MicroRNA-146a and its adapter proteins are affected by diabetes in rat s heart

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1 DOI: /BLL_2016_031 Brtisl Med J 2016; 117 (3) EXPERIMENTAL STUDY MicroRNA-146 nd its dpter proteins re ffected y dietes in rt s hert Eftekhr E 1, Doustki Zoli M 2, Ktei M 3, Ghdiri Soufi F 3 Deprtment of Biochemistry, Fculty of Medicine, Hormozgn University of Medicl Sciences, Bndr As, Irn. soufifg@tzmed.c.ir ABSTRACT OBJECTIVES: This study ws conducted to explore whether microrna-146 nd its dpter proteins (TNF-α receptor-ssocited fctor 6 (TRAF6) nd interleukin-1 receptor-ssocited kinse 1 (IRAK1)) re ffected y dietes in the rt hert. METHODS: Twelve mle Sprgue-Dwley rts were rndomized into control nd dietic groups (n = 6). Streptozotocin-nicotinmide experimentl model ws used to induce type 2 dietes. The gene expression of MicroRNA-146, nucler fctor-κb (NF-κB), IRAK1 nd TRAF6, s well s NF-κB ctivity, IRAK1 nd TRAF6 protein levels were mesured. Moreover, NF-κB ctivity ws mesured in response to mir-146 mimic trnsfection (20 nmol) in humn umilicl vein endothelil cells (HUVECs) under hyperglycemic condition (25 mm D-glucose for 24 h). RESULTS: The expression of MicroRNA-146 ws incresed in the hert tissue, 2 months fter dietes induction nd in HUVECs. Also, the mrna nd protein levels of NF-κB, IRAK1 nd TRAF6 were incresed in the hert of dietic rts. Moreover, trnsfection of mir-146 mimic prevented from signifi cnt increse of NF-κB ctivity in hyperglycemic HUVECs. CONCLUSION: Presumly, defect in the regultion of IRAK1 nd TRAF6 cn weken mir-146 regultory effect nd provides sitution for sustined ctivtion of NF-κB nd its trgets to promote crdic cells towrd normlities (Fig. 3, Ref. 28). Text in PDF KEY WORDS: dietes, hert, microrna-146, NF-κB, IRAK1, TRAF6. Introduction Dietes mellitus is serious glol helth prolem resulting from insulin insufficiency or inefficiency which, its prevlence is incresing drmticlly, nd due to the rising rte of oesity nd sedentry lifestyle, out 552 million people re estimted to e suffering from dietes mellitus worldwide y 2030 (1, 2). Dietes-induced hyperglycemi nd the corresponding glucotoxicity result in mcro nd microvsculr complictions such s dietic neuropthy, nephropthy, retinopthy, nd crdiomyopthy (3). 1 Deprtment of Biochemistry, Fculty of Medicine, Hormozgn University of Medicl Sciences, Bndr As, Irn, 2 Deprtment of Physiology, Fculty of Medicine, Hormozgn University of Medicl Sciences, Bndr As, Irn, nd 3 Stem cells Reserch Center, Hormozgn University of Medicl Sciences, Bndr As, Irn Address for correspondence: Dr. F.G. Soufi, PhD, Stem cells Reserch Center, Shhid Mohmmdi hospitl, Jomhouri Boulevrd, Bndr As, Postl Code: , Irn. Phone/Fx: Grnt support: The grnt of this study ws fully supported y Stem cells Reserch Center, Hormozgn University of Medicl Sciences, Bndr As, Irn. Acknowledgements: The grnt of this study ws supported y Stem cells Reserch Center, Hormozgn University of Medicl Sciences, Bndr As, Irn. Our dt in this work were derived from the thesis of Ms. Mrym Doustki Zoli for Mster of Science degree in physiology (thesis seril numer: 90-ph-3). Ptients with dietes hve significntly incresed risk of premture mortlity nd n incresed risk of microvsculr nd crdiovsculr complictions. (4). Despite numerous investigtions hve een directed towrd incresing our knowledge out the mechnisms involved in the pthogenesis of dietes-relted ngiopthies, the precise mechnism is still uncler, nd needs to e clrified y further studies. In recent yers severl studies hve demonstrted significnt ltertions in the expression level of microrna-146 (mir-146) in the lood nd peripherl tissues of oth dietic ptients nd nimls (5-15). MiR-146 like other memers of micrornas fmily is smll, noncoding, nd single strnd RNA tht modultes trget gene expression t post-trnscriptionl level y inding to trget messenger RNA (mrna), nd regulting its stility nd trnsltion (16). It hs een suggested tht mir-146 prticiptes in mny physiologicl nd pthologicl processes, in prt y regulting the ctivtion of mster pro-inflmmtory trnscription fctor, nucler fctor kpp B (NF-κB) (16). It is elieved tht ctivtion of NF-κB promotes the trnscription of the mir-146 gene tht, in turn down-regultes two key dpter molecules, TNF receptor ssocited fctor 6 (TRAF6) nd Interleukin-1 receptor-ssocited kinse 1 (IRAK1), to decrese NF-κB ctivity (17). However, this negtive feedck loop hs not een demonstrted y severl investigtions (12, 13, 18, 19). While decresed expression of mir-146 hs een reported in dietic mouse wounds (5), dietic rts ort, scitic nerve, Indexed nd strcted in Science Cittion Index Expnded nd in Journl Cittion Reports/Science Edition

2 Eftekhr E et l. MicroRNA-146 nd its dpter proteins re ffected y dietes in rt s hert xx kidney, hert nd dorsl root gngli (6 9), glycted lumin- nd high glucose-stimulted endothelil cells (10), nd in serum nd peripherl lood mononucler cells of type 1 nd 2 dietic ptients (11, 12), n incresed mir-146 expression hs lso een documented in dietic kidney (13), nd in plsm nd liml cornel epithelium comprtment of dietic ptients (14, 15). While growing evidence indictes tht mir-146 plys role in the pthogenesis of dietes relted complictions, there is little informtion ville concerning the expression level of this microrna nd its role in the regultion of NF-κB ctivity in dietic hert. In this context, only Feng et l hve previously reported tht mir-146 expression level decreses in the herts of type 1 nd type 2 dietic rts (8). Given tht the NF-κB plys key role in the pthogenesis of dietic hert complictions, this study ws conducted to explore whether microrna-146 (s n NF-кB regulting fctor) nd its dpter proteins (TRAF6 nd IRAK1) re ffected y the dietes in the rt hert. To evlute this hypothesis, we mesured the gene expression level of mir- 146, IRAK1, TRAF6, nd NF-κB, s well s NF-κB ctivity nd IRAK1 nd TRAF6 protein levels in the hert of dietic rts. Moreover, NF-κB ctivity ws mesured in response to mir-146 mimic trnsfection in humn umilicl vein endothelil cells (HU- VECs) under hyperglycemic condition. Mterils nd methods Animl experiment Twelve mle Sprgue-Dwley rts (3 months old with 314 ± 7 g of ody weight; Rzi Institute, Tehrn, Irn) were housed in stndrd cges (3 rts in ech), t room temperture (22 25 C) with 12:12-h light/drk cycles nd free ccess to food nd wter. The rts were rndomized into control nd dietic groups (n = 6). This study ws designed in ccordnce with US Ntionl Institutes of Helth (NIH puliction, No , revised 1996) nd AR- RIVE guidelines for the cre nd use of nimls pproved y the Ethics Committee for the Use of Animls in Reserch t Hormozgn University of Medicl Sciences (No: 93/3-1/6/13 Mr 2014) (20). Type 2 dietes ws induced y single dose intrperitonel injection of streptozotocin (50 mg/kg) dissolved in 0.1 M of citrte uffer (ph 4.5), 15 min fter the intrperitonel injection of nicotinmide (110 mg/kg; i.p.) in 12 h fsted rts (13). Citrte uffer ws injected to control rts. Dietes ws defined s fsting lood glucose ws higher thn 250 mg/dl on 2 consecutive dys, using glucometer (Arkry, Kyoto, Jpn) (13). One dy efore scrifice of rts, n orl glucose tolernce test (OGTT) ws performed s previously descried (13). The nimls were killed 2 months fter dietes induction nd the herts were collected nd stored t 70 C. All mnipultions were held in the morning. Cell culture nd trnsfection of mir-146 HUVECs were purchsed from the Ntionl Cell Bnk of Irn (NCBI, Psteur Institute, Tehrn) nd cultured in endothelil cell growth medium, EGM-2 Bullet kit (Lonz, Bsel, Switzerlnd), ccording to the mnufcturer s recommendtions. Twenty four hours efore trnsfection, the cells were pssged in 6-well pltes, t the density of cells/well in μl medium nd incuted t stndrd culture condition for 24 h. On the dy of trnsfection, cell culture medium ws replced, nd D-Glucose ws dded to the medium of control nd hyperglycemic groups (5 mm nd 25 mm, respectively) (8). D-Mnnitol ws used s osmotic control. Concurrently with the cretion of cellulr hyperglycemi, the HUVECs were trnsfected in prllel with hs-mir-146 mimic or scrmle (20 nmol/l) (Qigen, Crwley, UK) using HiPerFect regent (Qigen), ccording to the mnufcturer s protocol. All experiments were crried out for 24 h (8, 22). Trnsfection efficiency ws determined y rel-time RT-PCR. Importntly, ll dt nlyses were linded. Rel time RT-PCR nlysis Twenty four hours fter trnsfection, totl RNA ws isolted from HUVECs nd left ventriculr tissue using mirnesy Mini Kit (Qigen) ccording to the mnufcturer s protocol. RNA quntity nd qulity were determined using Nnodrop (Thermo scientific) nd grose gel electrophoresis (Bio-Rd), respectively. Totl RNA (1 μg) ws used for cdna synthesis using miscript II RT cdna synthesis Kit (Qigen) ccording to the mnufcturer s instructions. Ech cdna ws used s templte for seprte ssy for mir-146 nd mrnas (IRAK, TRAF6 nd NF-κB) quntittive rel-time RT-PCR y using miscript SYBR Green PCR Kit (Qigen). All rections were performed in duplicte on Corett Rotor- Gene RG-6000 (Austrli). The mount of PCR ws normlized to tht for housekeeping gene β ctin for mrna smples, nd U6 for mir-146 (Qigen). The 2 - ΔΔCt method ws used for reltive quntifiction of individul mrnas nd mir146 expression. The results hve een expressed s fold chnge differences compred to the relevnt controls. ELISA mesurements ELISA kits were used for determintion of cytoplsmic protein levels of TRAF6 (MyBioSource, Sn Diego, CA), IRAK1 (MyBioSource), nd phosphorylted NF-κB p65 s n index of NF-κB ctivity (Cymn chemicls, Ann Aror, MI), s well s plsm insulin level (MyBioSource). Protein determintion kit (Cymn chemicls) ws used for detection of cytoplsmic protein concentrtion. Sttisticl nlysis Dt re expressed s men ± SD, nd were nlyzed y repeted mesures ANOVA (for nlysis of the orl glucose tolernce test), one-wy ANOVA (for nlysis of dt otined from HUVECs) nd independent t test (for other prmeters), using the SPSS 21.0 softwre (IBM, Armonk, NY, USA). The Tukey post hoc test ws used to determine the differences etween groups with significnt p-vlues. p < 0.05 ws considered sttisticlly significnt. Results Blood glucose nd insulin, ody weight nd OGTT Fsting lood glucose nd insulin levels in the dietic rts were higher thn those in the control counterprts t two months 167

3 Brtisl Med J 2016; 117 (3) Fig. 1. Rel-time quntittive RT-PCR nlysis of mir-146 levels in nimls herts, 2 months fter induction of dietes (), nd in humn endothelil cells, HUVECs exposed to 5 mmol/l nd 25 mmol/l D-glucose nd osmotic control (5 mmol/l D-glucose + 20 mmol/l D- mnnitol) (). Brs represent Men ± SD, ** p < 0.01 vs controls. fter dietes induction ( ± vs ± mg/dl, p = for glucose, nd 5.07 ± 4.11 vs 9.42 ± 3.63 ng/ml, p = for insulin). Moreover, the dietic rts showed reduced ody weight thn the control rts ( ± vs ± g, p = 0.003). To confirm NIDDM, 24 h efore scrifice, the rts were enrolled for n OGTT. Before glucose intke (time 0), fsting lood glucose ws higher in the dietic group thn in the control counterprt ( ± 9.76 vs ± 7.77 mg/dl; p = 0.002). Sixty minutes fter glucose intke (2 g/kg), the lood glucose concentrtion in oth dietic nd control groups reched mximum levels ( ± nd ± mg/dl, respectively), nd then decresed to minimum levels t 120 minutes ( ± 9.84 vs ± 9.51; p < 0.001) however, it did not return to the seline level in dietic rts. mir-146 expression Rel-time PCR nlysis demonstrted tht 2 months of uncontrolled dietes cuses significnt upregultion of mir-146 in dietic rts herts (Fig. 1) (p = 0.003). Also, nlysis of HUVECs in 25 mm glucose confirmed significnt upregultion of mir-146 compred with 5 mm glucose (p = 0.009). No significnt effect ws seen fter incution with 25 mm D-Mnnitol (Fig. 1). c d Fig. 2. ELISA mesurements of NF-κB ctivity (), TRAF6 (c) nd IRAK1 (d) in the herts of dietic nd control rts. Section () represents ELISA mesurements of NF-κB ctivity in HUVECs during norml (5 mmol/l D-glucose) nd hyperglycemic (25 mmol/l D-glucose) condition, in the presence of mir-146 mimic or scrmle. Brs represent men ± SD; * p < 0.05, nd ** p < 0.01 vs controls. NF-κB ctivity nd TRAF6 nd IRAK1 protein levels Figure 2 indictes tht the NF-κB ctivity ws significntly incresed in the hert of dietic nimls when compred with the non-dietic rts (p = 0.023). Furthermore, 24 h hyperglycemi results in 3 fold increse in NF-κB ctivity in HUVECs (Fig. 2) (p = 0.007). Trnsfection of mir-146 mimic prevented from significnt increse of NF-κB ctivity in HUVECs (Fig. 2). Moreover, no significnt effect ws seen y trnsfection of scrmle microrna. ELISA mesurements demonstrted tht TRAF6 nd IRAK1 protein levels were significntly higher in the dietic herts thn 168

4 Eftekhr E et l. MicroRNA-146 nd its dpter proteins re ffected y dietes in rt s hert xx c Fig. 3. The mrna expression levels of NF-κB (), TRAF6 () nd IRAK1 (c) in the hert of dietic nd control rts. Dt re presented s men ± SD; * p < 0.05, nd ** p < 0.01 vs controls in the control group (Fig. 2c nd 2d) (p = nd p = 0.009, respectively). The mrna expression level of TRAF6, IRAK1 nd NF-κB Figure 3 represents the chnges of crdic TRAF6, IRAK1 nd NF-κB mrna expression level 2 months fter dietes induction. In comprison to the norml control rts, 2 months uncontrolled dietes enhnced TRAF6 nd NF-κB mrna expression levels in the hert of dietic rts (p = nd p = 0.01, respectively), while it hd no significnt effect on IRAK1 gene expression level (p = 0.09). Discussion The min finding of this study ws significnt upregultion of mir-146 in dietic hert, 2 months fter uncontrolled dietes. Also, remrkle upregultion of NF-kB nd TRAF6 mrna levels, nd increses in NF-kB p65 suunit, TRAF6 nd IRAK1 protein concentrtions were seen in the dietic herts. Moreover, it ws oserved tht trnsfection of mir-146 mimic prevented from significnt increse of NF-κB ctivity in HUVECs during hyperglycemic condition. In the present study, nicotinmide-stz model ws used for reltive destruction of pncretic β cells. This model of experimentl dietes is economicl, nd remins stle for long period with low mortlity rte (23, 24). Furthermore, this model induces type 2 dietes esily nd in short period of time, nd it closely resemles the dietic pttern seen in non-oese dietic ptients (24). The results otined y OGTT nd fsting lood insulin in this study re in line with previous performed works (21, 25), nd confirm the induction of type 2 dietes. It hs een widely ccepted tht sustined hyperglycemi enhnces some intrcellulr fctors including protein kinse C, rective oxygen spices, polyols, nd lso ctivtes hexosmines nd dvnced glyction end products pthwys converging to ctivte mster switch pro-inflmmtory trnscription fctor, NF-кB (26). This fctor, in turn ctivtes severl pro-inflmmtory cytokine genes, nd therey promotes the cells towrd inflmmtion nd finlly poptosis (26). Incresed NF-кB ctivity in hyperglycemic HUVECs nd in the hert of our dietic rts is in greement with erlier oservtions, nd confirms hyperglycemi-induced inflmmtion (26). It hs een proposed tht NF-кB regultes its own ctivtion in prt through seprte negtive feedck loops y trnsctivtion of severl micrornas such s mir-155, mir-34, mir-21, nd mir-146 (27). It is elieved tht NF-кB ctivtion upregultes mir-146 gene tht, upon processing nd mturtion, enters to cytoplsm nd prevents from trnsltion of IRAK1 nd TRAF6 mrnas to proteins (17, 27). These two dpter molecules ctivte inhiitory kinse B kinses (Ikks) which in turn, phosphorylte inhiitory kinse B (IkB) to relese NF-кB from its inhiition (17). Indeed, it hs een suggested tht mir-146 negtively regultes the NF-кB ctivtion y reduction of IRAK1 nd TRAF6 proteins (17). However, this negtive feedck loop hs not een demonstrted y some investigtions (12, 13, 18, 19). Overexpression of mir-146 in hyperglycemic HUVECs nd in the hert of our dietic rts presumly resulted from the NFкB ctivtion for negtive feedck purpose. Surprisingly, this overexpression of mir-146 ws not ccompnied y downregultion of IRAK1 nd TRAF6 mrnas nd proteins. One explntion for this oserved result my e the possiility tht IRAK1 nd TRAF6 to e under other levels of control, such s Toll-like receptor ctivtors like pro-inflmmtory cytokines (19, 22). Incresed mir-146 expression hs lso een documented in dietic kidney (13), nd in plsm nd liml cornel epithelium comprtment of dietic ptients (14, 15). On the other hnd, decresed expression of mir-146 hs een reported in dietic mouse wounds (5), dietic kidney nd dorsl root gngli (8, 9), glycted lumin- nd high glucose-stimulted endothelil cells (10), nd in serum nd peripherl lood mononucler cells of type 1 nd 2 dietic ptients (11, 12). Furthermore, we previously reported the down-regultion of mir-146 in dietic rts ort nd scitic nerve (6, 7). To the est of our knowledge, only Feng et l hve previously reported tht mir-146 expression level decreses in the herts of type 1 nd type 2 dietic rts (8). This 169

5 Brtisl Med J 2016; 117 (3) is the first presenttion of dt on up-regultion of mir-146 in dietic hert with significnt up-regultion of IRAK1 nd TRAF6 mrnas nd proteins. At present, the cuse of different ehvior of mir-146 in different tissues is uncler. But it my depend on tissue type, lood nd tissue cytokines concentrtions, timing, nd durtion of inflmmtion. This study demonstrted tht while mir-146 nd NF-кB re up-regulted in HUVECs under hyperglycemic condition, trnsfection of mir-146 mimic prevents from enhncement of NF-кB ctivity. This result supports the nti-inflmmtory role of mir-146 in the pthogenesis of dietes relted complictions. Overll, given the ctivtion of NF-κB to e key step in the progression of dietic complictions (26, 28), the results of present study my suggest tht presumly defect in the regultion of IRAK1 nd TRAF6 cn weken mir-146 regultory negtive feedck loop nd provides sitution for sustined ctivtion of NF-κB nd its trgets to promote cells towrd normlities; however, this suggestion needs to e confirmed y mesurement of IKKs ctivity or concentrtion. References 1. Klninov J, Jkus V, Glejtkov M, Kurck L, Sndorov E. Impct of glycemic control on dvnced glyction nd inflmmtion in overweight nd oese ptients with type 2 dietes mellitus. Brtisl Lek Listy 2014; 115 (8): Mrtín-Timón I, Sevillno-Collntes C, Segur-Glindo A, Del Cñizo-Gómez FJ. Type 2 dietes nd crdiovsculr disese: Hve ll risk fctors the sme strength? World J Dietes 2014; 5 (4): Kwhito S, Kitht H, Oshit S. Prolems ssocited with glucose toxicity: Role of hyperglycemi-induced oxidtive stress.world J Gstroenterol 2009; 15 (33): Arsln M, Comu FM, Kip G, Alkn M, Kirz HA, Ozer A, Sivgin V. Effect of dexmedetomidine on erythrocyte deformility during ischemi reperfusion injury of hert in dietic rts. Brtisl Lek Listy 2014; 115 (8): Xu J, Wu W, Zhng L et l. The role of microrna-146 in the pthogenesis of the dietic wound-heling impirment: correction with mesenchyml stem cell tretment. Dietes 2012; 61 (11): Emdi SS, Soufi FG, Khmneh AM, Alipour MR. MicroRNA-146 expression nd its intervention in NF-кB signling pthwy in dietic rt ort. Endocr Regul 2014; 48 (2): Yousefzdeh N, Alipour MR, Soufi FG. Deregultion of NF-кB-miR- 146 negtive feedck loop my e involved in the pthogenesis of dietic neuropthy. J Physiol Biochem 2015; 71 (1): Feng B, Chen S, McArthur K et l. mir-146-medited extrcellulr mtrix protein production in chronic dietes complictions. Dietes 2011; 60 (11): Wng L, Chopp M, Szld A et l. The role of mir-146 in dorsl root gngli neurons of experimentl dietic peripherl neuropthy. Neuroscience 2014; 259: Wng HJ, Hung YL, Shih YY, Wu HY, Peng CT, Lo WY. MicroR- NA- 146 decreses high glucose/thromin-induced endothelil inflmmtion y inhiiting NAPDH oxidse 4 expression. Meditors Inflmm 2014; 2014: Bldeón R L, Weigelt K, de Wit H et l. Decresed serum level of mir-146 s sign of chronic inflmmtion in type 2 dietic ptients. PLoS One 2014; 9 (12): e Blsurmnym M, Arvind S, Gokulkrishnn K et l. Impired mir-146 expression links suclinicl inflmmtion nd insulin resistnce in Type 2 dietes. Mol Cell Biochem 2011; 351 (1 2): Alipour MR, Khmneh AM, Yousefzdeh N, Mohmmd-nejd D, Soufi FG. Upregultion of microrna-146 ws not ccompnied y downregultion of pro-inflmmtory mrkers in dietic kidney. Mol Biol Rep 2013; 40 (11): Rong Y, Bo W, Shn Z et l. Incresed microrna-146 levels in plsm of ptients with newly dignosed type 2 dietes mellitus. PLoS One 2013; 8 (9): e Winkler MA, Di C, Ljuimov AV, Sghizdeh M. Trgeting mir- 146 to tret delyed wound heling in humn dietic orgn-cultured cornes. PLoS One 2014; 9 (12): e Cheng HS, Njock MS, Khyzh N, Dng LT, Fish JE. Noncoding RNAs regulte NF-κB signling to modulte lood vessel inflmmtion. Front Genet 2014; 5: S R, Sorensen DL, Booth SA. MicroRNA-146: A Dominnt, Negtive Regultor of the Innte Immune Response. Front Immunol 2014; 5: Liu Z, Xio B, Tng B et l. Up-regulted microrna-146 negtively modulte Helicocter pylori-induced inflmmtory response in humn gstric epithelil cells. Microes Infect 2010; 12 (11): Zilhi E, Trr T, Ppp G, Griger Z, Sipk S, Zeher M. Incresed microrna-146/, TRAF6 gene nd decresed IRAK1 gene expressions in the peripherl mononucler cells of ptients with Sjögren s syndrome. Immunol Lett 2012; 141 (2): Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altmn DG. Improving ioscience reserch reporting: the ARRIVE guidelines for reporting niml reserch. PLoS Biol 2010; 8 (6): e Mohmmdshhi M, Hidri F, Soufi FG. Chronic resvertrol dministrtion improves dietic crdiomyopthy in prt y reducing oxidtive stress. Crdiol J 2014; 21 (1): Ru CS, Yng JC, Chen YC et l. Lipopolyscchride-induced microrna-146 trgets CARD10 nd regultes ngiogenesis in humn umilicl vein endothelil cells. Toxicol Sci 2014; 140 (2): Szkudelski T. Streptozotocin-nicotinmide-induced dietes in the rt. Chrcteristics of the experimentl model. Exp Biol Med (Mywood) 2012; 237 (5): Islm MS, Wilson RD. Experimentlly induced rodent models of type 2 dietes. Methods Mol Biol 2012; 933: Plsmy P, Surmnin S. Resvertrol, nturl phytolexin, normlizes hyperglycemi in streptozotocin-nicotinmide induced experimentl dietic rts. Biomed Phrmcother 2008; 62 (9): Fiorentino TV, Priolett A, Zuo P, Folli F. Hyperglycemi-induced oxidtive stress nd its role in dietes mellitus relted crdiovsculr diseses. Curr Phrm Des 2013; 19 (32): M X, Becker Buscgli LE, Brker JR, Li Y. MicroRNAs in NFkpp B signling. J Mol Cell Biol 2011; 3 (3): Ptel S, Sntni D. Role of NF-kpp B in the pthogenesis of dietes nd its ssocited complictions. Phrmcol Rep 2009; 61 (4): Received June 5, Accepted Novemer 11,

EFFECTS OF AN ACUTE ENTERIC DISEASE CHALLENGE ON IGF-1 AND IGFBP-3 GENE EXPRESSION IN PORCINE SKELETAL MUSCLE

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