn group.H.Z.Toklu et al./International Immunopharmacology 6(2006)156–169162and Dectin-1[45–47].Recently,it was reported that h -glucan induces cardioprotection against ischemic injury through a TLR-mediated pathway [49].The results of the previous studies about the oxidative/antioxidative activity of h -glucans are controversial.Some studies have demonstrated that glucans stimu-late cytokine release,cause generation of reactive oxygen metabolites (ROM)and nitric oxide and release arachidonic acid metabolites [32,33].On the other hand,some other researchers have claimed that protective effect was due to their antioxidant capacity [38,39].In our study h -glucan treatment was found to be effective against oxidative injury of the burned skin and the remote tissues.It is likely that h -glucans could have influenced the immune cells activated by an oxidative damage due to a thermal challenge.Admin-istration of glucans either systemically or as an oint-ment in the presence of a thermal insult inhibited theC2550756 h 48 hBurn******+++++++++++M D A (n m o l /g )ileumC123G S H (µ m o l /g )Burn6 h 48h******++++*****C102030406 h 48 hBurnM P O (U /g )**+++++++vehicle-treated oral glucan-treatedlocal glucan-treateda)b)c)Fig.5.The effect of oral or local h -glucan treatment on the malondialdehyde (MDA;an index of lipid peroxidation),glutathione (GSH;a key antioxidant)and myeloperoxidase (MPO;indirect measurement of neutrophil infiltration)levels in the ileum.MDA,GSH and MPO levels were determined by spectrophotometric methods 6or 48h after burn trauma (n =8in each group).Results were analysed in one-way ANOV A and expressed as mean F SEM.*p b 0.05,**p b 0.01,***p b 0.001:compared with the control (C)group.++p b 0.01,+++p b 0.001:compared with the respective vehicle-treated burn group.H.Z.Toklu et al./International Immunopharmacology 6(2006)156–169163TNF-a response,suggesting a mechanism in blunting the host’s inflammatory response to injury through the inhibition of cytokine activity [48].Consistently,Nakagawa et al.[25]and Solyths and Quinn [30]have also shown that cytokine release can be modu-lated by h -glucans.Experimental and clinical studies have shown that any harmful tissue event,such as infection,trauma or anoxia,is perceived by macrophages and monocytes,which in turn secrete cytokines such as IL-1and TNF-a .These cytokines activate inflammatory cells (neu-trophils,macrophages/monocytes,platelets,masto-cytes)that release large amounts of toxic ROMs,which cause cellular injury via several mechanisms,including peroxidation of membrane lipids as well as oxidative damage of proteins and DNA [50].Lipid peroxidation is believed to be a major cause of destruction and damage to cell membranes,sinceM D A (n m o l /g )kidneyG S H (µ m o l /g )C6 h 48hBurnM P O (U /g )a)b)c)Fig.6.The effect of oral or local h -glucan treatment on the malondialdehyde (MDA;an index of lipid peroxidation),glutathione (GSH;a key antioxidant)and myeloperoxidase (MPO;indirect measurement of neutrophil infiltration)levels in the kidney.MDA,GSH and MPO levels were determined by spectrophotometric methods 6or 48h after burn trauma (n =8in each group).Results were analysed in one-way ANOV A and expressed as mean F SEM.*p b 0.05,**p b 0.01,***p b 0.001:compared with the control (C)group.+p b 0.05,++p b 0.01,+++p b 0.001:compared with the respective vehicle-treated burn group.H.Z.Toklu et al./International Immunopharmacology 6(2006)156–169164polyunsaturated fatty acids of the cellular membranes are degraded and disrupted by this process.Membrane peroxidation can lead to changes in membrane fluidity and permeability and also to enhanced rates of protein degradation,and these will eventually lead to cell lysis [51].In the present study,the levels ofMDA,Fig.7.Evaluation of morphological changes and collagen fibers by Masson’s trichrome (Â66)and Haematoxylin–Eosin (Â132)staining method.(a)Burn group (6h):epidermal desquamation due to extensive degeneration (white arrow),disintegrated collagen fibers in edematous dermis (arrowhead),vasocongestion;hair follicular and sebaceous glandular degeneration (double asterisk),Masson’s trichrome.Inset:extensive leukocyte infiltration (arrow).H &E.(b)Burn+oral h -glucan treatment group (6h):regeneration at epidermis and dermis region with thick collagen fibers;slight edema at the dermal region.Masson’s trichrome.Inset:slight leukocytic infiltration (arrow).H &E.(c)Burn+local h -glucan treatment group (6h):recovery (white arrow)at epidermis and dermis with regularly arranged collagen fibers and vasocongestion;slightly degenerated sebaceous glands and hair follicles (double asterisk)with peripheral edema (asterisk).Masson’s trichrome.Inset:few number of leukocytes (arrow),H &E.(d)Burn group (48h):epidermal loss due to extensive degeneration (white arrow),vasocongestion and edematous dermis (asterisk)with loosely arranged collagen fibers,extensive degeneration at both hair follicles and sebaceous glands.Masson’s trichrome.Inset:leukocyte infiltration (arrow),H &E.(e)Burn+oral h -glucan treatment group (48h):prominent keratinization at the epidermis with normal skin layers;thick collagen fibers in the dermis,regeneration at hair follicles and sebaceous glands.Masson’s trichrome.Inset:scanty leukocytic infiltration (arrow),H &E.(f)Burn+local h -glucan treatment group (48h):restored keratinization at the epidermis reflecting a normal histology with regular arrangement of collagen fibers and normal morphology of hair follicles and sebaceous glands.Masson’s trichrome.Inset:slight leukocyte infiltration (arrow),H &E.H.Z.Toklu et al./International Immunopharmacology 6(2006)156–169165an end product of lipid peroxidation,are significantly increased in all the tissues.This observation is in agreement with the previous studies,in which ele-vated levels of lipid products were increased from 40%to 80%above basal values as a result of oxida-tive stress [52,53].Our results also demonstrate that locally or systemically given h -glucan inhibits MDA elevations significantly in the distant tissues,while burned skin is better protected against oxidant injury by topical h -glucan.Thus,as an antioxidant,h -glucan could preserve cellular integrity and protect against both local and distant organ damage due to thermal trauma.Glutathione is an important constituent of intracel-lular protective mechanisms against several noxious stimuli including oxidative stress,while the reduced GSH is the main component of endogenous non-pro-tein sulfhydryl pool that scavenges free radicals in the cytoplasm [54,55].Because of their exposed sulfhy-dryl groups,non-protein sulfhydryls bind a variety of electrophilic radicals and metabolites that may be damaging to cells [56].It has been proposed that antioxidants,which maintain the concentration of reduced GSH,may restore the cellular defense mechanisms,block lipid peroxidation,and thus pro-tect against the oxidative tissue damage.In accor-dance with these findings,our results also verify that h -glucan maintained GSH levels,while it pro-tected against sepsis-induced oxidative tissue damage.Observations suggest that ROMs play a role in the recruitment of neutrophils into the injured tissue,but activated neutrophils are also a potential source of ROM [57,58].In the presence of neutrophil-derived myeloperoxidase (MPO),ROMs can generate hypo-cholorus acid (HOCl)and initiate the deactivation of antiproteases and activation of latent proteases,whichFig.8.Activated mast cells visualized by toluidine blue staining method (Â66).High activity of mast cells in burn groups (a,d).Decreased activity of mast cells in burn+oral (b,e)and burn+local (c,f)h -glucan treatment groups.。