Journal of Chemical and Pharmaceutical Research

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1 Avilble on line Journl of Chemicl nd Phrmceuticl Reserch ISSN No: CODEN(USA): JCPRC5 J. Chem. Phrm. Res., 2011, 3(3):52-63 Effect of Lovsttin on Lipoprotein Lipid Peroxidtion nd Antioxidnt Sttus in Inflmmtion Induced Hyperlipidemic Rts Smir Chhetri, Amir Khn b*, N S Rthore e, Fouzi Ishq c, Abhy S Chndel nd Deepti Mlhotr Dept. of Biotechnology, HNB Grhwl University, Sringr, Uttrkhnd, Indi b Dept. of Biotechnology nd Biomedicl Science, DIBNS, Dehrdun, Uttrkhnd, Indi c Dept. of Environmentl Science, Gurukul Kngri University, Hridwr, Uttrkhnd, Indi d Dept. of Biotechnology, Shri Guru Rm Ri (P.G) College Dehrdun Indi e Dept. of Biotechnology, Grphic Er University, Dehrdun Indi ABSTRACT Crdiovsculr diseses (CVD) re the mjor contributor to the globl burden of disese. Coronry hert disese, cerebrovsculr disese, hypertension, rtery disese, rheumtic hert disese, congenitl hert disese nd filure of hert cme under the ctegory of CVD. Epidemiologicl studies hve suggested link between therosclerosis, infection nd inflmmtion. Atherosclerosis is multifceted disese process with severl different well defined risks fctors, such s hypercholesterolemi, hypertension nd dibetes. In this study we investigte the efficcy of hypolipidemic, nti-therogenic nd ntioxidnt gent Lovsttin by nlyzing ll the prmeters in plsm, Totl lipid, TC, TG, VLDL-C, LDL-C, non-hdl-c, MDA nd Heptic TG, TC s well s heptic ntioxidnt enzymes (Ctlse, Superoxide dismutse, Glutthione peroxidse nd Glutthione reductse). All the plsm lipids prmeters s well s heptic ntioxidnt enzymes level were significntly incresed/decresed in inflmmtion induced hyperlipidemic (IIH-C) rts. After 4-weeks dministrtion of Lovsttin significntly restore the bove ltered prmeters. In conclusion, Lovsttin my be useful in the prevention nd tretment of inflmmtion induced hyperlipidemi, CVD nd therosclerosis. Keywords: CVD, Hypercholesterolemi, Hypolipidemic, Inflmmtion, Atherosclerosis Lovsttin nd Heptic ntioxidnt enzymes. INTRODUCTION Crdiovsculr diseses (CVD) re the mjor contributor to the globl burden of disese. (CVD) is the number one cuse of deth worldwide [1, 2, 3] nd is projected to remin the leding cuse of deth. Hence, this disese gretly contributes to the rising costs of helth cre in the world. It 52

2 is mjor public-helth chllenge, especilly in low nd middle income countries, where 80 % of these deths occur [4]. Coronry hert disese, cerebrovsculr disese, hypertension, rtery disese, rheumtic hert disese, congenitl hert disese nd filure of hert cme under the ctegory of CVD. It is widely recognized tht therosclerosis is n inflmmtory disorder. Moreover, recent epidemiologicl studies hve strongly suggested tht disorders tht led to systemic inflmmtion increses the risk of developing CVD. Atherosclerosis is multifceted disese process with severl different well defined risks fctors, such s hypercholesterolemi, hypertension nd dibetes. Hyperlipidemi refers to incresed levels of lipids (fts) in the blood, including cholesterol nd triglycerides. Approximtely two-thirds of the blood cholesterol is found in the LDL frction nd higher LDL cholesterol concentrtions hve been ssocited with n incresed incidence of coronry rtery disese. Infection nd inflmmtion induce the systemic host response known s cute phse response (APR), nd produce mny bnormlities tht could increse the risk of developing therosclerosis. In niml models, Infection nd inflmmtion re produced by dministrtion of turpentine or croton oil (cute loclized sterile inflmmtion). Ech of these stimuli is well-chrcterized inducer of APR. [5, 6, 7, 8, 9] APR represents the initil line of defense ginst injury s well s bcteril nd prsitic infections. The APR hs been shown to reduce the expression of certin conjugtion enzymes s well s ntioxidnt enzymes. Cytokines, secreted from mcrophges in response to infection nd inflmmtion, hve been implicted in the suppression of ctivity of these enzymes in ddition to their role in oxidtive stress. These enzymes re importnt in defending the body ginst free rdicls s well s toxic substnces by converting them to form tht cn be redily excreted. Therefore, ny chnges in these enzymes could be potentilly detrimentl to the host by ltering these defense mechnisms. Norml cellulr metbolism involves the production of ROS [10], low levels of ROS re vitl for proper cell functioning, while excessive in vivo genertion of these products cn dversely ffect cell functioning [11, 12]. The body hs the bility to produce endogenous ntioxidnts such s Superoxide dismutse, Ctlse nd Glutthione peroxidse. Under norml circumstnces, there is blnce between these endogenous ntioxidnts nd the production of free rdicls in the body. It hs been reported tht endotoxin injection cused renl tissue dmge nd decresed the SOD, Gpx nd CAT ctivities compred to control rts [13, 14]. A significnt decrese in heptic GSH s well s in enzymtic ctivities of SOD, CAT, Gpx nd GST were observed in d-glctosmine/lipopolyscchride-intoxicted rts s compred with the levels of norml control rts [15]. Oxidtive modifiction of lipoproteins is believed to ply centrl role in the pthogenesis of therosclerosis [16, 17] becuse plsm contins severl ntioxidnts [18] nd lipoproteins with oxidtive dmge hve been isolted from therosclerotic lesions,[16, 17]enzymtic ntioxidnt defenses include Superoxide dismutse (SOD), Glutthione peroxidse (GPx), Ctlse (CAT). To protect the cells nd orgn systems of the body ginst rective oxygen species, humns hve evolved highly complex ntioxidnt protection system. These include ntioxidnt enzymes tht ctlyze free rdicl quenching rections, nd diet-derived ntioxidnts. Lipid-lowering drugs such s sttins hve lso been shown to ntgonize inflmmtion [4, 19, nd 20] they lower cholesterol by inhibiting the enzyme HMG-CoA reductse, which is the rte-limiting enzyme of the mevlonte pthwy of cholesterol synthesis. Inhibition of this enzyme in the liver results in decresed cholesterol synthesis s well s incresed synthesis of LDL receptors, resulting in n incresed clernce of low-density lipoprotein (LDL) from the bloodstrem. In this study we investigte the efficcy of nti-therogenic hypolipidemic nd ntioxidnt gent Lovsttin by nlyzing ll the prmeters in plsm TC, VLDL-C, LDL-C, HDL-C, nd its sub frction HDL2-C nd HDL3-C, MDA, Heptic TG, TC nd ntioxidnt enzymes (Ctlse, Superoxide dismutse, Glutthione peroxidse nd Glutthione reductse). 53

3 EXPERIMENTAL SECTION Chemicls:-1- Chloro 2, 4-Dinitrobenzene ws purchsed from Centrl drug house, Pvt. Ltd. (Indi). All other chemicls used for this study were of nlyticl grde nd obtined from HIMEDIA (Indi), Sisco (Indi), Ashirwd (Indi), Sigm-Aldrich (USA), Miles (USA), Acros (USA) nd Lovsttin drug ws supplied s gift from Simir Innoform Pvt. Ltd. Chenni, Indi. Estimtion: Frctiontion of Plsm lipoprotein such s LDL[21], HDL nd its frctions-hdl 2, HDL 3[ 22], Plsm FRAP[23], determintion of triglyceride nd totl cholesterol in liver homogente[24], ctivities of ntioxidnt enzymes such s Ctlse[25], Superoxide dismutse[26], Glutthione peroxidse [27] nd Glutthione reductse [28] in liver homogente were mesured by following known procedures. Experimentl Design: The experimentl study ws pproved by the Dolphin Institute of Biomedicl nd Nturl Sciences, Dehrdun, Uttrkhnd, where the study ws conducted. Helthy mle lbino rts, weighing bout g were purchsed from Indin Veterinry Reserch Institute (IVRI), Breilly (Indi), were mintined to niml house environmentl condition prior to the experiment. For the present study, nimls were divided into following 3 groups: Norml Control (NC); six rts were given 1.0 ml sline/rt/dy through gstric intubtion for 4 weeks, inflmmtion induced hyperlipidemic Control (IIH-C) rts; six rts in this group were dministered 1.0 ml sline/rt/dy through gstric intubtion for 4 weeks, Inflmmtion induced hyperlipidemic Lovsttin treted rts (IIH-LT); six rts in this group were given 1.0 mg Lovsttin/rt/dy through gstric intubtion for 4 weeks. Diet/Drug Administrtion:-The rts were given pelleted rt chow. Mintennce nd tretment of ll the nimls ws done in ccordnce with the principles of Institutionl Animl Ethics Committee constituted s per the directions of the Committee for the Purpose of Control nd Supervision of Experiments on Animls (CPCSEA), Indi. Six rts in IIH-LT group were given 1.0 mg Lovsttin/rt/dy, through gstric intubtion for 4 weeks. Induction of Inflmmtion: - Inflmmtion ws induced in IIH-C nd IIH-LT group by the subcutneous injection of turpentine (0.5ml/rt) in the dorsolumbr region nd left for five hours. Collection of Blood nd Plsm: For the estimtion of different prmeters, overnight fsted rts in ech group were nesthetized nd blood drwn from crdic puncture, nd were collected in heprinised tube. Plsm ws seprted from blood by centrifugtion t 2500 rpm for 30 min. Totl cholesterol nd triglyceride estimtion in liver homogente:- Liver were excised nd chilled in ice cold sline. Weight of ll liver ws tken only fter drying the tissue. The volume of ech homogente ws recorded nd centrifuged t 1000 rpm for 10 min t 4 0 C. After centrifugtion, portion of ech homogente from liver thus obtined ws used for the estimtion of totl cholesterol nd triglyceride content in it. Stticl evlution: This ws done by employing two-tiled student t-test s describe by Bennet nd Frnklin (1967). P vlues less thn 0.02 were considered significnt. 54

4 RESULT Effects of Lovsttin on verge body weight in ech group of rts:-tble-1 depicts the verge body weight (g) of N-C, IIH-C, IIH-LT ws 165g, 170g nd 176g, wheres, the verge body weight of N-C, IIH-C, IIH-LT rts showed significnt gin of 34%, 13% nd 42% respectively fter 4 weeks of tretment. These results demonstrte tht in inflmmtion induced hyperlipidemic lovsttin treted (IIH-LT) rts the gin in body weight fter 4 weeks were significntly higher thn N-C rts. Tble-1 Averge body weight in ech group of rts before nd fter 4 weeks of Lovsttin tretment Averge body weight/rt (g) Group Before tretment After Tretment N-C ±2.13 * ±12.23 (+34.87%) IIH-C ±4.11 * ±9.45 (+13.79%) b IIH-LT ±4.72 * ±11.15 (+42.10%) *Vlues re men ± SD from 6 rts in ech group. N- C norml Control; IIH-C inflmmtion induced hyperlipidemic control rts; IIH-LT fed 1mg Lovsttin/rts/dy for 4 weeks, Significntly different from N-C t b p< Significntly different from IIH-C t p< b mg/dl TL TG TC Prmeters Norml Control Hyperlipidemic Control Lovsttin Treted Fig.1 Impct of Lovsttin on, plsm Totl lipid (TL), Triglycerides (TG) nd Totl Cholesterol (TC) in inflmmtion induced hyperlipidemic rts fter 4 weeks of tretment. * Vlues re men (mg/dl) ± SD from pooled plsm of 6 rts in ech group. N-C, norml control; IIH-C, Inflmmtion induced hyperlipidemic control rts; IIH-LT fed 1 mg Lovsttin/rt/dy for 4 weeks. Significntly different from N-C t p < 0.001, Significntly different from IIH-C t p<0.001 nd b p<0.05. Effects of Lovsttin on plsm totl lipid (TL), triglycerides (TG) nd totl cholesterol (TC) in inflmmtion induced hyperlipidemic rts fter 4 weeks of tretment: As seen in Fig 1, ll the plsm lipids prmeters were significntly incresed in Inflmmtion induced hyperlipidemic (IIH-C) rts, when compred to N-C vlues. Totl lipids (TL), triglycerides (TG) nd totl cholesterol (TC) significntly incresed from 380, 52, nd 79 mg/dl in N-C to 483, 102, 55

5 nd 151 mg/dl, respectively, in IIH-C group. After 4 weeks of Lovsttin tretment, levels of TL, TG, nd TC were significntly decresed by 6 %, 33 %, nd 42 %, respectively, when compred to corresponding N-C vlues. These results demonstrte tht 4-week tretment of inflmmtion induced hyperlipidemic rts with 1.0 mg Lovsttin medited significnt reduction in bove lipid prmeters. Effects of Lovsttin on plsm lipoprotein frction nd the rtios of LDL-C/HDL-C nd HDL-C/TC: As seen in Fig 2, plsm VLDL-C, LDL-C nd non-hdl-cholesterol (non-hdl- C) levels were significntly incresed from 9 mg/dl, 52 mg/dl nd 63 mg/dl in N-C to 20 mg/dl (112%), 110 mg/dl (111 %) nd 125 mg/dl (98 %) respectively in IIH-C. After 4 weeks of Lovsttin tretment, both VLDL-C, LDL-C nd non-hdl-c levels showed significnt reduction 39 %, 49 % nd 44 %, respectively, in IIH-LT. Wheres HDL-C, HDL 2 -C nd HDL 3 - C levels were decresed from 19, 6 nd 10 mg/dl in IIH-C to 16 mg/dl (15 %), 4 mg/dl (34 %) nd 9 mg/dl (5 %), respectively, in IIH-C vlues. After 4 weeks of Lovsttin tretment (IIH-LT) HDL-C, HDL 2 -C nd HDL 3 -C levels showed significnt increse of 54 %, 122 % nd 40 %, respectively, when compred to corresponding vlues in IIH-C. These results demonstrte tht Lovsttin is effective in reducing VLDL-C nd LDL-C levels. On the other hnd, in comprison to IIH-C vlues, tretment of Inflmmtion induced hyperlipidemic rts with Lovsttin medited significntly higher increse in HDL-C, HDL 2 -C nd HDL 3 -C concentrtion mg/dl b VLDL-C LDL-C HDL-C HDL2-C HDL3-C Non-HDL-C Prmeters Norml Control Hyperlipidemic Control Lovsttin Treted Fig 2 Impcts of Lovsttin on plsm VLDL-C, LDL-C, HDL-C, HDL2-C, HDL3-C nd Non-HDL-C in inflmmtion induced hyperlipidemic rts fter 4 weeks of tretment Vlues re men (mg/dl) ± SD from pooled plsm of 6 rts in ech group, N-C, norml control; IIH-C, Inflmmtion induced hyperlipidemic control rts; IIH-LT fed 1 mg Lovsttin/rt/dy for 4 weeks, Significntly different from N-C t p<0.001 nd b p<0.02, Significntly different from IIH-C t p< On the other hnd, LDL-C/HDL-C nd HDL-C/TC rtios were clculted from the dt presented in Tble 2 nd 3. LDL-C/HDL-C rtio ws significntly incresed from 2.73 in N-C to 6.83 (150 %) in IIH-C group, when compred to rtio in N-C. After 4 weeks of tretment, the increse in LDL-C/HDL-C rtio ws significntly prevented nd decresed to 2.22 in IIH-LT, which is close to norml control vlue. HDL-C/TC rtio ws significntly decresed from in N-C to (55 %) in IIH-C group, s seen in Tble 2. Lovsttin tretment to these rts 56

6 significntly prevented the increse in HDL-C/TC rtios nd fully restored them to rtio vlue similr to N-C. Tble 2 Impcts of Lovsttin on the rtio of LDL-C/HDL-C, HDL-C/TC, in inflmmtion induced hyperlipidemic rts fter 4 weeks of tretment. Prmeters N-C IIH-C IIH-LT LDL-C/HDL-C 2.73±0.023 ** 6.83±0.043 ** 2.22±0.011 ** ( %) ( %) HDL-C/TC 0.240±0.027** 0.106±0.018** 0.285±0.019** ( %) b ( %) For the clcultion of rtio, dt hve been tken from fig 1 nd 2. Vlues re men (mg/dl) ± SD from pooled plsm of 6 rts in ech group, N-C, norml control; IIH-C, Inflmmtion induced hyperlipidemic control rts; IIH-LT fed 1 mg Lovsttin/rt/dy for 4 weeks, Significntly different from N-C t p<0.001, Significntly different from IIH-C t p< µmole/dl Totl Antioxidnts CD LHPO MDA Prmeters Norml Control Hyperlipidemic Control Lovsttin Treted Fig.3 Impct of Lovsttin on plsm totl ntioxidnt, Conjugted diene (CD), Lipid hydroperoxide (LHPO) nd Mlondildehyde (MDA) contents in inflmmtion induced hyperlipidemic rts fter 4 weeks of tretment Vlues re men (µmole/dl) ± SD from pooled plsm of 6 rts in ech group. N-C norml Control; IIH-C inflmmtion induced hyperlipidemic control rts; IIH-LT feed 1mg Lovsttin /rts/dy for 4 weeks. Significntly different from N-C t p< Significntly different from IIH-C t p< Effects of Lovsttin on plsm totl ntioxidnts nd lipid peroxidtion products:-fig-3 depicts the ntioxidnt impct of lovsttin on plsm concentrtions of totl ntioxidnts, conjugted diene (CD), lipid hydroperoxide (LHPO) nd mlondildehyde (MDA) in inflmmtion induced hyperlipidemic rts. In IIH-C rts, plsm totl ntioxidnts level ws reduced from control vlue of 53 to 38 (27%) µmole/dl. Tretment of IIH-LT rts with Lovsttin for 4 weeks resulted in significnt increse of totl ntioxidnts levels by 13 % when compred to IIH-C vlue. The oxidtive stress induced in IIH-C rts significntly enhnced plsm lipid peroxidtion products, such s conjugted diene, lipid hydroperoxide nd MDA. Formtion of conjugted diene, lipid hydroperoxide nd MDA in plsm ws incresed from 10.97, 1.26 nd 1.29 in N-C to (31 %), 1.98 (57 %) nd 2.96 (129 %) µmole/dl, 57

7 respectively, in IIH-C. After Lovsttin tretment, in IIH-LT, significnt decrese of 9 %, 22 %nd 35 % ws seen in the formtion of conjugted diene, lipid hydroperoxide nd MDA, respectively, when compred to corresponding vlues in IIH-C rts. These results demonstrte tht in IIH-C rts, due to increse in oxidtive stress, totl ntioxidnts level ws decresed, wheres, concentrtion of plsm conjugted diene, lipid hydroperoxide nd MDA were significntly incresed. Tocotrienols tretment significntly restored the totl ntioxidnts level nd blocked the increse in plsm conjugted diene, lipid hydroperoxide nd MDA to level close to corresponding norml vlues. Lovsttin effect on Triglycerides (TG), Totl Cholesterol (TC) nd vrious Lipid peroxidtion products in the Liver homogente:-as seen in Tble 3, heptic levels of triglyceride (TG) nd totl cholesterol (TC) were significntly incresed in inflmmtion induced hyperlipidemic control rts (IIH-C) by 38 % nd 113 % respectively, when compred to corresponding vlues in N-C. Feeding of lovsttin inflmmtion induced rts for 4 weeks ws ssocited with significnt decline in liver TG nd TC levels by 9 % nd 29% respectively, in IIH-LT. On the other hnd, formtion of conjugted diene, lipid hydroperoxide nd MDA in liver of inflmmtion induced hyperlipidemic (IIH-C) rts ws significntly incresed by 47%, 34 % nd 42 %, respectively. Feeding of lovsttin to IIH-LT rts for 4 weeks, ws ssocited with significnt decline in the formtion of liver conjugted diene, lipid hydroperoxide nd MDA by 26 %, 21 % nd 20 % respectively, when compred to corresponding vlues in IIH-C group. These results demonstrte tht incresed levels of TG, TC, conjugted diene, lipid hydroperoxide nd MDA in liver of inflmmtion induced hyperlipidemic rts were significntly reduced fter 4 weeks of lovsttin tretment. Tble 3 impct of Lovsttin on triglycerides, totl cholesterol nd vrious lipid peroxidtion products in the Liver homogente fter 4 weeks tretment of inflmmtion induced hyperlipidemic rts. Prmeter NC IIH-C IIH-LT Triglycerides* 0.493±0.001 * 0.685±0.006 * 0.623±0.003 * (+38.94%) (-9.05%) Totl cholesterol* 1.34± ± ±0.011 ( %) (-29.37%) Conjugted diene** 4.97±0.020 * 7.32±0.020 * 5.35±0.005 * (+47.28%) (-26.91%) Lipid Hydroperoxide** 0.952±0.001 MDA** 2.25± ± ±0.018 (+34.45%) (-21.71%) 3.20± ±0.011 (+42.22%) (-20.31%) d *Vlues re men mg/100mg protein ± SD from homogen te of pooled liver of 6 rts in ech group. ** Vlues re men µmole/dl ± SD from homogente of pooled Liver of 6 rts in ech group. N-C norml control; IIH-C inflmmtion induced hyperlipidemic control rts; IIH-LT feed 1 mg of Lovsttin/rts/dy for 4 weeks, significntly different from N-C t p<0.001 nd b p<0.001, significntly different from IIH-C t p< Effects of Lovsttin on the vrious ntioxidnt enzymes ctivities in the liver homogente:- As seen in Tble 4, Ctlse (CAT) ctivity in liver ws significntly decresed from vlue of 3.07 unit in N-C to 2.30 (37 %) in IIH-C, respectively. Administrtion of Lovsttin to inflmmtion induced hyperlipidemic lovsttin treted rts (IIH-LT) resulted in significnt increse in liver CAT ctivities by 3.20 (39 %) unit, respectively. However, in comprison to corresponding tissue vlues of norml control rts (N-C), the decline in heptic Superoxide dismutse (SOD) ctivity of inflmmtion induced hyperlipidemic (IIH-C) rts ws 26 %.Tretment of lovsttin to inflmmtion induced hyperlipidemic lovsttin treted (IIH- 58

8 LT) rts resulted in significnt increse in heptic SOD ctivity by 22 %, respectively from norml vlue. In inflmmtion induced rts, Glutthione peroxidse (Gpx) ctivity in liver ws significntly incresed from vlue of 48 units in N-C to 61 (27 %) units, in IIH-C rts. As evident, fter 4 weeks of tretment with lovsttin, Gpx ctivity in liver ws significntly decresed by 32 %, when compred to corresponding tissue vlues in IIH-C group. On the other hnd, in smoke exposed rts, the enzymtic ctivities of heptic Glutthione reductse (Gred) ws decresed significntly by 37 %, when compred to corresponding vlues of N-C rts. Feeding of lovsttin to inflmmtion induced hyperlipidemic rts significntly blocked the decrese in heptic Gred ctivities nd incresed them to similr vlue of 36 %, Compred to corresponding vlues of Gred ctivities in N-C. Administrtion of lovsttin to smoke exposed rts significntly prevented the decrese in Gred ctivity nd incresed to level, which is similr to norml vlue. In summry, heptic CAT, SOD, Gpx nd Gred enzymes, which constitute mutully supportive tem of defense ginst ROS, re significntly decresed in inflmmtion, induced hyperlipidemic rts. However, lovsttin tretment to inflmmtion induced hyperlipidemic rts substntilly quenches these free rdicls (ROS), thus positively normlizing the bove enzyme levels. Tble 4 Impct of Lovsttin on Liver Ctlse, Superoxide dismutse, Glutthione peroxidse nd Glutthione reductse ctivities in inflmmtion induced hyperlipidemic rts fter 4 weeks of tretment One unit(u/mg protein) of enzyme ctivity is defined s the µmoles of H 2 O 2 decomposed/min/mg protein. One unit (U/mg protein) of enzyme ctivity is defined s the mount of enzyme required to inhibit O.D. t 560 nm of chromogen production by 50%in one minute. β One unit (U/mg protein) of enzyme ctivity is defined s nmole oxidized Glutthione formed /min/mg homogente protein. One unit (U/mg protein) of enzyme ctivity is defined s nmole NADPH oxidized/min/mg PMS protein. Group Ctlse Superoxide Glutthione Glutthione dismutse peroxidse β Reductse N-C 3.70±0.123* 0.755± ±1.02* 8.45±0.198 IIH-C 2.30±0.201* ( %) 0.552±0.002 (-26.88%) 61.56±1.41* (+27.08%) 5.26±0.231 (-37.75%) IIH-LT 3.20±0.013* ( %) 0.665±0.005 ( %) 41.38±1.74* (-32.78%) 7.18±0.199 (+36.50%) *Vlues re men ± SD from homogente or PMS frctio n of pooled liver of 6 rts in ech group, N-C, norml control; IIH-C inflmmtion induced hyperlipidemic control rts nd IIH-LT fed 1 mg Lovsttin/rts/dy for 4 weeks. Significntly different from N-C t p< Significntly different from IIH-C t p< DISCUSSION Severl epidemiologicl studies suggest link between infection/inflmmtion nd therosclerosis. The present study demonstrtes the extensive protherogenic chnges tht occurred s prt of the host response to turpentine (cute loclized sterile inflmmtion) dministrtion, on vriety of prmeters, like, plsm nd lipoprotein lipids in plsm, liver lipid peroxidtion products, liver nd plsm totl ntioxidnt. Pretretment of stressed rts with Lovsttin significntly reduced the overll oxidtive burden nd effectively meliorted the bove ltered prmeters, thus, indicting potent theroprotective effect of Lovsttin. The chnge in lipids nd lipoproteins re similr to those proposed to promote therogenesis, they my initite or ggrvte therosclerosis if the course of infection or inflmmtion is prolonged [30, 31]. Our results demonstrte significnt increse in plsm totl lipids (27%), TG (94%) nd TC (90%) in turpentine (IIH-C) stressed rts. A similr increse in serum TG in LPS treted hmsters or rts were previously reported [5]. In nother report n increse in plsm TG level ws seen during inflmmtion, induced by turpentine oil in pigs [32]. The increse in plsm TG 59

9 levels is pprently due to n increse in VLDL (112%) which cn be the result of either incresed VLDL production or decresed VLDL clernce. Similr to plsm lipids, VLDL-C, LDL-C nd therogenic non-hdl-c levels were lso incresed in stressed nimls, indicting tht the increse in plsm TC is pprently due to increse in VLDL-C nd LDL-C concentrtions. On the other hnd, low decrese of 15 % in plsm ntitherogenic HDL-C level of IIH-C rts ws seen. Similr to plsm TG nd TC in liver ws lso significntly incresed in IIH-C rts. Low density lipoproteins re composed of distinct subclsses tht differ in size, density, chemicl composition, nd their ssocition with crdiovsculr disese [33, 34, nd 35]. It hs been estblished tht LDL-C/HDL-C nd HDL-C/TC rtios re good predictors for the presence nd severity of CAD [36]. LDL-C/HDL-C nd HDL-C/TC rtios were clculted from the dt presented in fig1 nd fig2. LDL-C/HDL-C rtio ws significntly incresed from 2.73 in N-C to 6.83 (150 %) in IIH-C group, when compred to rtio in N-C. After 4 weeks of tretment, the increse in LDL-C/HDL-C rtio ws significntly prevented nd decresed to 2.22 in IIH-LT, which is close to norml control vlue. On the other hnd, HDL- C/TC rtio ws significntly decresed from in N-C to (55 %) in IIH-C group. Lovsttin tretment to these rts significntly prevented the increse in HDL-C/TC rtios nd fully restored them to rtio vlue of which is close to N-C. In ddition, the rtios relted to HDL-C in Lovsttin treted rts were positively modulted nd restored similr to norml control vlue, indicting normliztion of cholesterol levels ssocited with the bove lipoproteins Oxidtive modifiction of lipoproteins is believed to ply centrl role in the pthogenesis of therosclerosis [16, 17] Similr to plsm TG nd TC in liver ws lso significntly incresed in IIH-C rts. Therefore, tocotrienols my exert their cholesterol lowering effect in inflmmtion /infection induced hyperlipidemic rts in similr mnner s previously reported for hyperlipidemic nimls [37] nd humns [38, 39]. Mechnism wise, s previously shown in HepG2 cells, s well s in normolipidemic nd hyperlipidemic rts, tocotrienols reduce cholesterol synthesis by suppressing HMG-CoA reductse ctivity, which in turn is reduced by decline in its protein mss [37, 40] The decline in protein mss my be chieved by inhibition of HMG-CoA reductse synthesis nd/or enhnced degrdtion. Consistent with in vivo results in rts [37], γ-tocotrienol hs been shown to medite the suppression of enzymtic ctivity nd protein mss of HMG-CoA reductse in HepG2 cells through decresed synthesis (57 % of control) nd enhnced degrdtion (2.4-fold versus control) of the enzyme [40]. In ddition, γ- tocotrienol ws shown to up regulte LDL receptor in mmmlin cells nd my be implicted in prt for the reduction of pob-lipoprotein in vivo [40]. Thus, tocotrienols reduce cholesterol formtion in mmmlin cells by suppressing HMG-CoA reductse ctivity through two ctions: decresing the efficiency of trnsltion of HMG-CoA reductse mrna nd incresing the controlled degrdtion of HMG-CoA reductse protein, post-trnscriptionlly [40]. In ddition, nother report indictes tht γ-tocotrienol influences pob secretion by both co trnsltionl nd posttrnsltionl processes involving decresed rte of pob trnsloction nd ccelerted degrdtion of pob in HepG2 cells. This ctivity correlted with decrese in free nd esterified cholesterol [41]. Tken together, the informtion indictes n ssocition between the suppression of heptic cholesterol synthesis nd pob secretion, nd the observed lowering of pob nd LDL-C levels in niml nd humn models [41]. Our dt show tht systemic oxidtion of lipid/lipoprotein prticles occurs s prt of the host response to infection nd inflmmtion. Conjugted diene (which mesure the initil phse of lipid peroxidtion), lipid hydroperoxide (intermedite product of lipid peroxidtion) nd MDA (which mesure the degrdtion phse of lipid peroxidtion) in plsm nd liver re significntly incresed in rts fter, turpentine dministrtion. The increse in plsm lipid peroxidtion products is ssocited with significnt decline in totl ntioxidnts cpcity of plsm. Administrtion of turpentine (cute loclized sterile inflmmtion) generlly leds to fulminnt relese of rective oxygen species (ROS) [42, 43]. Our results indicte significnt decrese in plsm lipid peroxidtion 60

10 products with concomitnt nd significnt increse in plsm totl ntioxidnts in IIH-C group, pretreted with 1.0 mg Lovsttin/dy for 28 dys before turpentine injection. The cute phse response represents the initil line of defense ginst injury s well s bcteril nd prsitic infections. Turpentine which elicits the APR hs been shown to reduce the expression of certin conjugtion enzymes s well s ntioxidnt enzymes. Cytokines, secreted from mcrophges in response to infection nd inflmmtion, hve been implicted in the suppression of ctivity of these enzymes in ddition to their role in oxidtive stress. These enzymes re importnt in defending the body ginst free rdicls s well s toxic substnces by converting them to form tht cn be redily excreted. Therefore, ny chnges in these enzymes could be potentilly detrimentl to the host by ltering these defense mechnisms. Norml cellulr metbolism involves the production of rective oxygen species (ROS) [10], low levels of ROS re vitl for proper cell functioning, while excessive in vivo genertion of these products cn dversely ffect cell functioning [11, 12] Mlondildehyde (MDA) is one of the finl products of lipid peroxidtion in humn cells, nd n increse in ROS cuses overproduction of MDA, which is considered surrogte mrker of oxidtive stress [44, 45]. The mjor intrcellulr ntioxidnt enzyme, superoxide dismutse (SOD), specificlly converts superoxide rdicls to hydrogen peroxide, [46] nd ctlse (CAT) s well s glutthione peroxidse (Gpx) detoxifies hydrogen peroxide to wter [47]. Gpx protects ginst free rdicl injury by reducing the peroxide concentrtion vi glutthione dependent reduction process, thereby reducing the mount of peroxides vilble to produce cellulr dmge. Reduced glutthione is mjor intrcellulr nonenzymtic ntioxidnt. It hs mny biologicl functions, including mintennce of membrne protein nd lipoprotein SH groups in the reduced form, the oxidtion of which cn otherwise cuse ltered cellulr structure nd function. Glutthione cycle opertes in the erythrocytes for the disposl of H 2 O 2 generted in the cell supplementing the function of ctlse. GSH nd H 2 O 2 re twin substrtes for glutthione peroxidse. GSH is formed from its oxidized form, GSSG by the enzyme glutthione reductse (Gred), which requires NADPH s cofctor. Therefore, s the blnce between free rdicl production nd ntioxidnt defenses is lost, the resultnt oxidtive stress through series of events deregultes the cellulr functions leding to vrious pthologicl conditions. An ntioxidnt compound might contribute prtil or totl llevition of such dmge. An impired ROS scvenging function hs been linked to the decresed ctivity of enzymtic nd nonenzymtic scvengers of free rdicls. Our results demonstrte tht, ctlse ctivity in liver ws significntly decresed from vlue of 3.70 unit in N-C to 2.30 (37 %) in IIH-C, respectively. Administrtion of Lovsttin to Inflmmtion induced hyperlipidemic Lovsttin treted rts (IIH-LT) resulted in significnt increse in liver ctlse ctivities by 3.20 (39 %) unit, respectively. However, in comprison to corresponding tissue vlues of norml control rts (N-C), the decline in heptic SOD ctivity of Inflmmtion induced hyperlipidemic (IIH-C) rts ws 26 %. Tretment of Lovsttin to Inflmmtion induced hyperlipidemic Lovsttin treted (IIHC-LT) rts resulted in significnt increse in heptic SOD ctivity by 22 %, respectively from norml vlue. These results re consistent showing decrese in CAT ctivity nd n increse in Gpx ctivity in LPS treted rts [48]. Lovsttin given 28 dys before the onset of infection nd inflmmtion significntly improved the integrity of erythrocytes membrne s shown by improved protection ginst lipid peroxidtion s well s reversl of SOD, CAT, Gpx, nd Gred to ner norml level. The bove results, which represent n initil demonstrtion, indicte tht the host response to infection nd inflmmtion induces severl chnges: hyperlipidemi, enhnced lipid peroxidtion in plsm nd tissues, with depletion in plsm totl ntioxidnts, nd overll wekening of ntioxidnt defense system. LDL prticles with greter tendency to become oxidized might thus be more likely to prticipte in protherogenic events. Incresed oxidtion of LDL density subfrctions tht occurs during infection nd inflmmtion could be one of the mechnisms tht my promote therosclerosis in ptients with chronic infection nd inflmmtory diseses. Orl pretretment of rts with 61

11 Lovsttin for 28 dys significntly prevented the turpentine induced dverse effects nd meliorted the levels of ll the evluted prmeters. Our results strongly suggest tht the llevition of inflmmtory conditions is due to potent lipid lowering nd free rdicl scvenging properties of Lovsttin nd, thus, cn be useful in the therpy of systemic inflmmtory process which might induce therosclerosis. Bsed on these findings, the ntiinflmmtory potentil of Lovsttin looks promising nd more comprehensive studies should be undertken to determine their ctul mode of ction. In conclusion, considering the strong hypolipidemic/theroprotective nd ntioxidnt, nd possibly nti-inflmmtory ctions of Lovsttin, intke of Lovsttin my be useful in the prevention nd tretment of infection/inflmmtion induced hyperlipidemi nd therosclerosis. REFERENCES [1] CD Mthers; A Lopez; C Stein; D MFt; C Ro; M Inoue. In Disese Control Priorities Project Working 2001 Pper 18. Bethesd, MD. [2] CJ Murry; AD Lopez. Globl Comprtive Assessments in the Helth Sector Disese Burden, Expenditures, nd Intervention Pckges Genev., 1994, WHO. [3] The World Helth Report. Reducing Risks, Promoting Helthy Life. Genev., 2002, WHO. [4] EA Ens; S Yusuf; JL Meht. Am. J. Crdiol., (9): [5] KR Feingold; I Stprns; RA Memon. J. Lipid Res., 1992, 33: [6] NJ Todd; JT Whicher; C Westcott; A Gilbert. Clin. Chim. Act., 1990, 189: [7] TS Ro; JL Currie; AF Shffer; PC Iskson.. J. Phrmcol. Exp. Ther., 1994, 269: [8] BJ Vn Lenten; SY Hm; FC de Beer; DM Stfforini; TM McIntyre; SM Prescott; BN L Du; AM Fogelmn; M Nvb. J. Clin. Invest., 1995, 96: [9] VG Cbn; JN Siegel; SM Sbesin.. J. Lipid Res., 1989, 30: [10] JM McCord. Clin. Biochem., 1993, 26: [11] P Gregorevic; GS Lynch; D Willims. Eur. J. Appl. Physiol., 2001, 86:24 7. [12] CL Fttmn; LM Schefer; TD Oury. Free Rdic. Biol. Med.,2003, 35: [13] M Knter; O Coskun; F Armutcu; Y Uz Huly; G Kizily. Tohoku J. Exp. Med., 2005, [14] O Coskun; F Armutcu; M Knter; GM Kuzey. J. Appl. Toxicol., 2005, 25: 8 12 [15] V Viml; T Devki. Journl of Ethnophrmcology., 2004, 90: [16] JA Berliner; JW Heinecke.. Free Rdic. Biol. Med., : [17] D Steinberg.. J. Biol. Chem., 1997, 272: [18] B Frei. Crit. Rev. Food Sci. Nutr., 1995, 35: [19] JT Willerson; PM Ridker. Inflmmtion s crdiovsculr risk fctor. Circultion., 2004, 109:II2 II10. [20] MS Elkind. Inflmmtion, therosclerosis, nd stroke. Neurologist., 2006, 12: [21] H Wielnd; D Seidel. J. Lipid Reserch., 1989, 24: [22] W Ptsch; SA Brown; JD Morrisett; AM Jr Gotto; JR Ptsch.. Clin. Chem., 1989, 35: [23] IFF Benzie; JJ Strin. The FRAP ssy. Anlyticl Biochem., 1996, 239: [24] J Folch; N Lees; SG Stnley.. J. Biol. Chem., 1957, 226: [25] AK Sinh. Anl. Biochem., 1972, 47: [26] P Kkkr; B Ds; PN Viswnthn. Indin J. Biochem. Biophys., 1984, 21: [27] DG Hfemn; RA Sunde; WG Hoekstr. J. Nutr., 1974, 104: [28] I Crlberg; EB Mnnervik. J. Biol. Chem., : [29] CA Bennet; NL Frnklin. Sttisticl Anlysis in Chemistry nd Chemicl Industry. John- Wiley nd Sons Inc., New York., 1967 p

12 [30] W Khovidhunkit; RA Memon; KR Feingold; C Grunfeld. J. Infect. Dis., 2000, 181(Suppl3):S [31] W Khovidhunkit; MS Kim; RA Memon. J. Lipid Res., 2004, 45: [32] MA Nvrro; R Crpintero, S Acın. Immune-regultion of the polipoprotein A-I/C-III/A- IV gene cluster in experimentl inflmmtion. Cytokine., 2005, 31: [33] CD Grdner; SP Fortmnn; RM Kruss. JAMA., 1996, 276: [34] BA Griffin; DJ Freemn; GW Tit; J Thomson; MJ Cslke; CJ Pckrd; J Shepherd. Role of plsm triglyceride in the regultion of plsm low density lipoprotein (LDL) subfrctions: reltive contribution of smll, dense LDL to coronry hert disese risk. Atherosclerosis., 1994, 106: [35] B Lmrche; A Tchernof; S Moorjni. Smll, dense low-density lipoprotein prticles s predictor of the risk of ischemic hert disese in men. Circultion., 1997, 95: [36] H Drexel; W Frnz; RK Amnn; C Neuenschwnder; A Luethy; S Khn; F Follth. Am. J. Crdiol., 1992, 70: [37] M Minhjuddin; J Iqbl; ZH Beg. Current Adv. Atheroscler. Res., 1999, 2: [38] AA Qureshi; BA Brdlow; L Brce; J Mngnello; DM Peterson; BC Perce; JJK Wright; A Gpor; CE Elson. Response of hypercholesterolemi subjects to dministrtion of tocotrienols. Lipids., 1995, 30: [39] AA Qureshi; N Qureshi; JJK Wright. Am J Clin Nut., 1991, 53: 1021S-6S. [40] RA Prker; BC Perce; RW Clrk; DA Godn; JJK Wright. J. Biol. Che., : [41] A Theriult; Q Wng; A Gpor; K Adeli. Arterioscler. Thromb. Vsc. Biol., 1999, 19: [42] C Gby; I Kushner. N. Engl. J. Med., 1999, 340: [43] JL Meht; TGP Sldeen; K Rnd. J. Am. Coll. Crdio., 1998, 31: [44] F Nielsen; BB Mikkelsen; JB Nielsen; HR Andersen; P Grndjen. Clin. Chem., 1997, 43: [45] HH Drper; M Hdley. Mlondildehyde determintion s index of lipid peroxidtion. Methods Enzymol., 1990, 186: [46] HR Andersen; JB Nielsen; F Nielsen; P Grndjen. J.Clin. Chem., 1997, 43: S [47] M Inoue; IM Aris; JL Boyer; N Fusto; WB Jkoby; DA Schchter; DA Shfritz. The liver: biology nd pthobiology. Rven Press: New York, USA., 1994, [48] MB Yerer; S Aydoğn; R Srymen. Turk J. Biochem., 2006, 31: