Research Article The Proinflammatory Cytokine High...

Research ArticleThe Proinflammatory Cytokine High-Mobility Group Box-1Mediates Retinal Neuropathy Induced by Diabetes

Ahmed M Abu El-Asrar12 Mohammad Mairaj Siddiquei1 Mohd Imtiaz Nawaz1

Karel Geboes3 and Ghulam Mohammad1

1 Department of Ophthalmology College of Medicine King Saud University Riyadh Saudi Arabia2Department of Ophthalmology King Abdulaziz University Hospital Old Airport Road PO Box 245 Riyadh 11411 Saudi Arabia3 Laboratory of Histochemistry and Cytochemistry University of Leuven Leuven Belgium

Correspondence should be addressed to Ahmed M Abu El-Asrar abuasrarksuedusa

Received 15 July 2013 Revised 13 January 2014 Accepted 28 January 2014 Published 10 March 2014

Academic Editor Charles J Malemud

Copyright copy 2014 Ahmed M Abu El-Asrar et alThis is an open access article distributed under theCreative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

To test the hypothesis that increased expression of proinflammatory cytokine high-mobility group box-1 (HMGB1) in epiretinalmembranes and vitreous fluid from patients with proliferative diabetic retinopathy and in retinas of diabetic rats plays apathogenetic role in mediating diabetes-induced retinal neuropathy Retinas of 1-month diabetic rats and HMGB1 intravitreallyinjected normal rats were studied using Western blot analysis RT-PCR and glutamate assay In addition we studied the effect ofthe HMGB1 inhibitor glycyrrhizin on diabetes-induced biochemical changes in the retina Diabetes and intravitreal injection ofHMGB1 in normal rats induced significant upregulation of HMGB1 protein and mRNA activated extracellular signal-regulatedkinase 1 and 2 (ERK12) cleaved caspase-3 and glutamate and significant downregulation of synaptophysin tyrosine hydroxylaseglutamine synthetase and glyoxalase 1 Constant glycyrrhizin intake from the onset of diabetes did not affect the metabolic statusof the diabetic rats but it significantly attenuated diabetes-induced upregulation of HMGB1 protein and mRNA activated ERK12cleaved caspase-3 and glutamate In the glycyrrhizin-fed diabetic rats the decrease in synaptophysin tyrosine hydroxylase andglyoxalase 1 caused by diabetes was significantly attenuatedThese findings suggest that early retinal neuropathy of diabetes involvesupregulated expression of HMGB1 and can be ameliorated by inhibition of HMGB1

1 Introduction

Diabetic retinopathy (DR) a vision-threatening diseasehas classically been regarded as a disease of the retinalmicrovasculature and a consequence of vascular cell damageHowever recent studies proved that neurodegeneration andimpaired visual function are initiated early after the onset ofdiabetes and progress independently of the vascular lesions[1ndash4] However the molecular mechanisms underlying thediabetes-induced retinal neurodegeneration and dysfunctionare still not well understood

High-mobility group box-1 (HMGB1) is a nonhistoneDNA-binding nuclear protein that has been implicated indiverse intracellular functions including the stabilization ofnucleosomal structures and the facilitation of gene tran-scription Necrotic cell death can result in passive leakageof HMGB1 from the cell as the protein is then no longer

bound to DNA In addition HMGB1 can be actively secretedby different cell types including activated monocytes andmacrophages mature dendritic cells natural killer cellsand endothelial cells Extracellular HMGB1 functions as aproinflammatory cytokine and triggers the inflammatoryresponse through the activation of multiple receptors suchas the receptor for advanced glycation end products (RAGE)toll-like receptor-2 (TLR2) and TLR4 leading to activation ofthe transcription factors extracellular signal-regulated kinase1 and 2 (ERK12) and nuclear factor Kappa B (NF-120581B) whichmay alter gene transcription and lead to the upregulationof proinflammatory cytokines chemokines and adhesionmolecules and intensifies cellular oxidative stress [5ndash10]processes that may play a role in the pathogenesis of diabeticretinal neurodegeneration and dysfunction Strong evidenceindicates that chronic low-grade inflammation is implicated

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 746415 10 pageshttpdxdoiorg1011552014746415

2 Mediators of Inflammation

in the pathogenesis of DR [11 12] Recently it was demon-strated that HMGB1 is the main mediator bridging persistentneuroinflammation and chronic progressive dopaminergicneurodegeneration in neurodegenerative diseases such asParkinsonrsquos disease [13]

Recently HMGB1 has received particular attention withrespect to its pathological role in cerebral ischemia Afterischemic injury induced by transient middle cerebral arteryocclusion in mice and rats HMGB1 was found to betranslocated into the cytoplasmic compartment from nucleiand released into the extracellular space from neurons [14ndash18] In these studies extracellular HMGB1 plays a key rolein the development of neuronal injury through microglialactivation induction of apoptosis excitatory amino acidrelease and induction of proinflammatorymediators [14ndash19]Downregulation of HMGB1 or treatment with neutralizinganti-HMGB1 monoclonal antibody remarkably suppressedinfarct size activation of microglia and induction of proin-flammatorymarkers and inhibited the increased permeabilityof the blood-brain barrier [14 18]

In previous studies we demonstrated that HMGB1 andRAGE were expressed by vascular endothelial cells andstromal cells in fibrovascular epiretinal membranes frompatients with proliferative diabetic retinopathy (PDR) Inaddition we demonstrated increased levels of HMGB1 in thevitreous samples from patients with PDR and that there weresignificant positive correlations between the vitreous levels ofHMGB1 and the levels of the inflammatory biomarkers [20ndash22] Furthermore we demonstrated that diabetes inducedsignificant upregulation of the expression of HMGB1 andRAGE in the retinas of rats and mice and that intravitrealadministration of HMGB1 to normal rats induced activa-tion of inflammatory signaling pathways in the retina andincreased retinal vascular permeability [21 23]

Glycyrrhizin (GA) an ingredient of the licorice rootshas long been known to exhibit glucocorticoid-like anti-inflammatory actions by inhibiting 11120573-hydroxysteroid dehy-drogenase GA has been shown to have anti-inflammatoryand antiviral effects More recently GA has also been shownto bind to and inhibit chemoattractant mitogenic andcytokine-like activities of HMGB1 [24] In this study weexplored the hypothesis that HMGB1 plays a pathogeneticrole in mediating diabetes-induced retinal neuropathy Totest this hypothesis we investigated the expression of theneurodegeneration mediators and markers cleaved caspase-3 synaptophysin tyrosine hydroxylase (TH) glutamine syn-thetase (GS) glutamate and glyoxalase 1 (GLO 1) in theretinas of diabetic rats and in the retinas of normal ratsafter intravitreal administration of HMGB1 In additionwe analyzed whether constant GA intake suppresses retinalneuropathy induced by diabetes in rats

2 Materials and Methods

21 Animals

211 Induction of Diabetes and Glycyrrhizin Treatment Allprocedures with animals were performed in accordance with

the Association for Research in Vision and Ophthalmology(ARVO) statement for use of animals in ophthalmic andvision research andwere approved by the institutional animalcare and use committee of the College of Pharmacy KingSaud University Adult male Sprague Dawley rats of 8-9weeks of age (200minus220 g) were overnight fasted and strep-tozotocin (STZ) 55mgkg in 10mM sodium citrate bufferpH 45 (Sigma St Louis MO) was injected intraperitoneallyEqual volumes of citrate buffer were injected in nondiabeticanimals Rats were considered diabetic if their blood glucosewas greater than 250mgdL Age-matched normal rats servedas controls

Diabetic rats were divided into 2 groups the rats in groupI received normal drinking water without any supplementa-tion and group II received drinkingwater supplementedwithglycyrrhizic acid (150mgkgday Santa Cruz BiotechnologyInc Santa Cruz CA) immediately after establishment ofdiabetes throughout the course of the experiment Eachgroup had 8ndash12 rats After 4 weeks of diabetes the rats wereeuthanized by an overdose of chloral hydrate the eyes wereremoved and retina was isolated and frozen immediately inliquid nitrogen and stored atminus80∘C to be analyzed byWesternblot analysis or biochemical assay

212 Intravitreal Injection of HMGB1 Sprague Dawley rats(200minus220 g) were kept under deep anesthesia and sterilizedsolution of recombinant HMGB1 (5 ng5120583L RampD SystemsMinneapolis MN) was injected into the vitreous of the righteye as previously described by us [23] For the controlthe left eye received 5 120583L of sterile phosphate buffer saline(PBS) The animals were sacrificed 4 days after intravitrealadministration and the retinas were carefully dissectedsnap-frozen in liquid nitrogen and stored at minus80∘C to beanalyzed by Western blot analysis or biochemical assay Thebreakdown of blood-retina barrier induced by intravitrealinjection of HMGB1 might lead to raised levels of HMGB1 inthe serumTherefore we determined the levels of HMGB1 inequal amounts of serum from intravitreally injected rats andnormal rats using Western blot analysis

213 Western Blot Analysis Retinas were homogenized ina western lysis buffer (30mM Tris-HCl pH 74 250mMNa3VO4 5mM EDTA 250mM sucrose 1 Triton X-

100 with Protease inhibitor) The lysate was centrifuged at14000timesg for 10min at 4∘C and the supernatant was col-lected Protein content was assayed by DC protein assay (Bio-Rad Laboratories Hercules CA)The tissue lysate containing40ndash50120583g of protein was separated on 10 or 12 SDS-polyacrylamide gels and was transferred onto nitrocellulosemembranes The blots were blocked with 5 nonfat milkin TBST (20mM Tris-HCl pH 76 136mM NaCl and 01Tween-20)

For detection of HMGB1 phospho-ERK12 synapto-physin cleaved caspase-3 TH GLO1 and GS the membranewas incubated overnight at 4∘C with rabbit polyclonal anti-HMGB1 (1 1000 Cat number ab18256 Abcam UK) rabbitmonoclonal anti-phospho-ERK12 (05 120583gmL Cat numberMAB1018 RampD Systems) mouse monoclonal anti-ERK12

Mediators of Inflammation 3

(05 120583gmL Cat number MAB1576 RampD Systems) goatpolyclonal anti-synaptophysin (1 120583gmL Cat number AF-5555 RampD Systems) rabbit polyclonal anti-cleaved caspase-3 (1 300 Cat number SC-7148 Santa Cruz) mouse mon-oclonal anti-TH (05 120583gmL Cat number MAB7566 RampDSystems) rabbit polyclonal anti-GLO1 (1 200 Cat numberab96032 Abcam) and goat polyclonal anti-GS (1 500 sc-6640 Santa Cruz) After overnight incubation with primaryantibodies themembraneswerewashed four timeswithTBS-T (5min each) For synaptophysin and GS the membranewas incubated at room temperature for 15 h with anti-goat secondary horseradish peroxidase-conjugated antibody(1 2000 SC-2768 Santa Cruz) for HMGB1 phospo-ERK12cleaved caspase-3 and GLO1 with anti-rabbit secondaryhorseradish peroxidase-conjugated antibody (1 2000 SC-2004 Santa Cruz) and for ERK12 and TH with anti-mouse secondary horseradish peroxidase-conjugated anti-body (1 2000 SC-2005 Santa Cruz) After incubations withsecondary antibodies membranes were washed four timeswith TBS-T (5min each) and the immunoreactivity of bandswas visualized on a high-performance chemiluminescencemachine (G Box Chemi-XX8 from Syngene Synoptic LtdCambridge UK) by using enhanced chemiluminescence plusLuminol (sc-2048 Santa Cruz) and quantified by densit-ometric analysis using image processing and analysis inGeneTools (Syngene by Synoptic Ltd Cambridge UK) Forloading control the blots were stripped and detected witha mouse monoclonal anti-120573-actin (1 2000 SC-2048 SantaCruz) antibody For phosphor-ERK12 the loading controlwas total ERK12 All data from the three independentexperiments were expressed as a ratio to optical density

214 Real-Time Reverse Transcription Polymerase ChainReaction (RT-PCR) Total RNA was extracted from retinausing TRI reagent (Ambion TX) according to man-ufacturerrsquos protocol cDNA was synthesized from 1120583gRNA using a high capacity cDNA reverse transcrip-tion kit (Applied Biosystem CA) following manufac-turerrsquos instruction RT-PCR was performed using a SYBRgreen PCR master mix The PCR primers for rat wereHMGB1 forward 51015840-TGATTAATGAATGAGTTCGGGC-31015840reverse 51015840- TGCTCAGGAAACTTGACTGTTT-31015840 and 120573-actin forward 51015840-CCTCTATGCCAACACAGTGC-31015840 reverse51015840-CATCGTACTCCTGCTTGCTG-31015840 The standard PCRconditions included 2minutes at 50∘C and 10min at 95∘C fol-lowed by 40 cycles of extension at 95∘C for 15 seconds and oneminute at 60∘C Threshold lines were automatically adjustedto intersect amplification lines in the linear portion of theamplification curves and cycle to threshold (Ct) was recordedautomatically Data were normalized with 120573-actin mRNAlevel (housekeeping gene) and the fold change in gene expres-sion relative to normal was calculated using the ddCtmethod

215 Measurement of Glutamate in Rat Retinas Glutamatelevel was measured in retinal homogenate by using gluta-mate assay kit (Cat number ab83389 Abcam) according tothe manufacturerrsquos instruction Briefly 50 120583L of standardsand retinal homogenate (equal amount) was loaded in a

clear 96-well plate followed by addition of 100 120583L reactionmix solution having assay buffer glutamate developer andglutamate enzyme mix The plate was incubated for 30minutes at 37∘C (protected from light) and was read at450 nm inmicroplate reader (Stat Fax 4200microplate readerawareness technology Palm City FL) The concentration ofmeasured glutamate was expressed as nmol120583L120583g of protein

216 Statistical Analysis The Mann-Whitney test was usedto compare means from two independent groups A 119875 valueless than 005 indicated statistical significance SPSS version120 was used for the statistical analyses

3 Results

31 Severity of Hyperglycemia in Rats The body weights ofthe diabetic rats were lower and their blood glucose levelswere more than fourfold higher compared with age-matchednormal control rats (178 plusmn 22 versus 287 plusmn 28 g and 475 plusmn32 versus 111 plusmn 12mgdL resp) Treatment of the diabeticrats with GA for one month did not change these metabolicvariables in the diabetic rats (167 plusmn 25 versus 178 plusmn 22 g and449 plusmn 36 versus 475 plusmn 32mgdL resp)

32 Effect of Diabetes on Retinal Expression of Mediatorsand Markers of Neurodegeneration Western blot analysisdemonstrated significant upregulation of HMGB1 expressionin diabetic retinas compared to nondiabetic retinas Theexpression of HMGB1 protein in the retinas of diabeticrats was upregulated by about 66 as compared to theretinas of nondiabetic rats (Figure 1(a)) Diabetes signifi-cantly increased ERK12 activation in the retinas by about77 compared to nondiabetic controls (Figure 1(b)) Cleavedcaspase-3 the apoptosis executer enzyme was significantlyupregulated in diabetic retinas compared to nondiabeticcontrols Cleaved caspase-3 levels in the retinas of diabeticrats were increased by about 70 compared to nondiabeticcontrols (Figure 1(c)) The synaptic vesicle protein synap-tophysin and the dopaminergic amacrine cell marker THlevels obtained in diabetic animals were significantly lowerthan those of nondiabetic animals The levels decreased byabout 68 and 46 respectively (Figures 1(d) and 1(e)) GSan enzyme that converts glutamate into glutamine proteinexpression was significantly decreased in diabetic retinascompared to nondiabetic retinas GS levels decreased byabout 75 (Figure 1(f)) GLO1 an enzyme critical for thedetoxification of advanced glycation end products (AGEs)protein expression was significantly decreased in diabeticretinas compared to nondiabetic rats GLO1 expression indiabetic retinas decreased by about 51 (Figure 1(g)) Glu-tamate assay revealed that glutamate levels in the retinas ofdiabetic animals (004 plusmn 0015 nmol120583L120583g protein) were sig-nificantly higher than those in nondiabetic controls (0022 plusmn0004 nmol120583L120583g protein) (119875 = 0036) (Figure 1(h))

33 Effect of Intravitreal Administration of HMGB1 on RetinalExpression of Mediators and Markers of Neurodegeneration inNormal Rats There was no significant difference in serum


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Figure 1 Western blot analysis of high-mobility group box-1 (HMGB1) extracellular signal-regulated kinase 1 and 2 (ERK12) cleavedcaspase-3 synaptophysin tyrosine hydroxylase glutamine synthetase and glyoxalase 1 in rat retinas 120573-Actin was used as a housekeepingcontrol Protein expression of ERK12 activation (phosphorylation) was quantified by Western blot analysis using phospho- (P-) ERK12specific antibody and was adjusted to the protein levels of unphosphorylated ERK12 antibody in each sample There is a significant increasein the expression of HMGB1 (a) activated ERK12 (b) and cleaved caspase-3 (c) and a significant decrease in the expression of synaptophysin(d) tyrosine hydroxylase (e) glutamine synthetase (f) and glyoxalase 1 (g) in the retinas of diabetic rats compared to nondiabetic controlsGlycyrrhizic acid (GA) significantly attenuated diabetes-induced upregulation of HMGB1 (a) activated ERK12 (b) and cleaved caspase-3 (c)and diabetes-induced downregulation of synaptophysin (d) tyrosine hydroxylase (e) GS (f) and glyoxalase 1 (g) Glutamate assay revealeda significant increase in glutamate levels in the retinas of diabetic rats compared to nondiabetic controls The levels of glutamate in the GA-treated diabetic rats were significantly less than those in the untreated diabetic rats (h) Each experiment was repeated at least 3 times withfresh samples A representative set of samples is shown Results are expressed as mean plusmn SD of at least 6 rats in each group lowast119875 lt 005compared with nondiabetic control rats

119875 lt 005 compared with diabetic rats

levels of HMGB1 between rats intravitreally injected withHMGB1 (13564plusmn1438) andnormal rats (13488plusmn1101) (119875 =0225) Intravitreal injection of HMGB1 resulted in increasedHMGB1 expression by about 72 compared to the valuesobtained from the contralateral eye that received PBS alone(Figure 2(a)) In the same retinal samples HMGB1 injectionresulted in a 76 increase in ERK12 activation (Figure 2(b))31 increase in cleaved caspase-3 expression (Figure 2(c))65 decrease in synaptophysin expression (Figure 2(d)) 65decrease in TH expression (Figure 2(e)) 70 decrease in GSexpression (Figure 2(f)) and 71 decrease in GLO1 expres-sion (Figure 2(g)) Injection of HMGB1 tended to increaseretinal glutamate expression but this was not significant(0041 plusmn 0025 versus 0021 plusmn 0004 nmol120583L120583g protein 119875 =007) (Figure 2(h))

34 HMGB1 Inhibitor Glycyrrhizin Attenuates the Effect ofDiabetes Western blot analysis was used to assess the effectof GA on diabetes-induced alterations of HMGB1 ERK12activation cleaved caspase-3 synaptophysin TH GS andGLO1 Constant GA intake from the onset of diabetes signifi-cantly attenuated diabetes-induced upregulation of HMGB1ERK12 activation and cleaved caspase-3 by about 73 55

and 78 respectively (Figures 1(a) 1(b) and 1(c)) In theGA-fed diabetic rats the decrease in synaptophysin TH GS andGLO1 caused by diabetes was attenuated by about 58 5579 and 53 respectively (Figures 1(d) 1(e) 1(f) and 1(g))The levels of glutamate in theGA-treated diabetic rats (0024plusmn0002 nmol120583L120583g protein) were significantly less than thosein the untreated diabetic rats (004 plusmn 0015 nmol120583L120583gprotein) (119875 = 0045) (Figure 1(h))

35 Retinal Expression of HMGB1 mRNA The expression ofHMGB1 mRNA in the retinas of diabetic rats was increasedby about 4-fold compared to the retinas of nondiabeticrats Intravitreal injection of HMGB1 resulted in increasedHMGB1 mRNA expression by about 5-fold compared to thevalues obtained from the contralateral eye that received PBSalone GA intake significantly attenuated diabetes-inducedupregulation of HMGB1 mRNA by about 35-fold comparedto untreated diabetic rats (Figure 3)

4 Discussion

In the present study we investigated the pathological roleof HMGB1 in diabetes-induced retinal neuropathy Ourprevious studies showed that diabetes upregulates HMGB1


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Figure 2 Western blot analysis of rat retinas Intravitreal administration of high-mobility group box-1 (HMGB1) induced a significantupregulation of the expression of HMGB1 (a) activated extracellular signal-regulated kinase 1 and 2 (ERK12) (b) and cleaved caspase-3 (c)and a significant downregulation of the expression of synaptophysin (d) tyrosine hydroxylase (e) glutamine synthetase (f) and glyoxalase1 (g) compared to intravitreal administration of phosphate buffer saline (PBS) Glutamate assay revealed that injection of HMGB1 tended toincrease retinal glutamate expression but this was not significant (h) Each experiment was repeated at least 3 times with fresh samples Arepresentative set of samples is shown Results are expressed as mean plusmn SD of at least 6 rats in each group lowast119875 lt 005 compared with PBS

expression in the vitreous fluid and preretinal membranesfrom patients with PDR and its expression correlated withthe activity of the disease and the levels of inflammatorybiomarkers [20ndash22] In addition we demonstrated thatHMGB1 expression was upregulated in the retinas of diabeticmice and rats [21 23] In the current study we demonstratedthat diabetes induced significant upregulation of cleavedcaspase-3 and glutamate expression in the retinas of rats Onthe other hand diabetes induced significant downregulationof synaptophysin TH GS and GLO1 in the retinas of ratsFurthermore our data show that intravitreal injection ofHMGB1 in normal rats mimics the effect of diabetes TheHMGB1 inhibitor GA attenuated diabetes-induced upreg-ulation of HMGB1 protein and mRNA cleaved caspase-3 and glutamate and downregulation of synaptophysinTH GS and GLO1 in the retinas of rats Taken togetherthese findings suggest that HMGB1 contributes to retinalneuropathy induced by diabetes

Diabetes induces retinal neurodegeneration as evidencedby the presence of apoptotic cells in all retinal layers [4

25] Activation of caspase-3 is part of the mechanism ofapoptosis [25] Several studies demonstrated that expressionof active caspase-3 an indication of apoptosis was upregu-lated in the diabetic retinas of human subjects [4] and rats[25] In STZ-diabetic rat retinas caspase-3 immunoreactivitywas upregulated after 2 8 and 16 weeks of diabetes [25]However Feit-Leichman et al [26] demonstrated in STZ-diabetic mice upregulation of retinal caspase-3 activity at1 month after induction of diabetes which diminished tonormal and began to increase again after approximately 6months They concluded that diabetes-induced degenerationof retinal capillaries can develop independent of neuronalloss Synaptophysin is an integral protein of synaptic vesiclesIt possibly serves multiple functions in synaptic vesicleformation and exocytosis playing an important role in neu-rotransmitter delivery It is widely used as one of the synapticfunction markers and is also thought to be closely relatedto synaptogenesis and synaptic plasticity during neural tis-sue development Synaptophysin knockout mice exhibiteda significant decrease in synaptic vesicles in retinal rod


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Figure 3 Gene expression of high-mobility group box-1 (HMGB1)in the retinas was quantified by RT-PCR using a primer given in theMaterials and Methods section and was adjusted to the mRNA levelof120573-actin in each sample Eachmeasurement was performed at least3 times Results are expressed as mean plusmn SD of at least 6 rats in eachgroup GA glycyrrhizic acid PBS phosphate buffer saline lowast119875 lt 005compared with nondiabetic controlled rats lowastlowast119875 lt 005 comparedwith PBS 119875 lt 005 compared with diabetic rats

photoreceptors which disturbs neurotransmitter release andsynaptic network activity [27] Previous studies demonstratedthat 1 month of diabetes decreases retinal expression ofsynaptophysin [3 28ndash30] TH is the rate-limiting biosyntheticenzyme for dopamine synthesis Therefore the TH proteinlevel is a marker of dopaminergic amacrine cells in theretina [1 25 31] Several studies showed decreased THprotein levels in the diabetic retinas reflecting reductions inthe density of dopaminergic amacrine cells [1 31ndash33] Thesynaptically released glutamate is taken up by Muller cellswhere GS converts it into glutamine Several studies foundthat the expression of GS was significantly decreased in thediabetic rat retinas [34 35] These dysfunctions resulted inelevated glutamate levels in the diabetic retinas [34 36 37]which might induce retinal neurodegeneration via glutamateexcitotoxicity In diabetes there is accumulation of the AGEsprecursor methylglyoxal (MG) In diabetic retinopathy MG-derived AGEs are elevated in retina and are viewed to becausative in retinal injury and neurodegeneration [38ndash40]Normally MG is detoxified by the glyoxalase (GLO) enzymesystem composed of GLO1 andGLO2 [41] A previous reportdemonstrated that GLO1 expression is reduced in the diabeticrat retinas [42] It was also shown that GLO1 overexpressionin diabetic rats prevents hyperglycemia-induced formationof MG-derived AGEs in the neural retina prevents Mullerglia dysfunction and protects against capillary degenerativepathology [43]

In the present study we demonstrated that similar todiabetes intravitreal injection of HMGB1 caused a significantupregulation of HMGB1 protein and mRNA and activatedcleaved caspase-3 in the retina of normal rats In additioninjection of HMGB1 tended to increase retinal glutamateexpression but this was not significant In this regard therecent finding that HMGB1 promotes glutamate release fromgliosomes [19] suggests that its neurotoxicity is mediatedat least in part by increased release of glutamate and

enhanced excitotoxic neuronal death On the other handin vitro studies demonstrated that glutamate can inducethe release of HMGB1 from neuronal cells [14 15] Fur-thermore intravitreal administration of HMGB1 induceddownregulation of synaptophysin TH GS and GLO1 Thesefindings suggest that the early retinal neuropathy of diabetesinvolves the upregulated expression of HMGB1 Several stud-ies showed that HMGB1 is massively released immediatelyafter an ischemic insult and that it subsequently inducesneuroinflammation in the postischemic brain [14 15 17]To exert these activities HMGB1 must transit from thenucleus through the cytoplasm to the outside of the cellSeveral studies demonstrated that the inflammatory markerC-reactive protein [44] and the proinflammatory cytokinestumor necrosis factor-120572 (TNF-120572) [45] and interleukin-1120573(IL-1120573) [46] induce translocation of HMGB1 from nucleusto the cytoplasm and its subsequent extracellular releasefrom the cell In vitro studies demonstrated that extra-cellular HMGB1 induced the proinflammatory biomarkersTNF-120572 IL-1120573 interferon-120574 intercellular adhesion molecule-1 (ICAM-1) inducible nitric oxide synthase (iNOS) andcyclooxgenase-2 in cultured neurons astrocytes microgliaand endothelial cells [14 15 17] Moreover cerebral microin-jection of exogenous HMGB1 increased postischemic braininjury and upregulated the expression of iNOS and IL-1120573[17] These findings indicate that HMGB1 functions as anovel proinflammatory cytokine-like factor linking very earlystages of cerebral ischemic injury with the activation oflocal neuroinflammatory response in the postischemic brain[14 15] Furthermore it was also shown that extracellularHMGB1 released from the damaged brain neurons inducesneuronal apoptosis and that this involves HMGB1-RAGEinteraction [16] In our laboratory we recently demonstratedthat diabetes induced significant upregulation of retinalexpression of HMGB1 RAGE activated NF-120581B activatedERK12 and ICAM-1 and that intravitreal administration ofHMGB1 in normal rats mimics the effect of diabetes [23] Wealso showed that the HMGB1 inhibitor GA was effective inpreventing diabetes-inducedNF-120581B activation In the presentstudy GA attenuated diabetes-induced ERK12 activation Inaddition coimmunoprecipitation studies showed that dia-betes increases the interaction between HMGB1 and RAGEin the retina [23] These findings suggest that activationof HMGB1RAGE signaling axis with subsequent activationof NF-120581B and ERK12 is important in promoting diabetes-induced retinal neuropathy Recently it was reported thatchronic neuroinflammation may be a driving force of pro-gressive neurodegeneration and that HMGB1 provides thelink between chronic neuroinflammation and progressiveneurodegeneration in neurodegenerative diseases such asParkinsonrsquos disease [13]

To confirm the neuropathological implications ofHMGB1 in diabetic retinopathy the HMGB1 inhibitor GAwas administered orally after inducing diabetes in ratsGA is known to bind directly to HMGB1 and inhibit itschemoattractant and mitogenic activities [24] Interestinglyconstant intake of GA significantly reduced retinal HMGB1protein and mRNA expression induced by diabetes Inaddition GA attenuated diabetes-induced upregulation


of cleaved caspase-3 and glutamate and counteracted thedownregulation of synaptophysin TH GS and GLO1induced by diabetes without changing body weight orblood glucose levels Our results are consistent with pre-vious reports that demonstrated a neuroprotective effect ofGA in animal models of brain ischemia [47] intracerebralhemorrhage [48] and spinal cord ischemia [49] GA is knownto possess glucocorticoid-like anti-inflammatory propertiesdue to its inhibitory activity on 11120573-hydroxysteroiddehydrogenase [24] Ohnishi et al [48] demonstrated that theneuroprotective effect of GA on intracerebral hemorrhage-related pathogenic events was notmediated by glucocorticoidreceptors or modulation of nitric oxide production and wasreversed by exogenous HMGB1 application These findingssuggest that the protective effect of GA is mediated by itsanti-HMGB1 activity and is irrelevant to the glucocorticoidsystem It was demonstrated that the neuroprotective effectof GA was partly attributable to its inhibitory effect onHMGB1 release [47ndash49] Furthermore it was shown thatGA affords protection in postischemic brain and spinal cordvia its anti-inflammatory antiapoptotic antiexcitotoxic andantioxidative effects [47 49] Together these findings suggestthat early retinal neuropathy induced by diabetes is atleast in part attributable to diabetes-induced upregulation ofHMGB1 expression and that inhibiting the release of HMGB1with constant intake of GA results in less diabetes-inducedretinal neuropathy It is important to note that excessiveintake of licorice may cause hypermineralocorticoidism-likesyndrome characterized by sodium and water retentionpotassium loss edema increased blood pressure metabolicalkalosis and depression of rennin-angiotensin-aldosteronesystem [50 51]

In conclusion our data point to a potential novel andpivotal role for HMGB1 as a mediator of diabetes-inducedneuropathy in the retina The HMGB1 inhibitor GA attenu-ated diabetes-induced upregulation of HMGB1 and diabetes-induced retinal neuropathy Therefore GA and other agentstargeted to HMGB1 may provide novel therapeutic optionsfor diabetic retinopathy

Conflict of Interests

All the authors do not have any conflict of interests with anytrademark mentioned in the paper

Acknowledgments

The authors thank Ms Connie B Unisa-Marfil and MsMichelle B Rasonabe for secretarial work This work wassupported by Dr Nasser Al-Rasheed Research Chair in Oph-thalmology (Abu El-Asrar AM) andNational Plan for Scienceand Technology (NPST) King Saud University under theProject 12-MED2604-02

References

[1] M Seki T Tanaka H Nawa et al ldquoInvolvement of brain-derived neurotrophic factor in early retinal neuropathy of

streptozotocin-induced diabetes in rats therapeutic potential ofbrain-derived neurotrophic factor for dopaminergic amacrinecellsrdquo Diabetes vol 53 no 9 pp 2412ndash2419 2004

[2] A J Barber ldquoA new view of diabetic retinopathy a neu-rodegenerative disease of the eyerdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 27 no2 pp 283ndash290 2003

[3] M Sasaki Y Ozawa T Kurihara et al ldquoNeurodegenerativeinfluence of oxidative stress in the retina of a murine model ofdiabetesrdquo Diabetologia vol 53 no 5 pp 971ndash979 2010

[4] A M Abu El-Asrar L Dralands L Missotten I A Al-Jadaanand K Geboes ldquoExpression of apoptosis markers in the retinasof human subjects with diabetesrdquo Investigative Ophthalmologyand Visual Science vol 45 no 8 pp 2760ndash2766 2004

[5] J R Van Beijnum W A Buurman and A W GriffioenldquoConvergence and amplification of toll-like receptor (TLR) andreceptor for advanced glycation end products (RAGE) signalingpathways via high mobility group B1 (HMGB1)rdquo Angiogenesisvol 11 no 1 pp 91ndash99 2008

[6] C J Treutiger G E Mullins A-S M Johansson et alldquoHigh mobility group 1 B-box mediates activation of humanendotheliumrdquo Journal of Internal Medicine vol 254 no 4 pp375ndash385 2003

[7] C Fiuza M Bustin S Talwar et al ldquoInflammation-promotingactivity of HMGB1 on human microvascular endothelial cellsrdquoBlood vol 101 no 7 pp 2652ndash2660 2003

[8] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010

[9] M Takeuchi J-I Takino and S-I Yamagishi ldquoInvolvement ofthe toxic AGEs (TAGE)-RAGE system in the pathogenesis ofdiabetic vascular complications a novel therapeutic strategyrdquoCurrent Drug Targets vol 11 no 11 pp 1468ndash1482 2010

[10] D Tang R Kang H J Zeh 3rd andM T Lotze ldquoHigh-mobilitygroup box 1 oxidative stress and diseaserdquo Antioxidants andRedox Signaling vol 14 no 7 pp 1315ndash1335 2011

[11] A M Joussen V Poulaki M L Le et al ldquoA central role forinflammation in the pathogenesis of diabetic retinopathyrdquo TheFASEB Journal vol 18 no 12 pp 1450ndash1452 2004

[12] K Miyamoto S Khosrof S-E Bursell et al ldquoPreventionof leukostasis and vascular leakage in streptozotocin-induceddiabetic retinopathy via intercellular adhesion molecule-1 inhi-bitionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 19 pp 10836ndash10841 1999

[13] H-M Gao H Zhou F Zhang B C Wilson W Kam and J-S Hong ldquoHMGB1 acts on microglia Mac1 to mediate chronicneuroinflammation that drives progressive neurodegenerationrdquoJournal of Neuroscience vol 31 no 3 pp 1081ndash1092 2011

[14] J-B Kim S C Joon Y-M Yu et al ldquoHMGB1 a novel cytokine-like mediator linking acute neuronal death and delayed neu-roinflammation in the postischemic brainrdquo Journal of Neuro-science vol 26 no 24 pp 6413ndash6421 2006

[15] J Qiu M Nishimura Y Wang et al ldquoEarly release of HMGB-1 from neurons after the onset of brain ischemiardquo Journal ofCerebral Blood Flow andMetabolism vol 28 no 5 pp 927ndash9382008

[16] S-W Kim C-M Lim J-B Kim et al ldquoExtracellular HMGB1released by NMDA treatment confers neuronal apoptosisvia RAGE-p38 MAPKERK signaling pathwayrdquo NeurotoxicityResearch vol 20 no 2 pp 159ndash169 2011


[17] G Faraco S Fossati M E Bianchi et al ldquoHigh mobility groupbox 1 protein is released by neural cells upon different stressesand worsens ischemic neurodegeneration in vitro and in vivordquoJournal of Neurochemistry vol 103 no 2 pp 590ndash603 2007

[18] K Liu S Mori H K Takahashi et al ldquoAnti-high mobilitygroup box 1 monoclonal antibody ameliorates brain infarctioninduced by transient ischemia in ratsrdquoThe FASEB Journal vol21 no 14 pp 3904ndash3916 2007

[19] M Pedrazzi L Raiteri G Bonanno et al ldquoStimulation ofexcitatory amino acid release from adult mouse brain gliasubcellular particles by high mobility group box 1 proteinrdquoJournal of Neurochemistry vol 99 no 3 pp 827ndash838 2006

[20] A M A El-Asrar L Missotten and K Geboes ldquoExpressionof high-mobility groups box-1receptor for advanced glycationend productsosteopontinearly growth response-1 pathwayin proliferative vitreoretinal epiretinal membranesrdquo MolecularVision vol 17 pp 508ndash518 2011

[21] A M A El-Asrar M I Nawaz D Kangave et al ldquoHigh-mobility group box-1 and biomarkers of inflammation in thevitreous from patients with proliferative diabetic retinopathyrdquoMolecular Vision vol 17 pp 1829ndash1838 2011

[22] A M Abu El-Asrar M I Nawaz D Kangave M M Siddiqueiand K Geboes ldquoOsteopontin and other regulators of angiogen-esis and fibrogenesis in the vitreous from patients with prolif-erative vitreoretinal disordersrdquoMediators of Inflammation vol2012 Article ID 493043 8 pages 2012

[23] GMohammadMM Siddiquei AOthmanMAl-Shabraweyand A M Abu El-Asrar ldquoHigh-mobility group box-1 proteinactivates inflammatory signaling pathway components anddisrupts retinal vascular-barrier in the diabetic retinardquo Exper-imental Eye Research vol 107 pp 101ndash109 2013

[24] L Mollica F DeMarchis A Spitaleri et al ldquoGlycyrrhizin bindsto high-mobility group box 1 protein and inhibits its cytokineactivitiesrdquo Chemistry and Biology vol 14 no 4 pp 431ndash4412007

[25] M J Gastinger R S J Singh and A J Barber ldquoLoss ofcholinergic and dopaminergic amacrine cells in streptozotocin-diabetic rat and Ins2Akita-diabetic mouse retinasrdquo InvestigativeOphthalmology and Visual Science vol 47 no 7 pp 3143ndash31502006

[26] R A Feit-Leichman R Kinouchi M Takeda et al ldquoVasculardamage in a mouse model of diabetic retinopathy relation toneuronal and glial changesrdquo Investigative Ophthalmology andVisual Science vol 46 no 11 pp 4281ndash4287 2005

[27] I Spiwoks-Becker L Vollrath M W Seeliger G Jaissle L GEshkind and R E Leube ldquoSynaptic vesicle alterations in rodphotoreceptors of synaptophysin-deficient micerdquo Neurosciencevol 107 no 1 pp 127ndash142 2001

[28] H D Vanguilder R M Brucklacher K Patel R W Ellis W MFreeman andA J Barber ldquoDiabetes downregulates presynapticproteins and reduces basal synapsin i phosphorylation in ratretinardquo European Journal of Neuroscience vol 28 no 1 pp 1ndash11 2008

[29] T Kurihara Y Ozawa N Nagai et al ldquoAngiotensin II type1 receptor signaling contributes to synaptophysin degradationand neuronal dysfunction in the diabetic retinardquo Diabetes vol57 no 8 pp 2191ndash2198 2008

[30] Y Ozawa T Kurihara M Sasaki et al ldquoNeural degeneration inthe retina of the streptozotocin-induced type 1 diabetes modelrdquoExperimental Diabetes Research vol 2011 Article ID 108328 7pages 2011

[31] K Szabadfi T Atlasz P Kiss et al ldquoProtective effects of theneuropeptide PACAP in diabetic retinopathyrdquo Cell and TissueResearch vol 348 no 1 pp 37ndash46 2012

[32] C Wan N N Liu L M Liu N Cai and L Chen ldquoEffect ofadenovirus-mediated brain derived neurotrophic factor in earlyretinal neuropathy of diabetes in ratsrdquo International Journal ofOphthalmology vol 3 pp 145ndash148 2010

[33] F K Northington RWHamill and S P Banerjee ldquoDopamine-stimulated adenylate cyclase and tyrosine hydroxylase in dia-betic rat retinardquo Brain Research vol 337 no 1 pp 151ndash154 1985

[34] E Lieth K F LaNoue D A Antonetti and M Ratz ldquoDiabetesreduces glutamate oxidation and glutamine synthesis in theretinardquo Experimental Eye Research vol 70 no 6 pp 723ndash7302000

[35] Y Xu-hui Z HongW Yu-hong L Li-juan T Yan and L PingldquoTime-dependent reduction of glutamine synthetase in retinaof diabetic ratsrdquo Experimental Eye Research vol 89 no 6 pp967ndash971 2009

[36] E Lieth A J Barber B Xu et al ldquoGlial reactivity and impairedglutamate metabolism in short-term experimental diabeticretinopathy Penn State Retina Research Grouprdquo Diabetes vol47 pp 815ndash820 1998

[37] R A Kowluru R L Engerman G L Case and T S KernldquoRetinal glutamate in diabetes and effect of antioxidantsrdquoNeurochemistry International vol 38 no 5 pp 385ndash390 2001

[38] A Lecleire-Collet L H Tessier P Massin et al ldquoAdvancedglycation end products can induce glial reaction and neuronaldegeneration in retinal explantsrdquo British Journal of Ophthalmol-ogy vol 89 no 12 pp 1631ndash1633 2005

[39] J V Glenn and A W Stitt ldquoThe role of advanced glycation endproducts in retinal ageing and diseaserdquo Biochimica et BiophysicaActa vol 1790 no 10 pp 1109ndash1116 2009

[40] N Karachalias R Babaei-Jadidi N Ahmed and P J Thor-nalley ldquoAccumulation of fructosyl-lysine and advanced glyca-tion end products in the kidney retina and peripheral nerveof streptozotocin-induced diabetic ratsrdquo Biochemical SocietyTransactions vol 31 no 6 pp 1423ndash1425 2003

[41] M Shinohara P J Thornalley I Giardino et al ldquoOverex-pression of glyoxalase-I in bovine endothelial cells inhibitsintracellular advanced glycation endproduct formation andprevents hyperglycemia-induced increases in macromolecularendocytosisrdquo Journal of Clinical Investigation vol 101 no 5 pp1142ndash1147 1998

[42] A G Miller G Tan K J Binger et al ldquoCandesartan attenuatesdiabetic retinal vascular pathology by restoring glyoxalase-Ifunctionrdquo Diabetes vol 59 no 12 pp 3208ndash3215 2010

[43] A K Berner O Brouwers R Pringle et al ldquoProtectionagainstmethylglyoxal-derivedAGEs by regulation of glyoxalase1 prevents retinal neuroglial and vasodegenerative pathologyrdquoDiabetologia vol 55 no 3 pp 845ndash854 2012

[44] K-I Kawahara K K Biswas M Unoshima et al ldquoC-reactiveprotein induces high-mobility group box-1 protein releasethrough activation of p38MAPK in macrophage RAW2647cellsrdquo Cardiovascular Pathology vol 17 no 3 pp 129ndash138 2008

[45] S Willenbrock O Braun J Baumgart et al ldquoTNF-120572 inducedsecretion of HMGB1 from non-immune canine mammaryepithelial cells (MTH53A)rdquoCytokine vol 57 no 2 pp 210ndash2202012

[46] K Hayakawa K Arai and E H Lo ldquoRole of ERK MAP kinaseand CRM1 in IL-1120573-stimulated release of HMGB1 from corticalastrocytesrdquo GLIA vol 58 no 8 pp 1007ndash1015 2010


[47] S-W Kim Y Jin J-H Shin et al ldquoGlycyrrhizic acid affordsrobust neuroprotection in the postischemic brain via anti-inflammatory effect by inhibitingHMGB1 phosphorylation andsecretionrdquo Neurobiology of Disease vol 46 no 1 pp 147ndash1562012

[48] M Ohnishi H Katsuki C Fukutomi et al ldquoHMGB1inhibitor glycyrrhizin attenuates intracerebral hemorrhage-induced injury in ratsrdquoNeuropharmacology vol 61 no 5-6 pp975ndash980 2011

[49] G Gong L-B Yuan L Hu et al ldquoGlycyrrhizin attenuatesrat ischemic spinal cord injury by suppressing inflammatorycytokines andHMGB1rdquoActa Pharmacologica Sinica vol 33 no1 pp 11ndash18 2012

[50] F C Stormer R Reistad and J Alexander ldquoGlycyrrhizic acidin liquoricemdashevaluation of health hazardrdquo Food and ChemicalToxicology vol 31 no 4 pp 303ndash312 1993

[51] M M Celik A Karakus C Zeren et al ldquoLicorice inducedhypokalemia edema and thrombocytopeniardquoHumanampExper-imental Toxicology vol 31 pp 1295ndash1298 2012

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


in the pathogenesis of DR [11 12] Recently it was demon-strated that HMGB1 is the main mediator bridging persistentneuroinflammation and chronic progressive dopaminergicneurodegeneration in neurodegenerative diseases such asParkinsonrsquos disease [13]

Recently HMGB1 has received particular attention withrespect to its pathological role in cerebral ischemia Afterischemic injury induced by transient middle cerebral arteryocclusion in mice and rats HMGB1 was found to betranslocated into the cytoplasmic compartment from nucleiand released into the extracellular space from neurons [14ndash18] In these studies extracellular HMGB1 plays a key rolein the development of neuronal injury through microglialactivation induction of apoptosis excitatory amino acidrelease and induction of proinflammatorymediators [14ndash19]Downregulation of HMGB1 or treatment with neutralizinganti-HMGB1 monoclonal antibody remarkably suppressedinfarct size activation of microglia and induction of proin-flammatorymarkers and inhibited the increased permeabilityof the blood-brain barrier [14 18]

In previous studies we demonstrated that HMGB1 andRAGE were expressed by vascular endothelial cells andstromal cells in fibrovascular epiretinal membranes frompatients with proliferative diabetic retinopathy (PDR) Inaddition we demonstrated increased levels of HMGB1 in thevitreous samples from patients with PDR and that there weresignificant positive correlations between the vitreous levels ofHMGB1 and the levels of the inflammatory biomarkers [20ndash22] Furthermore we demonstrated that diabetes inducedsignificant upregulation of the expression of HMGB1 andRAGE in the retinas of rats and mice and that intravitrealadministration of HMGB1 to normal rats induced activa-tion of inflammatory signaling pathways in the retina andincreased retinal vascular permeability [21 23]

Glycyrrhizin (GA) an ingredient of the licorice rootshas long been known to exhibit glucocorticoid-like anti-inflammatory actions by inhibiting 11120573-hydroxysteroid dehy-drogenase GA has been shown to have anti-inflammatoryand antiviral effects More recently GA has also been shownto bind to and inhibit chemoattractant mitogenic andcytokine-like activities of HMGB1 [24] In this study weexplored the hypothesis that HMGB1 plays a pathogeneticrole in mediating diabetes-induced retinal neuropathy Totest this hypothesis we investigated the expression of theneurodegeneration mediators and markers cleaved caspase-3 synaptophysin tyrosine hydroxylase (TH) glutamine syn-thetase (GS) glutamate and glyoxalase 1 (GLO 1) in theretinas of diabetic rats and in the retinas of normal ratsafter intravitreal administration of HMGB1 In additionwe analyzed whether constant GA intake suppresses retinalneuropathy induced by diabetes in rats

2 Materials and Methods

21 Animals

211 Induction of Diabetes and Glycyrrhizin Treatment Allprocedures with animals were performed in accordance with

the Association for Research in Vision and Ophthalmology(ARVO) statement for use of animals in ophthalmic andvision research andwere approved by the institutional animalcare and use committee of the College of Pharmacy KingSaud University Adult male Sprague Dawley rats of 8-9weeks of age (200minus220 g) were overnight fasted and strep-tozotocin (STZ) 55mgkg in 10mM sodium citrate bufferpH 45 (Sigma St Louis MO) was injected intraperitoneallyEqual volumes of citrate buffer were injected in nondiabeticanimals Rats were considered diabetic if their blood glucosewas greater than 250mgdL Age-matched normal rats servedas controls

Diabetic rats were divided into 2 groups the rats in groupI received normal drinking water without any supplementa-tion and group II received drinkingwater supplementedwithglycyrrhizic acid (150mgkgday Santa Cruz BiotechnologyInc Santa Cruz CA) immediately after establishment ofdiabetes throughout the course of the experiment Eachgroup had 8ndash12 rats After 4 weeks of diabetes the rats wereeuthanized by an overdose of chloral hydrate the eyes wereremoved and retina was isolated and frozen immediately inliquid nitrogen and stored atminus80∘C to be analyzed byWesternblot analysis or biochemical assay

212 Intravitreal Injection of HMGB1 Sprague Dawley rats(200minus220 g) were kept under deep anesthesia and sterilizedsolution of recombinant HMGB1 (5 ng5120583L RampD SystemsMinneapolis MN) was injected into the vitreous of the righteye as previously described by us [23] For the controlthe left eye received 5 120583L of sterile phosphate buffer saline(PBS) The animals were sacrificed 4 days after intravitrealadministration and the retinas were carefully dissectedsnap-frozen in liquid nitrogen and stored at minus80∘C to beanalyzed by Western blot analysis or biochemical assay Thebreakdown of blood-retina barrier induced by intravitrealinjection of HMGB1 might lead to raised levels of HMGB1 inthe serumTherefore we determined the levels of HMGB1 inequal amounts of serum from intravitreally injected rats andnormal rats using Western blot analysis

213 Western Blot Analysis Retinas were homogenized ina western lysis buffer (30mM Tris-HCl pH 74 250mMNa3VO4 5mM EDTA 250mM sucrose 1 Triton X-

100 with Protease inhibitor) The lysate was centrifuged at14000timesg for 10min at 4∘C and the supernatant was col-lected Protein content was assayed by DC protein assay (Bio-Rad Laboratories Hercules CA)The tissue lysate containing40ndash50120583g of protein was separated on 10 or 12 SDS-polyacrylamide gels and was transferred onto nitrocellulosemembranes The blots were blocked with 5 nonfat milkin TBST (20mM Tris-HCl pH 76 136mM NaCl and 01Tween-20)

For detection of HMGB1 phospho-ERK12 synapto-physin cleaved caspase-3 TH GLO1 and GS the membranewas incubated overnight at 4∘C with rabbit polyclonal anti-HMGB1 (1 1000 Cat number ab18256 Abcam UK) rabbitmonoclonal anti-phospho-ERK12 (05 120583gmL Cat numberMAB1018 RampD Systems) mouse monoclonal anti-ERK12







3 Results





0

05

1

15

2

25

Control

Mea

n le

vel

HMGB-1

lowast

120573-Actin


(a)

000

020

040

060

080

100

120

140

160

Mea

n le

vel

P-ERK12

ERK12

Control

lowast


(b)

000020040060080100120140160180

Mea

n le

vel

Caspase-3

Control

lowast

120573-Actin


(c)

Synaptophysin

000

050

100

150

200

250

300

Mea

n le

vel

Control

lowast

120573-Actin


(d)

000005010015020025030035040045

Mea

n Le

vel

Control

Tyrosinehydroxylase

lowast

Diabetic

120573-Actin

Diabetic + GA

(e)

000020040060080100120140160180

Mea

n le

vel

Glutaminesynthetase

lowast

Control Diabetic

120573-Actin

Diabetic + GA

(f)

Figure 1 Continued


000

020

040

060

080

100

120M

ean

leve

l

Glyoxalase 1

Control Diabetic

120573-Actin

lowast

Diabetic + GA

(g)

0

001

002

003

004

005

006

Glu

tam

ate (

nmol

120583L120583

g pr

otei

n)

lowast


(h)







4 Discussion



HMGB-1

0

02

04

06

08

1

12

PBS HMGB-1

Mea

n le

vel

120573-Actin

lowast

(a)

P-ERK12

ERK12

0

05

1

15

2

25

PBS HMGB-1

Mea

n le

vel

lowast

(b)

Caspase-3

002040608

1121416

PBS HMGB-1

Mea

n le

vel

120573-Actin

lowast

(c)

Synaptophysin

0

05

1

15

2

25

PBS HMGB-1

Mea

n le

vel

120573-Actin

lowast

(d)

lowast

002040608

112141618

PBS HMGB-1

Mea

n le

vel

120573-Actin

Tyrosinehydroxylase

(e)

lowast

0

05

1

15

2

25

PBS HMGB-1

Mea

n le

vel

Glutaminesynthetase

120573-Actin

(f)

Glyoxalase 1

lowast

002040608

112141618

PBS HMGB-1

Mea

n le

vel

120573-Actin

(g)

0

001

002

003

004

005

006

007

PBS HMGB-1

Glu

tam

ate (

nmol

120583L120583

g pr

otei

n)(h)






0

1

2

3

4

5

6

7

8

HM

GB-

1 ge

ne ex

pres

sion

(fold

chan

ge)


lowastlowast

lowast

Diabetic + GA











Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















3 Results





0

05

1

15

2

25

Control

Mea

n le

vel

HMGB-1

lowast

120573-Actin


(a)

000

020

040

060

080

100

120

140

160

Mea

n le

vel

P-ERK12

ERK12

Control

lowast


(b)

000020040060080100120140160180

Mea

n le

vel

Caspase-3

Control

lowast

120573-Actin


(c)

Synaptophysin

000

050

100

150

200

250

300

Mea

n le

vel

Control

lowast

120573-Actin


(d)

000005010015020025030035040045

Mea

n Le

vel

Control

Tyrosinehydroxylase

lowast

Diabetic

120573-Actin

Diabetic + GA

(e)

000020040060080100120140160180

Mea

n le

vel

Glutaminesynthetase

lowast

Control Diabetic

120573-Actin

Diabetic + GA

(f)

Figure 1 Continued


000

020

040

060

080

100

120M

ean

leve

l

Glyoxalase 1

Control Diabetic

120573-Actin

lowast

Diabetic + GA

(g)

0

001

002

003

004

005

006

Glu

tam

ate (

nmol

120583L120583

g pr

otei

n)

lowast


(h)







4 Discussion



HMGB-1

0

02

04

06

08

1

12

PBS HMGB-1

Mea

n le

vel

120573-Actin

lowast

(a)

P-ERK12

ERK12

0

05

1

15

2

25

PBS HMGB-1

Mea

n le

vel

lowast

(b)

Caspase-3

002040608

1121416

PBS HMGB-1

Mea

n le

vel

120573-Actin

lowast

(c)

Synaptophysin

0

05

1

15

2

25

PBS HMGB-1

Mea

n le

vel

120573-Actin

lowast

(d)

lowast

002040608

112141618

PBS HMGB-1

Mea

n le

vel

120573-Actin

Tyrosinehydroxylase

(e)

lowast

0

05

1

15

2

25

PBS HMGB-1

Mea

n le

vel

Glutaminesynthetase

120573-Actin

(f)

Glyoxalase 1

lowast

002040608

112141618

PBS HMGB-1

Mea

n le

vel

120573-Actin

(g)

0

001

002

003

004

005

006

007

PBS HMGB-1

Glu

tam

ate (

nmol

120583L120583

g pr

otei

n)(h)






0

1

2

3

4

5

6

7

8

HM

GB-

1 ge

ne ex

pres

sion

(fold

chan

ge)


lowastlowast

lowast

Diabetic + GA











Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

05

1

15

2

25

Control

Mea

n le

vel

HMGB-1

lowast

120573-Actin


(a)

000

020

040

060

080

100

120

140

160

Mea

n le

vel

P-ERK12

ERK12

Control

lowast


(b)

000020040060080100120140160180

Mea

n le

vel

Caspase-3

Control

lowast

120573-Actin


(c)

Synaptophysin

000

050

100

150

200

250

300

Mea

n le

vel

Control

lowast

120573-Actin


(d)

000005010015020025030035040045

Mea

n Le

vel

Control

Tyrosinehydroxylase

lowast

Diabetic

120573-Actin

Diabetic + GA

(e)

000020040060080100120140160180

Mea

n le

vel

Glutaminesynthetase

lowast

Control Diabetic

120573-Actin

Diabetic + GA

(f)

Figure 1 Continued


000

020

040

060

080

100

120M

ean

leve

l

Glyoxalase 1

Control Diabetic

120573-Actin

lowast

Diabetic + GA

(g)

0

001

002

003

004

005

006

Glu

tam

ate (

nmol

120583L120583

g pr

otei

n)

lowast


(h)







4 Discussion



HMGB-1

0

02

04

06

08

1

12

PBS HMGB-1

Mea

n le

vel

120573-Actin

lowast

(a)

P-ERK12

ERK12

0

05

1

15

2

25

PBS HMGB-1

Mea

n le

vel

lowast

(b)

Caspase-3

002040608

1121416

PBS HMGB-1

Mea

n le

vel

120573-Actin

lowast

(c)

Synaptophysin

0

05

1

15

2

25

PBS HMGB-1

Mea

n le

vel

120573-Actin

lowast

(d)

lowast

002040608

112141618

PBS HMGB-1

Mea

n le

vel

120573-Actin

Tyrosinehydroxylase

(e)

lowast

0

05

1

15

2

25

PBS HMGB-1

Mea

n le

vel

Glutaminesynthetase

120573-Actin

(f)

Glyoxalase 1

lowast

002040608

112141618

PBS HMGB-1

Mea

n le

vel

120573-Actin

(g)

0

001

002

003

004

005

006

007

PBS HMGB-1

Glu

tam

ate (

nmol

120583L120583

g pr

otei

n)(h)






0

1

2

3

4

5

6

7

8

HM

GB-

1 ge

ne ex

pres

sion

(fold

chan

ge)


lowastlowast

lowast

Diabetic + GA











Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















000

020

040

060

080

100

120M

ean

leve

l

Glyoxalase 1

Control Diabetic

120573-Actin

lowast

Diabetic + GA

(g)

0

001

002

003

004

005

006

Glu

tam

ate (

nmol

120583L120583

g pr

otei

n)

lowast


(h)







4 Discussion



HMGB-1

0

02

04

06

08

1

12

PBS HMGB-1

Mea

n le

vel

120573-Actin

lowast

(a)

P-ERK12

ERK12

0

05

1

15

2

25

PBS HMGB-1

Mea

n le

vel

lowast

(b)

Caspase-3

002040608

1121416

PBS HMGB-1

Mea

n le

vel

120573-Actin

lowast

(c)

Synaptophysin

0

05

1

15

2

25

PBS HMGB-1

Mea

n le

vel

120573-Actin

lowast

(d)

lowast

002040608

112141618

PBS HMGB-1

Mea

n le

vel

120573-Actin

Tyrosinehydroxylase

(e)

lowast

0

05

1

15

2

25

PBS HMGB-1

Mea

n le

vel

Glutaminesynthetase

120573-Actin

(f)

Glyoxalase 1

lowast

002040608

112141618

PBS HMGB-1

Mea

n le

vel

120573-Actin

(g)

0

001

002

003

004

005

006

007

PBS HMGB-1

Glu

tam

ate (

nmol

120583L120583

g pr

otei

n)(h)






0

1

2

3

4

5

6

7

8

HM

GB-

1 ge

ne ex

pres

sion

(fold

chan

ge)


lowastlowast

lowast

Diabetic + GA











Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HMGB-1

0

02

04

06

08

1

12

PBS HMGB-1

Mea

n le

vel

120573-Actin

lowast

(a)

P-ERK12

ERK12

0

05

1

15

2

25

PBS HMGB-1

Mea

n le

vel

lowast

(b)

Caspase-3

002040608

1121416

PBS HMGB-1

Mea

n le

vel

120573-Actin

lowast

(c)

Synaptophysin

0

05

1

15

2

25

PBS HMGB-1

Mea

n le

vel

120573-Actin

lowast

(d)

lowast

002040608

112141618

PBS HMGB-1

Mea

n le

vel

120573-Actin

Tyrosinehydroxylase

(e)

lowast

0

05

1

15

2

25

PBS HMGB-1

Mea

n le

vel

Glutaminesynthetase

120573-Actin

(f)

Glyoxalase 1

lowast

002040608

112141618

PBS HMGB-1

Mea

n le

vel

120573-Actin

(g)

0

001

002

003

004

005

006

007

PBS HMGB-1

Glu

tam

ate (

nmol

120583L120583

g pr

otei

n)(h)






0

1

2

3

4

5

6

7

8

HM

GB-

1 ge

ne ex

pres

sion

(fold

chan

ge)


lowastlowast

lowast

Diabetic + GA











Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

5

6

7

8

HM

GB-

1 ge

ne ex

pres

sion

(fold

chan

ge)


lowastlowast

lowast

Diabetic + GA











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
















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