International Immunopharmacology

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1 Interntionl Immunophrmology 11 (211) Contents lists vilble t SiVerse SieneDiret Interntionl Immunophrmology journl homepge: Impt of the rotenoid stxnthin on phgoyti pity nd ROS/RNS prodution of humn neutrophils treted with free ftty ids nd high gluose Betriz Alves Guerr, Rosemri Otton Postgrdute Progrm Helth Sienes CBS, Cruzeiro do Sul University, So Pulo, SP, 3342, Brzil rtile info bstrt Artile history: Reeived 3 August 211 Aepted 4 Otober 211 Avilble online 18 Otober 211 Keywords: Astxnthin Free rdils Hyperlipidemi Hyperglyemi Leukoytes Oxidtive stress Bkground: The purpose of the present study ws to evlute the effet of rotenoid stxnthin (ASTA) on humn neutrophils treted with high gluose nd free ftty ids (FFA) on the phgoyti pity nd retive oxygen/nitrogen speies prodution. Methods: The following prmeters were evluted: phgoyti pity of neutrophils by using zymosn prtiles, intrellulr nd extrellulr superoxide nion (luigenin nd DHE probes), hydrogen peroxide (H 2 O 2 phenol red), nitri oxide (Griess regent) prodution, nd mximl tivity of G6PDH. Results: There ws deresed phgoyti pity of humn neutrophils treted with high gluose (3 mm) or FFA (.1 mm) nd prtil restoring of the phgoyti pity fter ASTA-tretment ws observed. ROS nd RNS prodution ws inresed in neutrophils due to both high gluose nd FFA. This inrese in ROS/ RNS prodution ws lso prtilly prevented by ASTA tretment. Both gluose nd FFA inresed the G6PDH tivity. We show tht ASTA provides modest improvement of ellulr funtions fter ells hve been treted with high gluose nd FFA. Conlusions: In summry, this study showed tht both high gluose nd mixture of FFA re potent induers of ROS/RNS prodution on neutrophils s observed by higher levels of superoxide nion, hydrogen peroxide nd NO prodution. Also, these metbolites derese the phgoyti pity of neutrophils nd inrese the G6PDH tivity. Overll, ASTA-tretment ws ble to redue prtilly ROS/RNS prodution by reduing the vilbility of NADPH nd reover phgoyti pity of neutrophils. 211 Elsevier B.V. Open ess under the Elsevier OA liense. 1. Introdution Dibetes is speil ondition where both gluose nd free ftty ids (FFA) re inresed in plsm. This ft ours by redued insulin tion on trget tissues or filure of insulin seretion by pnreti β-ells. Chroni exposure to high gluose nd FFA n use dmge in different ell types by vriety of mehnisms inluding gluolipotoxiity. Beuse n elevted level of gluose is one of the metboli hllmrks of dibetes, muh ttention hs been given to the sugr's oxidtive hemistry. One widely studied mehnism involves utoxidtion of gluose itself, whih genertes retive oxygen speies (ROS) suh s hydroxyl rdil nd lso ross-links proteins [1,2]. Other potentil mehnisms inlude gluose nd FFAstimulted mitohondril oxidtive phosphoryltion, prodution of retive intermedites by phgoyti white blood ells t sites of inflmmtion (e.g. therosleroti tissue), nd peroxidtion of polyunsturted ftty ids, ll of whih hve been implited in the genertion of retive intermedites in vitro [3]. In ft, it hs been Corresponding uthor t: Av. Regente Feijó, 1295, 3342, São Pulo, SP, Brzil. Tel./fx: E-mil ddress: rosemri.otton@ruzeirodosul.edu.br (R. Otton). reported tht gluose nd FFA initite the formtion of ROS in musles, dipoytes, pnreti β-ells nd other ells [4 6]. Ptients with dibetes re ffeted by frequent episodes of idosis used by inresing metbolism of ftty ids. This pthologil ondition is orrelted with the high inidene of vsulr disese, for whih there strong orreltion with tissue dmge medited by oxidtive stress [7 9]. However, there is onsiderble ontroversy regrding the nture, mgnitude, nd mehnisms of oxidtive stress in the dibeti stte. Overprodution of ROS or filure in intrellulr defenses ginst ROS hs been held s one mjor ontributor to long-term dibeti omplitions [6,1 14]. Peripherl blood ells suh s phgoytes (mrophges nd neutrophils) re well known to be tivted in dibetes nd thus produe lrge mounts of ROS. The mjor pthwy through whih neutrophils generte oxidnts begins with their membrne-bound NADPHoxidse, whih produes superoxide with subsequent hydrogen peroxide prodution [15,16]. The peroxide n then be used by nother phgoyte enzyme, myeloperoxidse. The onnetion between this pthwy nd dibetes my be tht, t lest in the rtery wll of dibeti nimls, hyperglyemi n tivte protein kinse C [17,18], whih leds to phgoyte tivtion, seretion of myeloperoxidse, nd oxidnt genertion. These hnges might enhne the prodution of superoxide nd hydrogen peroxide, whih myeloperoxidse Elsevier B.V. Open ess under the Elsevier OA liense. doi:1.116/j.intimp

2 B.A. Guerr, R. Otton / Interntionl Immunophrmology 11 (211) onverts into more potent ytotoxi oxidnts, suh s hypohlorous id. Superoxide nion n ret with nitri oxide (NO ) nd produes peroxinitrite, powerful oxidnt gent. Free ftty ids tht ommonly re overbundnt in dibetes n lso tivte phgoytes in vitro [19,2]. Unontrolled ROS prodution n use oxidtive dmge to vriety of biologi mromoleules suh s nulei ids (DNA, RNA), lipids, proteins nd rbohydrtes [21]. However, there re intrellulr ntioxidnt defense mehnisms whih involve both enzymti nd non-enzymti strtegies. Among the non-enzymti ompounds we n find rotenoids tht hve ntioxidnt properties nd therpeuti role in vrious diseses [22]. The ketorotenoid stxnthin (ASTA), 3,3 -dihydroxy-β,βrotene-4,4 -dione is ubiquitous in nture, espeilly in the mrine environment [23]. It is red pigment ommon in mny mrine nimls, suh s slmonids, shrimps, lobsters nd ryfish, ontributing to the pinkish-red olor of their flesh [24]. ASTA my t s strong ntioxidnt by donting the eletrons nd reting with free rdils to onvert them into more stble produt nd terminte free rdil hin retion in wide vriety of living orgnisms [25,26]. In reent yers, mjor number of studies on ASTA hve demonstrted its ntioxidnt effet, for exmple the quenhing effet on singlet oxygen, strong svenging effet on superoxide, hydrogen peroxide, nd hydroxyl rdils nd n inhibitory effet on lipid peroxidtion [27 3]. In ddition, severl other biologi tivities of ASTA, inluding nti-ner, nti-inflmmtory, ntidibeti, immunomodultory tivities nd neuroprotetive effet lso hve been reported [25,31]. Antioxidnts, inluding number of rotenoids, hve been hypothesized to inhibit oxidtive stress nd ply protetive role ginst hroni diseses suh s dibetes. The purpose of this study ws to evlute the effets of ASTA in the phgoyti pity nd ROS/RNS prodution indued by FFA nd high gluose in humn neutrophils. 2. Mterils nd methods 2.1. Regents Astxnthin (ASTA) nd most of the other hemils were purhsed from Sigm-Aldrih Chemil Compny (St. Louis, MO, USA), exepting the RPMI-164 ulture medium nd luigenin, whih me from Invitrogen (CA, USA). Common regents for buffers (e.g. PBS) nd regulr lbortory solutions were obtined from Lbsynth (Didem, SP, Brzil) Subjets The Ethil Committee of the Cruzeiro do Sul Univerity pproved the experimentl proedure of this study. Around 3 helthy dult women nd men (men ge 28.±9.) were inluded in the present study. The subjets reruited did not present ny systemi or topil therpeuti regimen, smoking history, lohol hbits, obesity or ny other systemi diseses t lest for the lst 2 months (bsed on n nmnesis protool) Cell isoltion nd ulture ondition Neutrophils were obtined through the olletion of humn peripherl blood by venipunture proedure in vuum/silionized tubes ontining.1 mm EDTA. Peripherl blood neutrophils were isolted under sterile onditions by using density grdient present in the regent Histopque 177 (Sigm-Aldrih), ording to the mnufturer's instrution. Briefly, blood (~2 ml) ws diluted (1:2) with phosphte-buffered sline PBS (133.8 mm NCl, 2.7 mm KCl,.9 mm KH 2 PO 4, 6.4 mm N 2 H 2 PO 2, ph 7.4) nd entrifuged for 3 min, 3 g t room temperture. The plsm nd the intermediry lyer were removed nd both neutrophils nd erythroytes were olleted from the sediment. Erythroytes were lysed with hemolysis solution (15 mm NH 4 Cl, 1 mm NHCO 3,.1 mm EDTA, ph 7.4) nd subsequently entrifuged for 1 min (6 g t 4 C). This proedure ws repeted twie for totl red blood ell lyses. Therefter, neutrophils were wshed with PBS followed by entrifugtion for 1 min (6 g t 4 C). After entrifugtion, the neutrophils were ounted in Neubuer hmber using Trypn blue (1%). Neutrophils (1 1 6 / ml) were ultured in 1 ml of RPMI-164 medium supplemented with 1% fetl bovine serum, 2 mm Hepes, 2 mm glutmine, nd ntibiotis (streptomyin 1 units/ml nd peniillin 2 units/ml). The ells were treted with.1 mm of mixture of free ftty id prepred s desribed in our previous study [32,33] nd with high gluose (3 mm) dded or not of 2 μm of ASTA solubilized in DMSO. Then, fter tretment we hd the following experimentl groups: ontrol (without tretment), high gluose, high gluose+asta, FFA nd FFA+ASTA. The ells were ultured t 5% CO 2 for up to 24 h t 37 C. After this period, the ells were olleted, entrifuged nd stored t 8 C to ssy enzyme tivity. For ute effets of FFA nd high gluose on ell ROS prodution, fter isoltion, neutrophils were resuspended in Tyrode's solution (137 mm NCl, 2.68 mm KCl,.49 mm MgCl 2, 12 mm NHCO 3,.36 mm NH 2 PO 4, 5.6 mm D-gluose, nd 5 mm id HEPES, ph 7.4) nd freshly used Phgoyti pity Neutrophils (1 1 6 ell/ml) were inubted for 3 min t 37 C in 1 ml RPMI 164 medium with opsonized zymosn prtiles nd treted with or without 2 μm of ASTA,.1 mm of FFA nd 3 mm of gluose. The prtiles (5 1 6 /ml) were opsonized by inubtion in the presene of ontrol serum for 3 min t 37 C. The ells were inubted with prtiles nd ounted fter ytoentrifugtion. The sore of phgoytosis ws expressed fter the ounting of one hundred ells. The number of prtiles phgoyted ws multiplied by one, two nd three, ording to riterion estblished to sore [34] Dihydroethidium ssy Dihydroethidium (DHE) is fluoresene probe used to mesure the intrellulr superoxide nion prodution. One inside the ell, DHE is rpidly oxidized to ethidium ( red fluoresent ompound) by superoxide nd/or H 2 O 2 (in the presene of peroxidse). Neutrophils (5 1 5 /well) were inubted with 5 μm DHE for 15 min t room temperture in the drk. At the beginning of the ssy, the ells were stimulted with phorbol myristte ette (PMA 2 ng/ well), promoter of respirtory burst in phgoytes nd treted with or without 2 μm of ASTA,.1 mm of FFA nd 3 mm of gluose in Tyrode's buffer for 3 min. The fluoresene ws nlyzed in miroplte reder (Ten, Slzburg, Austri) (396 nm wvelength exittion nd 59 nm wvelength emission). Results re expressed s reltive fluoresene units (RFU) Luigenin ssy The luigenin hemiluminesent probe ws utilized to mesure the extrellulr superoxide nion ontent minly produed through NADPH-oxidse tivtion. Luigenin releses energy in the form of light fter exittion by superoxide nion. The hemiluminesene produed ws monitored by luminometer for 2 min (Ten, Slzburg, Austri). Luigenin (5 μm) ws dded to ells (5 1 5 /well) treted with or without 2 μm of ASTA,.1 mm of FFA nd 3 mm of gluose in Tyrode's buffer. The experiments were rried out in the presene nd bsene of PMA (2 ng/well). The results re expressed s hemiluminesene reltive units. The sttistil nlysis ws performed by AUC lultion (re under the urve).

3 2222 B.A. Guerr, R. Otton / Interntionl Immunophrmology 11 (211) Hydrogen peroxide prodution Hydrogen peroxide (H 2 O 2 ) prodution ws mesured ording to Pik nd Mizel [35], bsed on horserdish peroxidses, whih tlyze the phenol red oxidtion by H 2 O 2. Neutrophils (5 1 5 /well) were inubted with or without 2 μm of ASTA,.1 mm of FFA nd 3 mm of gluose in Tyrode's buffer, mixed with.28 mm phenol red nd horserdish peroxidse (1 units/mg) t 37 C for 1 h. The prodution of H 2 O 2 ws mesured in the bsene nd presene of PMA (2 ng/well). The retion ws terminted by lkliniztion (ddition of 1 μl of NOH 1 M solution) nd bsorbne t 62 nm ws mesured to evlute H 2 O 2 onentrtion (ompred to stndrd urve) Nitri oxide prodution NO prodution ws performed ording to Ding et l. [36] through nitrite determintion. Nitri oxide is rpidly onverted into nitrite in queous solutions nd, therefore, the totl nitrite n be used s n inditor of nitri oxide onentrtion. The spetrophotometri nlysis of the totl nitrite ontent ws performed by using the Griess regent (1% sulfnili id,.1% N-1-nphthyl-ethylenedimine dihydrohloride) in superntnts. Neutrophils (5 1 5 /well) were ultured with or without 2 μm of ASTA,.1 mm of FFA nd 3 mm of gluose nd LPSstimulted (1 ng/well) for 4 h in RPMI-164 medium supplemented with 1% fetl bovine serum. Then, the sme volume of Griess ws dded to ells nd the bsorbne ws mesured in 55 nm. The nitrite onentrtion ws determined using sodium nitrite s stndrd ( 6 μm) Assy the gluose-6-phosphte dehydrogense (G6PDH) tivity Gluose-6-phosphte dehydrogense (G6PDH), EC 1.1.l.49, is key regultory enzyme of the oxidtive segment of the pentosephosphte pthwy nd produes reduing equivlents in the form of NADPH to meet some ellulr needs for redutive biosynthesis nd s ontribution to the mintenne of the ellulr redox stte [37]. The neutrophils (5 1 6 /well) fter ulture of 24 h were disrupted in the ph 8. extrtion buffer onsisting of 5 mm Tris HCl nd 1 mm EDTA. The tivity ws ssyed s desribed in [38] nd.5% (v/v) Triton-X-1 ws dded to the ssy system to omplete the extrtion of the enzyme. The following 334 μl ssy medium t ph 7.6 omposed of 8.6 mm Tris HCl, 6.9 mm MgCl 2,.4 mm NADP, 1.2 mm gluose-6-phosphte. The spetrophotometri mesurements were mde in miroplte reder (Ten, Slzburg, Austri). Enzyme tivity is expressed s nmol/ min/mg of protein Protein mesurement The mesurement of the speifi enzyme tivity of G6PDH ws entirely relted to protein onentrtions, whih were estimted by the Brdford method [39] using bovine serum lbumin s stndrd Sttistil nlysis All dt points re the men vlues with stndrd errors of t lest three independent experiments. The dt were nlyzed by one-wy ANOVA followed by the Tukey's post-test. The softwre employed for sttistil nlysis ws GrphPd Prism (version 4; GrphPd Softwre, Sn Diego, CA, USA). 3. Results 3.1. Phgoyti pity The phgoyti pity ws mesured by the inorportion of opsonized zymosn prtiles (Fig. 1). There ws signifint derese in the phgoyti pity of neutrophils fter the ells were treted with FFA nd gluose of 46% nd 39% respetively, when ompred with the ontrol group. ASTA tretment promoted signifint inrese in phgoytosis of neutrophils, s ompred with the FFA nd gluose group, by 51% nd 45%, respetively. The ddition of ASTA on the FFA group improved the phgoyti pity of neutrophils by 47% when ompred with the FFA group. The sme effet ws observed in the glu+asta group, whih presented n improvement of 22% s ompred with the gluose group Superoxide nion prodution There ws n inrese in intrellulr superoxide nion prodution s ssyed by DHE fter the tretment of the ells with gluose nd FFA to levels of ontrol-stimulted group, inditing tht both metbolites n indue neutrophil ROS prodution (Fig. 2A). When ompred with the bsl ontrol group, gluose nd FFA promoted n inrese of 98% nd 97%, respetively. In ells treted with ASTA, derese in superoxide nion prodution of bout 43% ws observed s ompred with the ontrol group. The ddition of ASTA ws not effetive in reduing the superoxide nion prodution indued by FFA nd gluose. On the ontrry, it promoted n inrese of 51% in superoxide prodution when ompred to the FFA group. The extrellulr superoxide nion prodution ws mesured by using luigenin probe (Fig. 2B). There ws diret indution of NADPH-oxidse by FFA whih promoted signifint inrese in O 2 prodution in ells. The tretment of FFA with ASTA ws unble to redue superoxide nion prodution Hydrogen peroxide prodution To promote hydrogen peroxide prodution, humn ontrol neutrophils were tivted with PMA (2 ng/well). The ells were then treted with or without 2 μm of ASTA nd 3 mm of gluose or.1 mm of FFA for 1 h. Hydrogen peroxide prodution ws inresed by 7%, 6%, 1% nd 65% in ASTA, gluose, FFA, nd FFA+ASTA respetively when ompred with the ontrol-unstimulted group. A derese of 34% in hydrogen peroxide prodution ws observed in the glu+asta Sore of phgoyti pity ,b,b ontrol ASTA gluose glu+asta FFA FFA+ASTA Fig. 1. The phgoyti pity ws mesured by inorportion of opsonized zymosn prtiles in humn neutrophils (1 1 6 /ml) treted with or without 2 μm of ASTA, 3 mm gluose nd.1 mm FFA. Results re expressed s men±sem of t lest 4 determintions. () Signifintly different ompred with the ontrol group (pb.1). (b) Signifintly different ompred with the respetive group without ASTA (pb.1). () Signifintly different ompred with the gluose group (pb.1). (d) Signifintly different ompred with the FFA group (pb.1). d

4 B.A. Guerr, R. Otton / Interntionl Immunophrmology 11 (211) A Reltive Fluoresene Unit B Chemiluminesene rbitrry unit ontrol ont+pma ASTA gluose glu+asta FFA FFA+ASTA ontrol ont+pma ASTA gluose glu+asta FFA FFA+ASTA group s ompred to gluose group nd of 37% in FFA+ASTA when ompred to the FFA group (Fig. 3) Gluose-6-phosphte dehydrogense tivity Time (minutes) Fig. 2. (A) Intrellulr (DHE) nd (B) extrellulr (luigenin) superoxide nion prodution in humn neutrophils. Cells (5 1 5 /well) were treted with or without 2 μm of ASTA nd tivted with PMA (2 ng/well) or 3 mm gluose or.1 mm FFA for 3 min. Results re expressed s men ±SEM of t lest 5 determintions. () Signifintly different ompred with the ont+pma group (pb.1). (b) Signifintly different ompred with the ASTA group (pb.1). The mximum G6PDH tivity ws ssessed by the redution of the o-ftor NADP+ to NADPH in humn neutrophils. G6PDH tivity ws inresed 97% nd 17% in gluose nd FFA groups respetively when ompred to the ontrol group. The ddition of ASTA in gluose nd FFA groups promoted derese of 39% nd 32% s ompred with gluose nd FFA respetively (Fig. 4). b 3.5. Nitri oxide prodution The NO prodution ws evluted in ells treted with gluose (Fig. 5A) nd FFA (Fig. 5B) in both bsl nd LPS-stimulted onditions. An inrese of 77%, 19% nd 146% ws observed in NO prodution in ASTA, gluose, nd glu+asta groups respetively when ompred with the ontrol-unstimulted group. After LPS-stimultion, the gluose nd glu+asta groups were inresed by 74% nd 5% respetively s ompred with the ontrol-stimulted group. The tretment of ells with FFA did not promote n inrese in NO prodution s observed fter the tretment of ells with high gluose. However, ASTA promoted n inrese of ~3% t both bsl nd LPS-stimultion onditions s ompred with the respetive ontrol group. 4. Disussion In the present study we observed deresed phgoyti pity of humn neutrophils (Fig. 1) when they were treted with high gluose (3 mm) or FFA (.1 mm) mimiking dibeti ondition in vitro. A prtil restoring of the phgoyti pity ws observed fter ASTA-tretment. Overll, ROS nd RNS prodution ws inresed in neutrophils due to both high gluose nd FFA. This inrese in ROS/RNS prodution ws lso prtilly prevented by ASTA tretment. Both gluose nd FFA inresed the G6PDH tivity (Fig. 4), probbly by inresing the flux of these metbolites throughout oxidtive pthwys whih result in inresed NADPH o-ftor ontent. While ASTA hs potent ntioxidnt effet s shown by other uthors, in this study we show tht ASTA hs modest improvement of ellulr funtions fter ells hs been treted with high gluose nd FFA. This effet ould be in prt due to high stress imposed to ells by the presene of high gluose nd FFA. ASTA ws unble to totlly brogte the huge oxidtive stress indued by these metbolites. Proposed mehnisms for the pthogenesis of dibeti omplitions inlude the formtion of dvned glyosyltion end produts (AGEs), oxidtive stress, rbonyl stress, inresed protein kinse C tivity, ltered growth ftor or ytokine tivities, nd mitohondril dysfuntion [3,4,41]. Two ftors tht strongly ffet the risk of dibeti omplitions re disese durtion nd degree of glyemi ontrol. These observtions hve given rise to the gluose hypothesis whih suggests tht gluose medites mny of the deleterious effets of dibetes [3]. However, strit glyemi ontrol lone does not prevent dibeti omplitions, suggesting the involvement of dditionl ftors. High levels of plsm FFA re lso implited in dibeti omplitions [6,42]. A vriety of types of reurrent infetions drws ttention to dibeti ptients, not beuse of its ese of quired infetions, but lso H 2 O 2 Prodution (µmol H 2 O 2 /5x1 5 ell) 3 2, 1 b ontrol ont+pma ASTA gluose glu+asta FFA FFA+ASTA G6PDH tivity (nmol/mim/mg protein) ,b,b d ontrol ASTA gluose glu+asta FFA FFA+ASTA Fig. 3. Hydrogen peroxide prodution evluted by phenol red oxidtion. Humn neutrophils (5 1 5 /well) were treted with or without 2 μm of ASTA nd tivted with PMA (2 ng/well) or 3 mm gluose or.1 mm FFA for 6 min. Results re expressed s men±sem of t lest 5 determintions. () Signifintly different ompred with the unstimulted-ontrol group (pb.1). (b) Signifintly different ompred with the gluose group (pb.1). () Signifintly different ompred with the FFA group (pb.1). Fig. 4. Mximum G6PDH tivity ssessed by the redution of the oftor NADP+ to NADPH in humn neutrophils (1 1 6 /ml) ultured for 24 h nd treted with or without 2 μm of ASTA, 3 mm gluose nd.1 mm FFA. Results re expressed s men±sem of t lest 5 determintions. () Signifintly different ompred with the ontrol group (pb.1). (b) Signifintly different ompred with the respetive group without ASTA (pb.1). () Signifintly different ompred with the gluose group (pb.1). (d) Signifintly different ompred with the FA group (pb.1).

5 2224 B.A. Guerr, R. Otton / Interntionl Immunophrmology 11 (211) due to the ft tht they nnot del well with invding pthogens. Even simple nd reltively benign pthogens n use severe infetions in these ptients [43]. Hyperglyemi nd high levels of FFA were shown to derese bteriidl tivity, impir phgoytosis nd derese the relese of lysosoml enzymes, nd redue prodution of retive oxygen speies by neutrophils of dibeti ptients [44 46]. In the present study gluose nd mixture of ftty ids used t.1 mm used mrked derese in phgoyti pity of humn neutrophils similr to the one observed in the dibeti ondition. ASTA ddition signifintly improves phgoyti pity of neutrophils treted with gluose nd FFA. A similr result ws obtined by our group previously [3]. Long-hin FFA nd some of their derivtives nd metbolites n modify intrellulr prodution of retive oxygen speies (ROS), in prtiulr superoxide nd H 2 O 2. In mitohondri, FFA exerts dul effet on ROS prodution. Beuse of slowing down the rte of eletron flow through Complexes I nd III of the respirtory hin due to n intertion within the omplex subunit struture, nd between Complexes III nd IV due to the relese of ytohrome from the inner membrne, FFA inreses the rte of ROS genertion in the forwrd mode of eletron trnsport [47]. FFA n lso modulte mitohondril ROS prodution by other mehnisms: () being protonophores, they bolish RET (reverse eletron trnsport) dependent on the superoxide genertion; (b) hving potentil bility to interfere with vrious enzymti proesses, FFA ould intivte the regenertion of redued glutthione (GSH) from its oxidized form (GSSG), thus impeding removl of H 2 O 2 by glutthione peroxidse; () the mphiphili nture of FFA fvors their inorportion into the inner mitohondril membrne with onsequent hnge of membrne fluidity [47]. FFA re lso long known s n tivtors of the NADPH oxidse-dependent superoxide prodution of neutrophils [48 51]. This non-mitohondril oxygen uptke is ompnied by the genertion of superoxide nd H 2 O 2 [52]. In our study there ws huge inrese in superoxide nion prodution in ells treted utely with high gluose nd FFA s ssyed by DHE nd luigenin probes (Fig. 2). The ddition of ASTA on the FFA group promoted higher inrese in superoxide prodution. Vsulr ells inrese their ROS genertion through the tivtion of NADPH oxidse indued by high gluose levels nd FFA [53]. The neutrophil respirtory burst seems to be relted diretly to glyemi ontrol with n inrese in protein kinse C (PKC) nd NADPH oxidse tivity [54 56]. Mehnistilly, hyperglyemi results in inresed phosphoryltion of p47 phox, leding to n inrese in the genertion of superoxide nion [54 56]. Reent works hve shown tht p47 phox, key protein in the ssembly of NAPDH oxidse, premturely trnslotes to the membrne nd ssoites with p22 phox in neutrophils from dibeti subjets [57]. In the present study the mehnism by whih FFA nd high gluose indue ROS prodution needs to be lrified. Neutrophils re dependent on the prodution of retive oxygen speies to exert their role in destroying pthogens during phgoytosis. But these retive oxygen speies being produed in lrge mounts n dmge the ell, minly beuse their membrnes hve lrge mount of polyunsturted ftty ids, whih re esily oxidized by these retive speies. Superoxide is rpidly onverted into hydrogen peroxide (H 2 O 2 ), either spontneously or enzymtilly by superoxide dismutse. H 2 O 2 is then redued to wter by tlse or onverted into hypohlorous id (HOCl) by myeloperoxidse [58]. H 2 O 2 n lso be trnsformed into hydroxyl rdils in the presene of trnsition metl ions [59]. In this study we observed tht the hydrogen peroxide prodution in neutrophils ws inresed (Fig. 3) when ells were treted with gluose nd FFA. Mny uthors hve reported tht stxnthin hs greter ffinity with hydrogen peroxide when ompred to superoxide [27 29]. Reently, Medo et l. [3] showed tht stxnthin signifintly redued the prodution of pro-inflmmtory ytokines, suh s tumor nerosis ftor-α nd interleukin-6 in lipopolyshride-stimulted neutrophils. The uthors lso showed tht stxnthin improved neutrophil phgoyti nd mirobiidl pity nd redued superoxide nion nd hydrogen peroxide prodution, whih ppered to be medited by lium relesed from intrellulr storges nd nitri oxide prodution. The intertion of gluose with proteins leding to the formtion of Amdori produts nd then AGEs is n importnt soure of free rdils in dibetes [4]. These AGEs, vi their reeptors (RAGE), intivte enzymes nd lter their strutures nd funtions [6], promote free rdil formtion [41,61], nd quenh nd blok ntiprolifertive effets of nitri oxide [62,63]. By inresing intrellulr oxidtive stress, AGE tivte the trnsription ftor NFκB (nuler ftor kpp B), thus promoting up-regultion of vrious NFκB ontrolled trget genes [64,65]. NFκB enhnes prodution of NO, whih is believed to be meditor of islet bet ell dmge [66]. We observed n inrese of 19% in NO prodution (Fig. 5A) in ells treted with high gluose. FFA ws not potent induer of NO prodution (Fig. 5B). Whether the NFκB pthwy is tivted in these ells remins to be eluidted. ASTA, s previously shown by our group [29,3,33], inresed NO prodution both in lymphoytes nd neutrophils, whih ws ssoited with benefiil nti-inflmmtory role of ASTA. In the present study ASTA lso promoted signifint inrese in NO. Hussein nd oworkers [25] lso found n inrese in NO prodution ssoited with redution of the rteril blood pressure in spontneously hypertensive rts, suggesting n ntihypertensive role of ASTA. Reently, Kim nd ollegues [67] found tht ASTA redued the tivtion of the trnsription ftor NF-κB nd deresed the IL-6 prodution in miroglil ells. Sine NFκB is the most importnt pthwy in immune ells, responsible of induing NO prodution by induing inos expression, we n speulte tht ASTA n promote NF-κB tivtion whih leds to n inrese in NO prodution. Defense mehnisms ginst free rdil-indued oxidtive stress involve: (i) preventive mehnisms, (ii) repir mehnisms, (iii) physil defenses, nd (iv) ntioxidnt defenses. Enzymti ntioxidnt A NO prodution (µm NNO 2 /5x1 5 ells) B NO prodution (µm NNO 2 /5x1 5 ells) ontrol ASTA gluose glu+asta ontrol ASTA gluose glu+asta b LPS-stimulted ells ontrol ASTA FFA FFA+ASTA ontrol ASTA FFA FFA+ASTA LPS-stimulted ells Fig. 5. Nitri oxide prodution evluted by Griess regent. Humn neutrophils (5 1 5 /well) were treted with or without 2 μm of ASTA nd stimulted with LPS (1 ng/well) or 3 mm gluose (A) or.1 mm FA (B) for 4 h. Results re expressed s men±sem of t lest 5 determintions. () Signifintly different ompred with respetive ontrol group (pb.1). (b) Signifintly different ompred with the ASTA group (pb.1). () Signifintly different ompred with the ontrol-stimulted group (pb.1). (d) Signifintly different ompred with the ASTA-stimulted group (pb.1). b, d

6 B.A. Guerr, R. Otton / Interntionl Immunophrmology 11 (211) defenses inlude superoxide dismutse (SOD), glutthione peroxidse (GPx), tlse (CAT). Non-enzymti ntioxidnts re represented by sorbi id (Vitmin C), α-toopherol (Vitmin E), glutthione (GSH), rotenoids, flvonoids, nd other ntioxidnts. Under norml onditions, there is blne between both the tivities nd the intrellulr levels of these ntioxidnts. This blne is essentil for the survivl of orgnisms nd their helth [68]. Some ntioxidnt enzyme systems need the oftor NADPH, whih is produed minly by the pentose pthwys through the enzyme gluose-6-phosphte dehydrogense (G6PDH). This oftor is extremely importnt for severl ellulr proesses. In this study we evluted the mximum tivity of G6PDH nd our findings show tht there ws n inrese in G6PDH tivity in ells treted with gluose nd FFA. Probbly, the high flux of these metbolites throughout the metboli pthwys ws responsible for inresed G6PDH tivity. In the pentose pthwy, higher G6PDH tivity n rise the level of NADPH, whih is n importnt o-ftor for ntioxidnts enzymes suh s glutthione peroxidse nd glutthione redutse. In ddition, NADPH is n essentil o-ftor for NADPH-oxidse, n importnt pthwy for ROS/RNS prodution. Tretment with ASTA ws ble to restore G6PDH tivity to ontrol levels, reduing the vilbility of NADPH for prtiiption in ROS prodution. Perhps, the ntioxidnt role of ASTA observed in this study is due to this speifi tion on pentose pthwy. In summry, this study showed tht both high gluose nd mixture of FFA re potent induers of ROS/RNS prodution on neutrophils s observed by higher levels of superoxide nion, hydrogen peroxide nd NO prodution. Also, these metbolites derese the phgoyti pity of neutrophils nd inrese G6PDH tivity. Overll, ASTA-tretment ws ble to redue prtilly the ROS/RNS prodution by reduing the vilbility of NADPH nd to reover phgoyti pity of neutrophils. 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