International Journal of Pharma and Bio Sciences

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1 Int J Phrm Bio Si 2013 Ot; 4(4): (B) Reserh Artile BioChemistry Interntionl Journl of Phrm nd Bio Sienes ISSN SILDENAFIL ALLEVIATES INSULIN SENSITIVITY VIA ATTENUATING OXIDATIVE STRESS AND PROINFLAMMATORY CYTOKINE PRODUCTION IN DIABETIC RATS N B S ABORYAG 1, A M MAHMOUD 2* AND S A RAMADAN 3 1 Physiology Deprtment, Fulty of Mediine, Zwi University, Liy. 2 Physiology Division, Zoology Deprtment, Fulty of Siene, Beni-Suef University, Egypt. 3 Physiology Deprtment, Fulty of Mediine, Beni-Suef University, Egypt. ABSTRACT The present study ws hypothesized to ssess the effet of sildenfil, phosphodiesterse inhiitor, on proinflmmtory ytokines nd hyperglyemi-medited oxidtive stress in type 2 dieti rts. Dietes ws indued y feeding rts with high ft diet for 2 weeks followed y n intrperitonel injetion of streptozotoin. In the dieti ontrol group, levels of gluose were signifintly inresed, while serum insulin level ws deresed. In ddition, serum TNF-α, IL-6, IL-1β, totl holesterol, triglyerides, LDL-holesterol nd vldl-holesterol were signifintly elevted in dieti rts. Moreover, hepti MDA ws signifintly inresed in dieti rts, while GSH ontent s well s GPx nd SOD tivities were signifintly deresed. Both doses of sildenfil llevited the ltered prmeters. Colletively, sildenfil exerts protetion to type 2 dieti rts y lleviting insulin sensitivity, potentiting the ntioxidnt defense system nd suppressing proinflmmtory ytokine prodution. KEYWORDS: Sildenfil, oxidtive stress, dietes mellitus, proinflmmtory ytokines. A M MAHMOUD Physiology Division, Zoology Deprtment, Fulty of Siene, Beni-Suef University, Egypt. This rtile n e downloded from B - 427

2 Int J Phrm Bio Si 2013 Ot; 4(4): (B) INTRODUCTION Dieti mellitus (DM) is one of the most importnt helth prolems in the world, espeilly in developing ountries 1. It is group of metoli diseses hrterized y hyperglyemi resulting from the defets in insulin seretion, insulin tion, or oth 2,3. The hroni hyperglyemi ondition in dietes is ssoited with long term dmge, dysfuntion, nd filure of vrious orgns, suh s eyes, kidneys, nerves, hert nd lood vessels 2,4. Aute inflmmtion is utilized y the immune system to effetively isolte nd eliminte pthogeni miroorgnisms. Among the most ommonly oserved ytokines in inflmmtory miroenvironments is tumor nerosis ftor (TNF)-α 5. This ytokine is produed y vriety of immune ells inluding mrophges nd lymphoytes 6 nd is pleiotropi in nture 7. TNF-α is lso produed y dipose tissue 8,9 nd it is thought to ply mjor role in the Metoli Syndrome (MS), whih is hrterized y insulin resistne nd inflmmtion 9. TNF-α n eliit n insulinresistnt stte, hrterized y n impired ility of insulin to suppress hepti gluose prodution nd to stimulte peripherl gluose uptke 10. Also, TNF-α is implited to inrese the irulting level of free ftty ids nd thus indiretly ontriutes to the pthogenesis of insulin resistne 11. Similrly, inreses in the proinflmmtory ytokine, IL-6, led to redution in insulin reeptor sustrte-1 (IRS- 1) tyrosine phosphoryltion, deresed ssoition etween the PI-3 kinse nd IRS- 1 nd n inhiition of insulin-dependent tivtion of Akt 12,13. In ddition, IL-6 hs lso een found to redue lipoprotein lipse (LPL) tivity in the dipose tissue of mie nd in 3 T3-L1 dipoytes in vitro 14. Sildenfil is drug ommonly used in the tretment of eretile dysfuntion. It inhiits the metolism of yli gunosine monophosphte (GMP), resulting in inresed relxtion of the smooth musle surrounding rterioles supplying the humn orpus vernosum 15. Reently, we hd ddressed the enefiil effets of sildenefil itrte in eti id-indued olitis in rts 16. Thus, the urrent study ws designed to investigte the meliortive potentil of sildenfil itrte on proinflmmtory ytokines nd hyperglyemi-medited oxidtive stress in type 2 dieti rts. MATERIALS AND METHODS (i) Chemils Streptozotoin (STZ) ws purhsed from Sigm Chemils Co., St. Louis, MO, USA. Sildenfil itrte ws supplied from Pfizer In. (Pfizer, Egypt), stored t 2-4 C nd proteted from sunlight. All other hemils were of nlytil grde nd were otined from stndrd ommeril supplies. (ii) Experimentl nimls Mle lino rts weighing out g were used s experimentl nimls in the present investigtion. The nimls were housed in stndrd polypropylene ges with stinless steel good erted overs nd mintined under ontrolled room temperture (22±2 C) with 12 hrs light - drk yle nd were fed stndrd diet of known omposition, nd wter d liitum. The nimls used in the present study were mintined in ordne with the priniples nd guidelines of the Cndin Counil on Animl Cre s outlined in Guide for the Cre nd Use of Lortory Animls 17. (iii) Indution of dietes mellitu Type 2 DM ws experimentlly indued y feeding high ft diet (HFD) for n initil period of 2 weeks followed y n interpritonel injetion of 35 mg/kg. wt streptozotoin dissolved in itrte uffer ph The omposition nd preprtion of HFD were desried elsewhere 19. Seven dys fter the injetion, rts were sreened for serum gluose levels. Rts hving serum gluose 200 mg/dl, fter 2 hours of gluose intke, were onsidered dieti.. (iv) Experimentl design The experimentl nimls were divided into four groups, eh group omprising six rts designted s follows: group 1 served s norml ontrol rts; group 2 served s dieti ontrol rts; group 3 served s dieti rts This rtile n e downloded from B - 428

3 Int J Phrm Bio Si 2013 Ot; 4(4): (B) dministered 5 mg/kg.wt sildenfil orlly for 4 weeks; nd group 4 served s dieti rts dministered 10 mg/kg.wt sildenfil orlly for 4 weeks. The dosge ws djusted every week ording to ny hnge in ody weight to mintin similr dose per kg ody weight of rt over the entire period of study for eh group. By the end of the experiment, nimls were srified nd lood smples nd liver were otined. (v) Biohemil studies The level of serum gluose ws estimted spetrophotometrilly ording to the method of Trinder 20, using ommeril dignosti kit (Spinret, Spin). Serum insulin nd diponetin levels were determined using speifi ELISA kit (R&D Systems, USA), ording to the mnufturer instrutions. Insulin resistne ws evluted y HOMA-IR 21 s follows: HOMA-IR=Fsting insulin (µu/ml) x Fsting gluose (mmol/l)/22.5. Serum totl holesterol 22, triglyerides 23 nd HDLholesterol 24 were ssyed using ommeril dignosti kits (Spinret, Spin). Serum vldl-holesterol onentrtion ws lulted ording to Noert 25 formul (vldlholesterol=triglyerides/5). Serum LDLholesterol level ws lulted from Friedewld 26 formul (LDL-holesterol = totl holesterol triglyerides/5 HDLholesterol). Serum levels of the proinflmmtory ytokines, TNF- α, IL-6 nd IL-1β, were determined y speifi ELISA kits ording to the mnufturer's instrutions (R&D Systems, USA). The onentrtion of proinflmmtory ytokines ws determined spetrophotometrilly t 450 nm. Stndrd plots were onstruted y using stndrd ytokines nd the onentrtions for unknown smples were lulted from the stndrd plot. Lipid peroxidtion, redued glutthione ontent, nd superoxide dismutse (SOD) nd glutthione peroxidse (GPx) tivities were lso mesured in liver homogente ording to the methods of Preuss et l 27, Beutler et l 28, Mrklund nd Mrklund 29 nd Kr nd Mishr 30, respetively. (vi)sttistil nlysis The dt were nlyzed using the one-wy nlysis of vrine (ANOVA) (PC-STAT, University of Georgi, 1985) followed y LSD test to ompre vrious groups with eh other. Results were expressed s men ± SE nd vlues of P>0.05 were onsidered nonsignifintly different, while those of P<0.05 nd P<0.01 were onsidered signifint nd highly signifint, respetively. RESULTS HFD followed y STZ produed very highly signifint elevtion (P<0.001; LSD) of oth fsting nd postprndil gluose levels s ompred with norml rts. Tretment of dieti rts with oth 5 nd 10 mg/kg sildenfil produed pronouned meliortion of the elevted serum lood gluose levels (Figure 1). This rtile n e downloded from B - 429

4 1 Int J Phrm Bio Si 2013 Ot; 4(4): (B) Norml Dieti Control Dieti + 5 mg Sildenfil Dieti + 10 mg Sildenfil Blood Gluose (mg/dl) Fsting lood gluose Postprndil lood gluose Figure 1 Effet of sildenfil on fsting nd postprndil lood gluose levels of norml, dieti ontrol nd dieti treted rts. The reorded vlues of fsted dieti rts showed highly signifint derese in serum insulin onentrtion s ompred with the norml ontrol rts. Tretment with oth 5 nd 10 mg/kg sildenfil produed n inrese in serum insulin onentrtion (Figure 2). 30 Norml Dieti + 5 mg Sildenfil Dieti Control Dieti + 10 mg Sildenfil Insulin (µiu /L) Figure 2 Effet of sildenfil on serum insulin onentrtion of norml, dieti ontrol nd dieti treted rts This rtile n e downloded from B - 430

5 1 Int J Phrm Bio Si 2013 Ot; 4(4): (B) Sildenfil t 10 mg dose ppered to e more effetive in inresing serum insulin level thn the 5 mg dose. On the other hnd, dieti rts showed signifint (p<0.01; LSD) elevtion of HOMA- IR tht ws deresed signifintly upon dministrtion of either low or high dose of sildenfil s illustrted in Figure Norml Dieti + 5 mg Sildenfil Dieti Control Dieti + 10 mg Sildenfil 10 HOMA-IR Figure 3 Effet of sildenfil on HOMA-IR of norml, dieti ontrol nd dieti treted rts Dt on the effet of sildenfil on lipid profile of dieti rts were presented in Tle 1. Group Tle 1 Lipid profile of norml, dieti nd dieti rts treted with sildenfil Prmeter Totl Cholesterol (mg/dl) Triglyerides (mg/dl) HDLholesterol (mg/dl) LDLholesterol (mg/dl) vldlholesterol (mg/dl) Norml ± ± ± ± ± 0.50 Dieti ontrol ± ± ± ± ± 1.64 Dieti + 5 mg/kg Sildenfil ± ± ± ± ± 2.06 Dieti + 10 mg/kg Sildenfil ± ± ± ± ± 0.90 F- pro P< P< P< P< P< LSD t 5% LSD t 1% Dt re expressed s Men ± SE. Numer of nimls in eh group is six. - Mens whih shre the sme supersript symol (s) re not signifintly different. Dieti rts exhiited highly signifint inrese (LSD; P<0.01) in serum totl holesterol, triglyerides, LDL- nd vldl-holesterols s ompred with the non-dieti group. Moreover, HDL-holesterol ws ffeted in n opposite mnner, s it ws highly signifint deresed (LSD; P<0.01) in dieti rts. The dministrtion of oth tested doses led to mrked meliortion of ll prmeters of the ltered lipid profile. The effet of sildenfil on serum TNF-α, IL-6 nd IL-1β of norml nd dieti rts ws illustrted in figures (4). This rtile n e downloded from B - 431

6 Int J Phrm Bio Si 2013 Ot; 4(4): (B) Norml Dieti + 5 mg Sildenfil Dieti Control Dieti + 10 mg Sildenfil Conentrtion (pg/ml) d 0 TNF-α IL-1β IL-6 Figure 4 Serum TNF-α, IL-1β nd IL-6 of norml, dieti ontrol nd dieti rts treted with sildenfil. In dieti rts, oth doses of sildenfil indued highly signifint (P<0.01) derese of TNFα,IL-6 nd IL-1β s ompred to the ontrol rts. Tle 2 Liver MDA, GSH, GPx nd SOD of norml, dieti nd dieti rts treted with sildenfil. MDA GSH GPx Group Prmeter SOD (U/g tissue) (nmol/100 mg tissue) (nmol/100 mg tissue) (U/g tissue) Norml ± 1.36 d ± ± ± 7.87 Dieti ontrol ± ± 1.62 d ± 2.18 d ± 3.67 Dieti + 5 mg/kg Sildenfil ± ± ± ± 8.14 Dieti + 10 mg/kg Sildenfil ± ± ± ± 7.58 F- pro P< P< P< P< 0.01 LSD t 5% LSD t 1% Dt re expressed s Men ± SE. Numer of nimls in eh group is six. - Mens whih shre the sme supersript symol (s) re not signifintly different. Tle 2 illustrtes the effet of sildenfil dministrtion on the levels of liver lipid peroxides nd ntioxidnt mrkers of dieti rts. The elevted level of lipid peroxides oserved in liver of dieti rts ws potentilly (p<0.01; LSD) improved y the tretment of dieti groups of rts with oth doses of sildenfil. Conversely, hepti GSH ontent nd GPx nd SOD tivities were signifintly delined in HFD/STZ dieti rts. Tretment of the dieti rts with doses of sildenfil potentilly inresed (p<0.01; LSD) GSH s well s ntioxidnt enzymes tivity, however the low dose of sildenfil non-signifintly (P>0.05) ffets the hepti SOD tivity. DISCUSSION HFD/STZ indued dieti rt is one of the niml models of humn dietes mellitus 19. The rts fed with HFD n result in insulinresistne minly through Rndle or gluose ftty id yle 31. It ws lso reported tht in This rtile n e downloded from B - 432

7 Int J Phrm Bio Si 2013 Ot; 4(4): (B) ddition to diret effet on gluose, whih is hrteristi of this model, other pthophysiologil hnges were seen, suh s insulin resistne in dipose tissue 19 nd dieti kidney lesions suh s glomeruloslerosis nd proteinuri 32. Also, HFD feeding to rodents ws shown to ffet the respirtory pity, retive oxygen speies (ROS) genertion, ftty id et oxidtion, mitohondril ADP/ATP trnslotor inhiition nd regultion of kinses involved in rohydrte nd lipid metolism 33. Sine the omintion of high ft diet-fed nd lowdose streptozotoin treted rt whih serves s n lterntive niml model for dietes hs een proved to e suitle for testing ntidieti gents 34, so this model ws hosen to rry out our evlutions. In the present study, dieti group rts exhiited signifintly elevted fsting nd postprndil lood gluose, nd HOMA-IR, ompnied with diminished serum insulin levels s ompred to norml rts. Hene, it is proposed tht insulin resistne hs een developed in these nimls. Therefore, this rt model exhiits hyperglyemi with insulin resistne tht would losely reflet the nturl history nd metoli hrteristis of dieti humns, nd it is further sensitive to phrmologil testing. Tretment of the dieti rts with either 5 or 10 mg sildenfil itrte potentilly improved the ltered lood gluose. These results my e due meliortion of insulin sensitivity. The urrent study is in greement with results of Ayl et l 35 who showed tht hroni inhiition of phosphodiesterse-5 improves insulin tion in mouse model of diet-indued oesity nd insulin resistne. One potentil mehnism y whih phosphodiesterse-5 inhiition my improve insulin tion is prevention of endothelil dysfuntion. Reent evidene supports the notion tht endothelil dysfuntion my e ustive of insulin resistne nd type 2 dietes 36. Endothelil dysfuntion is hrterized y derese in NO levels, reduing GMP prodution nd impiring musle gluose uptke 37. Thus, it is possile tht preventing derese in GMP levels y inhiiting phosphodiesterse-5 intervenes downstrem of the site of endothelil dysfuntion, resulting in improved insulin tion on musle gluose uptke. It is lso possile tht the enhned insulin tion in sildenfil-treted dieti rts resulted from n effet on the entrl nervous system. Sildenfil hs een shown to ross the loodrin rrier, nd phosphodiesterse-5 expression hs een deteted in the rin 38. Thus, signling through GMP in the entrl nervous system my ply role in the regultion of insulin tion nd energy homeostsis s stted y Ayl et l 35. Dieti dyslipidemi hs long een shown to hve strong reltion with oronry hert disese 39,40, whih is the most dngerous nd life thretening omplition of dietes nd the risk of oronry hert disese in dietes inreses two- or more folds 41. The rise in lood gluose ws ompnied with mrked inrese in totl holesterol, LDLholesterol, triglyerides nd redution in HDL-holesterol in HFD/STZ dieti rts. The ltered lipid nd lipoprotein profiles were signifintly reversed fter 4 weeks of oth 5 nd 10 mg sildenfil supplementtion to the dieti rts. The signifint ontrol in the serum lipid levels in treted dieti rts might hve een due to the inrese insulin sensitivity following sildenfil dministrtion. An importnt finding of the present study ws tht sildenfil, in dose dependent mnner, ttenuted prodution of the pro-inflmmtory ytokines, TNF-α, IL-6 nd IL-1β, whih re elieved to ply signifint role in the pthogenesis of DM. TNF-α ffets intrellulr insulin signling in ft, skeletl musle, endothelil ells, nd other insulinresponsive tissues y inhiiting kinse tivities in the insulin-signling pthwy 42. TNF-α hs een shown to inrese plsm triglyerides nd onentrtions of very low density lipoproteins 43, s well s lipolysis in mouse, rt, nd humn ft ells 44. TNF-α redues insulin-stimulted reeptor tyrosine kinse tivity t low onentrtions nd n lso derese the expression of the insulin reeptor IRS-1 nd GLUT-4 t higher onentrtions s well s inreses the phosphoryltion of serine 307 in IRS-1, thus impiring its ility to ind to the insulin reeptor nd initite downstrem signling 42. In ddition, inreses in IL-6 leds to redution in IRS-1 tyrosine phosphoryltion, This rtile n e downloded from B - 433

8 Int J Phrm Bio Si 2013 Ot; 4(4): (B) deresed ssoition etween the PI-3 kinse nd IRS-1 nd n inhiition of insulindependent tivtion of Akt 12,13. Thus, the oserved meliortion of insulin sensitivity my e medited vi ttenuting proinflmmtory ytokines y sildenfil. Inresed oxidtive stress nd impired ntioxidnt defense mehnism re importnt ftors in the pthogenesis nd progression of DM nd other oxidnt-relted diseses 3. The potentilly ntioxidtive effets of PDE-5 inhiition in dietes 40 ould e onfirmed in the present study. The dministrtion of oth doses of sildenfil to dieti group of rts signifintly reverted k the ltered levels of mlondildehyde nd the ntioxidnts, GSH, GPx nd SOD, whih in turn revel the ntioxidnt potentil of sildenfil. There is evidene tht iologil responses triggered y oxidtive produts re ssoited with lipid peroxidtion derivtives, whih re le to indue vrious pthogeni intrellulr signls involving lium, G-proteins, AMP, GMP, phospholipse C nd D, protein kinse C, ermide, nd MAP kinse sde leding to ellulr dysfuntion 46. Thus inresing yli nuleotides y use of PDE inhiitors ould overome to oxidtive stress-indued ellulr dysfuntions nd poptosis. Supporting this onlusion, Polte nd Shroder 47 reported n ntioxidnt property for nitri oxide (NO) donors in vsulr endothelium through onerted tion of GMP nd AMP. They showed tht S-nitroso-N-etyl-d,lpeniillmine (SNAP) protets from TNFmedited endothelil ell toxiity. The ytoprotetion y SNAP ws ompletely olished y the denylyl ylse inhiitor 2,5- dideoxydenosine nd mimiked y 8-romo AMP or forskolin. In onlusion, iohemil findings of the present study indite tht the PDE inhiitor sildenfil itrte exerts protetion to HFD/STZ dieti rts ginst hyperglyemi-medited oxidtive stress. This ould e due to the prevention or inhiition of lipid peroxidtion nd lleviting the ntioxidnt system. In ddition, the hypolipidemi nd insulin sensitizing effets of sildenfil my e ttriuted to its ntiinflmmtory effet. REFERENCES 1. Mnn P, Ds J, Ghosh J, Sil PC. Contriution of type 1 dietes to rt liver dysfuntion nd ellulr dmge vi tivtion of NOS, PARP, IκBα/NF-κB, MAPKs, nd mitohondri-dependent pthwys: Prophylti role of rjunoli id. Free Rdil Biology & Mediine, 48: , (2010). 2. Amerin Dietes Assoition. Dignosis nd lssifition of dietes mellitus. Dietes Cre, 30 (Suppl. 1): S42-S47, (2007). 3. Srvnn G, Ponmurugn P. Ameliortive potentil of Sllyl ysteine on oxidtive stress in STZ indued dieti rts. Chemio-Biologil Intertions, 15;189(1-2):100-6, (2011). 4. Rmhndrn S, Asokkumr K, Um Mheswri M, Rvi TK, Sivshnmugm AT, Srvnn S, Rjsekrn R, Dhrmn J. Investigtion of Antidieti, Antihyperlipidemi, nd In Vivo Antioxidnt Properties of Sphernthus indius Linn in Type 1 Dieti Rts: An Identifition of Possile Biomrkers. Evidene-Bsed Complementry nd Alterntive Mediine, 2011: , (2011). 5. Cvillon JM. Cytokines in inflmmtion. C R Senes So. Biol. Fil., 189: , (1995). 6. Antun-Puente B, Feve B, Fellhi S, Bstrd JP. Adipokines: the missing link etween insulin resistne nd oesity. Dietes nd Mtol, 34: 2-11, (2008). 7. Vssiliou E, Jing H, Gne D. Prostglndin E2 inhiits TNF prodution in murine one mrrow-derived dendriti ells. Cell Immunol., 223: , (2008). 8. Montgue CT. Adipose depot-speifi effets of PPAR gmm gonists: onsequene of differentil expression of PPAR gmm in dipose tissue depots? Dietes Oes. Met., 4(6): , (2002). This rtile n e downloded from B - 434

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