20 - World Journal of Gastroenterology

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Grattagliano I et al . Silybin and liver steatosisconjugated antibodies: goat anti-mouse IgG (1:5000) for1 h at room temperature. Membranes were incubatedwith ECF detection system (Amersham, GE Health-Care, Piscataway, NJ) and read with the Versa Doc imagingsystem (Bio-Rad, Barcelona, Spain).HistologyLiver specimens were fixed in 10% neutral bufferedformalin and paraffin embedded. Five sections of 4 μmthickness from each sample were cut and stained with toluidinblue-periodic acid-schiff stain. Histologic featureswere examined on five low-power fields per specimen,and a semi-quantitative estimation of the empty vacuoles(fat) was performed by measuring the fat percentage distributionon the surface areas.ChemicalsTotal OXPHOS Rodent Antibody Cocktail was purchasedfrom Mitosciences Inc. (Cambridge, MA). Secondaryalkaline phosphatase-conjugated antibodies werepurchased from Jackson ImmunoResearch Laboratories,Inc. (Cambridgeshire, United Kingdom). All other chemicalsused were of the highest purity available and werepurchased from Sigma-Aldrich Chemical Co. (Barcelona,Spain or Milan, Italy).Statistical analysisAll data are expressed as mean ± SD. The Mann-Whitneyrank sum test was used to compare groups. For multiplecomparisons, the ANOVA on ranks analysis of variancefollowed by Dunn’s method was used. The nonparametricSpearman rank order correlation was used to relatebiochemical parameters. P < 0.05 defined significance.RESULTSEffect of CD and HFD on general parameters and liverhistologyFeeding CD or HFD was associated with a progressiveand significant increase of liver weight but not of bodyweight at days 30 and 60, respectively, as compared withcontrol rats (Table 1). In rats with fatty liver, ALT levelsincreased progressively. At days 30 and 60, both CD andHFD rats showed a two-to-threefold increase.Rats on CD and HFD showed remarkable liver steatosis(Figure 1 and Table 1). There was a progressivehepatic fat infiltration ranging from large droplets at day14 to disseminated and confluent vacuoles resulting inmicrovesicular steatosis at day 30 (CD) and day 60 (HFD).Single cell necrosis was rarely noted. No evidence for inflammationand/or fibrosis was present.Effect of CD on oxidative and nitrosative stressparametersIn the liver, CD was associated with a higher content ofGSH, thioredoxin, and nitrosothiols, while GPx activitywas higher at day 7 and then lower at day 30 (Table 2).MDA-TBA levels were five-fold higher from 9.1 ± 1.2nmol/g to 75.6 ± 5.4 nmol/g at day 30 (Figure 2). SerumMDA-TBA and nitrosothiols levels as well as nitrotyrosineand K-18 (Table 2) were higher in CD rats.Effect of HFD on oxidative and nitrosative stressparametersIn the liver, HFD determined a progressive decrease inthe content of GSH, thioredoxin and nitrosothiols (Table3). GPx activity was initially unchanged, then increasedand next decreased (Table 3); MDA-TBA were doubledat day 60 (Figure 2). Serum levels of MDA-TBA, K-18,nitrotyrosine and nitrosothiols were significantly higherin HFD rats (Figure 3).Effect of HFD on mitochondrial oxidativephosphorylation complexesSamples from liver, heart and skeletal muscle were comparedregarding the content in subunits of the mitochondrialrespiratory chain. Western blotting yielded bandsto all OXPHOS subunits studied, although the bandcorresponding to complex Ⅲ was very difficult to detectin the three tissues and hence was not quantified by densitometry.Band density for each protein was normalizedfor the corresponding β-actin band.In the liver (Figure 4), the HFD caused an increase inthe amount of the Complex Ⅰ subunit NDUFB8 (at day60) and Complex Ⅳ subunit Ⅰ, COX Ⅰ (at day 30) anda decrease of Complex Ⅱ subunit 30kDa, CII-30 (days14 and 60) and ATP synthase subunit α, CV-α (day 14).Minor changes in the heart and skeletal muscle proteinswere observed (data not shown).Effect of Realsil on general parameters, liver histology,stress markers, and mitochondrial proteinsRealsil administration was associated with a lower increaseof liver weight and a less pronounced increase ofserum transaminase levels in rats (Table 1). As shown(Figure 1 and Table 2), Realsil resulted in a lower extentof fat infiltration in both CD and HFD rats. In CD rats,Realsil determined an improvement in the number anddiameter of fat droplets. In HFD rats, Realsil was associatedwith a lower diameter of fat droplets; the number ofdroplets per histological section was lower at day 30 buthigher at day 60, as a likely consequence of a less confluentphenomenon.Realsil was protective against CD and HFD inducedoxidative and nitrosative changes both in the liver and inplasma. The improvement in such parameters was moreevident for CD rats. In particular, the decrease of liverGSH and nitrosothiols was kept to 50% compared withuntreated rats, while the changes in MDA-TBA, thioredoxin,and GPx were less marked although significant(Table 2). The same parameters were less affected byRealsil in HFD rats (Table 3 and Figure 3). In fact, thehepatic concentrations of GSH, MDA-TBA, GPx, andnitrosothiols did not differ significantly compared tountreated HFD rats. Only thioredoxin levels were maintainedto a higher level by Realsil administration. In theseWJG|www.wjgnet.com3010 May 28, 2013|Volume 19|Issue 20|
Grattagliano I et al . Silybin and liver steatosisACD - day 30 CD + Realsil - day 30BHFD - day 30 HFD + Realsil - day 30CHFD - day 60 HFD + Realsil - day 60Figure 1 Light micrographs of rat liver stained with toluidin blue-periodic acid-schiff stain. A: Liver from rat fed a choline deficient diet (day 30); B: Liver fromrat fed a high fat diet (day 30); C: Liver from rat fed a high fat diet (day 60). Right column without and left column with administration of Realsil (Magnification: 200 ×).CD: Choline deficiency; HFD: High fat diet.rats, Realsil halved the variations of serum MDA-TBAand nitrotyrosine levels, while it was less effective on K-18(222 ± 15 IU/L vs 242 ± 23 IU/L at day 60 in treatedand untreated HFD rats, respectively) and nitrosothiols(72 ± 10 nmol/L vs 81 ± 9 nmol/L at day 60 in treatedand untreated HFD rats, respectively) levels.In HFD animals receiving also Realsil, several changesoccurred in liver mitochondria (Figure 4). At day 14, administrationof Realsil resulted in a significant increasein the protein levels of NDUFB8 subunit, which wereslightly decreased (although not statistically significant)by the diet alone. On the other hand, Realsil in HFDanimals further decreased the amount of CII-30 (day14), NDUFB8 (day 30), and COX Ⅰ (day 60). At day 60,Realsil had protective effect on the Complex Ⅱ subunitCII-30 by increasing the amount of the protein that wasdecreased by the diet alone. Interestingly, in the muscle,at day 60 Realsil reversed the effect induced by HFD inCII-30 subunit and in COX Ⅰ subunit and, at day 30, decreasedCV-α subunit protein amount.DISCUSSIONObesity is a health problem in developed countries [18-21]and is related to insulin resistance, dyslipidemia, type 2diabetes, hypertension, and liver steatosis (NAFLD) [22] .Simple steatosis is considered a benign condition withlow risk of evolution, while its inflammatory form(NASH) is considered a risk factor for liver cirrhosis.Mechanisms governing hepatocellular fat depositioninvolve metabolic pathways partly depending onthe up-regulation of peroxisome proliferator-activatedreceptor and on the consequent activation of adipocytedifferentiation programs [23] . The relationship betweenWJG|www.wjgnet.com3011 May 28, 2013|Volume 19|Issue 20|
Page 1 and 2: ISSN 1007-9327 (print)ISSN 2219-284
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