Effects of rosuvastatin and/or -carotene on non-alcoholic fatty liver in rats

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1 Reserch in Phrmceuticl Sciences, August 215; 1(4): Received: Sept 214 Accepted: Nov 214 School of Phrmcy & Phrmceuticl Sciences Isfhn University of Medicl Sciences Originl Article Effects of rosuvsttin nd/or -crotene on non-lcoholic ftty liver in rts Syed H. Seif el-din 1,*, Ngl M. El-Lkkny 1, Aeer A. El-Nggr 1, Olft A. Hmmm 2, Hekm A. Ad El-Ltif 3, Aff A. Ain-Shok 4, nd Ftm A. Eeid 1 1 Deprtment of Phrmcology, Theodor Bilhrz Reserch Institute, Wrk El-Hdr, Im, P.O. Box 3, Giz 12411, Egypt. 2 Deprtment of Pthology, Theodor Bilhrz Reserch Institute, Wrk El-Hdr, Im, P.O. Box 3, Giz 12411, Egypt. 3 Deprtment of Phrmcology, Fculty of Phrmcy, Umm Al-Qur University, Sudi Ari. 4 Deprtment of Phrmcology, Fculty of Phrmcy, Ciro University, Giz, Egypt. Astrct The prevlence of non-lcoholic ftty liver disese () hs mrkedly incresed, especilly in ptients exhiit one or more fetures of the metolic syndrome. This study investigtes the effect of rosuvsttin (RSV) nd/or -crotene ( C) in -induced rts. Rts were clssified into nine groups; norml (I), -induced with high-ft diet (; II), switched to regulr diet (; III), - or - treted with RSV (IV, V), C (VI, VII) or oth RSV+ C (VIII, IX), respectively. After four weeks, rts were scrificed to otin serum smples nd liver tissues. Liver histology, lipid profile, liver oxidtive stress mrkers, nd dipocytokines were mesured. Liver sections of rts with - reveled stetosis, lose of heptic rchitecture, inflmmtion nd heptocyte vcuoltion with high percentge of cell firosis. Serum levels of ALT, AST, ALP, gmm glutmyl trnsferse (GGT) nd lipid profile (triglycerides, cholesterol, LDL nd VLDL) were significntly incresed (P<.5) compred with norml. Also, heptic mlondildehyde level nd serum leptin, tumor necrosis fctor-lph (TNF- ) nd trnsforming growth fctor- 1 (TGF- 1) were incresed. Menwhile, superoxide dismutse (SOD) ctivity, GSH content in liver, serum HDL nd diponectin were decresed (P<.5) vs norml. These chnges were oserved to lesser extent in - group. Administrtion of RSV or/nd C lmost improved ll previously mentioned prmeters. Moreover, heptic stetosis ws decresed nd inflmmtion ws mrkedly meliorted with reduction of TNF- nd TGF-. These results were more pronounced in the groups VIII nd IX vs ech drug lone. In conclusion RSV nd βc could e eneficil for the tretment nd prevention of. Comined RSV with C is more effective thn RSV lone. Keywords: ; Rosuvsttin; -crotene; TNF- ; TGF- 1; Oxidtive stress INTRODUCTION Non-lcoholic ftty liver disese (), regrded s the heptic mnifesttion of the metolic syndrome, currently represents the most common cuse of chronic liver disese (1). rnges from simple heptic ft ccumultion (stetosis) to non-lcoholic stetoheptitis (NASH), where ft is ccompnied y heptocyte injury, nd necroinflmmtion. This condition poses n incresed risk of cirrhosis nd heptocellulr crcinom (1). Depending on the ssessment tools, the prevlence of in dults rnges etween 2% nd 3% (2), reching up to 46% in some studies (3). Furthermore, hs ecome the most common cuse of chronic liver disese in children nd dolescents, with prevlence of 3 1%, nd even up to 7% in oese children (2,4). The drmtic increse in prevlence of oesity, metolic syndrome, nd hs een linked to the excess cloric intke due to incresed consumption of processed food nd everges, coupled with more sedentry lifestyle (5). The condition is strongly *Corresponding uthor: S.H. Seif el-din Tel: , Fx: Emil: s.seifeldin@tri.gov.eg

2 S.H. Seif el-din et l. / RPS 215; 1(4): ssocited with overweight nd insulin resistnce, thus the promoting of chnges in lifestyle, e.g. focusing on regulr exercise nd reduction in ft nd crohydrte intke, is key fctor in the tretment of ptients with (6). Approximtely 7% of ptients with NASH lso hve concurrent dyslipidemi (7) mking tretment with lipid-lowering mediction pper to e resonle pproch. Rosuvsttin (RSV) is lipid-lowering gent tht competitively inhiits 3-hydroxy-3- methylglutryl coenzyme A reductse. RSV exhiits the highest efficcy in the reduction of low-density lipoprotein (LDL), totl cholesterol (TC), nd triglycerides (TG) compred with other sttins t comprle doses (8). It hs een reported tht RSV meliortes heptic insulin resistnce in rodents nd humns (9). In ddition, RSV reduces the risk of crdiovsculr disese, decrese vsculr rective oxygen species (ROS) genertion independently of cholesterol reduction (1) nd exerts severl pleiotropic effects, ut lso chieve significnt improvement in endothelil function effect (11). In ftty liver disese, ROS induces lipid peroxidtion cting on polyunsturted ftty cids nd producing highly rective ldehydic derivtives (e.g. mlondildehyde, MDA) which hve long-term dverse effects on liver cells (12). There is chrcteristic cytokine pttern in liver stetosis which includes incresing tumor necrosis fctor-lph (TNF- ), trnsforming growth fctor- 1 (TGF- 1) nd interleukin 6, ut decresing diponectin, ghrelin nd leptin (13). -crotene ( C), lipid-solule ntioxidnt, hs n importnt function s precursor of vitmin A, nd it hs direct impct on cholesterol synthesis (14). Vitmin A prticiptes in severl functions tht re primordil to the humn system, cting in visul cuity, in cellulr prolifertion nd differentition, nd immunologicl ctivity (15). Retinol nd crotenoids, especilly C, hve received prominent position for their role ginst ROS, protecting the orgnism ginst oxidtive stress (OS) nd, consequently, preventing dmges nd tissue lesions relted to severl chronic diseses (16). Considering the role of OS in pthogenesis nd the potent ction of C s precursor of vitmin A in the fight ginst ROS, it is prole tht individuls with will hve lower levels of this vitmin, s OS much increses the intke of sustnces with ntioxidnt function (17). Moreover, C serves s prehormone tht, through metolism, is converted into retinoic cid, which functions s lignd, regulting the expression of genes involved in metolic processes. The efficcy nd sfety profile of phrmcotherpy in the tretment of hs yet een remined uncertin (18) nd since there is no effective specific therpy for, this study ws conducted to evlute whether the ddition of C to RSV could improve chrcteristics in rt model sujected to different life styles. MATERIALS AND METHODS Animls Seventy-two mle Sprgue-Dwley rts, weighting 15 2 g, were provided y the Schistosome Biology Supply Center of Theodor Bilhrz Reserch Institute, Giz, Egypt. The rts were housed in cges with free ccess to food (stndrd commercil pelleted diet, El-Khir Compny for oils nd sop) nd wter for one week efore initition of the experiment in n ir-conditioned niml house t 2 22 C. The experimentl study protocol ws conducted t the niml unit ccording to the interntionl ethicl guidelines tht ws pproved y Institutionl Animl Ethics Committee of Theodor Bilhrz Reserch Institute. Experimentl design A rt model of ws induced y high-ft diet contining 25% fts, 1% cholesterol (Win l Lortory chemicls), nd.25% ile slts (Alph chemik, Indi) for 12 weeks (19). After 12 weeks, rts in groups were dministered orlly with RSV (Astrzenec-Egypt) t dose of 1 mg/kg/dy or/nd C (Ar Co. for Phrmceuticls nd Medicinl Plnts; MEPACO-Egypt) t dose of 7 mg/kg every lterntive dy, for 4 weeks (strting from the 13 th week to the 16 th week). Rts in the - treted groups were switched nd 276

3 Effects of rosuvsttin, β-crotene, nd dietry control on mintined on the stndrd chow diet (weeks 13-16). After estlishing (fter 12 weeks) rts were rndomly divided into nine groups (eight rts ech); norml control (I), -induced with high-ft diet (; II), switched to regulr diet (; III), - or - treted with RSV (IV, V), C (VI, VII) or the comined RSV+ C (VIII, IX) respectively. Rts in the norml group were mintined on the stndrd chow diet for 16 weeks. At the end of the study, ll rts were weighted nd then scrificed y rpid decpittion fter 12 h of fsting to insure tht the chnge in lipid profile nd other serum iomrkers is due to or drug tretment nd not to instnt food intke. Blood smples were collected for iochemicl ssys. The liver ws immeditely removed nd weighted fter rinsing with icecold sline, nd smpled for ssessment of liver reduced glutthione (GSH), superoxide dismutse (SOD), lipid peroxidtion s mlondildehyde (MDA), nd for histologicl study. Liver weight index (%) ws clculted s liver weight/ody weight 1. Biochemicl nlysis Blood iochemicl prmeters including; serum lnine minotrnsferse (ALT), sprtte minotrnsferse (AST), lkline phosphtse (ALP), gmm glutmyl trnsferse (GGT), triglycerides (TG), totl cholesterol (TC), HDL, LDL nd VLDL were ssyed spectrophotometriclly using the commercilly ville kits. Serum levels of leptin nd TNF- (RyBiotech Inc., Norcross, GA, USA), diponectin (Assypro, St. Louis, MO, USA), nd TGF- 1 (ebioscience, Vienn, Austri) were mesured using enzyme-linked immunosorent ssy (ELISA) kits. Assy of oxidtive stress nd lipid peroxidtion Liver tissues were immeditely cooled on ice nd.5 g of liver ws homogenized (Pro Scientific Inc., U.S.A. homogenizer) in 4 volumes (w/v) of ice-cold.1 M KH 2 PO 4 uffer contining 1 mm EDTA djusted to ph 7.4 nd centrifuged t 1, g for one hour t 4 C. The superntnt ws collected nd kept t 8 C for susequent nlysis for 277 determintion of GSH (2), SOD ctivity (21). Degree of lipid peroxidtion in liver tissue homogentes ws determined in terms of thiorituric cid-rective sustnces (TBARs) s expressed y MDA formtion (22). Histologicl studies The sections of liver tissues were fixed in 1% uffered formldehyde, nd then emedded in prffin wx. A 5 μm-thick section cut from prffin-emedded lock nd t distnce of 25 μm from the preceding section ws stined with H&E nd Msson s trichrome. Stetosis ws ssessed y morphologicl semi-quntittive pproch nd grded s follows: mild=5 3%, moderte=3 6%, nd severe >6% of heptocytes ffected (23). The specimens were lso exmined for histologicl fetures including; llooning degenertion, heptocytes with cytoplsmic vcuoltion, cidophilic necrosis, sinusoidl firosis nd polymorph nucler infiltrtion. Sttisticl nlysis All vlues re expressed s men ± SEM. Clcultions were crried out using SPSS, softwre pckge version 16. (Chicgo, IL, USA). Groups of dt were compred with the one-wy nlysis of vrince (ANOVA) followed y post-hoc LSD test for multiple comprison mong groups of tretments with the nd norml control groups. P vlues of.5 were considered sttisticlly significnt. RESULTS Effects on liver gross mnifesttions The ody weight, liver weight nd liver index in - or - groups were significntly incresed (P<.5) compred with those of norml control group. Compred with -, there is no significnt difference in ody nd liver weight, nd liver index in the group treted with RSV- (Tle 1). Menwhile, tendency for normliztion with significnt decrese (P<.5) in the previously mentioned prmeters in the groups treted with C or RSV either lone or in comintion nd the group treted with RSV- reltive to their corresponding control group.

4 S.H. Seif el-din et l. / RPS 215; 1(4): Effects on heptic function, lipid profile nd dipocytokine mrkers The serum levels of ALT, AST, ALP, GGT, TG, TC, LDL nd VLDL s well s the levels of the dipocytokines; leptin, TNF- nd TGF- 1 were significntly incresed (P<.5) in - group nd to lesser extent in - group when compred with norml control group (Tle 2; Figs. 1 nd 2). At the sme time, there ws significnt decrese (P<.5) in the levels of HDL nd diponectin. When compred with corresponding control group, serum levels of ALT, AST, ALP, GGT, TG, TC, LDL, VLDL, leptin, TNF-α nd TGF- 1 were VLDL, leptin, TNF-α nd TGF- 1 were significntly decresed (P<.5) in the groups treted with either RSV lone or in comintion with C. Menwhile, n insignificnt decresing tendency in lmost ll these prmeters ws oserved in the group treted with C lone especilly in lipid profile nd dipocytokines. On the other hnd, serum levels of HDL nd diponectin were significntly incresed (P<.5) in either RSV or C-treted groups nd their comintion except C- (for HDL) or C- (for diponectin) compred with their corresponding control groups (Tle 2, Figs. 1 nd 2). Tle 1. Effect of rosuvsttin nd -crotene lone or in comintion on liver gross mnifesttions in induced rts. Groups Body weight (g) Liver weight (g) Liver weight index (%) Norml ± ± ±.1 control 236. ± ± ±.5 RSV ± ± ±.8 C ± ± ±.6 RSV+ C ± 8.22 c 9. ±.54 c 5.9 ±.2 control ± ± ±.3 RSV ± ± ±.3 C ± ± ±.5 RSV+ C ± ± ±.2 Vlues re presented s men SEM, (n=8). ; P<.5 significnt difference from norml. ; P<.5 significnt difference from corresponding control. c ; P<.5 significnt difference from corresponding RSV. Tle 2. Effect of rosuvsttin nd -crotene lone or in comintion on lipid profile in -induced rts. Groups Triglycerides (mg/dl) Totl cholesterol (mg/dl) HDL (mg/dl) LDL (mg/dl) VLDL (mg/dl) Norml ± ± ± ± ±.97 control 21. ± ± ± ± ± 2.14 RSV ± ± ± ± ± 1.14 C ± ± ± ± ± 1.17 RSV+ C ± ± ± ± ± 1.32 control ± ± ± ± ± 1.83 RSV ± ± ± ± ±.95 C ± ± ± ± ± 1.96 RSV+ C ± ± ± ± ± 1.12 HDL; high-density lipoprotein, LDL; low-density lipoprotein, VLDL; very low-density lipoprotein Vlues re presented s men SEM, (n=8). ; P<.5 significnt difference from norml. ; P<.5 significnt difference from corresponding control. 278

5 Effects of rosuvsttin, β-crotene, nd dietry control on Tle 3. Effect of rosuvsttin nd -crotene lone or in comintion on oxidtive stress prmeters nd lipid peroxidtion in -induced rts. Groups GSH (mmol/g liver) SOD (U/g liver) TBARs (nmol/g liver) Norml 6.96 ± ± ±.63 control 3.5 ± ± ± 1.39 RSV 3.5 ± ± ±.9 C 3.92 ± ± ± 1.7 RSV+ C 3.91 ± ± c ± 1.43 c control 3.73 ± ± ± 1.17 RSV 4.3 ± ± ± 1. C 4.4 ± ± ± 1.24 RSV+ C 4.64 ± ± ± 1.35 GSH; reduced glutthione, SOD; superoxide dismutse, TBARs; thiorituric cid-rective sustnces Vlues re presented s men SEM, (n=8). ; P<.5 significnt difference from norml. ; P<.5 significnt difference from corresponding control. c ; P<.5 significnt difference from corresponding RSV. ALT (U/L) A Norml AST (U/L) B Norml ALP (IU/L) C Norml c GGT (U/L) D Norml Fig. 1. Effect of rosuvsttin nd -crotene lone or in comintion on liver functions in -induced rts. A; ALT, B; AST, C; ALP, D; GGT. Vlues re presented s men SEM, (n=8). ; P<.5 significnt difference from norml. ; P<.5 significnt difference from corresponding control. Effects on oxidtive stress nd lipid peroxidtion Compred with norml control group, the heptic GSH content nd SOD ctivities in - group were significntly decresed (P<.5), menwhile MDA level ws significntly incresed. These chnges were to less extent in - group. Compred with the corresponding control groups, oxidtive stress nd lipid peroxidtion in the heptic tissue were restored y decresing SOD ctivities nd MDA levels significntly (P<.5) upon dministrtion of either RSV or C lone or oth except the RSV- nd C- treted groups for MDA (Tle 3). 279

6 S.H. Seif el-din et l. / RPS 215; 1(4): In the RSV+ C group, ll the mesured serumic nd heptic mrkers gve etter results over those of single treted group whether mintined on or switched to regulr diet. Moreover, the est results were otined in ll groups switched to regulr diet. Histopthologicl evlution In norml control group, the liver loules were distinct (preserved loulr rchitecture) nd the liver cell cords rrnged regulrly (Fig. 3A), wheres, the liver in - group (Fig. 3B) showed typicl stetosis ( , micro nd mcrostetotic chnges), loulr infiltrtion ( ) y mononucler cell nd lymphocyte, heptocytes with vcuoltion ( ), ft droplets ccumultion, spotty necrosis nd focl necrosis. These findings suggest tht the niml model of ws successfully estlished. However, the degree of heptic injury including stetosis, heptocytes with cytoplsmic vcuoltion nd loulr inflmmtion were to less degree in - group ( , nd respectively; Tle 4; Fig. 3D). Tretment of -induced rts mintined on either or, mrkedly ttenuted stetosis with either RSV or C lone or their comintion ( nd RSV; nd C; nd RSV+ C, respectively), inflmmtion ( nd RSV; nd C; nd RSV+ C, respectively) nd heptocytes with cytoplsmic vcuoltion ( nd RSV; nd C; nd RSV+ C, respectively) 4 weeks post tretment (Tle 4; Fig. 3(E J)). In the RSV+ C comined group, stetosis nd extension of loulr inflmmtion were relieved significntly over those of ech treted group, nd in ll - groups over those groups mintined on (Tle 4; Fig. 3 (I nd J)). Adiponectin (ng/ml) A Leptin (pg/ml) B Norml Norml TNF-lph (pg/ml) C TGF-et1 (pg/ml) D Norml Norml Fig. 2. Effect of rosuvsttin nd -crotene lone or in comintion on dipocytokine mrkers, A; diponectin, B; leptin, C; TNF-α nd D; TGF- 1 in -induced rts. Vlues re presented s men SEM, (n=8). ; P<.5 significnt difference from norml. ; P<.5 significnt difference from corresponding control. 28

7 Effects of rosuvsttin, β-crotene, nd dietry control on c d e f g h i j Fig. 3. Liver sections from; norml untreted rts, ; 1; composed of hexgonl or pentgonl loules with centrl veins (lck rrow) nd portl trct (right green rrow) emedded in connective tissue. Heptocytes re rrnged in trecules running rdintly from the centrl vein nd re seprted y sinusoids (yellow rrow) contining Kuppfer cells, ; 2; - rts, c; showing lost heptic rchitecture, formtion of firous sept (lue rrow) moderte mcro 5% (lck rrow) nd micro (right green rrow) stetotic chnges, mny heptocytes with cytoplsmic vcuoles (yellow rrow), nd to less extent in - rts, d; RSV--treted rts, e; showing loss of heptic rchitecture 62.5%, mcro (lck rrow) nd micro (yellow rrow) 37.5% stetotic chnges, RSV--treted rts, f; showing intct heptic rchitecture 75%, lrge lipid droplets s mcro (lck rrow) 75% stetotic chnges, C-treted rts, g; showing prtil loss of heptic rchitecture 37.5%, mcro (lck rrow) nd micro (yellow rrow) 5% stetotic chnges, C--treted rts, h; showing intct heptic rchitecture 75%, micro (yellow rrow) nd mcro (lck rrow) 5% stetotic chnges, RSV+ C--treted rts, i; showing preserved heptic rchitecture 62.5%, mcro (lck rrow) 37.5% stetotic chnges, nd RSV+ C--treted rts, j; showing norml preserved heptic rchitecture 1%, liver pper within norml limit with micro (lck rrow) lipid droplets (H&E, 2). 281

8 S.H. Seif el-din et l. / RPS 215; 1(4): Tle 4. Effect of rosuvsttin nd -crotene lone or in comintion on histologicl prmeters of liver in induced rts. Groups Heptic rchitecture preserved lost % Stetosis Stetosis type Microvesiculr Mcrovesiculr Inflmmtion Heptocyte vcuoltion Firosis Non Present -control 3/8 5/8 31. ± /8 4/ ± ± /8 6/8 RSV 3/8 5/ ± /8 5/ ± ± 3.4 3/8 5/8 C 5/8 3/ ± 6.7 4/8 4/8 15. ± ± /8 2/8 RSV+ C 5/8 3/ ± 2.6 c 5/8 3/8 11. ± ± 4.28 c 5/8 3/8 -control 6/8 2/ ± /8 5/ ± ± /8 2/8 RSV 6/8 2/ ± /8 6/ ± ± /8 1/8 C 6/8 2/ ± 4.5 4/8 4/ ± ± /8 4/8 RSV+ C 8/8 / ± /8 3/8 5. ± ± /8 /8 Vlues re presented s men SEM, (n=8). ; P<.5 significnt difference from corresponding control. ; P<.5 significnt difference from corresponding RSV. c ; P<.5 significnt difference from corresponding C. DISCUSSION represents wide spectrum of disorders, the hllmrk of which is heptic stetosis. ws considered enign condition, ut is now incresingly recognized s mjor cuse of liver-relted moridity nd mortlity (24). Although the exct physiopthology of is not fully understood (25), it ws descried s two hit model. The first hit is supposed to e the increse of free ftty cids in heptocytes, which results in decrese of -oxidtion, which ggrvtes ccumultion of ftty cids nd insulin resistnce. The second step includes ll mechnisms contriuting to the genertion of proinflmmtory cytokines, oxidtive species nd therey enhnces lipid peroxidtion of the heptocyte memrne (26) nd development of inflmmtion nd firosis (27). In this study hypercholestermic model of ws estlished y feeding nimls with for 12 weeks (19) followed y for 4 weeks concomitnt with drugs dministrtion. Incresed ody weight, liver weight nd liver weight index, elevted serum liver enzymes, lipid profile levels, oxidtive stress, enhnced lipid peroxidtion nd ltered liver histopthology including stetosis, inflmmtion nd firosis were oserved in the rts, nd to less extent in - rts. These normlities were significntly improved 4 weeks fter tretment with either RSV or C or oth. Body weight significntly incresed with the high-ft diet, this might e due to metolic imlnce of crohydrte, protein, nd ft. Tretment of - with RSV resulted in significnt increse in ody weight versus norml ut not significnt with - control; this ws in greement with Vlero-Muñoz nd coworkers (28) who reported tht RSV did not modify ody weight or the weight of the dipose pckges in rt. Tretment with C lone or in comintion with RSV resulted in significnt decrese of ody weight, liver weight nd its index. This ws in ccordnce with Chung nd collegues (29) who oserved tht most of the crotenoids ppered to e inversely correlted to ft mss, suggesting tht during oesity crotenoids re sequestered in dipose tissue, decresing their plsm concentrtions. Moreover, Vn Helden nd coworkers (3) showed tht the nti-oesity effect of C hs een demonstrted to e linked to its provitminic A effect. However, heptic index ws higher in the group with stetosis s compred to the RSV or C-treted nd norml groups, which mens tht C nd, t lest in prt, RSV cts y decresing ft

9 Effects of rosuvsttin, β-crotene, nd dietry control on ccumultion in the liver nd ft weight, nd therefore decreses heptic index. In the present investigtion, the oserved elevtion in ll liver enzymes in model is in greement with Al-Dosri nd collegues (31) who reported tht there were incresed plsm ctivities of AST, ALT, ALP nd GGT in -fed rts. ALT nd AST re iomrkers in the dignosis of heptic dmge ecuse they re relesed into the circultion fter heptocellulr dmge (32). Moreover, dt lso showed significnt increse in serum TG, TC, LDL, VLDL, leptin s well s TNF- nd TGF- 1 levels ccompnied with significnt decrese in serum HDL nd diponectin in the group s in previous reports (33 35). Rizos nd coworkers (36) reported tht in hyperlipidemic ptient cliniclly significnt elevtions in AST/ALT vlues were defined s >3-fold the upper limit of norml. This ws in greement with the result of this study where AST/ALT ws >3- fold the upper limit. Body ft distriution ppers to e even more importnt thn the totl mount of dipose tissue, nd viscerl ft mss is strongly linked to insulin resistnce nd (37). Viscerl ft relesed FFAs re trnsported to the liver y the portl vein nd my contriute to heptic stetosis, production of triglyceride rich VLDL nd elevted -oxidtion (38). These normlities were significntly improved, nd heptic stetosis ws significntly decresed in ll RSV nd Ctreted groups nd to less extent in the group treted with C lone. The results of the present study showed tht RSV tretment in model resulted in normliztion of GGT nd significnt decrese in ALT, AST nd ALP. This ws in greement with Antonopoulos nd collegues (39) nd Ahmed nd coworkers (4) who reported tht the use of RSV hs een improved liver normlities in ptients. Moreover, RSV tretment reduced the stetosis likely due to decresed input of FFAs nd n incresed output of FFAs through incresed -oxidtion. In ddition, RSV tretment in - model showed normliztion in lipid profile; TG, TC, LDL, VLDL nd HDL. This ws in ccordnce with Awd nd Kmel (41) who indicted tht RSV meliorted heptic injury, inflmmtion nd lipid perixodtion, nd in contrst with those of Ansri nd coworkers (42) who reported tht orl dministrtion of RSV t 1 mg/dy in rts fed with resulted in significnt elevtion of HDL. Kostpnos nd collegues (43) showed tht RSV tretment t 1 mg/dy in ptients ws ssocited with significnt hypotriglyceridemic effect, reduction in the cholesterol concentrtion of ll LDL prticles. Moreover, Mlgurner nd coworkers (44) reported tht RSV improves lipid profiles, heptic prmeters, nd the histology of HCV. Furthermore, RSV limits the effects of cytokines, improving insulin resistnce nd TNF- produced y monocytes nd mcrophges, which impirs insulin signling nd plys crucil role in NASH progression (45). Dt from the present investigtion showed tht RSV tretment incresed diponectin level significntly ut not leptin compred with control. On contrry to this, De ls Hers nd collegues (46) nd Vlero-Muñoz nd coworkers (28) reported tht RSV decresed leptin levels nd did not modify diponectin levels in treted rts. Hsegw nd collegues (47) reported tht TGF- 1 medites the trnsformtion of quiescent heptic stellte cells into myofirolsts-like cells with n incresed production of extr cellulr mtrix proteins including type I collgen. Activted kupffer cells nd heptic stellte cells secrete TGF- 1 nd its plsm concentrtion is elevted in NASH ptients compred to ptients with heptic stetosis nd helthy sujects, suggesting tht this cytokine is involved in firogenesis in NASH. Yng nd coworkers (48) reported tht diponectin lso regultes heptic expression of TGF- 1, pro-firotic fctor involved in heptic stellte cell ctivtion tht plys n importnt role in neofirogenesis of. In this study, the improvement in TNF- nd TGF- 1 with RSV- rts ws not significnt from its - control, while tendency for normliztion ws chieved using RSV- with respect to norml. This is in greement with De ls Hers nd collegues (46) who oserved tht RSV tretment did not ffect the 283

10 S.H. Seif el-din et l. / RPS 215; 1(4): elevted expression of TNF- in dipose tissue of rts with respect to controls. Also, M nd coworkers (49) reported tht RSV in dose dependent mnner reduces TGF- 1 expression nd inhiits the development of myocrdil firosis in dietic rts. In the current study, C supplementtion resulted in significnt decrese in liver enzymes except ALP from control. This is in greement with the study of Vrdi nd collegues (5) who reported tht C given for 21 dys efore the methotrexte ppliction provided significnt protection from the heptotoxicity of methotrexte. On the other hnd, it showed no significnt chnges in lipids serum mrkers from control. These results re in ccordnce with Nierenerg nd coworkers (51) who oserved tht high intkes of C hd no effect on serum lipid concentrtions in humn eings. This could e due to C not ccumultes in rts tissues when fed t physiologic levels (52,53). Moreover, the codministrtion of RSV+ C herein, resulted in further improvements in liver enzymes nd lipid profile versus ech drug lone. In this context, Rydén nd collegues (54) reported tht there is risk to misinterpret the crotenoids sttus in individuls with cholesterol-lowering therpy. In this study, dministrtion of C lone did not produce significnt reduction in diponectin or leptin levels from control. This is conversely to Cns nd coworkers (55) who oserved tht there were inverse correltions etween C nd dominl ft mss t seline. Menwhile, co-dministrtion of RSV with C resulted in significnt increse in diponectin concomitnt with reduction in leptin nd TGF- 1 nd normliztion in TNF- level versus nd norml control. In this context, Kmeji nd collegues (56) reported tht the ntiinflmmtory effects of C in dipocytes were suggested to rise through limittion of TNF- -medited down-regultion of genes linked to dipocyte iology. Oxidtive stress is elieved to ply n importnt role in pthogenesis of. It hs een shown tht chronic oxidtive stress, generted through oxidtion of cytotoxic free ftty cids, my led to cytokine upregultion nd depletion of heptic ntioxidnt levels (57). In ddition, enhnced lipid peroxidtion leds to the genertion of y-products, such s MDA, which hve een shown to further stimulte cytokine production (58). In the current study, there ws significnt decrese in GSH content nd SOD ctivity in rts nd significnt increse in MDA levels. These findings re in greement with those of Khn nd coworkers (59) who reported tht liver GSH nd SOD ctivity ws decresed in hypercholesterolemic rts, while MDA content ws significntly higher in comprison with norml control group. As, hypercholesterolemi induces cellulr oxidtive stress, to protect cells or tissues ntioxidnt systems hve to fight ginst this (6). RSV tretment showed ntioxidnt properties s it normlized SOD ctivity nd significntly decresed MDA levels in rts fed with. These findings re in ccordnce with those of Ansri nd collegues (42) who reported tht orl dministrtion of RSV t 1 mg/dy in rts fed with resulted in significnt elevtion in ntioxidnt properties of crdic SOD ctivity nd GSH content ccompnied with significnt decrese in crdic TBARs level. Dt in the present study showed tht C tretment significntly incresed SOD ctivity wheres the increse in GSH ws not significnt from control. These results re in ccordnce with Vrdi nd coworkers (5) who oserved tht the oxidtive dmge incresing with methotrexte in liver tissue ws prevented y dosing C for 21 dys prior to methotrexte dministrtion. Also, El- Demerdsh nd collegues (61) reported tht C plys n importnt role in protecting cell memrne ginst oxidtive dmge ecuse of its property of scvenging lipid nd peroxyl rdicls. Moreover, Shih nd collegues (62) oserved tht C ttenuted the diet-induced oxidtive stress in rts fed with high ft, highcholesterol diet, which ws due in prt to up regulted ntioxidnt defenses. At the sme time, the significnt decrese in MDA level from control fter C dministrtion 284

11 Effects of rosuvsttin, β-crotene, nd dietry control on herein is in greement with Berrymn nd coworkers (63) who reported tht the ddition of C returned MDA levels ck to norml in the liver of dietic rts. CONCLUSION The current study shows tht weight loss through dietry control is one of the most effective therpeutic strtegies nd is the first step in mngement (64). As hypertriglyceridemi nd hypercholesterolemi is often ssocited with, hence the rtionle for using the effective lipid lowering gent RSV in its mngement. In ddition, C recognized s the most potent retinol precursor nd provitmin A (65), which responsile for most of its ntioxidtive nd phrmcologicl effects. Accordingly, the comined dministrtion of RSV nd C in this study hs n enhncement effect in modulting the iochemicl nd histologicl mrkers relted to disese compred to either drug lone. This my e due to their synergistic ntioxidtive, nti-inflmmtory nd lipid lowering effects. Moreover, comined tretment with RSV nd C hs modulting effect on the extent of ftty infiltrtion nd on lipid peroxidtion compred with ech drug lone. Reduction of leptin, TGF- 1, TNF- nd oxidtive stress remins the est-trgeted tretment to dte for. ACKNOWLEDGMENTS This work ws supported y the internl reserch project 95/A for sic nd pplied reserch, grnt from Theodor Bilhrz Reserch Institute. REFERENCES 1. Chlsni N, Younossi Z, Lvine JE, Diehl AM, Brunt EM, Cusi KN, et l. The dignosis nd mngement of non-lcoholic ftty liver disese: prctice guideline y the Americn Gstroenterologicl Assocition, Americn Assocition for the Study of Liver Diseses, Americn College of Gstroenterology nd the Americn Gstroenterologicl Assocition. Am J Gstroenterol. 212;142: Bellentni S, Scglioni F, Mrino M, Bedogni G. Epidemiology of non-lcoholic ftty liver disese. Dig Dis. 21;28: Willims CD, Stengel J, Asike MI, Torres DM, Shw J, Contrers M, et l. Prevlence of nonlcoholic ftty liver disese nd non-lcoholic stetoheptitis mong lrgely middle-ged popultion utilizing ultrsound nd liver iopsy: prospective study. Gstroenterology. 211;14: Loom R, Sirlin CB, Schwimmer JB, Lvine JE. Advnces in peditric non-lcoholic ftty liver disese. Heptology. 29;5: Anni FA. Non-lcoholic ftty liver disese nd fructose: d for us, etter for mice. J Heptol. 211;55: Bjornsson E, Angulo P. Non-lcoholic ftty liver disese. Scnd J Gstroenterol. 27;42: Hyogo H, Tzum S, Arihiro K, Iwmoto K, Neshim Y, Inoue M, et l. Efficcy of torvsttin for the tretment of nonlcoholic stetoheptitis with dyslipidemi. Metolism. 28;57: Vijn S, Hywrd RA. Phrmcologic lipidlowering therpy in type 2 dietes mellitus: ckground pper for the Americn College of Physicins. Ann Intern Med. 24;14: Frulo JC, Souz-Mello V, Aguil MB, Mndrimde-Lcerd CA. Beneficil effects of rosuvsttin on insulin resistnce, diposity, inflmmtory mrkers nd non-lcoholic ftty liver disese in mice fed on high-ft diet. Clin Sci (Lond). 212;123: Puccetti L, Psqui AL, Scrpini F, Cppellone R, Ghezzi A, Cecctelli L, et l. Sttins discontinution in complint chronic users induces therothromotic profile despite seline clinicl setting nd tretments. Int J Crdiol. 211;3: Prson HK, Bundy MA, Dulin CB, Boyd AL, Pulson JF, Vinik AI. Pleiotropic effects of rosuvsttin on microvsculr function in type 2 dietes. Dietes Met Syndr Oes. 21;3: Dy CP. From ft to inflmmtion. Gstroenterology. 26;13: Kolk M, Westerck J, Velgpudi VR, Wågsäter D, Yetukuri L, Mkkonen J, et l. Adipose tissue inflmmtion nd incresed cermide content chrcterize sujects with high liver ft content independent of oesity. Dietes. 27;56: Burri BJ. Lycopene nd humn helth. In: Meskin MS, Bidlck WR, Dvies AJ, Omye ST, editors. Phytonutrients in helth nd disese. Boc Rton: CRC Press; 22. p Institute of Medicine (IOM). Vitmin A. In: Dietry reference intkes for vitmin A, vitmin K, rsenic, oron, chromium, copper, iodine, iron, mngnese, molydenum, nickel, silicon, vndium, nd zinc. Wshington: Ntionl Acdemy Press; 21. p Delport R, Uink JB, Humn JA, Becker PJ, Myurgh DP, Vermk WJ. Antioxidnt vitmins nd coronry rtery disese risk in South Africn mles. Clin Chim Act. 1998;278:55 6.

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