http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–11 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2015.1060247

ORIGINAL ARTICLE

Protective effect of ursodeoxycholic acid, resveratrol, and N-acetylcysteine on nonalcoholic fatty liver disease in rats Mahmoud Hussein Hassan Ali1, Basim Anwar Shehata Messiha1, and Hekma Abdel-Tawab Abdel-Latif2 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Beni-Sueif University, Beni-Sueif, Egypt and 2Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo, Egypt

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Abstract

Keywords

Context: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Resveratrol (RSV) and N-acetylcysteine (NAC) are safe representatives of natural and synthetic antioxidants, respectively. Objective: The objective of this study was to evaluate protective effects of RSV and NAC, compared with ursodeoxycholic acid (UDCA), on experimental NAFLD. Materials and methods: NAFLD was induced by feeding rats a methionine choline-deficient diet (MCDD) for four cycles, each of 4 d of MCDD feeding and 3 d of fasting. Animals were divided into normal control, steatosis control, and five treatment groups, receiving UDCA (25 mg/kg/d), RSV (10 mg/kg/d), NAC (20 mg/kg/d), UDCA + RSV, and UDCA + NAC orally for 28 d. Liver integrity markers (liver index and serum transaminases), serum tumor necrosis factor-a (TNF-a), glucose, albumin, renal functions (urea, creatinine), lipid profile (total cholesterol; TC, triglycerides, high density lipoproteins, low density lipoproteins; LDL-C, very low density lipoproteins, leptin), and oxidative stress markers (hepatic malondialdehyde; MDA, glutathione; GSH, glutathione-S-transferase; GST) were measured using automatic analyzer, colorimetric kits, and ELISA kits, supported by a liver histopathological study. Results: RSV and NAC administration significantly improved liver index (RSV only), alanine transaminase (52, 52%), TNF-a (70, 70%), glucose (69, 80%), albumin (122, 114%), MDA (55, 63%), GSH (160, 152%), GST (84, 84%), TC (86, 86%), LDL-C (83, 81%), and leptin (59, 70%) levels compared with steatosis control values. A combination of RSV or NAC with UDCA seems to ameliorate their effects. Discussion and conclusion: RSV and NAC are effective on NAFLD through antioxidant, antiinflammatory, and lipid-lowering potentials, where as RSV seems better than UDCA or NAC.

Dyslipidemia, inflammation, methionine– choline-deficient diet, oxidative stress, steatohepatitis, steatosis

Introduction Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease, affecting up to 20% of the population in Western countries, and 70–80% of obese individuals (Chitturi et al., 2007; Radcke et al., 2015). NAFLD is now recognized as the most common cause of cryptogenic cirrhosis implicated in 21–63% of patients in one study (Czaja, 2011; Singh et al., 2013). Skelly et al. (2001) reported that 34% of British patients suffering from idiopathic abnormal liver function tests were later diagnosed as nonalcoholic steatohepatitis (NASH) persons by liver biopsy. In the United States, most patients with unexplained aminotransferase elevation are considered to have NAFLD (Clark et al., 2003; Vital et al., 2013). There are two major risk factors for NAFLD, namely obesity and type II diabetes, with chances of NAFLD increased due to sedentary lifestyle and improper calorie consumption in the daily dietary intake.

Correspondence: Basim Anwar Shehata Messiha, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Beni-Sueif University, Beni-Sueif, Egypt. E-mail: [email protected]

History Received 16 February 2015 Revised 23 April 2015 Accepted 5 June 2015 Published online 1 July 2015

A fact that 90% of patients with NAFLD have at least one characteristic feature of metabolic syndrome and about 33% have the diagnosis of metabolic syndrome determines NAFLD as a hepatic manifestation of metabolic syndrome (Marchesini et al., 2003). NAFLD encompasses a spectrum of distinct histological entities with different natural histories and outcomes, ranging from simple fat accumulation in hepatocytes to liver steatosis accompanied with necroinflammatory component that may have associated fibrosis. Simple steatosis is defined as a benign form of NAFLD with minimal risk of progression, in contrast to NASH which progresses to cirrhosis in up to 20% of patients, subsequently leading to liver failure or hepatocellular carcinoma (Dongiovanni et al., 2014). The rationale for investigating potential NAFLD treatment modalities comes from understanding the proposed mechanisms in the pathogenesis of the disease. As the currently accepted theory suggests, the first step in disease genesis is liver fat accumulation induced by changes in lipid metabolism favoring excessive triglyceride accumulation in hepatocytes, as a result of insulin resistance. The second step is believed to be increased oxidative stress within the hepatocytes, which is characterized by excessive

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production of reactive oxygen species (ROS) by mitochondria and the cytochrome P-450 (CYP450) system of the liver. Subsequently, ROS, through lipid peroxidation, pro-inflammatory cytokine induction and Fas ligand induction, promotes progression from steatosis to steatohepatitis, fibrosis, and finally cirrhosis (Chan et al., 2014; Chitturi & Farrell, 2001; Day & James, 1998; Tsochatzis et al., 2009). Therefore, medical regimens have targeted insulin resistance, metabolic syndrome (primarily obesity and dyslipidemia), and oxidative stress. Ursodeoxycholic acid (UDCA), a hydrophilic bile acid, is used in this experiment as a reference treatment for NAFLD, and is known to act by competitive displacement of hepatotoxic hydrophobic endogenous bile acids that facilitate apoptosis, minimizing their toxicity and leading to a decrease in oxidative stress and hepatic injury (Mahmoud & Elshazly, 2014). Resveratrol (RSV), a phytoalexin polyphenolic compound, is used and found in the skin of red grapes [Vitis vinifera (Vitaceae)] and other fruits, as well as in the roots of Japanese knotweed [Polygonum cuspidatum (Polygonaceae)]. It acts as a strong antioxidant, reported to inhibit low-density lipoprotein oxidation and to ameliorate oxidative stress induced in vivo and in vitro (Al Maruf & O’Brien, 2014; Pangeni et al., 2014; Suwalsky et al., 2015). RSV acts also by lowering hepatic fat content, reducing cholesterol levels and inhibiting platelet aggregation (Prasad, 2012). RSV was particularly selected in the current study as a representative of naturally occurring antioxidants. It was recently reported to be safely used in many pathological disorders (Aires & Delmas, 2015). Finally N-acetylcysteine (NAC), a precursor of glutathione (GSH) which is a major endogenous antioxidant, has been in clinical use for the management of acetaminophen poisoning (Buckley et al., 1999). The protective effects of NAC are evident in chronic diseases where decreased GSH or oxidative stress such as NASH occur (Ronis et al., 2005). NAC, as a source of sulfhydryl groups, stimulates GSH synthesis, enhances GSH-S-transferase (GST) activity and acts as a scavenger of free radicals, thus getting rid of ROS (Wang et al., 2014). It was selected in the current study as a representative of synthetic antioxidants, as it was welldocumented as a safe beneficial agent in a variety of pathologies (Rushworth & Megson, 2014). Based on the aforementioned background, UDCA, RSV and NAC were used in the present investigation to manage NAFLD in adult male Wistar rats as preventive treatments by administering them concurrently with the methionine–choline deficient diet (MCDD). Our purpose was to investigate whether these agents decrease progression to NASH in our animal model of steatosis, and whether this therapeutic approach may result in improvement of hepatocellular integrity and functions, inflammation, oxidative injury, dyslipidemia, and secondary nephropathy.

Materials and methods Chemicals and diagnostic kits All chemicals used for this experiment were of analytical grade. RSV was purchased from Human Nutramax Inc.

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(Hunan, China). UDCA and NAC were purchased from Mutual Chemical Ltd. (Henan, China). Laboratory parameters such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose, albumin, urea, creatinine, total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and very low-density lipoprotein cholesterol (VLDL-C) were measured using an automatic analyzer (Roche/Hitachi Modular Analytics, Roche Diagnostics, Mannheim, Germany) at 37  C. Rat tumor necrosis factoralpha (TNF-a) ELISA kit was purchased from Thermo Scientific, Inc., Waltham, MA (Catalog no.: ENER3TNFA). Malondialdehyde (MDA), GSH, and GST kits were purchased from Bio Diagnostic, Dokki, Giza (Egypt). Rat Leptin ELISA kit was purchased from Crystal Chem, Inc., Downers Grove, IL (Catalog no. 90040). Experimental procedure Forty-two weanling Wistar male albino rats, purchased from the Modern Veterinary Office (Mohamed El-Ghazaly, Cairo, Egypt) consumed a standard laboratory rat diet (Modern Veterinary Office) and water ad libitum until they weighed 200–225 g. Wistar strain was selected in this study because they are more susceptible to fatty liver compared to other strains like Fischer or Sprague–Dawley strains due to interstrain variations in metabolic pattern (Kacew & Festing, 1996; Latha et al., 2010). Thereafter, rats were divided into weight-matched pairs and allocated to seven experimental groups of six rats each. Animals were housed in individual stainless steel wire-bottomed cages with upper water supply to avoid coprophagy (Sukemori et al., 2006) in a room kept at 22–25  C with 12-h light/dark cycles. All animal housing and handling were conducted in compliance with the Beni-Suef University guidelines and in accordance with the research protocols established by the Animal Care Committee of the National Research Center (Cairo, Egypt) which followed the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication no. 85–23, revised 1985). Regarding the experimental design, rats were distributed into seven groups: a normal control group, a steatosis group, and five treatment groups (UDCA as a reference standard, RSV, UDCA + RSV, NAC, UDCA + NAC). Method of steatosis induction While the normal group was given standard diet and free access to water without any restrictions, the steatosis group and all treatment groups were given MCDD consisting of corn starch, casein protein, and sheep fat in the ratio 5:4:1, respectively, with free access to drinking water for 4 consecutive days and fasting for the next 3 consecutive days (with free access to drinking water) per week. This animal model of steatosis was based mainly on the model reported by Delzenne et al. (1997) with diet fed being MCDD instead of a high-fructose fat-free diet (Kucera & Cervinkova, 2014) because on using fructose alone liver inflammation does not occur in a pattern consistent with human fatty liver (Lee et al., 2007). UDCA, RSV, and NAC were dissolved in water and given daily by the oral route through an orogastric catheter at

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DOI: 10.3109/13880209.2015.1060247

doses of 25, 10, and 20 mg/kg/d, respectively (Bujanda et al., 2008; Mas et al., 2008; Samuhasaneeto et al., 2007). The same doses of test agents were used in monotherapies and in combinations. The study lasted for four cycles of feeding and fasting, i.e., 28 d. On day 28, all rats were weighed, anesthetized with isoflurane, and whole blood was collected from the orbital sinus into heparinized microcentrifuge tubes. Rats then underwent midline laparotomy and the liver was removed rapidly and stored at 80  C until needed. Serum preparation

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Blood samples collected in microcentrifuge tubes were centrifuged at 3000 rpm for 20 min. The obtained serum was stored at 20  C until used for biochemical assays. Liver tissue preparation The isolated liver was washed in cold saline, dried with ashless filter paper, and weighed. Hepatic index was calculated by having the ratio of liver weight to rat weight  100. Liver was homogenized (10% w/v) in ice-cold Tris-HCl buffer (0.1 M; pH ¼ 7.4). The homogenate was centrifuged at 3000 rpm for 15 min at 4  C and the resultant hepatic lysed cells supernatant was used for different estimations. Histopathological study Liver was weighed and liver tissue samples, taken at the time of sacrifice, were immediately fixed in 10% buffered formalin in normal saline, dehydrated in gradual ethanol (50–100%), cleared in xylene then embedded in paraffin. Liver sections (4–5 mm) were prepared and stained with hematoxylin–eosin dye using standard techniques for photomicroscopic observations (Banchroft & Steven, 1983). Sections of samples were viewed and evaluated by two experienced pathologists blinded to the experiment. Agreement between both pathologists was prominent and distinguished. Assessment of parameters Body weight was measured according to the method developed by Bujanda et al. (2008) and hepatic index calculation was in compliance with the method developed by Yang et al. (2005). Serum ALT and AST activities were measured by the method developed by Reitman and Frankel (1957). Determination of serum TNF-a level was performed according to the method described by Bonavida (1991). Serum glucose level was determined using the method of Trinder (1969). Serum albumin was measured according to the method of Doumas et al. (1971). Determination of serum urea was based on the method of Fawcett and Scott (1960). Serum creatinine was measured as described earlier (Bartles et al., 1972). Hepatic MDA was quantified using the thiobarbituric acid reaction as described by Ohkawa et al. (1979). Determination hepatic GSH content was done by the method developed by Aykac et al. (1985). Activity of hepatic GST was determined by the method developed by Habig et al. (1974). Measurement of the serum TC was performed by the method described to Meiattini et al. (1978). Estimation of TG and HDL-C was performed as described by Bucolo and David (1973) and Grove (1979), respectively. The levels of serum

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LDL-C and VLDL-C were estimated according to the equation of Friedewald et al. (1972), where serum LDLC ¼ TC–HDL–(TG/5), while VLDL-C ¼ TG/2.175. Serum leptin level was determined by ELISA assay according to the instructions of the manufacturer as described earlier (Maffei et al., 1995). Bacterial translocation detection Before sacrifice, portal blood, peripheral blood, and perihepatic (mesenteric) lymph nodes samples were taken under sterile conditions, cultured in MacConkey agar (Oxoid, Thermo Fisher Scientific Inc., Waltham, MA), Columbia sheep blood (Oxoid, Thermo Fisher Scientific Inc., Waltham, MA), and Esculin-Bile-Azide agar (Sigma-Aldrich, St. Louis, MO), and incubated at 37  C for 48 h. A Positive culture of mesenteric lymph nodes meant bacterial translocation existed while a positive culture of portal and peripheral blood samples meant a systemic infection is evident (Bujanda et al., 2008). Statistical analysis Results were expressed as mean ± standard error of the mean (SEM). All statistical analyses were performed by one-way analysis of variance (ANOVA) followed by the Games– Howell post hoc test using the statistical package for social sciences (SPSS; version 19.0) computer software program (SPSS Inc., Chicago, IL) with a value of p50.05 considered statistically significant.

Results Effect of test agents and their combinations on rat body weight, liver index, serum ALT, and AST activities, and serum TNF-a level At the time of sacrifice, body weights of the rats in the steatosis, UDCA, RSV, and UDCA + RSV were significantly lower than normal control rat body weights, although starting rat weights were almost the same in all treatment groups. Moreover, final rat body weights were higher than initial starting weights in all groups except for the steatosis group (data not included). Rat liver index decreased significantly in the steatosis group, and was significantly improved only in the RSV group, even back to normal value. However, liver index in the UDCA group was also not significantly different from that of the normal control group. Serum ALT and AST levels significantly increased in the steatosis group compared with the normal control group. Serum ALT activities were significantly improved in all treatment groups, but serum AST activities were not corrected in either of the treatment group. Serum TNF-a level increased significantly in the steatosis group as compared with the normal control group, and was significantly decreased in all treatment groups compared with the steatosis group but not normalized (Table 1). Effect of test agents and their combinations on rat serum glucose, albumin, urea, and creatinine levels Serum glucose level was significantly increased in the steatosis group compared with the normal control group.

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Table 1. Effect of UDCA, RSV, and NAC, alone or in combinations, on the rat body weight, liver index, serum ALT, and AST activities, and serum TNF-a level in adult male Wistar albino rats with experimentally induced fatty liver. Parameters Treatment

Body wt. (g)

Liver index (absolute)

ALT (IU/L)

AST (IU/L)

TNF-a (pg/ml)

Normal control Steatosis control UDCA RSV UDCA + RSV NAC UDCA + NAC

250 ± 3.00 213 ± 3.15* 220 ± 2.66* 218 ± 2.17* 222 ± 2.96* 233 ± 4.52 234 ± 2.39

3.29 ± 0.10 2.54 ± 0.08* 2.96 ± 0.17 3.04 ± 0.10@ 2.42 ± 0.06* 2.99 ± 0.08* 2.88 ± 0.09*

39.2 ± 1.47 163.0 ± 3.57* 85.0 ± 3.63*@ 85.5 ± 3.57*@ 102.0 ± 6.11*@ 84.0 ± 7.75*@ 87.3 ± 3.19*@

46.2 ± 2.17 130.1 ± 2.76* 129.3 ± 5.54* 125.2 ± 6.48* 139.4 ± 7.21* 129.0 ± 4.00* 114.1 ± 4.06*

460 ± 38 1997 ± 71* 1627 ± 51*@ 1402 ± 37*@ 1531 ± 62*@ 1406 ± 70*@ 1717 ± 67*@

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Values represent means of six rats ± SE. Statistical analysis was performed using the one-way ANOVA test followed by the Games–Howell multiple comparisons test. *Significantly different from the respective normal control values at p50.05. @Significantly different from the respective fatty liver value at p50.05.

Table 2. Effect of UDCA, RSV and NAC, alone or in combinations, on the serum glucose, albumin, urea and creatinine levels in adult male Wistar albino rats with experimentally induced fatty liver. Parameters Treatment

Glucose (mg/dl)

Albumin (mg/dl)

Urea (mg/dl)

Creatinine (mg/dl)

Normal control Steatosis control UDCA RSV UDCA + RSV NAC UDCA + NAC

74.8 ± 3.66 122.0 ± 1.20* 94.8 ± 4.95@ 84.8 ± 2.12@ 86.7 ± 4.5@ 98.2 ± 2.30*@ 86.7 ± 6.46@

4.15 ± 0.09 3.35 ± 0.08* 3.87 ± 0.10@ 4.08 ± 0.06@ 3.86 ± 0.08@ 3.83 ± 0.08@ 3.74 ± 0.04*@

28.2 ± 0.91 31.3 ± 1.09 29.0 ± 1.13 29.2 ± 1.30 30.0 ± 0.93 28.2 ± 0.95 30.0 ± 1.03

0.66 ± 0.02 0.80 ± 0.02* 0.74 ± 0.03 0.73 ± 0.03 0.69 ± 0.03 0.74 ± 0.03 0.75 ± 0.02

Values represent means of six rats ± SE. Statistical analysis was performed using the one-way ANOVA test followed by the Games–Howell multiple comparisons test. *Significantly different from the respective normal control values at p50.05. @Significantly different from the respective fatty liver value at p50.05.

This elevated value was significantly decreased in all treatment groups. These reductions were even back to normal level except for the NAC group. Serum albumin level was significantly reduced in the steatosis group compared with the normal control group, and was significantly increased in all treatment groups compared with the steatosis group. Except for UDCA + NAC group, serum albumin level was not significantly different from the normal control level. Serum urea was not significantly changed in either the steatosis group or any of the treatment groups. Alternatively, serum creatinine was significantly increased in the steatosis group. In all treatment groups, serum creatinine levels were not significantly different from the normal control level (Table 2). Effect of test agents and their combinations on rat hepatic MDA levels, GSH levels, and GST activity Hepatic MDA content in the steatosis group was significantly higher than normal level. This elevation was significantly decreased in all treatment groups, being normalized in the UDCA and RSV groups. UDCA alone showed significantly better improvement of MDA content compared with either

UDCA + RSV or UDCA + NAC. Hepatic GSH content was significantly decreased in the steatosis group compared with the normal control group. Hepatic GSH content was significantly increased in the UDCA, RSV, and NAC groups, but not in the UDCA + RSV or UDCA + NAC groups, compared with the steatosis group. Similarly, hepatic GST activity was significantly increased in the steatosis group compared with the normal control group, and was significantly corrected in the UDCA, RSV, and NAC groups, but not in the UDCA + RSV or UDCA + NAC groups (Table 3). Effect of test agents and their combinations on rat serum TC, TG, HDL-C, LDL-C, VLDL-C, and leptin levels Serum TC, TG, HDL-C, LDL-C, VLDL-C, and leptin levels were significantly increased in the steatosis group compared with the corresponding normal control group levels. Serum TC and LDL-C levels were significantly corrected in all treatment groups except for the UDCA group. Serum TG and VLDL-C levels were significantly corrected only in the NAC group. Serum HDL-C was not corrected in any of the treatment groups compared with the steatosis group, although its values in the UDCA and RSV groups were not

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significantly different from the normal control value. Finally, the elevated serum leptin level was corrected in all treatment groups except for the UDCA + NAC group (Table 4).

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Effect of test agents and their combinations on rat liver histopathology Regarding the results of the histopathological study, Figure 1 (Photos A–I) shows liver sections from the normal group, steatosis group, UDCA group, RSV group, UDCA + RSV group, NAC group, and UDCA + NAC group. Photo A (a normal control section) shows the central vein (CV) and hepatocytes (H) arranged in radiating cords. Hepatic cords are separated by blood sinusoids (S) carrying blood towards CV. Von Kupffer cells are seen scattered along the walls of sinusoids. Photo B (a normal control section) shows a typical portal tract containing structures in a fibrous stroma, consisting of a terminal branch of the portal vein (PV), a terminal branch of the hepatic artery (A), and a bile ductile (B). Surrounding the portal tract are anastomosing

Table 3. Effect of UDCA, RSV and NAC, alone or in combinations, on the hepatic MDA levels, GSH levels, and GST activity in adult male Wistar albino rats with experimentally induced fatty liver. Parameters Treatment Normal control Steatosis control UDCA RSV UDCA + RSV NAC UDCA + NAC

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plates of hepatocytes between which are the hepatic sinusoids. Photo C (a steatosis control section) shows multifocal hepatocellular necrosis marked by inflammatory cell infiltration with visible congested hepatic sinusoids. Photo D (a steatosis control section) shows hemorrhagic necrosis near the portal tract with marked dilatation of the hepatic sinusoids. Pyknotic nuclei within intact hepatocytes are observed. Photo E (an UDCA treatment section) shows slightly dilated CV with preserved normal hepatic architecture and normal H. Hepatic S show minimal congestion. Photo F (a RSV treatment section) shows normal H with preserved hepatic architecture. Hepatic S shows slight congestion and dilatation. Slight Von Kupffer cell activation is also noticed. Photo G (an NAC treatment section) shows dilated CV with slight congestion. Hepatocytes appear normal with preserved hepatic architecture. Slightly congested and dilated hepatic sinusoids are observed with slight activation of Von Kupffer cells noticed. Photo H (an UDCA + RSV treatment section) shows congested CV with congested and dilated hepatic S. Normal H is seen with preserved hepatic architecture. Photo I (an UDCA + NAC treatment section) shows congested dilated CV with slightly congested dilated hepatic S. Slight Von Kupffer cell activation is also noticed with H appearing normal with preserved hepatic architecture. Bacterial translocation and systemic infection

MDA (nmol/g tissue)

GSH (mg/g tissue)

GST (IU/g tissue)

18.54 ± 3.35 56.75 ± 1.40* 31.50 ± 1.43@ 31.32 ± 1.52@ 43.10 ± 1.05*@# 35.92 ± 2.38*@ 42.50 ± 2.74*@#

76.1 ± 2.63 44.5 ± 2.26* 62.7 ± 2.39*@ 71.3 ± 1.45@ 46.8 ± 1.89*# 67.5 ± 3.35@ 52.8 ± 6.31*

60.5 ± 4.51 158.0 ± 4.32* 108.2 ± 6.60*@ 132.8 ± 1.60*@ 140.5 ± 1.76*# 133.3 ± 3.42*@ 141.4 ± 3.78*#

Values represent means of six rats ± SE. Statistical analysis was performed using the one-way ANOVA test followed by the Games– Howell multiple comparisons test. *Significantly different from the respective normal control values at p50.05. @Significantly different from the respective fatty liver value at p50.05. #Significantly different from the respective UDCA standard value at p50.05.

After the incubation period, cultures of all samples taken were negative in all the seven groups regarding bacterial translocation.

Discussion The NAFLD has been described recently as the hepatic manifestation of metabolic syndrome that can lead to NASH, cirrhosis, and hepatocellular carcinoma (Sugimoto & Takei, 2011). Hepatic lipid accumulation due to insulin resistance referred to as ‘‘the first hit’’, or oxidative stress referred to as ‘‘the second hit’’, may trigger this transition from steatosis to NASH and finally ultimate fibrosis (Berlanga et al., 2014; Jaeschke, 2011; Parekh & Anania, 2007; Samuhasaneeto et al., 2007). The present investigation aims at reducing this

Table 4. Effect of UDCA, RSV, and NAC, alone or in combinations, on the lipid profile of adult male albino rats with experimentally induced fatty liver. Parameters Treatment Normal control Steatosis control UDCA RSV UDCA + RSV NAC UDCA + NAC

TC (mg/dl)

TG (mg/dl)

HDL-C (mg/dl)

LDL-C (mg/dl)

VLDL-C (mg/dl)

Leptin (ng/ml)

108 ± 2.84 175 ± 2.61* 172 ± 5.31* 151 ± 1.69*@ 149 ± 3.50*@ 150 ± 3.23*@ 145 ± 4.40*@#

59 ± 2.18 122 ± 2.60* 115 ± 6.46* 102 ± 4.92* 110 ± 3.79* 100 ± 3.79*@ 107 ± 6.62*

32 ± 0.67 37 ± 0.48* 38 ± 1.55 36 ± 1.41 40 ± 1.06* 38 ± 1.08* 40 ± 0.67*@

64 ± 3.22 113 ± 2.75* 111 ± 4.62* 94 ± 2.51*@ 87 ± 3.68*@# 91 ± 4.05*@ 82 ± 5.01@#

27.2 ± 10.02 56 ± 1.19* 53 ± 2.97* 47 ± 2.26* 51 ± 1.74* 46 ± 1.74*@ 49 ± 3.04*

2.59 ± 0.27 6.22 ± 0.28* 4.05 ± 0.12*@ 3.69 ± 0.06@ 4.38 ± 0.22*@ 4.33 ± 0.32*@ 5.62 ± 0.39*

Values represent means of six rats ± SE. Statistical analysis was performed using the one-way ANOVA test followed by the Games–Howell multiple comparisons test. *Significantly different from the respective normal control values at p50.05. @Significantly different from the respective fatty liver value at p50.05. #Significantly different from the respective UDCA standard value at p50.05.

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Figure 1. Photomicrographs of liver sections obtained from different groups and stained with routine hematoxylin and eosin (H&E) stain: Photo A: A normal rat section, showing central vein (CV) and hepatocytes (H) arranged in radiating cords. Hepatic cords are separated by blood sinusoids (S) carrying blood towards central vein (CV). Von Kupffer cells are seen scattered along the walls of sinusoids (arrows). Photo B: A normal rat section, showing typical portal tract containing three main structures in a fibrous stroma, a terminal branch of portal vein (PV), a terminal branch of hepatic artery (A) and a bile ductule (B). Surrounding portal tracts are anastomosing plates of hepatocytes (H) between which are the hepatic sinusoids (S). Photo C: An experimentally induced steatosis section, showing slightly dilated congested central veins, and vacuolated hepatocytes can be observed with intervening dilated congested hepatic sinusoids (S). Normal architectural pattern is distorted. Photo D: An experimentally induced steatosis section, showing the presence of small fatty vesicles filling the cytoplasm of the hepatocyte (foamy hepatocyte; black arrows), with central rounded nucleus can be seen. Some hepatocytes show pyknotic nuclei (white arrows). Hepatic sinusoids are widely dilated and congested. Photo E: An UDCA treatment section, showing slightly dilated central vein (CV) and congested hepatic sinusoids (S) are observed. Normal hepatic architecture is preserved. Most of hepatocytes are normal except few centrilobular hepatocytes showing cytoplasmic vacuolations. Tiny cytoplasmic vacuolations are seen within some hepatocytes, while others show slight granularity. Congested and slightly dilated hepatic sinusoids can also be noticed. Photo F: A RSV treatment section, showing normal hepatocytes (H) with preserved hepatic architecture. Congested hepatic sinusoids are still noticed (S). Photo G: An UDCA + RSV treatment section, showing many vacuolated centrilobular hepatocytes (arrows). Congested dilated hepatic sinusoids are seen (S) with activated Von Kupffer cells. Photo H: An NAC treatment section, showing congested central vein (CV) and slightly dilated congested hepatic sinusoids (S) with activated Von Kupffer cells. Photo I: An UDCA + NAC treatment section, showing congested dilated central veins (CV) and slightly dilated congested hepatic sinusoids (S).

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DOI: 10.3109/13880209.2015.1060247

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Figure 1. Continued.

progression using three selected test agents, namely UDCA, RSV, and NAC. Although fatty liver was proven to be induced efficiently by feeding experimental animals with high fat diet, we applied here a somewhat novel and attractive model based on deficiency of the essential components methionine and choline in diet, which was also applied by other authors (Ding et al., 2015; Jha et al., 2014). This model is also relevant to human NAFLD as it was proven recently that a complex interplay among methionine, choline, and other factors is essential for proper lipid metabolism in the liver, and that deficiency of methionine or choline in human diet is a leading factor to fatty liver progression (Tarantino, 2014; Veena et al., 2014). In the present investigation, only NAC and UDCA + NAC groups showed final body weights not significantly different from normal control final weight (Table 1), which complies with the findings of one study regarding NAC effect (Alfawwaz & Alhamdan, 2006), while disagrees with the results of another study regarding RSV effect (Bujanda et al., 2008). The metabolic derangements induced by MCDD were reported to cause significant weight loss relative to hepatic steatosis (Costa et al., 2014). The significantly lower liver index in the steatosis group may be attributed to liver shrinkage and hepatocellular necrosis. Liver index was significantly higher than the steatosis group only in the RSV group (Table 1). Such protective effect of RSV on

steatohepatitis may be attributed to its antioxidant and antiinflammatoty potentials (Al Maruf & O’Brien, 2014; Bujanda et al., 2008; Pangeni et al., 2014), promoting regeneration of old damaged hepatocytes. Serum ALT and AST are among the most sensitive biochemical markers investigated in the diagnosis of liver diseases and the best indicators of liver necrosis (Amacher et al., 2013). Interestingly, serum ALT level was higher than serum AST level in the steatosis group (Table 1), where the elevated serum ALT level, but not AST, was corrected by either drug treatment. In agreement, a previous study recorded lower serum ALT level with no effect on serum AST level with UDCA treatment (Wang et al., 2012) or NAC treatment (Khoshbaten et al., 2010). Another study agreed with our results regarding RSV lowering serum ALT level, but disagreed on its effect on serum AST level (Kopec´ & Piatkowska, 2013). AST is present in most body tissues, while ALT is found in its highest concentrations in the liver and so is more specific to the liver (Pratt & Kaplan, 2000). According to one study, cirrhosis was present when serum AST level went higher than serum ALT level and when the ratio AST/ALT exceeded 1 (Anglo et al., 1999). Additionally, it has been reported that NAFLD is considered a direct cause for mild aminotransferase elevation (Diehl et al., 1988; Kim et al., 2013), and that improvement in ALT levels may indicate improvement in steatosis and inflammation but not advanced fibrosis (Adams et al., 2005). Based on these

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findings, our model is a steatohepatitis model that did not progress to cirrhosis, in which liver was significantly protected by all treatment agents and their combinations. Serum TNF-a showed the highest level in the steatosis group and significantly improved in all treatment groups (Table 1). When NASH occurs, TNF-a is excessively produced by activated immune cells such as macrophages, causing the activation of hepatic stellate cells and eventually liver fibrosis (Osawa et al., 2013). In agreement, previous studies have linked the production of TNF-a to NAFLD (Belonovskaia et al., 2013; Silva et al., 2014). UDCA is believed to decrease the inflammatory effect of TNF-a and even its production by decreasing the levels of the substrates of gene ADAM metallopeptidase domain 17 (ADAM17), a TNF-a converting enzyme (Buryova et al., 2013). RSV is a potent Sirtulin 1 activator, and is, therefore, recognized as a natural anti TNF-a agent (Zhu et al., 2011). NAC acts by down-regulating the production of TNF-a and its soluble receptors (Cu et al., 2009). In agreement with our results, the UDCA + NAC combination had the least lowering potential regarding TNF-a level and was not as effective as UDCA alone (Belonovskaia et al., 2013). It should be mentioned that cultures of samples from portal blood, peripheral blood, and perihepatic lymph nodes in our model were negative in all groups, proving that expression of hepatic TNF-a was not due to bacterial translocation but due to MCDD (Pinzone et al., 2012). Hyperglycemia and hypoalbuminemia were associated with NAFLD in previous studies (Ota et al., 2007; Vidyashankar et al., 2013), which comes in agreement with our results (Table 2). The hyperglycemic effect of MCDD was proven to promote inflammatory responses through increasing mitochondrial superoxide production, as well as activation of nuclear factor-kappa B (NFkB) and Protein Kinase C (Chiu & Taylor, 2011). Additionally, insulin resistance which resulted from hyperglycemia was reported to be linked to NAFLD (Ota et al., 2007). Moreover, impaired hepatocyte functions could mediate serum hypoalbuminemia which is a somewhat late marker of hepatocyte dysfunction (Weib et al., 2014). Serum glucose level was normalized in all treatment groups except for the NAC group, while serum albumin level was normalized in all treatment groups except for the UDCA + NAC group (Table 2). This comes in agreement with previous authors regarding serum glucose (Lukivskaya et al., 2004), while in disagreement of other regarding serum albumin (Tsochatzis et al., 2013). In support, it was reported that non-UDCA bile acids regulate metabolism by binding to the nuclear hormone farnesoid X receptor (FXR) and to a transmembrane bile acid receptor TGR5. Activation of FXR was reported to improve insulin sensitivity and to decrease circulating glucose and lipid levels (Zhang & Edwards, 2008), while TGR5 is an important regulator of glucose homeostasis and lipid metabolism and its activation could stimulate energy expenditure and protect against obesity (Chen et al., 2011). Concerning the effect of RSV on serum glucose and albumin, previous studies agreed to our results regarding both parameters (Das et al., 2010; Movahed et al., 2013). Other studies have also noted that RSV improved insulin sensitivity in experimental mice (Carreras et al., 2015). Mechanisms may include RSV

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activation of AMP-activated protein kinase C and SIRT1 (Deng et al., 2007; Pfluger et al., 2008; Tamaki et al., 2014). Concerning the effect of RSV on hypoalbuminemia, previous authors reported that serum albumin concentration was increased by RSV in the course of treatment of nephritic syndrome in rats (Nihei et al., 2001). An antidiabetic potential for NAC was reported experimentally in different animal models (Bajaj & Khan, 2012; Murata et al., 2003). In previous studies, NAC could raise serum albumin back to normal levels, being a reservoir for cysteine (Alfawwaz & Alhamdan, 2006; Higashi et al., 1977). Renal dysfunction is a major complication that accompanies NAFLD (Mikolasevic et al., 2013), which agrees our results (Table 2). Serum creatinine is increasingly being incorporated into prognostic models for patients with decompensated cirrhosis (Czaja, 2011; De Souza et al., 2014). In a previous study, RSV was reported to exert its nephroprotective effect through nitric oxide (NO) release thus lowering serum creatinine level and attenuating renal dysfunction (Chander & Chopra, 2006; Xia et al., 2014). NAC was reported to decrease renal injury secondary to hepatic injury, attributed to its oxygen free radical scavenging activity as well as its vasodilator effect (Seifi et al., 2012). Results of the present study showed that MCDD significantly increased hepatic MDA content and significantly decreased hepatic GSH store (Table 3), indicating oxidative stress (Georgieva, 2005; Janero, 1990). Hepatic MDA was significantly corrected in all treatment groups, while hepatic GSH was significantly corrected in the UDCA, RSV, and NAC groups, but not in UDCA + RSV or UDCA + NAC groups. This comes in agreement with previous studies on UDVA (Sokolovic et al., 2013), RSV (Anderson & Prolla, 2009; Olas et al., 2006; Zheng et al., 2012), and NAC (Sahin & Alatas, 2013). Interestingly, UDCA alone showed better lowering in MDA level when compared with UDCA + RSV or UDCA + NAC, but not when compared with RSV or NAC alone, indicating somewhat antagonistic effect between UDCA and RSV or NAC. Similarly, UDCA, RSV, or NAC alone significantly increased hepatic GSH which did not occur with UDCA + RSV or UDCA + NAC, further suggesting the presence of the aforementioned antagonism. Despite searching literature, this antagonism remains not clearly explained. However, UDCA was reported to cause pharmacokinetic interactions with other drugs based on two facts. First, UDCA can directly bind other agents in the gut retarding their absorption and effect, specially knowing that UDCA was administered concurrently orally with RSV or NAC in our model. Second, UDCA is an inducer of metabolizing enzymes in the gut and the liver (Hempfling et al., 2003). This is particularly important regarding RSV concerning the fact that RSV is extensively metabolized in the gut and the liver (Maier-Salamon et al., 2013). Previous investigations showed increased hepatic GST activity in rats with NAFLD (Hardwick et al., 2010; Lodhi et al., 2014), which comes in support to our results (Table 3). Interestingly, total serum GST activity declines in conditions of oxidative stress but it should be mentioned that there are several isoforms of GST. During oxidative stress, the cationic form present in the liver is induced while the anionic form in

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DOI: 10.3109/13880209.2015.1060247

other tissues is suppressed, with total GST activity decreasing (Hardwick et al., 2010). Similar to findings of hepatic MDA and GSH measurements in this study, the elevated hepatic GST activity was improved by UDCA, RSV, or NAC treatments but not with UDCA + RSV or UDCA + NAC treatments, further supporting antagonistic effects between RSV or NAC with UDCA. Our findings of dyslipidemic markers (Table 4) come in agreement with most previous investigations except for HDL-C results, as most authors reported HDL-C to decrease in liver injury (Huang et al., 2014; Kang & Koppula, 2015; Szkudelska et al., 2009). Alternatively, a recent research agrees with our result based on the fact that HDL-C can be stimulated in response to high TNF-a level (Martius et al., 2014). Dyslipidemia associated with MCDD-induced NAFLD may be due to insulin resistance (Siddiqui et al., 2015). NASH was reported to be coupled with increased cholesterol content in hepatocytes and hence increased lipoprotein production and export to circulation, increasing atherogenic risk thereafter (Ma¨nnisto¨ et al., 2014). Leptin is an adipokine produced from adipocytes, and its elevation in serum is an indication of steatosis induction (Giby & Ajith, 2014). In agreement, the cholesterol-lowering effect of UDCA is well-documented (Braga et al., 2009; Wang et al., 2012). Similarly, RSV was reported to improve dyslipidemia in hypercholesterolemic mice (Xie et al., 2013) and to suppress leptin production from rat adipocytes (Szkudelska et al., 2009). Additionally, NAC was reported to exert hypolipidemic effect in experimental mice (Korou et al., 2010). Suppression of serum lipoproteins and leptin is not only a result of the hepatoprotective potential of test agents but also a hepatoprotective mechanism as well, as dyslipidemia increases atherogenic risk (Kumar & Singh, 2010), while leptin enhances oxidative and nitrosative stress and also stimulates Kupffer cells (Chatterjee et al., 2013). It may be concluded that complications of NAFLD induced by MCDD, like hepatocyte injury, inflammation, insulin resistance, renal insufficiency, oxidative stress, or dyslipidemia may be effectively prevented by UDCA, RSV, NAC, or their combinations. Antioxidant, anti-inflammatory, and lipid-lowering potential are suggested to be behind their protective effects. Efficiencies of RSV or NAC seem to be comparable with UDCA, with RSV being somewhat better. Combination of UDCA with either RSV or NAC does not seem to improve their hepatoprotective potential. These results are promising for further clinical trials with RSV or NAC on different metabolic syndromes.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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New approaches in managing steatohepatitis

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Protective effect of ursodeoxycholic acid, resveratrol, and N-acetylcysteine on nonalcoholic fatty liver disease in rats.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Resveratrol (RSV) and N-acetylcysteine (NAC) are safe representativ...
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