Role of Grape Seed Proanthocyanidins in the Suppression of High Calorie Diet-Induced Hepatic Injury and Apoptosis

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1 Interntionl Journl of Science nd Reserch (IJSR), Indi Online ISSN: Role of Grpe Seed Pronthocynidins in the Suppression of High Clorie Diet-Induced Heptic Injury nd Apoptosis Bskrn Yoglkshmi 1, Crni Venktrmn Anurdh 2 1, 2 Deprtment of Biochemistry nd Biotechnology, Annmli Univerity, Annmli Ngr , Chidmbrm, Indi Abstrct: Heptocyte oxidtive stress nd poptosis re considered to be the key components in the pthogenesis of nonlcoholic stetoheptitis. In this study we investigted the effect of grpe seed pronthocynidins (GSP) nd metformin (MET), lone nd in combintion, on high fructose/ft diet (HFFD) induced stetoheptitis, with focus on oxidtive stress nd poptosis mrkers. NASH ws induced in mle lbino Wistr rts by feeding HFFD for 45 dys. Either GSP (100 mg/kg b. w), MET (50 mg/kg b. w) or both were dministered s therpeutic options. HFFD-feeding cused increse in lipid peroxides, protein crbonyls, propoptotic proteins, (bx, cspses 3 nd 9) nd decline in nti-poptotic protein (bcl2). Further, growth nd DNA dmge (GADD) 45β, n NF-κB regulted nti-poptotic fctor, ws lso reduced in HFFD-fed rts. These chnges were reversed more effectively by GSP dministrtion thn by MET. Combined dministrtion of GSP nd MET showed synergistic effect in improving cell survivl nd hence could be considered for controlling HFFD-induced heptocyte poptosis. Keywords: Grpe seed pronthocynidins, heptic injury, oxidtive stress, poptosis. 1. Introduction The key events tht contribute to the initition nd progression of NAFLD re summrized in multi-hit model of NASH pthogenesis by Polyzos nd others [1]. According to this model, dysregulted metbolism of free ftty cids (FFAs) is considered to be the first-hit of NAFLD pthogenesis, which leds to insulin resistnce nd ft ccumultion in the liver. Inflmmtory response, oxidtive stress, nd poptosis, serve s following-hits tht contribute to the ongoing inflmmtion tht leds to non-lcoholic stetoheptitis (NASH). Emerging dt suggest tht oxidtive stress nd poptosis plys criticl role in NAFLD-induced liver injury nd in the progression from stetosis to NASH nd then to cirrhosis [2, 3]. Moreover, the degree of poptosis is closely ssocited with the severity of NASH nd the stge of fibrosis [4]. Thus, inhibition of oxidtive stress nd poptosis in the liver my be useful tretment strtegy of NAFLD. Apoptosis is morphologiclly distinct, gene-directed form of cell deth chrcterized by cytoplsmic frgmenttion nd nucler condenstion tht contributes to both physiologicl nd pthologicl processes [5]. Emerging evidence suggest tht high rte of poptotic response is ssocited with oxidtive stress occurring in NASH [6]. The process of poptosis is regulted by severl proteins with either inhibiting or promoting ctions. Bcl2 fmily of proteins tht reside in mitochondri re the key plyers in poptosis. Bx protein, member of the bcl-2 fmily cts s propoptotic fctor nd promotes poptosis wheres bcl2 protein inhibits poptosis. Bcl-2 is potent, evolutionrily conserved, ntipoptotic protein which inhibits cell deth by reducing the genertion of rective oxidnts, which re requisite for the completion of the poptotic progrm [7]. The rtio of bx to bcl-2 determines the susceptibility of cell to poptosis [8]. Bcl2 is trnscriptionlly regulted by nucler fctor-kpp B (NF-κB). The nti-poptotic/ the protective effect of NF-kB hs been proposed to result from the inhibition of JNK signling by n NF-κB responsive gene product, growth nd DNA dmge (GADD)45β [9]. Severl protese fmilies re implicted in poptosis, the most prominent being cspses [10]. Opening of mitochondrion permebility trnsition pores due to oxidtive stressresults in ctivtion of procspse 9 to cspse 9 which then ctivtes its key downstrem molecule cspse 3 for executing poptosis. The propototic fctor bxis responsible for the conversion of procspse 9 to cspse 9 [11]. Recent evidence suggests tht the ntipoptotic fctor bcl-2 is downstrem deth substrte for cspses nd is inctivted by cspses [12]. Incresed poptosis hs been suggested in niml models of NASH [6] nd NASH ptients [13]. However, role of bx nd bcl-2 s custive fctor in diet induced NAFLD hve not been studied. Mngement of poptosis is considered s n effective wy to tret NAFLD. Grpe seed pronthocynidins (GSP) is known well for its ntioxidnt potentil nd hs been investigted to combt vrious disese conditions including insulin resistnce. In this study, we hve investigted the ltertions of bx/bcl-2 rtio in reltion to chnges in the poptosis co-ordintion enzymes, cspses-3 nd 9, in the liver of rts with NAFLD. Also, we investigted the effect of GSP, potent ntioxidnt nd metformin (MET), potent insulin sensitizer, lone nd in combintion on oxidtive stress nd poptosis mrkers on HFFD-induced NAFLD rts. 406

2 Interntionl Journl of Science nd Reserch (IJSR), Indi Online ISSN: Methodology 2. 1 Animls Diet nd Experimentl design Albino mle Wistr rts of body weight g were obtined from nd mintined t the Centrl Animl House, Deprtment of Experimentl Medicine, Rjh Muthih Medicl College nd Hospitl, Annmli Ngr. The nimls were housed under controlled temperture (22±2 ºC) without limittion of ccess to wter nd chow. The study ws conducted in ccordnce with the guidelines of the Committee for the Purpose of Control nd Supervision on Experiments on Animls (CPCSEA). All procedures were pproved by the Institutionl Animl Ethicl Committee (IAEC). The nimls were fed with either norml rt chow (control) or high ft-fructose diet (HFFD). Norml chow consisted of 60% strch, % protein nd 4. 38% ft, the cloric content of which ws cl/100 g. HFFD ws prepred fresh in our lbortory every week which provided cl/100 g. HFFD contined 45% fructose, 20% ft (10% beef tllow, 10% groundnut oil) nd 22. 5% csein. A totl of 6 groups were treted s follows: Group 1 were fed with norml rt chow (CON), group 2 were fed with HFFD, group 3 were fed with HFFD nd supplemented with GSP by gstric intubtion (100 mg/kg b. w/dy, HFFD+GSP), group 4 rts were fed with HFFD nd dministered with MET through gstric intubtion (50 mg/kg b. w/dy, HFFD+MET), group 5 rts were fed with HFFD nd dministered GSP first followed by MET t n intervl of 3-4 hrs once dily (HFFD+GSP+MET) through gstric intubtion, nd group 6 rts were fed with norml rt chow nd dministered GSP (CON+GSP). Food nd wter were provided d libitum to the nimls. The totl experimentl durtion ws 45 dys. GSP nd MET were given for the lst 15 dys of the experimentl period. At the end of experimentl period, the nimls in ech group (n=6) were fsted overnight nd scrificed by cervicl decpittion to obtin blood nd liver. Smples were stored t -80ºC until further nlysis Liver injury mrkers nd oxidtive stress To ssess the liver injury, ctivities of sprtte trnsminse (AST), lnine trnsminse (ALT), lkline phosphtse (ALP), gmm glutmyltrnsferse (GGT) nd bilirubin levels were ssyed using kits obtined from Agppe dignostics, Kerl, Indi. Oxidtive stress mrkers like thiobrbituric cid rective substnce (TBARS), lipid hydroperoxides (LHP) nd protein crbonyl (PCO) were mesured in plsm ccording to respective stndrd procedures [14, 15, 16] 2. 3 Immunoblotting Homogentes contining equl mounts of protein were resolved by 8-12% SDS PAGE nd processed for Western blot nd electrotrnsferred onto polyvinylidene fluoride membrnes. The membrnes were then incubted overnight t 4 C with ntibodies specific to bx, bcl2, cspse 3 nd 9. The membrnes were wshed with TBST nd incubted with respective secondry ntibody for 2 h t room temperture. Protein bnd detection ws performed by enhnced chemiluminescence ssy (Thermo Scientific Super Signl West Pico chemiluminescent substrte, Rockford, USA) s per the mnufcturer s instructions. Blots were subsequently stripped, reprobed nd processed for visulizing β-ctin. The bnd density ws normlized with tht of β-ctin. Quntittive comprisons of protein expression between vrious groups were performed using Imge J softwre (from US Ntionl Institutes of Helth) qpcr Totl cellulr RNA ws extrcted from liver using TriZol regent. The concentrtion nd purity of RNA preprtion were checked by mesuring the bsorbnces t 260 nd 280 nm. Totl RNA (2. 0 μg) ws reverse trnscribed to cdna in rection mixture contining 1 μl of Oligo (dt) primer (0. 2 μg/ml), 1 μl of RNse inhibitor (10 U/ml), 1 μl of 0. 1 M DTT, 4 μl of RT Buffer (5X), 2. 0 μl of 30 mm dntp mix (7. 5 mm ech), 0. 5 μl of M-MuLV Reverse Trnscriptse (50 U/μl) nd mde up to 20 μl with sterile wter nd kept t 37 C for 1 h nd then heted t 95 C for 2 min. PCR mplifiction ws performed in mixture contining 100 μgcdna, 1 μl ech of 50 pm GADD45β forwrd (3 - gctggcctgcggg-5 ) nd reverse (3 - ggttcggtggggcctct-5 ) primer (Sigm Aldrich, St Louis, MO, USA), 10 μl SYBR green mster mix with finl volume of 20 μl mde up using nuclese free wter. The thermocycling conditions were s follows, initil denturtion 95 C for 10 min, denturtion 95 C for 15 s, nneling 60 ± 3 C for 30 s nd extension 72 C for 30 s for 40 cycles. The rections were run in triplicte for ech smple. The Ct vlues obtined for ech gene ws normlized with tht of GAPDH gene using the formul 2- Ct. The reltive quntity ws expressed in br grphs s fold chnge with respect to control fter normliztion with GAPDH for ech gene Histology Histologic nlysis of liver were performed fter liver tissue smples were fixed t room temperture in formlin nd embedded in prffin. The liver ws sectioned using microtome (3-5 μm) nd mounted on glss slides, Tble 1: Activities of liver function enzymes in experimentl nimls Prmeters CON HFFD HFFD+GSP AST (IU/L) ALT (IU/L) ALP (IU/L) GGT (IU/L) Bilirubin (mg/dl) 44. 6± ± ±2. 05 HFFD+ HFFD+GSP CON+GSP MET +MET ±8. 96 b 64. 8±3. 95 c 78. 6±4. 30 d 53. 9±3. 27 e 45. 3± ±5. 00 b 71. 6±4. 43 c 86. 5± ± ±2. 88 d e ±10. 5 b 75. 1±4. 30 c 92. 3± ± ±2. 21 d e 21. 8± ±2. 64 b 32. 6±1. 39 c 36. 1±1. 72 d 29. 6±2. 09 c 21. 6± ± ±0. 12 b 1. 18±0. 06 c 2. 01± ± ±0. 04 d e CON- Control, HFFD- High ft, fructose diet, HFFD+GSP- High ft, fructose diet + grpe seed pronthocynidins, HFFD+MET - High ft, fructose diet + metformin, HFFD+GSP+MET - High ft, fructose diet + grpe seed pronthocynidins + metformin, CON+GSP Control + 407

3 Interntionl Journl of Science nd Reserch (IJSR), Indi Online ISSN: grpe seed pronthocynidins. Vlues re mens ±SD of 6 rts from ech group. Vlues tht ber different lphbets in their superscript differ significntly from ech other. One wy ANOVA followed by TMRT. A vlue with p<0. 05 is considered sttisticlly significnt. Tble 2: Levels of oxidtive stress mrkers in plsm of experimentl nimls HFFD+GSP CON+ Prmeters CON HFFD TBARS(A) LHP(B) PCO(C) HFFD+ GSP 1. 68±0. 19 b HFFD+ MET 2. 79± c d + MET GSP 0. 96± ± ± ±0. 03 e ± ± ± b 05 c 2. 12±0. 11 d 1. 05±0. 11 e 0. 91± ± ± ± b 18 c 6. 81±0. 58 d 4. 26±0. 39 e 2. 21±0. 12 Comprisons nd significnce s given in Tble 1. A= µmoles/dl; B= nmoles/dl; C=nmol/dL. Figure 2: mrna expression of GADD45β, (A) qpcr qulifiction grph. (B) Fold chnge with respect to control clculted using. Dt re expressed s mens + S. D. of 4 rts from ech group. Sttisticl significnce between the groups, denoted by different lphbets, ws determined by one-wy ANOVA of significnce set t P< Sttisticl nlysis Vlues re presented s mens ± SD of 6 rts for biochemicl nlysis nd 4 rts for histology, qpcr nd immune blotting nlysis. Dt nlysis ws performed with the use of SPSS sttisticl softwre The sttisticl significnce of differences between groups ws determined by one-wy nlysis of vrince (ANOVA). ANOVA followed by the Turkey s multiple comprison tests (TMRT). Vlue with p<0. 05 ws considered sttisticlly significnt. 3. Results 3. 1 Liver injury mrkers Figure 1: Histology Using Reticulin The ctivities of AST, ALT, ALP nd GGT nd the level of bilirubin were found to be significntly incresed in the HFFD rts when compred with tht of CON nd tretment groups. All the three tretment groups showed significnt reduction of injury mrkers of which the combintion tretment showed the mximum reduction compred to tht of HFFD group of rts. The vlues did not differ significntly between CON nd CON+GSP groups (Tble 1) Oxidtive stress mrkers The levels of TBARS, LHP nd PCO in plsm were found to be significntly incresed in HFFD fed rts, compred to CON rts. GSP nd MET significntly reduced these oxidtive stress mrkers compred with HFFD. Combined dministrtion of GSP nd MET showed better reduction in oxidtive stress thn individul tretments (Tble 2) Histologicl nlysis of fibrosis (A) Figure 1 depicts the histologicl sections of liver upon reticulin stining for observing fibrotic chnges. Fig. 1 (A) nd (F) represent sections from CON nd CON+GSP respectively showing norml network of reticulin between heptocytes. HFFD fed rts show severe chnges with lrgely ccumulted reticulr fibers long the cells (B). GSP dministrtion to HFFD fed rts reduced these fibers effectively s compred to HFFD fed rts (C). (D) represents liver sections from HFFD+MET group of rts showing moderte deposition of reticulin. HFFD+GSP+MET tretment hve reduced the deposition of fibrils to ner norml rnge (E) mrna expression of GADD45β (B) mrna expression nlysis of the gene GADD45β showed decrese in HFFD fed rts compred to CON rts. HFFD fed rts showed decrese by fold compred to CON. All the three tretments showed improved expression of GADD45β 408

4 Interntionl Journl of Science nd Reserch (IJSR), Indi Online ISSN: (0.74 fold by GSP, 0.51 fold by MET nd 0.89 fold by GSP+MET dministrtion) (Figure 2) Protein expression of bx nd bcl2 Bx, the propoptotic mrker of bcl2 fmily ws found to be incresed by fold in HFFD fed rts compred to CON rts. GSP, MET nd combined tretment reduced the expression s compred to HFFD but the levels were higher by 2. 07, nd fold respectively compred to CON. Menwhile, the nti-poptotic mrker bcl2 ws decresed by 0. 5 fold upon HFFD feeding considering CON s 1. All the three tretment groups showed incresed expression s compred to HFFD. HFFD+GSP, HFFD+MET nd HFFD+GSP+MET group of rts showed n ner norml expression (0. 76, nd folds respectively) compred to CON group of rts (Figure 3B) Bx/Bcl2 rtio Bx/Bcl2 rtio ws clculted to find out the susceptibility of the cells to poptosis. HFFD fed rts showed mximum susceptibility to poptosis with n increse of fold expression considering control s 1. GSP, MET nd combintion reduced this susceptibility nd showed n expression level of 2. 76, nd folds respectively (Figure 4) bilirubin were higher in plsm of HFFD-fed rts compred to control diet fed rts. Administrtion of GSP nd MET, Either lone or in combintion reduced the levels of these mrkers in plsm suggesting tht cellulr injury hs been repired. Figure 3: Protein expression of Bx nd Bc12. Lne 1-CON, Lne 2- HFFD, Lne 3- HFFD+GSP, Lne 4- HFFD+MET, Lne 5- HFFD+GSP+MET, Lne 6- CON+ GSP. Dt re expressed s mens + S. D. of 4 rts from ech group. Sttisticl significnce between the groups, denoted by different lphbets, ws determined by one-wy ANOVA of significnce set s P< Protein expression of cspse 3 nd 9 HFFD fed rts showed significnt increse in the expression of cspse 3 (Figure 5A) nd 9 (Figure 5B) to nd fold respectively s compred with CON. Supplementtion of GSP, MET nd combintion tretment reduced the expression of cspse 3 to 1. 51, nd fold respectively nd cspse 9 by 1. 77, nd fold respectively compred to CON. 4. Discussion Our study reports for the first time tht heptocyte poptosis is induced in rts by HFFD feeding in rts. HFFD consumption resulted in incresed propoptotic Bx, decresed nti-poptotic Bcl2, which further incresed the expression of cspse 3 nd 9. GADD45β, n NF-kB regulted nti-poptotic fctor ws found to be reduced indicting tht NF-κB hs been switched to propoptosis mode. Upon GSP dministrtion, expression of Bx ws decresed with simultneous improvement in Bcl2 expression. MET ws less efficient thn GSP in shifting this blnce but showed significnt improvement towrds ntipoptosis. Combined dministrtion of GSP with MET shown synergistic effects in improving the cell survivl rte s evidenced by the decresed Bx/Bcl2 rtio. Consumption of westernized diets high in fructose nd ft leds to severe oxidtive injury tht culmintes in poptosis. Mny studies in niml models hve proved tht feeding high fructose nd/or ft diet for over month results in heptic stetosis which further progresses to stetoheptitis with the involvement of cellulr injury nd poptosis [6, 17]. Liver injury on mrker enzymes (AST, ALT, ALP nd GGT) nd Figure 4: Effect of GSP nd MET on Bx/Bc12 rtio. Dt re expressed s mens + S. D. of 4 rts from ech group. Sttisticl significnce between the groups, denoted by different lphbets, ws determined by one-wy ANOVA of significnce set t P<0. 05 Figure 5: Effects of GSP nd MET on protein expression of cspse 3 nd 9 in liver of experimentl nimls. Lne 1- CON, Lne 2- HFFD, Lne 3- HFFD+GSP, Lne 4- HFFD+MET, Lne 5- HFFD+GSP+MET, Lne 6- CON+ GSP. Dt re expressed s mens + S. D. of 4 rts from ech 409

5 Interntionl Journl of Science nd Reserch (IJSR), Indi Online ISSN: group. Sttisticl significnce between the groups, denoted by different lphbets, ws determined by one-wy ANOVA of significnce set t P<0. 05 Oxidtive insults emnting from within the cell cn threten homeostsis if they re not ppropritely resolved nd hence prolonged oxidtive injury is usully followed by poptosis. Excessive levels of ROS cn led to cellulr injury nd deth by ltering cellulr mcromolecules including DNA, proteins nd lipids. In the present study, HFFD feeding resulted in lipid nd protein dmge s evidenced by incresed peroxidtive products like TBARS, LHP nd PCO. Results obtined with respect to tretment studies show tht ll the three tretments re effective in removing the oxidtive insult nd to protect the cells from further injury nd poptosis. In the current study, there is strong increse in bx/bcl-2 rtio tht fvors poptosis with dvncing NAFLD. The functionlly linked enzyme to poptosis, cspse-3, ws lso incresed upon HFFD feeding. These results showed tht in NAFLD induced by HFFD feeding, cspse-3 my ctivte cell deth vi the mitochondril signls, i. e., bx ctivtion nd bcl2 suppression. GSP nd MET inhibit the ctivtion of bx nd lso improve expression of bcl2 thus decresing the bx/bcl2 rtio. This ws ccompnied by decresed cspse 3 nd 9 ctivtion nd thus GSP nd MET ct by suppressing the mitochondril triggered poptosis. NF-κB/Rel trnscription fctors re lso involved in controlling poptosis. Inctive form of NF-κB resides in the cytosol of resting cells trnsloctes to nucleus upon ctivtion by vrious fctors like oxidtive stress nd induce the trnscription of vrious proteins involved in both pro nd nti-poptosis [18]. The nti-poptotic ctivity of NFκBinvolves suppression of c-jun N-terminl kinse (JNK). GADD45β, one of the direct trgets of NF-κB, binds directly to JNK-inducing kinse MKK-7 nd blocks the ctlytic ctivity of MKK-7 which is necessry for ctivtion of JNK [19]. HFFD feeding cused decrese in the mrna expression of GADD45β indicting tht nti-poptotic effect of NF-κB is suppressed by HFFD feeding. Interestingly, GSP significntly improved the expression of GADD45β. Combined dministrtion of both hs showed synergistic effects. These results suggested tht GSP reduces JNK medited killing. Mtrix ccumultion or fibrosis of the liver occurs in response to ctivted stellte cells tht produces mtrix molecules in response to oxidtive stress nd inflmmtion [20]. Incresed mtrix ccumultion leds to cirrhosis, condition tht reflects unsuccessful repir of NASH. In our study, HFFD feeding to rts for 45 dys resulted in incresed fibrous network long the lining of heptocytes which ws significntly reduced in ll three tretment groups. In conclusion, the key findings of our study re tht HFFD induces liver cell injury nd poptosis tht cn be llevited by GSP nd MET. Decrese in the expression of bx/bcl2 rtio nd the suppression of cspses 3 nd 9 could be the mechnisms involved. GSP lso improved GADD45β, NFκB regulted nti-poptotic protein fctor showing tht GSP cts vi NF-κB signls to suppress poptosis. On the other hnd, MET reduces the bx/bcl2 rtio significntly but not s effectively s GSP. The mild suppression of GADD45β induced by MET ws lso not significnt. Hence it is evident tht MET induces cell survivl by inducing bcl2 nd not by GADD45β. Combintion of GSP nd MET showed mximum improvement in cell survivl by reducing bx/bcl2 rtio nd by improving GADD45β. Combined dministrtion of GSP nd MET holds better promise for developing ntipoptotic gents ginst NASH, but the underlying mechnisms re yet to be determined. 5. Acknowledgement We sincerely thnk nd cknowledge the Indin Council of Medicl Reserch (ICMR), New Delhi, for providing finncil ssistnce in the form of Senior Reserch Fellowship (SRF) to the first uthor Yoglkshmi. B. References [1] S. A. Polyzos, J. Kountours, C. Zvos, "Nonlcoholic ftty liver disese: the pthogenetic roles of insulin resistnce nd dipocytokines", Curr. Mol. Med., vol. 9, , [2] M. Mrzioni, S. S. Glser, G. Alpini, nd G. D. LeSge, Role of poptosis in development of primry biliry cirrhosis, Digestive nd Liver Disese, vol. 33, pp , [3] N. Alkhouri, C. Crter-Kent, nd A. E. Feldstein, Apoptosis in nonlcoholic ftty liver disese: dignostic nd therpeutic implictions, Expert Review of Gstroenterology nd Heptology, vol. 5, pp , [4] E. Feldstein, A. Cnby, P. Angulo et l., Heptocyte poptosis nd Fs expression re prominent fetures of humn nonlcoholic stetoheptitis, Gstroenterology, vol. 125, pp , [5] M. C. Cummings, C. M. Winterford nd N. I. Wlker, Apoptosis, Am. J. Surg. 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6 Interntionl Journl of Science nd Reserch (IJSR), Indi Online ISSN: determinnt in p53-induced poptosis in neurons, J. Neurosci., vol. 19, pp , [12] E. H. Cheng, D. G. Kirsch, R. J. Clem, R. Rvi,M. B. Kstn,A. Bedi,K. Ueno nd J. M. Hrdwick, Conversion of bcl-2 to bx like deth effector by cspses, Science,vol. 278, pp , [13] P. S. Ribeiro,H. Cortez-Pinto,S. Sol, R. E. Cstro,R. M. Rmlho,A. Bptist,M. C. Mour, M. E. Cmilo nd C. M. Rodrigues, Heptocyte poptosis, expression of deth receptors, nd ctivtion of NF-kppB in the liver of nonlcoholic nd lcoholic stetoheptitis ptients, Am. J. Gstroenterol., vol. 99, pp , [14] Z. Y. Jing,J. V. Hunt, nd S. P. Wolf, Detection of lipid hydroperoxides using Fox method, Anl. Biochem.,vol. 202, pp , [15] R. L. Levine, D. Grlnd, C. N. Oliver, A. Amici,I. Climent, A. G. Lenz,B. W. Ahn, S. Shltiel, E. R. Stdtmn, Determintion of crbonyl content in oxidtively modified proteins, Methods Enzymol.,vol. 186, pp , [16] W. G. Niehus ndb. Smuelsson, Formtion of mlonldehyde from phospholipid rchidonte during microsoml lipid peroxidtion, Eur. J. Biochem.,vol. 6, pp , [17] Q. M. Anstee ndr. D. Goldin, Mouse models in nonlcoholic ftty liver disese nd stetoheptitis reserch Int. J. Exp. Pthol., vol. 87, pp. 1-16, [18] A. S. Bldwin, The NF-kpp B nd I kpp B proteins: new discoveries nd insights,annu. Rev. Immunol., vol. 14, pp , [19] S. Pp, F. Zzzeroni, C. Bubici, S. Jywrden, K. Alvrez, S. Mtsud, D. U. Nguyen, C. G. Phm, A. H. Nelsbch, T. Melis, E. D. Smele, W. Tng, L. D. Admio nd G. Frnzoso, Gdd45β medites the NFκB suppression of JNK signling by trgeting MKK7/JNKK2, Nture cell biology, vol. 6, pp , 2004 [20] R. Btller nd D. A. Brenner, Liver fibrosis, J. Clin. Invest. vol. 115, pp , Author Profile Dr. C. V. Anurdh completed her M. Phil nd Ph. D in Biochemistry t PG Institute of Bsic Medicl Sciences, University of Mdrs, Trmni Cmpus, Tmil Ndu, Indi. At present she is Professor in the Deprtment of Biochemistry nd Biotechnology, Annmli University. She hs over 26 yers of reserch experience nd 120 reserch publictions in peer reviewed journls. Her re of reserch include the pthogenesis nd tretment of insulin resistnce nd type 2 dibetes focusing on the role of insulin sensitizers from nturl sources nd their potentil to combt metbolic bnormlities like obesity, type 2 dibetes nd nonlcoholic stetoheptitis. B. Yoglkshmi completed her M. Phil nd is pursuing Ph. D under the guidnce of Dr. C. V. Anurdh. Her reserch re includes type 2 dibetes nd ftty liver. 411