Increased expression of receptor for advanced glycation end-products worsens focal brain ischemia in diabetic rats*

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1 NEURAL REGENERATION RESEARCH Volume 7, Issue 13, My Cite this rticle s: Neurl Regen Res. 2012;7(13): Incresed expression of receptor for dvnced glyction end-products worsens focl brin ischemi in dibetic rts* Ying Xing, Jinting He, Weidong Yu, Lingling Hou, Jijun Chen Chin-Jpn Union Hospitl, Jilin University, Chngchun , Jilin Province, Chin Abstrct A rt model of dibetes mellitus ws induced by high ft diet, followed by focl induced using the thred method fter 0.5 month. Immunohistochemistry showed tht expression of receptor for dvnced glyction end-products ws higher in the ischemic cortex of dibetic rts compred with non-dibetic rts with. Western blot ssy reveled incresed phosphorylted c-jun N-terminl kinse expression, nd unchnged phosphorylted extrcellulr signl-regulted protein kinse protein expression in the ischemic cortex of dibetic rts compred with non-dibetic rts with. Additionlly, phosphorylted p38 mitogen-ctivted protein kinse protein ws not detected in ny rts in the two groups. Severity of limb hemiplegi ws worse in dibetic rts with compred with ischemi lone rts. The results suggest tht incresed expression of receptor for dvnced glyction end-products cn further ctivte the c-jun N-terminl kinse pthwy in mitogen-ctivted protein kinse, thereby worsening brin injury ssocited with focl in dibetic rts. Key Words receptor for dvnced glyction end-products; focl ; dibetes mellitus; mitogen-ctivted protein kinse; c-jun N-terminl kinse; signl trnsduction; neurl regenertion Abbrevitions RAGE, receptor for dvnced glyction end-products; JNK, c-jun N-terminl kinse; MAPK, mitogen-ctivted protein kinse Ying Xing, M.D., Associte professor, Associte chief physicin, Mster s supervisor, Chin-Jpn Union Hospitl, Jilin University, Chngchun , Jilin Province, Chin Corresponding uthor: Jijun Chen, M.D., Associte professor, Chief physicin, Mster s supervisor, Chin-Jpn Union Hospitl, Jilin University, Chngchun , Jilin Province, Chin xingying1970@163.com Received: Accepted: (N /YJ) Xing Y, He JT, Yu WD, Hou LL, Chen JJ. Incresed expression of receptor for dvnced glyction end-products worsens focl in dibetic rts. Neurl Regen Res. 2012;7(13): INTRODUCTION Receptor for dvnced glyction end-products (RAGE), member of the immunoglobulin superfmily, is extensively distributed t the surfce of mononucler mcrophges, endothelil cells, neurons, smooth muscle cells, nd mesngil cells [1]. As cell signl trnsduction membrne receptor, it cn bind vriety of lignds, such s dvnced glyction end-product, myloid-β, S100/clgrnulin, high-mobility group box 1 protein, nd β-sheet fibril [1-2]. Under norml conditions, RAGE expression is low, but its expression is significntly incresed when tissues or cells re stressed [3-5]. Shoji et l [6] found high expression of RAGE in vsculr endothelil cells in dibetic rts [7-10]. Recent studies hve lso shown tht RAGE mrna expression ws incresed in rts with brin ischemi [11-14]. A previous study from our group demonstrted incresed free rdicl concentrtion, which cn induce RAGE expression, in dibetes mellitus complicted by focl [15]. It is possible tht RAGE expression my increse following cute in sufferers of dibetes mellitus. MAPKs pthwy is n importnt signl trnsduction system in mmmlin cells [16-18]. It plys roles in cell growth, doi: /j.issn

2 development, differentition, prolifertion, nd poptosis. In the mmml, there re three mjor MAPK subgroups, extrcellulr signl-regulted protein kinse, c-jun N-terminl kinse (JNK), nd p38 mitogen-ctivted protein kinse (MAPK). Activted JNK cn bind to RAGE nd induce cell poptosis, thereby ggrvting pthologic injury [19-21]. RAGE is promoter of MAPK signl trnsduction [22] ; therefore chnges in RAGE expression cn ffect the expression of downstrem genes [23]. Thus, the purpose of the present study ws to investigte the role of the RAGE-MAPKs pthwy in dibetes mellitus complicted by focl in rts.. The pllor re ws significntly enlrged t 6 hours nd ws further worsened t 24 hours fter. The ischemic re ws significntly lrger in the dibetes mellitus plus brin ischemi group compred with tht in the group t 24 hours fter ischemi (P < 0.05; Figure 1, supplementry Figure 1 online). A RESULTS Quntittive nlysis of experimentl nimls Of the 80 selected rts, 40 were rndomly selected nd used to estblish dibetes mellitus model induced by high ft diet. During model estblishment, rts with blood glucose > 30 mm were excluded nd supplemented with other nimls. After 15 dys, focl ws induced by the thred method. Rts with subrchnoid hemorrhge, intropertive convulsion, nd conscious disturbnce were excluded nd supplemented with other nimls. Eight rts from ech of the group nd dibetes mellitus plus group were selected prior to nd 1, 3, 6, nd 24 hours post-ischemi for nlysis. Neurologicl function of dibetic rts with focl brin ischemi The Long neurologicl function test showed tht neurologicl function impirment ws worse in dibetic rts with compred with those in the brin ischemi only group, t 24 hours fter focl brin ischemi (P < 0.05; Tble 1). Tble 1 Neurologicl function scores (score) t 24 hours fter focl Group Medin Men rnk Brin ischemi Dibetes mellitus plus Dt re expressed s men rnk of eight rts in ech group. Intergroup comprison of enumerted dt ws conducted using nonprmetric test. P < 0.05, vs. group. Infrct size of dibetic rts with focl Brin tissues remined unchnged 1 hour fter brin ischemi. Frgmented pllor re ws observed in the left hemisphere with n uncler boundry 3 hours fter B Figure 1 Brin tissue sections of rts t 24 hours fter (2,3,5-triphenyltetrzolium chloride stining). Norml brin tissues were red, but the ischemic brin tissues were pllid. (A) Brin ischemi group; (B) dibetes mellitus plus brin ischemi group. Corticl RAGE expression in dibetic rts with focl Immunohistochemistry showed low level of RAGE expression in the cerebrl cortex of the nd dibetes mellitus plus groups prior to onset of. RAGE expression ws significntly incresed in both groups t 3 hours fter, nd cell bodies of RAGE-positive cells were smller. At 6 hours, RAGE expression ws decresed, nd RAGE-positive cells were stined lightly nd hd smll cell bodies. At 24 hours, RAGE expression ws elevted, cell bodies of RAGE-positive cells were smller, nd the intercellulr spce ws significntly enlrged, compred to non-ischemic rts (P < 0.05). Moreover, RAGE expression ws higher in the dibetes mellitus plus group compred with tht in the lone group (P < 0.05, except 24-hour group; Figures 2, 3, supplementry Figure 2 online). Corticl phosphorylted-jnk (p-jnk), phosphorylted-erk (p-erk) nd phosphorylted-p38mapk (p-p38mapk) in dibetic rts with focl Western blot ssy showed tht p-jnk expression ws 1001

incresed in the cerebrl cortex of rts in the dibetes mellitus plus group, which peked t 3 hours nd grdully decresed therefter.

Corticl p-erk expression remined unchnged in both groups fter (P > 0.05), with no sttisticl difference between groups (P > 0.05). No p-p38mapk expression ws detected in the cerebrl cortex in ny rts from the two groups (Figure 4; Tble 2).

β-ctin 43 kd C Figure 4 Phosphorylted c-jun N-terminl kinse (p-jnk), phosphorylted extrcellulr signl-regulted protein kinse (p-erk), nd phosphorylted p38 mitogen-ctivted protein kinse (p-p38-mapk)

3 incresed in the cerebrl cortex of rts in the dibetes mellitus plus group, which peked t 3 hours nd grdully decresed therefter. The expression of p-jnk begn to increse t 3 hours fter in the lone group, but ws significntly less thn tht in the dibetes mellitus plus group (P < 0.05). Corticl p-erk expression remined unchnged in both groups fter (P > 0.05), with no sttisticl difference between groups (P > 0.05). No p-p38mapk expression ws detected in the cerebrl cortex in ny rts from the two groups (Figure 4; Tble 2). Dibetes mellitus plus brin ischemi group Time fter ischemi (hour) Brin ischemi group Time fter ischemi (hour) p-jnk 54 kd β-ctin 43 kd p-erk 45 kd A B β-ctin 43 kd p-p38-mapk 35 kd β-ctin 43 kd C Figure 4 Phosphorylted c-jun N-terminl kinse (p-jnk), phosphorylted extrcellulr signl-regulted protein kinse (p-erk), nd phosphorylted p38 mitogen-ctivted protein kinse (p-p38-mapk) expression in the cerebrl cortex of rts (western blot). Figure 2 Receptor for dvnced glyction end-products expression in the cerebrl cortex of dibetic rts with brin ischemi (immunohistochemicl stining, 200). Arrows represent positive cells for receptor for dvnced glyction end-products. (A) Prior to ; (BC) 1 nd 3 hours fter brin ischemi. Number of RAGE positive cells (/200-fold field) Pre-ischemi Brin ischemi group Dibetes mellitus plus group Time post-ischemi (hour) Figure 3 Receptor for dvnced glyction end-products (RAGE) expression in the cerebrl cortex of rts. Dt re expressed s men ± SD of eight rts in ech group. Intergroup differences were compred using pired t-test. P < 0.05, vs. group. Tble 2 Phosphorylted c-jun N-terminl kinse (p-jnk) nd phosphorylted extrcellulr signl-regulted protein kinse (p-erk) expression in cerebrl cortex of rts (bsorbnce rtio to β-ctin) Time postischemi (hour) Brin ischemi DISCUSSION p-jnk Dibetes mellitus plus Brin ischemi p-erk Dibetes mellitus plus ± ± ± ± ±0.07 b 1.41±0.12 b 0.61± ± ±0.23 b 1.16±0.16 b 0.76± ± ±0.21 b 0.94±0.14 b 0.73± ±0.17 Dt re expressed s men ± SD of eight rts in ech group t ech time point. Intergroup differences were compred using pired t-test. p-jnk expression significntly chnged between two groups t different time points: P < 0.05, vs. group; b P < 0.05, vs. previous time point. However, p-erk expression remined unchnged in the two groups t different time points (P > 0.05). High bsorbnce indictes high protein expression. The present study used n niml model of focl brin ischemi nd dibetes mellitus plus focl, nd showed tht corticl RAGE expression incresed post-ischemi, consistent with previous results [24]. Moreover, corticl RAGE expression ws higher in the 1002

4 dibetes mellitus plus focl group compred with those in the focl lone group. RAGE is membrne receptor involved in pthologicl processes. Results from the present study suggest tht RAGE prticiptes in the process of dibetes mellitus plus increses in brin injury. RAGE expression ws reduced t 6 hours fter ischemi in focl lone or dibetes mellitus plus focl, possibly becuse fter ischemi for 6 hours, n endogenous protection mechnism ws triggered; t 24 hours, protection dispered, nd RAGE expression significntly incresed, thereby worsening the injury. As membrne receptor of signl trnsduction, RAGE expression increses cn enhnce downstrem signl trnsduction pthwys. One or severl signl trnsduction pthwys re triggered during different pthologicl processes. In the present study, western blot showed tht corticl p-jnk expression incresed t 3 hours fter, but ws restored by 6 hours. Expression of p-jnk begn to increse 1 hour fter brin ischemi in dibetic rts, peked t 3 hours, nd grdully decresed. p-jnk expression ws erlier nd greter in the dibetes mellitus plus group compred with focl lone. As p-jnk prticiptes in cell poptosis, the injury in the dibetes mellitus plus group ws worse, consistent with neurologicl function scores. Corticl p-erk expression remined unchnged between the two groups, nd no p-p38mapk expression ws detected in ny group. This suggests tht overctivtion of the JNK pthwy in dibetes mellitus ggrvtes. JNK ctivtion is mjor signl trnsduction pthwy of cell poptosis. Thus, dibetes mellitus plus brin ischemi cn increse cell poptosis nd worsen brin injury. In conclusion, incresed RAGE expression in the cerebrl cortex of rts with dibetes mellitus complicted by focl cn further ctivte the JNK pthwy in MAPKs, thus worsening brin injury. MATERIALS AND METHODS Design A rndomized, controlled, niml experiment. Time nd setting The experiment ws performed t the Lbortory of Morphology, Normn Bethune College of Medicine, Jilin University, Chin from August 2008 to Mrch Mterils A totl of 80 mle Wistr rts ged 67 weeks weighing g were provided by the Animl Experimentl Center, Bsic Medicl College of Jilin University (license No. SCXK (Ji) ). They were housed t 20 ± 2 C, with humidity t 4050%, nd nturl illumintion. Experimentl procedures were performed in ccordnce with the Guidnce Suggestions for the Cre nd Use of Lbortory Animls, issued by the Ministry of Science nd Technology of the People s Republic of Chin [25]. Methods Estblishment of dibetic model The rts were fed high ft diet comprising 40% crbohydrte, 13% protein, 40% ft nd 7% other ingredients, nd llowed free ccess to wter. After 4 weeks, they were intrperitonelly injected with 30 mg/kg streptozotocin (Sigm, St. Louis, MO, USA), nd blood ws extrcted from the til vein fter 7 dys. Successful estblishment of dibetic model ws determined by fsting blood glucose 16.7 mm [26-27]. Estblishment of focl model The rts were housed for 0.5 months fter the induction of dibetes mellitus, followed by focl s previously described [28]. Briefly, the rts were nesthetized by intrperitonel injection with 10% chlorl hydrte (Shnghi Yope Biotech, Shnghi, Chin), nd medin incision ws mde t the neck. The left common crotid rtery ws dissected nd internl nd externl crotid rteries were isolted towrds the hed long the left common crotid rtery. The pterygopltine rtery ws isolted nd the root ws ligted. An incision ws cut t the bifurction of the internl nd externl crotid rteries, nd 0.28 mm nylon thred ws inserted to the crnium, t depth of 18 ± 0.5 mm, until it reched the left middle cerebrl rtery, to induce focl. After the rts recovered from nesthesi, neurologicl function ws observed using the Long 5-score scle [28] to evlute model success (supplementry Video 1 online). Rts with scores of 0, 4, or tht subsequently died were excluded. The Long scle ws used 24 hours following model estblishment to evlute neurologicl function. 2,3,5-triphenyltetrzolium chloride stining for confirming focl After focl, one rt from ech group ws selected t the sme time point, nesthetized, nd perfused with norml sline. The intct brin ws hrvested, quick-frozen t 20 C for 20 minutes, nd coronlly sectioned every 2 mm, for five sections in totl. The sections were plced in 2% 2,3,5-triphenyltetrzolium chloride solution (Wuhn Boster, Wuhn, Chin) nd incubted t 37 C in the drk for 30 minutes. The sections were flipped every 1003

5 510 minutes to llow them to fully contct the stining solution. Norml brin tissues were stined red, nd ischemic brin tissues stined pllor. Immunohistochemistry for RAGE expression in ischemic cerebrl cortex The rts were nesthetized with diethyl ether nd plced on n operting bord. A medin incision ws mde t the bdomen to expose the hert. The right uricle ws cut open, nd perfused with 100 ml norml sline. The ischemic cortex ws hrvested, fixed in 10% formlin for 24 hours, prffin embedded, sectioned into 4 μm thick sections, stined with hemtoxylin nd eosin, nd ttched to polylysine-coted slides. The sections were dewxed with xylene nd hydrted through series of decresing ethnol concentrtions. The sections were then incubted with rbbit nti-rage polyclonl ntibody (1:100; Abcm, Cmbridge, UK) overnight t 4 C nd subsequently wshed with phosphte buffered sline, 5 minutes 3. Sections were then treted with biotin lbeled got nti-rbbit IgG (Abcm) t 37 C for 10 minutes, followed by horserdish peroxidse-lbeled streptvidin solution t 37 C for 10 minutes. The sections were visulized using diminobenzidine (Boster), counterstined with hemtoxylin, nd differentited with hydrochloric cid nd ethnol. Sections were then wshed with tp wter for 1 minute, nd dehydrted through series of incresing ethnol concentrtions, nd mounted. Five rndom fields of view from lesioned tissues were observed by light microscopy (Olympus, Tokyo, Jpn) to quntify men positive cells. Western blot for p-p38mapk, p-erk, nd p-jnk in the ischemic cerebrl cortex The ischemic cerebrl cortex ws cut into pieces, mixed with lyste t rtio of μl lyste per 200 mg tissue, homogenized, nd centrifuged t g for 35 minutes. The superntnt ws hrvested nd prepred for use in seprtion gel. Culture superntnt ws mixed with 5 sodium dodecyl sulfte smple buffer solution t rtio of 4:1, boiled for 35 minutes, cooled to room temperture, nd centrifuged t g for 30 seconds. The superntnt ws plced in wells, 50 μl per well, nd electrophoreticlly trnsferred to nitrocellulose filter t 200 ma, dried t room temperture for 3060 minutes, nd blocked with 20 ml blocking solution for 3 hours. The filter ws then incubted with rbbit nti-p-p38-mapk, p-erk, nd p-jnk polyclonl ntibodies (1:500; Beijing Biosynthesis Biotechnology, Beijing, Chin) nd mouse nti-rt β-ctin monoclonl ntibody (1:500; Shnghi Xingsheng Biotechnology, Shnghi, Chin) overnight t 4 C, followed by horserdish peroxidse lbeled got nti-rbbit, mouse ntibody (1:500; Boster). The products were plced in hybridiztion bg, nd shken with cellulose membrne for 14 hours. The membrne ws wshed with Tris buffered sline nd visulized with diminobenzidine. Bnd bsorbnce ws nlyzed using Bndscn (Shnghi Tinneng Softwre, Shnghi, Chin), nd the bsorbnce rtio of trget bnd to β-ctin represented the level of p-p38-mapk, p-erk, nd p-jnk expression. Sttisticl nlysis Dt were expressed s men ± SD nd nlyzed using SPSS 11.0 softwre pckge (SPSS, Chicgo, IL, USA). Intergroup differences were compred using pired t-test nd nonprmetric rnk sum test where pproprite. The vlue of P < 0.05 ws considered sttisticlly significnt. Acknowledgments: We thnk Kexin Hung nd Bo Shi, Deprtment of Pthology, Normn Bethune College of Medicine, Jilin University, Chin for technicl support. Funding: This study ws supported by the Science nd Technology Development Foundtion of Jilin Province, No Author contributions: Ying Xing conceived nd designed the study, revised the mnuscript, nd ws in chrge of funds. Jinting He nlyzed experimentl dt nd prepred the mnuscript. Weidong Yu provided nd integrted experimentl dt. Lingling Hou conducted sttisticl nlysis. Jijun Chen guided the study. Conflicts of interest: None declred. Ethicl pprovl: This study received permission from the Animl Ethics Committee of Jilin University, Chin. Supplementry informtion: Supplementry dt ssocited with this rticle cn be found in the online version by visiting nd entering Vol. 7, No. 13, 2012 fter selecting the NRR Current Issue button on the pge. REFERENCES [1] Mosquer JA. Role of the receptor for dvnced glyction end products (RAGE) in inflmmtion. Invest Clin. 2010; 51(2): [2] Rong LL, Yn SF, Wendt T, et l. RAGE modultes peripherl nerve regenertion vi recruitment of both inflmmtory nd xonl outgrowth pthwys. FASEB J. 2004;18(15): [3] Kim SW, Lim CM, Kim JB, et l. Extrcellulr HMGB1 relesed by NMDA tretment confers neuronl poptosis vi RAGE-p38 MAPK/ERK signling pthwy. Neurotox Res. 2011;20(2): [4] Hssid BG, Nir MN, Ducruet AF, et l. Neuronl RAGE expression modultes severity of injury following trnsient focl cerebrl ischemi. J Clin Neurosci. 2009;16(2):

6 [5] Kle AZ, Schmidt AM, Hudson BI. Alterntive splicing of RAGE: roles in biology nd disese. Front Biosci. 2011; 17: [6] Shoji T, Koym H, Moriok T, et l. Receptor for dvnced glyction end products is involved in impired ngiogenic response in dibetes. Dibetes. 2006;55(8): [7] Sullivn KA, Feldmn EL. New developments in dibetic neuropthy. Curr Opin Neurol. 2005;18(5): [8] Tn AL, Forbes JM, Cooper ME. AGE, RAGE, nd ROS in dibetic nephropthy. Semin Nephrol. 2007;27(2): [9] Schleicher E, Friess U. Oxidtive stress, AGE, nd therosclerosis. Kidney Int Suppl. 2007;(106):S [10] Niiy Y, Abumiy T, Shichinohe H, et l. Susceptibility of brin microvsculr endothelil cells to dvnced glyction end products-induced tissue fctor upregultion is ssocited with intrcellulr rective oxygen species. Brin Res. 2006;1108(1): [11] M L, Crter RJ, Morton AJ, et l. RAGE is expressed in pyrmidl cells of the hippocmpus following moderte hypoxic-ischemic brin injury in rts. Brin Res. 2003; 966(2): [12] Chng JS, Wendt T, Qu W, et l. Oxygen deprivtion triggers upregultion of erly growth response-1 by the receptor for dvnced glyction end products. Circ Res. 2008;102(8): [13] Ymgishi S. Role of dvnced glyction end products (AGEs) nd receptor for AGEs (RAGE) in vsculr dmge in dibetes. Exp Gerontol. 2011;46(4): [14] Zhng F, Bnker G, Liu X, et l. The novel function of dvnced glyction end products in regultion of MMP-9 production. J Surg Res. 2011;171(2): [15] Niiy Y, Abumiy T, Shichinohe H, et l. Susceptibility of brin microvsculr endothelil cells to dvnced glyction end products-induced tissue fctor upregultion is ssocited with intrcellulr rective oxygen species. Brin Res. 2006;1108(1): [16] Kyyli US, Pennell CM, Trujillo C, et l. Cytoskeletl chnges in hypoxic pulmonry endothelil cells re dependent on MAPK-ctivted protein kinse MK2. J Biol Chem. 2002;277(45): [17] Trini C, Pedt F, Ciprini S, et l. P2 receptor ntgonists prevent synptic filure nd extrcellulr signl-regulted kinse 1/2 ctivtion induced by oxygen nd glucose deprivtion in rt CA1 hippocmpus in vitro. Eur J Neurosci. 2011;33(12): [18] Chen X, Tin Y, Yo L, et l. Hypoxi stimultes prolifertion of rt neurl stem cells with influence on the expression of cyclin D1 nd c-jun N-terminl protein kinse signling pthwy in vitro. Neuroscience. 2010; 165(3): [19] Dhnsekrn DN, Reddy EP. JNK signling in poptosis. Oncogene. 2008;27(48): [20] Kog S, Kojim S, Kishimoto T, et l. Over-expression of mp kinse phosphtse-1 (MKP-1) suppresses neuronl deth through regulting JNK signling in hypoxi/ re-oxygention. Brin Res. 2012;1436: [21] Alikhni M, Mclelln CM, Rptis M, et l. Advnced glyction end products induce poptosis in fibroblsts through ctivtion of ROS, MAP kinses, nd the FOXO1 trnscription fctor. Am J Physiol Cell Physiol. 2007; 292(2):C [22] Rsheed Z, Akhtr N, Hqqi TM. Advnced glyction end products induce the expression of interleukin-6 nd interleukin-8 by receptor for dvnced glyction end product-medited ctivtion of mitogen-ctivted protein kinses nd nucler fctor-κb in humn osteorthritis chondrocytes. Rheumtology (Oxford). 2011;50(5): [23] Stern DM, Yn SD, Yn SF, et l. Receptor for dvnced glyction endproducts (RAGE) nd the complictions of dibetes. Ageing Res Rev. 2002;1(1):1-15. [24] Zong H, Mdden A, Wrd M, et l. Homodimeriztion is essentil for the receptor for dvnced glyction end products (RAGE)-medited signl trnsduction. J Biol Chem. 2010;285(30): [25] The Ministry of Science nd Technology of the People s Republic of Chin. Guidnce Suggestions for the Cre nd Use of Lbortory Animls [26] Rees DA, Alcoldo JC. Animl models of dibetes mellitus. Dibet Med. 2005;22(4): [27] Islm MS, Loots du T. Experimentl rodent models of type 2 dibetes: review. Methods Find Exp Clin Phrmcol. 2009;31(4): [28] Long EZ, Weinstein PR, Crlson S, et l. Reversible middle cerebrl rtery occlusion without crniectomy in rts. Stroke. 1989;20(1): (Edited by Go JL, Go HW/Su LL/Song LP) 1005

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