Acta Physiologica Hungarica, Volume 101 (3), pp. 341–352 (2014) DOI: 10.1556/APhysiol.101.2014.3.9
Naltrexone attenuates endoplasmic reticulum stress induced hepatic injury in mice A Moslehi1, F Nabavizadeh1, AR Dehpou2, SM Tavanga3, G Hassanzadeh4, A Zekri5, H Nahrevanian6, H Sohanaki7 1 Department of Physiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 3 Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran 4 Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 5 Department of Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 6 Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran 7 Department of Physiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
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Received: December 18, 2013 Accepted after revision: April 14, 2014 Endoplasmic reticulum (ER) stress provides abnormalities in insulin action, inflammatory responses, lipoprotein B100 degradation and hepatic lipogenesis. Excess accumulation of triglyceride in hepatocytes may also lead to disorders such as non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Opioid peptides are involved in triglyceride and cholesterol dysregulation, inflammation and cell death. In this study, we evaluated Naltrexone effects on ER stress induced liver injury. To do so, C57/BL6 mice received saline, DMSO and Naltrexone, as control groups. ER stress was induced by tunicamycin (TM) injection. Naltrexone was given before TM administration. Liver blood flow and biochemical serum analysis were measured. Histopathological evaluations, TNF-α measurement and Real-time RT-PCR were also performed. TM challenge provokes steatosis, cellular ballooning and lobular inflammation which significantly reduced in Naltrexone treated animals. ALT, AST and TNF-α increased in the TM group and improved in the Naltrexone plus TM group. Triglyceride and cholesterol levels decreased in TM treated mice with no increase in Naltrexone treated animals. In the Naltrexone plus TM group, gene expression of Bax/Bcl-2 ratio and caspase3 significantly lowered compared with the TM group. In this study, we found that Naltrexone had a notable alleviating role in ER stress induced steatosis and liver injury. Keywords: endoplasmic reticulum stress, liver, naltrexone, steatosis, tunicamycin
Endoplasmic reticulum (ER) plays a critical role in lipid synthesis, nascent protein folding and Ca+2 ions storage (13). In special circumstances, such as pharmacological stimuli, oxidative stress, viral infections and dietary demands, ER homeostasis can disrupt and create ER stress phenomenon. ER stress subsequently induces protein unfolding and misfolding in the ER lumen. Accumulation of unfolded and misfolded proteins in ER brings about an adaptive program called “unfolded protein response” (UPR) to restore ER homeostasis (21, 26). It has been known that activation of ER homeostasis pathway can provide steatosis by controlling decreased very low-density lipoprotein (VLDL) level, increased degradation of lipoprotein B100 and hepatic lipogenesis through Sterol Regulatory Element-Binding Protein1-c (SRBP1-c) (9, 20, 29). Chronic ER stress and UPR triggering have also been related
Corresponding author: Fatemeh Nabavizadeh, Professor of Physiology Department of Physiology, School of Medicine, Tehran University of Medical Sciences Tehran, 1417613151, Iran Phone: +98(21)88989485; Fax: +98(21)88989485; E-mail: [email protected] 0231–424X/$ 20.00 © 2014 Akadémiai Kiadó, Budapest
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to abnormalities in insulin function, inflammatory and immune responses and apoptosis (8, 30). ER stress has been induced in various tissues of obese mice and humans (16, 30, 34). Liver is an essential organ in whole body homeostasis, especially the metabolic type. Excess accumulation of triglycerides in hepatocytes may lead to a broad spectrum of hepatic disorders such as nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), fibrosis and possibly cirrhosis (10). Furthermore, several reports indicate ER stress contribution in NAFLD pathogenesis and presence of ER stress markers in the liver of patients suffering from NAFLD display (20, 34). Tunicamycin (TM), a bacterial nucleoside antibiotic, can induce pharmacologic ER stress and stimulate unfolded and misfolded protein accumulation in ER. Numerous documents demonstrated that TM challenge could increase gene expression of GRP78 and Chop, as ER stress markers (10, 36). Other studies also revealed that ER stress induction by hemocystein and TM led to hepatic steatosis in in vitro and in vivo models (24, 36). Endogenous opioids affect three classes of receptors: µ, δ and ĸ receptors. Although opioid peptides were first recognized in brain, they are also found in other tissues such as gut, spleen, stomach, heart, pancreas and liver (38). Opioid peptides are up regulated in diseases states such as liver dysfunction, endotoxic shock, trauma and cholestasis (7, 11, 14). Morphine administration is shown to induce oxidative stress and increase superoxide dismutase and catalase activities and apoptosis in liver tissue (32). Furthermore, several studies implicate a role for opioid peptides in total cholesterol, VLDL and low-density lipoprotein (LDL) cholesterol dysregulation (5). However, Naltrexone, a long-acting opioid receptor blocker can reverse fas-mediated cell death and cholestatic liver injury and decrease plasma and tissue triglyceride and cholesterol levels (3, 5, 19). It has been shown that blocking opioid system can reduce plasma enzymatic activity and attenuate hepatic matrix metalloproteinase-2 (MMP-2) in the liver (7). Regarding the leaving track of opioid system in, ER stress induced hepatic injury; this study was designed with the aim of searching for Naltrexone potential benefits on ER stress induced liver injury in a mouse model hepatic injury. Materials and Methods Reagents Tunicamycin and Naltrexone was purchased from Sigma-Aldrich (St. Louis, USA). Animals Ten to twelve weeks old C57/BL6 male mice weighing 23–25 g (Pasture Institute, Tehran, Iran), were used in the present study. Animals were kept in a temperature-controlled room and 12: 12, light/ dark cycle with free access to standard laboratory chow and water. They were treated according to the ethical committee of Tehran University of Medical Sciences (TUMS). Experimental procedures Subjects were randomly divided into 5 equal groups (N = 6): Group (A); saline control, received normal saline (0.2 ml i.p.), Group (B); vehicle control, received DMSO (0.2 ml i.p.), Group (C); Naltrexone-treated animals, received Naltrexone (20 mg/kg/d i.p.) (7), Group (D), TM-treated animals, received single dose of TM (2 mg/kg body weight) to induce ER stress (25), Group (E); Naltrexone plus TM, received single dose of Naltrexone (20 mg/kg i.p.) 30 min before TM administration (7, 27). Acta Physiologica Hungarica 101, 2014
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Liver blood flow measurement Thirty hours post-TM injection (25), animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) (28). The abdomen was excised via midline incision and the liver exposed. Laser Doppler blood flowmetry (Moor Instrument, VMS-LDF, UK) probe was placed on the left liver lobe and fixed with a probe holder to measure the liver blood flow (LBF) as perfusion unit and reported in baseline percentage. Immediately after LBF measurement, the liver was removed and weighed. Median lobe was then dissected and fixed in 10% buffered formaldehyde solution. Paraffin blocks were then prepared for histopathological findings. Other lobes were dissected and kept at –70 °C for tumor necrosis factor alpha (TNF-α) and gene expression evaluations. Cardiac blood samples were drawn at the end of each experiment. Serum lipids and enzymes measurement Serum was collected from centrifuged blood samples to measure total cholesterol, triglyceride, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Histological study Liver tissue sections were stained with hematoxylin and eosin (H&E). Histopathological evaluations were performed by an expert pathologist blind to the experiment to determine hepatic steatosis, hepatocytes ballooning and lobular inflammation. Histological findings were scored based on Kleiner et al. scoring system, as; steatosis (0–3), lobular inflammation (0–3) and hepatocellular ballooning (0–2) (23). Tissue TNF-α determination Weighed samples of tissues (approximately 0.5 g) were homogenized using an electrical homogenizer (Model RS541-242, RS Components, Corby, UK). Then supernatants were assayed for TNF-α production using an immunometric enzyme immunoassay (EIA) kit (Cat. No. ADI-900-047-Enzo Life Sciences). Real-time RT-PCR First, the total RNA was extracted from frozen tissue samples using RNX-plus Kit (cinnagen, Iran) according to the manufacturer’s instruction. Then its concentration was measured by Nanodrop spectrophotometer. Reverse transcription was also performed using Pocket Script RT perMix (BioNeer, USA). Complementary DNAs (cDNA) were made from mRNA templates for quantitative RT-PCR. Real-time PCR analysis was done with Accu Power 2×Green star qPCR Master Mix (BioNeer, USA) using GAPDH (Glyceraldehyde-3phosphate dehydrogenase) as an internal control. The amount of target mRNA was determined from the appropriate standard curve and normalized relative to the amount of GAPDH mRNA. The primers sequences were as follows: Bax: Forward: 5’-AGACAGGGGCCTTTTTGCTA-3’ Size: 137 Reverse: 5’-AATTCGCCGGAGACACTCG-3’ Bcl-2: Forward: 5’-CTTTGAGTTCGGTGGGGTCA-3’ Size: 153 Reverse: 5’-AGTTCCACAAAGGCATCCCA-3’ Caspase3: Forward: 5’-AAGATACCGGTGGAGGCTGA-3’ Size: 102 Reverse: 5’-AAGGGACTGGATGAACCACG-3’ GAPDH: Forward: 5’-TGGCCTTCCGTGTTCCTAC-3’ Size: 178 Reverse: 5’-GAGTTGCTGTTGAAGTCGCA-3’ Acta Physiologica Hungarica 101, 2014
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Statistical analysis Data were expressed as mean ± SEM. Statistical analysis was performed by one-way analysis of variances (ANOVA) and Tukey’s post hoc test using SPSS for windows version 11.5. P < 0.05 considered to be statistically significant. Results Body and liver weight measurement Mice body weights were measured before and at the end of the protocol. Data showed that in the TM and Naltrexone plus TM groups, body weight significantly decreased in comparison with other groups. Conversely, liver weight increased and liver index (body weight/liver weight×100) strikingly increased in the TM group compared with control ones. But liver index significantly reversed in the Naltrexone plus TM group (Table І). Table І. Body weight, liver weight and liver index in different experimental groups Body weight changes (g)
Liver weight (g)
Liver index (%)
Saline
Groups
0.1 ± 0.08
1.3 ± 0.03
5.5 ± 0.13
DMSO
0. 8 ± 0.04
1.28 ±0.03
5.4 ± 0.2
Naltrexone
0.6 ± 0.08
1.28 ± 0.03
5.6 ± 0.1
TM
–2.3 ± 0.24*
1.42 ± 0.037*
7.09 ± 0.2*
Naltrexone plus TM
–1.9 ± 0.23
1.32 ± 0.02
5.8 ± 0.3#
*p < 0.01 compared to saline, DMSO and naltrexone groups; #p < 0.01 compared to TM group (N = 6), TM (Tunicamycin)
Effects of Naltrexone on histological changes As noted in Figure 1 the liver appearance was pale and partly white in the TM group, but in the Naltrexone plus TM group it was red and partly normal. Histological evaluations of the liver sections showed no sign of steatosis or inflammation in saline, DMSO and Naltrexone groups, whereas severe steatosis, cellular ballooning and lobular inflammation observed in the TM group compared to control animals. Fibrosis was also seen in the TM group. In the Naltrexone-treated TM group, steatosis, ballooning and lobular inflammation significantly decreased compared with the TM group; as such in zone 1 (periphery of the portal tract) steatosis score 2 and in zone 2 and zone 3 (around the central vein) steatosis score 1 and 0, respectively (Fig. 2 and Table ІІ). Effects of Naltrexone on biochemical assays Blood sample analysis revealed that serum ALT and AST increased in TM treated mice in comparison to saline, DMSO and Naltrexone groups (ALT: 288 ± 9 vs, 62 ± 9.2, 55.5 ± 6.1 and 66.2 ± 13.3 U/L, p < 0.01, respectively; AST: 396 ± 94.7 vs 152 ± 20.9 U/L, p < 0.01; 111 ± 35.9 U/L, p < 0.006 and 131 ± 42.3 U/L, p < 0.01, respectively). ALT and AST were also decreased in the Naltrexone plus TM group compared with the TM group (ALT: 143 ± 21.7 vs 288 ± 9 U/L, p < 0.001 and AST: 135 ± 13.7 vs 396 ± 94.7 U/L, p < 0.02, respectively) (Fig. 3).
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(one-way ANOVA followed by Tukey’s post hoc test)
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Fig. 1. The liver of TM-challenged mice was exposed and photographed. TM group: showing pale and partly white liver, naltrexone plus TM group: showing red and partly normal liver Fig 2
Fig. 2. Histological findings of liver tissues after H&E staining (magnification; ×200) in different experimental groups. A; Saline: Normal liver histology, B; DMSO: Normal liver architecture, C; Naltrexone: Normal liver histology, D; TM (Tunicamycin): showing steatosis, ballooning degradation and lobular inflammation, E; Naltrexone plus TM: showing lower steatosis and lobular inflammation
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Table ІІ. Histopathological findings in experimental groups Groups
Saline
DMSO
Naltrexone
100(6)
100(6)
100(6)
TM
Naltrexone plus TM
Steatosis (%) Grade 0 Grade 1
66(4)
Grade 2
33(2)
Grade 3
100(6)
Ballooning (%) Grade 0
100(6)
100(6)
100(6)
Grade 1
33(2) 100(6)
66(4)
Lobular inflammation (%) Grade 0
100(6)
100(6)
100(6)
Grade 1
66(4)
Grade 2
100(6)
33(2)
All cases were scored and analyzed according to Kleiner et al. scoring system proposed (23). (N = 6), TM (Tunicamycin)
Fig. 3. Liver enzymes levels in different experimental groups. Mean ± SEM, N = 6, *p < 0.01, p < 0.006, p < 0.01 compared to saline, DMSO and Naltrexone groups, respectively. &p < 0.001 compared to TM group. #p < 0.01 compared to saline, DMSO and Naltrexone groups. † p < 0.02 compared to TM group (one-way ANOVA followed by Tukey’s post hoc test)
Serum triglyceride and cholesterol levels in TM group significantly lowered compared with saline, DMSO and Naltrexone groups (triglyceride: 40.6 ± 5.6 vs 80.6 ± 6.6 mg/dl, p < 0.005; 75 ± 6.3 mg/dl, p < 0.004 and 76.6 ± 6.6 mg/dl, p < 0.002, respectively; cholesterol: 18.6 ± 1.7 vs 76 ± 1.3 mg/dl, p < 0.002; 66.3 ± 2.6 mg/dl, p < 0.002 and 60 ± 4.1 mg/dl, p < 0.001, respectively). Treatment with Naltrexone did not increase serum triglyceride and cholesterol levels (Fig. 4). Acta Physiologica Hungarica 101, 2014
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Fig. 4. Lipid plasma levels in different experimental groups. Mean ± SEM, N = 6, *p < 0.005, p < 0.004 and p < 0.002 compared to saline, DMSO and Naltrexone groups, respectively. #p < 0.001 compared to saline, DMSO and Naltrexone groups (one-way ANOVA followed by Tukey’s post hoc test)
Evaluation of LBF Our findings showed that LBF significantly decreased in the TM group compared to saline, DMSO and Naltrexone groups (25.6 ± 4.4 vs 100, p < 0.04; 107.2 ± 2.7, p < 0.01 and 97.1 ± 8.5, p < 0.01 respectively). Treatment with Naltrexone, however increased LBF but not in a significant way (Fig. 5).
Fig. 5. Liver blood flow (LBF) in different experimental groups. Mean ± SEM, N = 6, Perfusion unit (PU), *p < 0.04, p < 0.01 and compared to saline, DMSO and Naltrexone groups, respectively (one-way ANOVA followed by Tukey’s post hoc test)
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Assessment of Bax, Bcl-2 and caspase 3 mRNA expressions in hepatocytes RT-PCR results depicted that TM markedly increased hepatocytes mRNA expressions of caspase 3 compared with control groups (saline: 1.34 ± 0.04 vs 1 ± 0.07; DMSO: 1.34 ± 0.04 vs 1.12 ± 0.02 and Naltrexone: 1.34 ± 0.04 vs 1.05 ± 0.04, p < 0.05), while Naltrexone significantly reduced mRNA expression in the naltrexone plus TM group (1.34 ± 0.04 vs 0.54 ± 0.06, p < 0.05) compared with the TM group (Fig. 6). The ratio of Bax/Bcl-2 mRNA expression significantly increased in the TM group in comparison with control animals (saline: 2.33 ± 0.21 vs 1 ± 0.07, DMSO: 2.33 ± 0.21 vs 0.97 ± 0.05 and Naltrexone: 2.33 ± 0.21 vs 1.24 ± 0.09, respectively) and strikingly decreased in the Naltrexone plus TM group (2.33 ± 0.21 vs 0.36 ± 0.06, p < 0.05) (Fig. 7).
Fig. 6. mRNA expression of caspase 3 in different experimental groups. Mean ± SEM, N = 6, *p < 0.05 compared to saline, DMSO and Naltrexone groups, respectively. p < 0.05 compared to TM (Tunicamycin) group (one-way ANOVA followed by Tukey’s post hoc test)
#
Fig. 7. mRNA expression of Bax/Bcl-2 ratio in different experimental groups. Mean ± SEM, N = 6, *p < 0.05 compared to saline, DMSO and Naltrexone groups, respectively. p < 0.05 compared to TM (Tunicamycin) group (one-way ANOVA followed by Tukey’s post hoc test)
#
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Measurement of TNF-α in liver tissue Our results showed that TNF-α significantly increased in the TM group compared to saline, DMSO and Naltrexone groups (46.3 ± 4.8 vs 20.2 ± 0.8, pg/gr. wet weight, p < 0.001; 19.2 ± 1.4, pg/gr. wet weight, p = 0.00 and 20.8 ± 4.4, pg/gr. wet weight, p < 0.001, respectively) and decreased in the Naltrexone plus TM group (46.3 ± 4.8 vs 29.4 ± 4.2, pg/gr. wet weight, p < 0.05) (Fig. 8).
Fig. 8. TNF-α level in different experimental groups. Mean ± SEM, N = 6, Pg/gr wet weight, *p < 0.001, p = 0.0 and p < 0.001 compared to saline, DMSO and Naltrexone groups, respectively. #p < 0.05 compared to TM (Tunicamycin) group (one-way ANOVA followed by Tukey’s post hoc test)
Discussion In this study, we showed that Naltrexone could reduce liver steatosis, inflammation, plasma ALT and AST levels and alleviate gene expression of caspase 3 and Bax/Bcl-2 ratio in TM challenge mice. Naltrexone, as a potent opioid receptor blocker, has previously been shown to have anti-inflammatory and anti-apoptotic effects (7, 14). To the best of our knowledge, this is the first report in which, attenuating effects of Naltrexone on ER stress induced liver injury has been presented in an animal model. Our histological results anticipatorily showed lipid accumulation, steatosis, hepatocyte ballooning and lobular inflammation 30 h after TM challenge. Despite body weight loss in the TM group, liver weight and liver index significantly rose. This may be owing to lipid droplet accumulation in the liver tissue based on many previously published (10, 39), but, Naltrexone administration strikingly decreased liver steatosis and liver index. Christopher et al. (12) also reported TM incremental effects on liver weight and triglyceride contents, but, mouse fed with supplemented taurine reduced liver weight and triglyceride contents. It has been demonstrated that rats treated with morphine sulphate (10, 20 and 30 days) revealed severe centrilobular congestion, portal fibrosis, inflammatory infiltration, lipid accumulation and total protein elevation in hepatocytes (2). Lowering triglyceride and cholesterol serum levels in the TM-treated animals may be due to lipid transfer from plasma to liver and Acta Physiologica Hungarica 101, 2014
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subsequent liver weight gain in TM-treated group. Consistent with our results, Lee et al. (25) also showed steatosis induction, hepatic triglyceride increase and plasma triglyceride, total cholesterol, high-density lipoprotein (HDL), and VLDL decrease in TM-treated mice. Interestingly, in our experiment, Naltrexone kept lipid plasma low in the Naltrexone plus TM group. Bryant et al. (3, 4, 5) had previously reported that immobilization stress or morphine exposure elevated plasma LDL, VLDL, total cholesterol, β lipoprotein and liver and aortic cholesterol levels which could be reversed by Naltrexone administration. In another study, Budzyński et al. (6) also showed that pharmacotherapy with Naltrexone could decrease plasma total cholesterol, triglyceride, LDL and VLDL in abstinent alcoholic patients. Our findings are also congruent with these studies and confirm Naltrexone positive effects on ER stress induced steatosis and plasma lipids. It seems that Naltrexone has a notable potential role on ER stress induced hepatic steatosis. Further studies are needed to clarify possible pathways for these Naltrexone functions. In the present study, we demonstrated that ER stress induction could increase ALT, AST and TNF-α level which may be interpreted as inflammation and hepatocellular death, however, treatment with Naltrexone could ameliorate ALT, AST and liver inflammation which may be delineated its liver conservatory role. In ER stress challenged mice, interleukin 4 (IL-4), IL-2 and interferon-gamma (INF-γ) gene expression together with TNF-α and IL-6 induction have also been increased (25, 40). Fuchs et al. (10) established that serum levels of ALT, AST and alkaline phosphates (ALP) markedly rose in wild type and adipose triglyceride lipase knockout mice. In several documents, it has been shown that Naltrexone as an opioid receptor antagonist could attenuate some features of inflammation. For example, Wang et al. (37) reported that Naltrexone decreased ALT, AST and TNF-α in lipopolysaccharide/D-galactosamine-induced hepatitis in mice. In another report Payabvash et al. (32) revealed that blockade of opioid receptors with Naltrexone declined ALT, AST, ALP and TNF-α in a rat model of chronic cholestasis. Taken together, it is highly probable that these findings delineate Naltrexone inhibitory and stimulatory effects on proinflammatory or anti-inflammatory cytokines such as ILs, TNF-α or liver redox state (7, 14, 32). Still, the precise mechanism remains to be clarified. In this experiment, TM induced ER stress significantly diminished liver blood flow. Naltrexone plus TM could slightly improve it but not in a significant manner. Amin et al. (1) indicated that blood flow post ER stress reduced in hind-limb of type II diabetic mice and recovered with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor. Impaired liver blood flow in ER stress conditions may be due to lipid accumulation and is accompanied by liver microcirculation sclerosis, though ample evidence is required to support this hypothesis. To our knowledge, morphine can increase gene expressions of Fas, Fas ligand (FasL), caspase 8 and caspase 9 while Naltrexone down regulates those (35). It has previously been stated that Naltrexone can prevent hepatocyte apoptosis and liver injury in chronic cholestatic rats (22, 32, 33). Greeneltch et al. (15) also reported morphine induced mRNA expression of Fas, FasL and TNF-related apoptosis-inducing ligand (TRAIL) and increased Bax, IL-4 and IL-13 production (35). All these studies suggested the opioids involvement in apoptosis phenomenon. In the current study, we showed that TM challenge could lead to a significant increase in mRNA expressions of Bax/Bcl-2 ratio and caspase 3; while, Naltrexone treatment significantly reduced their expressions. Previous reports have revealed Bcl-2 family members involvement in ER stress-induced cell death. Overexpression of Bcl-2 inhibits ER stressinduced apoptosis via changing mitochondrial membrane potential (17, 18). It seems that Naltrexone can decrease gene expression of Bax and caspase3 and indirectly vary mitochondrial membrane permeability, cytochrome c release and cell death process. On the other hand, Acta Physiologica Hungarica 101, 2014
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decreased TNF-α level may also be involved in this function. Yet, more studies are needed to clarify the precise Naltrexone ameliorating mechanism in ER stress induced apoptosis. In conclusion, our study demonstrated the palliative role of Naltrexone in ER stress induced steatosis in mouse liver tissues and its anti-inflammatory effects in an animal model of TM challenge. This may indicate that Naltrexone has considerable role in ER stress induced injury and liver steatosis. Acknowledgement This study was supported by a grant from Tehran University of Medical Sciences (TUMS), Tehran, Iran.
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Naltrexone attenuates endoplasmic reticulum stress induced hepatic injury in mice A Moslehi1, F Nabavizadeh1, AR Dehpou2, SM Tavanga3, G Hassanzadeh4, A Zekri5, H Nahrevanian6, H Sohanaki7 1 Department of Physiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 3 Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran 4 Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 5 Department of Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 6 Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran 7 Department of Physiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
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Received: December 18, 2013 Accepted after revision: April 14, 2014 Endoplasmic reticulum (ER) stress provides abnormalities in insulin action, inflammatory responses, lipoprotein B100 degradation and hepatic lipogenesis. Excess accumulation of triglyceride in hepatocytes may also lead to disorders such as non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Opioid peptides are involved in triglyceride and cholesterol dysregulation, inflammation and cell death. In this study, we evaluated Naltrexone effects on ER stress induced liver injury. To do so, C57/BL6 mice received saline, DMSO and Naltrexone, as control groups. ER stress was induced by tunicamycin (TM) injection. Naltrexone was given before TM administration. Liver blood flow and biochemical serum analysis were measured. Histopathological evaluations, TNF-α measurement and Real-time RT-PCR were also performed. TM challenge provokes steatosis, cellular ballooning and lobular inflammation which significantly reduced in Naltrexone treated animals. ALT, AST and TNF-α increased in the TM group and improved in the Naltrexone plus TM group. Triglyceride and cholesterol levels decreased in TM treated mice with no increase in Naltrexone treated animals. In the Naltrexone plus TM group, gene expression of Bax/Bcl-2 ratio and caspase3 significantly lowered compared with the TM group. In this study, we found that Naltrexone had a notable alleviating role in ER stress induced steatosis and liver injury. Keywords: endoplasmic reticulum stress, liver, naltrexone, steatosis, tunicamycin
Endoplasmic reticulum (ER) plays a critical role in lipid synthesis, nascent protein folding and Ca+2 ions storage (13). In special circumstances, such as pharmacological stimuli, oxidative stress, viral infections and dietary demands, ER homeostasis can disrupt and create ER stress phenomenon. ER stress subsequently induces protein unfolding and misfolding in the ER lumen. Accumulation of unfolded and misfolded proteins in ER brings about an adaptive program called “unfolded protein response” (UPR) to restore ER homeostasis (21, 26). It has been known that activation of ER homeostasis pathway can provide steatosis by controlling decreased very low-density lipoprotein (VLDL) level, increased degradation of lipoprotein B100 and hepatic lipogenesis through Sterol Regulatory Element-Binding Protein1-c (SRBP1-c) (9, 20, 29). Chronic ER stress and UPR triggering have also been related
Corresponding author: Fatemeh Nabavizadeh, Professor of Physiology Department of Physiology, School of Medicine, Tehran University of Medical Sciences Tehran, 1417613151, Iran Phone: +98(21)88989485; Fax: +98(21)88989485; E-mail: [email protected] 0231–424X/$ 20.00 © 2014 Akadémiai Kiadó, Budapest
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to abnormalities in insulin function, inflammatory and immune responses and apoptosis (8, 30). ER stress has been induced in various tissues of obese mice and humans (16, 30, 34). Liver is an essential organ in whole body homeostasis, especially the metabolic type. Excess accumulation of triglycerides in hepatocytes may lead to a broad spectrum of hepatic disorders such as nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), fibrosis and possibly cirrhosis (10). Furthermore, several reports indicate ER stress contribution in NAFLD pathogenesis and presence of ER stress markers in the liver of patients suffering from NAFLD display (20, 34). Tunicamycin (TM), a bacterial nucleoside antibiotic, can induce pharmacologic ER stress and stimulate unfolded and misfolded protein accumulation in ER. Numerous documents demonstrated that TM challenge could increase gene expression of GRP78 and Chop, as ER stress markers (10, 36). Other studies also revealed that ER stress induction by hemocystein and TM led to hepatic steatosis in in vitro and in vivo models (24, 36). Endogenous opioids affect three classes of receptors: µ, δ and ĸ receptors. Although opioid peptides were first recognized in brain, they are also found in other tissues such as gut, spleen, stomach, heart, pancreas and liver (38). Opioid peptides are up regulated in diseases states such as liver dysfunction, endotoxic shock, trauma and cholestasis (7, 11, 14). Morphine administration is shown to induce oxidative stress and increase superoxide dismutase and catalase activities and apoptosis in liver tissue (32). Furthermore, several studies implicate a role for opioid peptides in total cholesterol, VLDL and low-density lipoprotein (LDL) cholesterol dysregulation (5). However, Naltrexone, a long-acting opioid receptor blocker can reverse fas-mediated cell death and cholestatic liver injury and decrease plasma and tissue triglyceride and cholesterol levels (3, 5, 19). It has been shown that blocking opioid system can reduce plasma enzymatic activity and attenuate hepatic matrix metalloproteinase-2 (MMP-2) in the liver (7). Regarding the leaving track of opioid system in, ER stress induced hepatic injury; this study was designed with the aim of searching for Naltrexone potential benefits on ER stress induced liver injury in a mouse model hepatic injury. Materials and Methods Reagents Tunicamycin and Naltrexone was purchased from Sigma-Aldrich (St. Louis, USA). Animals Ten to twelve weeks old C57/BL6 male mice weighing 23–25 g (Pasture Institute, Tehran, Iran), were used in the present study. Animals were kept in a temperature-controlled room and 12: 12, light/ dark cycle with free access to standard laboratory chow and water. They were treated according to the ethical committee of Tehran University of Medical Sciences (TUMS). Experimental procedures Subjects were randomly divided into 5 equal groups (N = 6): Group (A); saline control, received normal saline (0.2 ml i.p.), Group (B); vehicle control, received DMSO (0.2 ml i.p.), Group (C); Naltrexone-treated animals, received Naltrexone (20 mg/kg/d i.p.) (7), Group (D), TM-treated animals, received single dose of TM (2 mg/kg body weight) to induce ER stress (25), Group (E); Naltrexone plus TM, received single dose of Naltrexone (20 mg/kg i.p.) 30 min before TM administration (7, 27). Acta Physiologica Hungarica 101, 2014
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Liver blood flow measurement Thirty hours post-TM injection (25), animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) (28). The abdomen was excised via midline incision and the liver exposed. Laser Doppler blood flowmetry (Moor Instrument, VMS-LDF, UK) probe was placed on the left liver lobe and fixed with a probe holder to measure the liver blood flow (LBF) as perfusion unit and reported in baseline percentage. Immediately after LBF measurement, the liver was removed and weighed. Median lobe was then dissected and fixed in 10% buffered formaldehyde solution. Paraffin blocks were then prepared for histopathological findings. Other lobes were dissected and kept at –70 °C for tumor necrosis factor alpha (TNF-α) and gene expression evaluations. Cardiac blood samples were drawn at the end of each experiment. Serum lipids and enzymes measurement Serum was collected from centrifuged blood samples to measure total cholesterol, triglyceride, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Histological study Liver tissue sections were stained with hematoxylin and eosin (H&E). Histopathological evaluations were performed by an expert pathologist blind to the experiment to determine hepatic steatosis, hepatocytes ballooning and lobular inflammation. Histological findings were scored based on Kleiner et al. scoring system, as; steatosis (0–3), lobular inflammation (0–3) and hepatocellular ballooning (0–2) (23). Tissue TNF-α determination Weighed samples of tissues (approximately 0.5 g) were homogenized using an electrical homogenizer (Model RS541-242, RS Components, Corby, UK). Then supernatants were assayed for TNF-α production using an immunometric enzyme immunoassay (EIA) kit (Cat. No. ADI-900-047-Enzo Life Sciences). Real-time RT-PCR First, the total RNA was extracted from frozen tissue samples using RNX-plus Kit (cinnagen, Iran) according to the manufacturer’s instruction. Then its concentration was measured by Nanodrop spectrophotometer. Reverse transcription was also performed using Pocket Script RT perMix (BioNeer, USA). Complementary DNAs (cDNA) were made from mRNA templates for quantitative RT-PCR. Real-time PCR analysis was done with Accu Power 2×Green star qPCR Master Mix (BioNeer, USA) using GAPDH (Glyceraldehyde-3phosphate dehydrogenase) as an internal control. The amount of target mRNA was determined from the appropriate standard curve and normalized relative to the amount of GAPDH mRNA. The primers sequences were as follows: Bax: Forward: 5’-AGACAGGGGCCTTTTTGCTA-3’ Size: 137 Reverse: 5’-AATTCGCCGGAGACACTCG-3’ Bcl-2: Forward: 5’-CTTTGAGTTCGGTGGGGTCA-3’ Size: 153 Reverse: 5’-AGTTCCACAAAGGCATCCCA-3’ Caspase3: Forward: 5’-AAGATACCGGTGGAGGCTGA-3’ Size: 102 Reverse: 5’-AAGGGACTGGATGAACCACG-3’ GAPDH: Forward: 5’-TGGCCTTCCGTGTTCCTAC-3’ Size: 178 Reverse: 5’-GAGTTGCTGTTGAAGTCGCA-3’ Acta Physiologica Hungarica 101, 2014
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Statistical analysis Data were expressed as mean ± SEM. Statistical analysis was performed by one-way analysis of variances (ANOVA) and Tukey’s post hoc test using SPSS for windows version 11.5. P < 0.05 considered to be statistically significant. Results Body and liver weight measurement Mice body weights were measured before and at the end of the protocol. Data showed that in the TM and Naltrexone plus TM groups, body weight significantly decreased in comparison with other groups. Conversely, liver weight increased and liver index (body weight/liver weight×100) strikingly increased in the TM group compared with control ones. But liver index significantly reversed in the Naltrexone plus TM group (Table І). Table І. Body weight, liver weight and liver index in different experimental groups Body weight changes (g)
Liver weight (g)
Liver index (%)
Saline
Groups
0.1 ± 0.08
1.3 ± 0.03
5.5 ± 0.13
DMSO
0. 8 ± 0.04
1.28 ±0.03
5.4 ± 0.2
Naltrexone
0.6 ± 0.08
1.28 ± 0.03
5.6 ± 0.1
TM
–2.3 ± 0.24*
1.42 ± 0.037*
7.09 ± 0.2*
Naltrexone plus TM
–1.9 ± 0.23
1.32 ± 0.02
5.8 ± 0.3#
*p < 0.01 compared to saline, DMSO and naltrexone groups; #p < 0.01 compared to TM group (N = 6), TM (Tunicamycin)
Effects of Naltrexone on histological changes As noted in Figure 1 the liver appearance was pale and partly white in the TM group, but in the Naltrexone plus TM group it was red and partly normal. Histological evaluations of the liver sections showed no sign of steatosis or inflammation in saline, DMSO and Naltrexone groups, whereas severe steatosis, cellular ballooning and lobular inflammation observed in the TM group compared to control animals. Fibrosis was also seen in the TM group. In the Naltrexone-treated TM group, steatosis, ballooning and lobular inflammation significantly decreased compared with the TM group; as such in zone 1 (periphery of the portal tract) steatosis score 2 and in zone 2 and zone 3 (around the central vein) steatosis score 1 and 0, respectively (Fig. 2 and Table ІІ). Effects of Naltrexone on biochemical assays Blood sample analysis revealed that serum ALT and AST increased in TM treated mice in comparison to saline, DMSO and Naltrexone groups (ALT: 288 ± 9 vs, 62 ± 9.2, 55.5 ± 6.1 and 66.2 ± 13.3 U/L, p < 0.01, respectively; AST: 396 ± 94.7 vs 152 ± 20.9 U/L, p < 0.01; 111 ± 35.9 U/L, p < 0.006 and 131 ± 42.3 U/L, p < 0.01, respectively). ALT and AST were also decreased in the Naltrexone plus TM group compared with the TM group (ALT: 143 ± 21.7 vs 288 ± 9 U/L, p < 0.001 and AST: 135 ± 13.7 vs 396 ± 94.7 U/L, p < 0.02, respectively) (Fig. 3).
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(one-way ANOVA followed by Tukey’s post hoc test)
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Fig. 1. The liver of TM-challenged mice was exposed and photographed. TM group: showing pale and partly white liver, naltrexone plus TM group: showing red and partly normal liver Fig 2
Fig. 2. Histological findings of liver tissues after H&E staining (magnification; ×200) in different experimental groups. A; Saline: Normal liver histology, B; DMSO: Normal liver architecture, C; Naltrexone: Normal liver histology, D; TM (Tunicamycin): showing steatosis, ballooning degradation and lobular inflammation, E; Naltrexone plus TM: showing lower steatosis and lobular inflammation
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Table ІІ. Histopathological findings in experimental groups Groups
Saline
DMSO
Naltrexone
100(6)
100(6)
100(6)
TM
Naltrexone plus TM
Steatosis (%) Grade 0 Grade 1
66(4)
Grade 2
33(2)
Grade 3
100(6)
Ballooning (%) Grade 0
100(6)
100(6)
100(6)
Grade 1
33(2) 100(6)
66(4)
Lobular inflammation (%) Grade 0
100(6)
100(6)
100(6)
Grade 1
66(4)
Grade 2
100(6)
33(2)
All cases were scored and analyzed according to Kleiner et al. scoring system proposed (23). (N = 6), TM (Tunicamycin)
Fig. 3. Liver enzymes levels in different experimental groups. Mean ± SEM, N = 6, *p < 0.01, p < 0.006, p < 0.01 compared to saline, DMSO and Naltrexone groups, respectively. &p < 0.001 compared to TM group. #p < 0.01 compared to saline, DMSO and Naltrexone groups. † p < 0.02 compared to TM group (one-way ANOVA followed by Tukey’s post hoc test)
Serum triglyceride and cholesterol levels in TM group significantly lowered compared with saline, DMSO and Naltrexone groups (triglyceride: 40.6 ± 5.6 vs 80.6 ± 6.6 mg/dl, p < 0.005; 75 ± 6.3 mg/dl, p < 0.004 and 76.6 ± 6.6 mg/dl, p < 0.002, respectively; cholesterol: 18.6 ± 1.7 vs 76 ± 1.3 mg/dl, p < 0.002; 66.3 ± 2.6 mg/dl, p < 0.002 and 60 ± 4.1 mg/dl, p < 0.001, respectively). Treatment with Naltrexone did not increase serum triglyceride and cholesterol levels (Fig. 4). Acta Physiologica Hungarica 101, 2014
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Fig. 4. Lipid plasma levels in different experimental groups. Mean ± SEM, N = 6, *p < 0.005, p < 0.004 and p < 0.002 compared to saline, DMSO and Naltrexone groups, respectively. #p < 0.001 compared to saline, DMSO and Naltrexone groups (one-way ANOVA followed by Tukey’s post hoc test)
Evaluation of LBF Our findings showed that LBF significantly decreased in the TM group compared to saline, DMSO and Naltrexone groups (25.6 ± 4.4 vs 100, p < 0.04; 107.2 ± 2.7, p < 0.01 and 97.1 ± 8.5, p < 0.01 respectively). Treatment with Naltrexone, however increased LBF but not in a significant way (Fig. 5).
Fig. 5. Liver blood flow (LBF) in different experimental groups. Mean ± SEM, N = 6, Perfusion unit (PU), *p < 0.04, p < 0.01 and compared to saline, DMSO and Naltrexone groups, respectively (one-way ANOVA followed by Tukey’s post hoc test)
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Assessment of Bax, Bcl-2 and caspase 3 mRNA expressions in hepatocytes RT-PCR results depicted that TM markedly increased hepatocytes mRNA expressions of caspase 3 compared with control groups (saline: 1.34 ± 0.04 vs 1 ± 0.07; DMSO: 1.34 ± 0.04 vs 1.12 ± 0.02 and Naltrexone: 1.34 ± 0.04 vs 1.05 ± 0.04, p < 0.05), while Naltrexone significantly reduced mRNA expression in the naltrexone plus TM group (1.34 ± 0.04 vs 0.54 ± 0.06, p < 0.05) compared with the TM group (Fig. 6). The ratio of Bax/Bcl-2 mRNA expression significantly increased in the TM group in comparison with control animals (saline: 2.33 ± 0.21 vs 1 ± 0.07, DMSO: 2.33 ± 0.21 vs 0.97 ± 0.05 and Naltrexone: 2.33 ± 0.21 vs 1.24 ± 0.09, respectively) and strikingly decreased in the Naltrexone plus TM group (2.33 ± 0.21 vs 0.36 ± 0.06, p < 0.05) (Fig. 7).
Fig. 6. mRNA expression of caspase 3 in different experimental groups. Mean ± SEM, N = 6, *p < 0.05 compared to saline, DMSO and Naltrexone groups, respectively. p < 0.05 compared to TM (Tunicamycin) group (one-way ANOVA followed by Tukey’s post hoc test)
#
Fig. 7. mRNA expression of Bax/Bcl-2 ratio in different experimental groups. Mean ± SEM, N = 6, *p < 0.05 compared to saline, DMSO and Naltrexone groups, respectively. p < 0.05 compared to TM (Tunicamycin) group (one-way ANOVA followed by Tukey’s post hoc test)
#
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Measurement of TNF-α in liver tissue Our results showed that TNF-α significantly increased in the TM group compared to saline, DMSO and Naltrexone groups (46.3 ± 4.8 vs 20.2 ± 0.8, pg/gr. wet weight, p < 0.001; 19.2 ± 1.4, pg/gr. wet weight, p = 0.00 and 20.8 ± 4.4, pg/gr. wet weight, p < 0.001, respectively) and decreased in the Naltrexone plus TM group (46.3 ± 4.8 vs 29.4 ± 4.2, pg/gr. wet weight, p < 0.05) (Fig. 8).
Fig. 8. TNF-α level in different experimental groups. Mean ± SEM, N = 6, Pg/gr wet weight, *p < 0.001, p = 0.0 and p < 0.001 compared to saline, DMSO and Naltrexone groups, respectively. #p < 0.05 compared to TM (Tunicamycin) group (one-way ANOVA followed by Tukey’s post hoc test)
Discussion In this study, we showed that Naltrexone could reduce liver steatosis, inflammation, plasma ALT and AST levels and alleviate gene expression of caspase 3 and Bax/Bcl-2 ratio in TM challenge mice. Naltrexone, as a potent opioid receptor blocker, has previously been shown to have anti-inflammatory and anti-apoptotic effects (7, 14). To the best of our knowledge, this is the first report in which, attenuating effects of Naltrexone on ER stress induced liver injury has been presented in an animal model. Our histological results anticipatorily showed lipid accumulation, steatosis, hepatocyte ballooning and lobular inflammation 30 h after TM challenge. Despite body weight loss in the TM group, liver weight and liver index significantly rose. This may be owing to lipid droplet accumulation in the liver tissue based on many previously published (10, 39), but, Naltrexone administration strikingly decreased liver steatosis and liver index. Christopher et al. (12) also reported TM incremental effects on liver weight and triglyceride contents, but, mouse fed with supplemented taurine reduced liver weight and triglyceride contents. It has been demonstrated that rats treated with morphine sulphate (10, 20 and 30 days) revealed severe centrilobular congestion, portal fibrosis, inflammatory infiltration, lipid accumulation and total protein elevation in hepatocytes (2). Lowering triglyceride and cholesterol serum levels in the TM-treated animals may be due to lipid transfer from plasma to liver and Acta Physiologica Hungarica 101, 2014
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subsequent liver weight gain in TM-treated group. Consistent with our results, Lee et al. (25) also showed steatosis induction, hepatic triglyceride increase and plasma triglyceride, total cholesterol, high-density lipoprotein (HDL), and VLDL decrease in TM-treated mice. Interestingly, in our experiment, Naltrexone kept lipid plasma low in the Naltrexone plus TM group. Bryant et al. (3, 4, 5) had previously reported that immobilization stress or morphine exposure elevated plasma LDL, VLDL, total cholesterol, β lipoprotein and liver and aortic cholesterol levels which could be reversed by Naltrexone administration. In another study, Budzyński et al. (6) also showed that pharmacotherapy with Naltrexone could decrease plasma total cholesterol, triglyceride, LDL and VLDL in abstinent alcoholic patients. Our findings are also congruent with these studies and confirm Naltrexone positive effects on ER stress induced steatosis and plasma lipids. It seems that Naltrexone has a notable potential role on ER stress induced hepatic steatosis. Further studies are needed to clarify possible pathways for these Naltrexone functions. In the present study, we demonstrated that ER stress induction could increase ALT, AST and TNF-α level which may be interpreted as inflammation and hepatocellular death, however, treatment with Naltrexone could ameliorate ALT, AST and liver inflammation which may be delineated its liver conservatory role. In ER stress challenged mice, interleukin 4 (IL-4), IL-2 and interferon-gamma (INF-γ) gene expression together with TNF-α and IL-6 induction have also been increased (25, 40). Fuchs et al. (10) established that serum levels of ALT, AST and alkaline phosphates (ALP) markedly rose in wild type and adipose triglyceride lipase knockout mice. In several documents, it has been shown that Naltrexone as an opioid receptor antagonist could attenuate some features of inflammation. For example, Wang et al. (37) reported that Naltrexone decreased ALT, AST and TNF-α in lipopolysaccharide/D-galactosamine-induced hepatitis in mice. In another report Payabvash et al. (32) revealed that blockade of opioid receptors with Naltrexone declined ALT, AST, ALP and TNF-α in a rat model of chronic cholestasis. Taken together, it is highly probable that these findings delineate Naltrexone inhibitory and stimulatory effects on proinflammatory or anti-inflammatory cytokines such as ILs, TNF-α or liver redox state (7, 14, 32). Still, the precise mechanism remains to be clarified. In this experiment, TM induced ER stress significantly diminished liver blood flow. Naltrexone plus TM could slightly improve it but not in a significant manner. Amin et al. (1) indicated that blood flow post ER stress reduced in hind-limb of type II diabetic mice and recovered with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor. Impaired liver blood flow in ER stress conditions may be due to lipid accumulation and is accompanied by liver microcirculation sclerosis, though ample evidence is required to support this hypothesis. To our knowledge, morphine can increase gene expressions of Fas, Fas ligand (FasL), caspase 8 and caspase 9 while Naltrexone down regulates those (35). It has previously been stated that Naltrexone can prevent hepatocyte apoptosis and liver injury in chronic cholestatic rats (22, 32, 33). Greeneltch et al. (15) also reported morphine induced mRNA expression of Fas, FasL and TNF-related apoptosis-inducing ligand (TRAIL) and increased Bax, IL-4 and IL-13 production (35). All these studies suggested the opioids involvement in apoptosis phenomenon. In the current study, we showed that TM challenge could lead to a significant increase in mRNA expressions of Bax/Bcl-2 ratio and caspase 3; while, Naltrexone treatment significantly reduced their expressions. Previous reports have revealed Bcl-2 family members involvement in ER stress-induced cell death. Overexpression of Bcl-2 inhibits ER stressinduced apoptosis via changing mitochondrial membrane potential (17, 18). It seems that Naltrexone can decrease gene expression of Bax and caspase3 and indirectly vary mitochondrial membrane permeability, cytochrome c release and cell death process. On the other hand, Acta Physiologica Hungarica 101, 2014
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decreased TNF-α level may also be involved in this function. Yet, more studies are needed to clarify the precise Naltrexone ameliorating mechanism in ER stress induced apoptosis. In conclusion, our study demonstrated the palliative role of Naltrexone in ER stress induced steatosis in mouse liver tissues and its anti-inflammatory effects in an animal model of TM challenge. This may indicate that Naltrexone has considerable role in ER stress induced injury and liver steatosis. Acknowledgement This study was supported by a grant from Tehran University of Medical Sciences (TUMS), Tehran, Iran.
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