Select Page

2017; 4(10):266–274

REVIEW ARTICLES

PDF logo-blue 30

 

Oxidative Stress and Endoplasmic Reticulum Stress as Potential Therapeutic Targets in Non-Alcoholic Fatty Liver Disease

 

Pablo Lizana, Melissa Galdames, and Ramón Rodrigo

Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Independencia 1027, CP 8380453, Santiago, Chile

Correspondence: rrodrigo@med.uchile.cl (R.R.)

Lizana P et al. Reactive Oxygen Species 4(10):266–274, 2017; ©2017 Cell Med Press

http://dx.doi.org/10.20455/ros.2017.847

(Received: March 23, 2017; Revised: May 12, 2017; Accepted: May 12, 2017)

ABSTRACT | Non-alcoholic fatty liver disease (NAFLD) is nowadays recognized as a common cause of chronic liver disease and aminotransferase elevation. Its incidence has been increasing through the last few years, raising a global prevalence of approximately 25%. The etiopathogenic mechanisms of this disease are not fully understood, but it has been related with various pathologies that compound the metabolic syndrome. Oxidative stress and endoplasmic reticulum (ER) stress have been recognized as key mechanisms in NAFLD pathogenesis. In this review, an updated overview of the role of oxidative stress and ER stress in the progression of NAFLD is provided. Besides, some current treatments focused on the above mechanisms are presented, with the objective to discuss new therapeutic strategies that could help physicians on their daily clinical practice.

KEYWORDS | Endoplasmic reticulum stress; Hepatocellular carcinoma; Non-alcoholic fatty liver disease; Non-alcoholic steatohepatitis; Oxidative stress; Reactive oxygen species

ABBREVIATIONS | ALT, alanine transferase; ER, endoplasmic reticulum; HCC, hepatocellular carcinoma; JNK, c-Jun N-terminal kinases; MS, metabolic syndrome; NAFL, non-alcoholic fatty liver; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; PJ, pomegranate juice; RNS, reactive nitrogen species; ROS, reactive oxygen species; T2DM, type 2 diabetes mellitus; UPR, unfolded protein response; XBP-1, X-box binding protein 1


CONTENTS

1. Introduction

2. General Overview of NAFLD

3. Pathophysiology of NAFLD

3.1. Role of Oxidative Stress

3.2. Role of Endoplasmic Reticulum Stress

4. Current Therapeutic Strategies

5. Novel Therapeutic Approaches

6. Concluding Remarks


1. INTRODUCTION

Non-alcoholic fatty liver disease (NAFLD) has been recognized as a major cause of liver disease worldwide. Recently, the development of cirrhosis in the absence of alcohol exposure has been identified as a frequent and important cause of elevation of aminotransferases [1‒3]. The prevalence of NAFLD worldwide has been increasing through the last years and it is thought to be on the rise, being nowadays reported, just in the United States, a prevalence between 10% and 30%, with similar rates reported from Europe and Asia [3].

Although the etiopathogenic mechanisms of this disease are not fully understood, individuals with components of metabolic syndrome (MS), such as obesity, insulin resistance, and hyperlipidemia, have an increased risk of developing non-alcoholic fatty liver (NAFL) [4]. The pathogenesis of this disease must be understood as a multifactorial process, with oxidative stress being one of the key mechanisms involved in the progression from NAFL to non-alcoholic steatohepatitis (NASH), and finally to cirrhosis and hepatocellular carcinoma (HCC).

Oxidative stress is derived from the loss in the balance of the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and the consumption of these reactive species by antioxidants produced inside the hepatocyte. Disruption of this balance that favors the formation and accumulation of ROS and RNS finally leads to hepatotoxicity. This occurs as a result of the direct attack by these species to the essential biomolecules in the hepatocytes, producing an impairment in their biological functions and compromising the cell viability [3, 5, 6].

The aim of this review is to present an updated overview about the pathogenesis and novel therapies of NAFLD based on the discussion that oxidative stress and reticulum stress act as key mechanisms involved in the progression of NAFLD. Accordingly, the identification of the oxidant and antioxidant factors that are pathologically disrupted in NAFLD could help improve not only the current therapeutic strategies, but also give rise to new prophylactic approaches that could be used by physicians in a near future for the effective management of this disease. In addition, novel therapeutic approaches based on the pathophysiology presented in this review will be discussed.

2. GENERAL OVERVIEW OF NAFLD

NAFLD was firstly described in the 1980s in subjects with an alcohol intake between or lower than 10 and 40 g/day or, as used in some studies, no more than 40 g of alcohol per week. Nowadays, this is the most common type of chronic liver disease in the western countries and it includes the whole spectrum of static steatosis to NASH that could progress to cirrhosis and HCC [1, 2, 7, 8].

NAFLD is usually asymptomatic, although some patients might consult for weakness with or without hepatomegaly in the physical exam. The laboratory tests could show elevation of the aminotransferases, an increase in the alkaline phosphatases, and an elevation of the gamma-glutamyl transpeptidase. Moreover, some studies have suggested that NASH patients tend to be older than the patients with NAFL and to have a higher homeostatic model assessment, indicating insulin resistance. On the other hand, NASH-HCC patients tend to be older than NASH patients and have the lowest platelet counts and alanine transferase (ALT) levels of the spectrum [1, 8]. In addition, these patients show an increase in the levels of triglycerides (TG), cholesterol, and free fatty acids in comparison with patients without NASH [9, 10].

Histologically, NAFLD is categorized into NAFL, in which it can be found steatosis without hepatocellular injury. NASH is characterized by the presence of hepatic steatosis and inflammation with hepatocyte injury (ballooning), which is a cardinal histologic feature of lipotoxic hepatic injury, and has been also correlated with the disease severity. MS is also correlated with the histological severity [1, 7, 11, 12].

Some studies have recognized NASH as a more advanced stage of NAFLD. This is based, among others, on the higher probabilities to develop more serious diseases, and the higher cardiovascular risk of the patients and NASH mortality rate (25.6 per 1.000 person-year). Its progression rate to cirrhosis is 20‒30% in 10 years versus the 1% found in static steatosis [1, 8]. Recently, there have been studies indicating that both NAFL and NASH are likely to have different genetic backgrounds and lipid contents, thus suggesting that the pathogenesis of steatosis in simple fatty liver and NASH is different, and disease-specific treatments are therefore required [8].

3. PATHOPHYSIOLOGY OF NAFLD

Several factors are involved in the pathogenesis and progression of NAFLD, such as lipotoxicity, oxidative stress, mitochondrial dysfunction, and immune dysregulation [3]. All of these factors act concomitantly in the hepatocyte to generate the liver damage seen in more advanced stages of this disease, with lipotoxicity-induced oxidative stress and the endoplasmic reticulum (ER) stress being the ones that appear to be the central drivers in this progression [3, 7]. Actually, the pathogenesis of NAFLD is based in a modified “two hits” hypothesis [13]. According to this hypothesis, the “first hit” refers to the generation of an steatotic liver, which is produced by the pathologic accumulation of TG as a result of an increased free fatty acid (FFA) influx. The “second hit” is produced later with the action of many other pathologic factors, such as cytokines, adipokines, oxidative stress, and mitochondrial dysfunction, which together cause the progression to NASH and fibrosis [14]. Nowadays, some genetic factors have been identified as key mechanisms in the pathogenesis of NAFLD, such as a patatin-like phospholipase 3 (PNPLA3) gene polymorphism, which has been proposed to have a key role in the development of NASH [15].

A “third hit” has also been described, which is related to the repairing response induced when the adaptive mechanisms that protect hepatocytes from the fatty acid-mediated lipotoxicity are overwhelmed. The repairing response involves the activation of hepatic stellate cells to myofibroblasts, which differentiate to replace dead hepatocytes. These cells also produce factors that attract various kinds of inflammatory cells to the liver, finally leading to NASH and fibrosis [16]. In addition, a “multifactorial hit” has been proposed, which adds the interaction of the inflammasome within the hepatocyte with the gut microbiota dysbiosis [17]. All of these mechanisms and factors have been considered crucial in the NAFLD pathogenesis (Figure 1).

FIGURE 1. Pathophysiology of NAFLD based on the modified “two hits” hypothesis. The pathologic accumulation of FFA in the liver leads to steatosis (first hit). The metabolism of the lipids within the hepatocytes produces a disturbance in the prooxidant/antioxidant balance, favoring the accumulation of ROS. This imbalance causes endoplasmic reticulum stress and mitochondrial dysfunction, among other outcomes, leading to oxidative stress (second hit). The oxidative stress produces necrosis and an increase of the cytoplasmic Ca2+, thereby activating the apoptotic pathways, leading to inflammation and NASH (third hit). NAFLD, non-alcoholic fatty liver disease; FFA, free fatty acids; ROS, reactive oxygen species; NASH, non-alcoholic steatohepatitis; SERCA, sarco-endoplasmic reticulum Ca2+-ATPase.

3.1. Role of Oxidative Stress

Normally, ROS are physiologically generated as an intrinsic property of any kind of aerobic organism, with the mitochondria being the most important source of their production. About 5% of the oxygen used in the metabolism produces some of the most relevant ROS affecting the cell homeostasis. The rest of oxygen is metabolized directly to water [18]. Within the hepatocytes, ROS are generated by the free fatty acid metabolism of organelles, such as peroxisome and ER (besides the mitochondria), with this process being proposed to be responsible for the initiating necroinflammation [8]. Hydroxyl radicals, singlet oxygen molecules, superoxide anions, and hydrogen peroxide are the most relevant ROS that have been associated with NAFLD pathogenesis, with hydroxyl radical being considered one of the strongest oxidants in nature [19, 20].

The prooxidant-favored dysregulation in NAFLD leads the above ROS to attack essential hepatocyte biomolecules, such as lipids, proteins, and DNA. In fact, a clinical trial showed that patients with type 2 diabetes mellitus (T2DM) and NASH have increased levels of oxidative stress markers. It was found that protein oxidation and lipid peroxidation, malondialdehyde (MDA), and 8-isoprostane are higher in T2DM than controls [5]. Another clinical trial showed that patients with NASH-HCC have significantly lower levels of antioxidant molecules, such as reduced glutathione and superoxide dismutase (SOD), compared with both patients only with NAFL and controls [8].

3.2. Role of Endoplasmic Reticulum Stress

Oxidative stress affects the ER, a major organelle that controls the production of cholesterol and lipid membrane biosynthesis, and also participates in the calcium homeostasis [21]. In fact, free cholesterol can cause ER stress in NASH liver by inhibition of the sarco-endoplasmic reticulum Ca2+-ATPase [22]. ER stress is elevated by accumulation of fatty acids and is involved in the pathogenesis of NASH via activation of the fibrotic and inflammatory responses [22, 23]. The initiation of ER stress is thought to be due to conditions associated with protein overload or an increased amount of unfolded proteins. These changes have both physiological and pathological roles causing, in severe cases of stress, an accumulation of these unfolded proteins [7, 24]. Based on the organelle’s role, another activator of ER stress is the high-fat diet [25].

The evidence demonstrating that ER stress is a common feature in NAFLD is increasing [26]. Some studies in mice have demonstrated that the treatment with pharmacological ER stress inducers, such as thapsigargin and tunicamycin, leads to lipid accumulation in the liver [21], and while a moderately induced ER stress causes adaptation and recovery of homeostasis, a severe or prolonged ER stress can ultimately lead to inflammation and apoptosis [7].

It has been suggested that activation of ER stress may trigger various inflammatory pathways, such as c-Jun N-terminal kinases (JNK) and nuclear factor-kappa B signaling pathways, further enhancing NASH progression [26, 27]. Moreover, an adaptive machinery—the unfolded protein response (UPR)—organized by ER transmembrane receptors, is kept inactive under unstressed conditions. The enhancement in the release of GRP78 from the transmembrane receptors leads to their activation with the subsequent use of single mechanism that induces transcription factors and upregulates UPR target genes [21, 28].

The activation of the inositol requiring kinase 1 (which is part of UPR) splices X-box binding protein 1 (XBP-1) mRNA and activates UPR pathway, all of which can cause an inflammatory response and apoptosis [29, 30]. The deletion of XBP-1 inhibits lipid accumulation in mice [31], and there is evidence showing a correlation between deficiency of hepatic XBP-1 and NASH development in humans [32]. XBP-1 also increases JNK phosphorylation, which is followed by apoptosis. This event is correlated with a decrease of XBP-1 and NASH which, according to the authors, proves the role of ER stress in the pathogenesis of the disease [21, 32]. It has been proposed that other molecules, and post-transcriptional and post-translational modifications could contribute to explaining the role of ER stress in NAFLD and its progression, although further investigations are still lacking [21].

Reducing inflammation ameliorates ER stress-induced liver injury. For example, IL-1β-deficient mice show reduced inflammation, hepatocyte death, and liver damage in an ER stress-induced steatohepatitis model [24]. The fibrotic process, which is responsible for the progression of NAFLD, has been proposed as an effect of the activation of the inflammasome within the hepatocytes. Although it is not well understood how this happens [21], the fibrotic process could be due to the upregulation of profibrotic gene SMAD-2 causing the fibrosis, which is likely to contribute to the progression of NAFLD [23, 33].

4. CURRENT THERAPEUTIC STRATEGIES

Nowadays, the management of NAFLD is based on the treatment of the liver disease as well as the associated metabolic comorbidities, such as obesity, hyperlipidemia, insulin resistance and T2DM [11]. Several studies have demonstrated that lifestyle modification may reduce aminotransferases and improve hepatic steatosis when measured either by ultrasound [34] or through magnetic resonance imaging and spectroscopy [35]. Some pharmacological-dietary combined treatments have also been proposed. For example, one study reported that a therapy with orlistat (an enteric lipase inhibitor) in conjunction with lifestyle modification improved ALT levels and steatosis [36]. Nevertheless, taking into account that patients with NAFLD without steatohepatitis have excellent prognosis from liver standpoint, treatments aimed at improving liver disease should be limited to those with NASH [11].

5. NOVEL THERAPEUTIC APPROACHES

Many natural compounds, such as vitamin E and polyphenols, represent potential therapeutic candidates, essentially due to their antioxidant, anti-inflammatory, and antifibrotic properties [37]. For example, there is a study showing that mice with a high-fat and sugar diet in association with pomegranate juice (PJ) consumption, had a decrease of body weight gain, food intake and serum levels of lipids, leptin, and glucose compared with high-fat and high sugar diet model. The outcomes, according to the authors, were a consequence of an upregulation of the hepatic mRNA of different factors, such as hormone-sensitive lipase, pyruvate kinase, fatty acid synthetase, and adiponectin, among others. The results also indicate that PJ reduces the gene expression of hepatic proinflammatory and profibrotic cytokines. The changes presented above are likely to “restore” the metabolic imbalance and favor an anti-inflammatory context with a lower progression of NAFLD. Furthermore, these results could also justify the reduction of TG and hepatic steatosis, generating a reduction in the score of the disease in mice with PJ intake [38].

During the last several years, different natural and pharmacological therapies related to oxidative stress and ER stress have been proposed, after being tested in mice and/or humans. Some of the studied compounds/modalities were: PJ, vitamins E, C, D, and A, toyocamycin, curcumin, and Meretrix meretrix oligopeptides, among others. The hepatic outcomes of the different experiments usually include promising results, such as a decrease of apoptosis, steatosis, proinflammatory and profibrotic gene expression, and an improvement of the redox imbalance [38‒42]. All these outcomes were associated with mechanisms that, in general, include changes in the gene expression (Table 1).

TABLE 1. Novel therapeutic approaches targeting NAFLD oxidative stress and endoplasmic reticulum stress
Ref Compound Dose Duration Model Specie/n Major Outcomes Mechanisms
[39] Vitamin E + symbiotic supplement 400 IU/d of vitamin E + 2 capsules of symbiotic supplement per day 8 weeks Randomized double-blind controlled clinical trial Human/60 ↓Serum levels of ALT, AST, ALP, TG, LDL-c

↓Steatosis, ↓Fibrosis

↓Necroinflammation

↓ROS

↓TNFα

↓TGFβ

[38] Polyphenols (Pomegranate juice) 60 ± 5 ml/day 7 weeks Randomized

HFD

Induced NAFLD

Mice/10 ↓Steatosis, ↓Ballooning

↓Lobular inflammation

↓Portal inflammation

↓Proinflammatory and profibrotic gene expression

↓Serum levels of ALT, AST, TG

↓Expression of TNFα, IL-1β, IL-6

↓TGF-β

[40] Toyocamycin 0.25 mg/kg/day 2 weeks Palmitic acid diet.

Induced NAFLD

Mice/36 ↓Apoptosis, ↓steatosis

↓Mitochondrial dysfunction

↓ER stress

↓Serum levels of ALT, AST, TG, cholesterol

↓XBP-1 expression

↓Bim protein

↓Bax activation.

[41] Polyphenols

(Curcumin)

2 g/kg/day 24 weeks Humans: had NAFLD before the study

Mice: HFD- induced NAFLD

Humans/120

Mice/32

↓Serum levels of cholesterol

↓ weight gain

↓steatosis

↓Inflammation

↓ballooning

↓Leptin-induced TNFα and IFNγ production

↓Linoleic acid-induced ROS generation

[42] Meretix meretrix oligopeptides 10 mg/ml

 

20 mg/ml

24 hours In vitro human cells Liver cells/96-well plates ↓Apoptosis, ↓ROS ↑SOD

↓Mitochondrial dysfunction

↓JNK pathway activation

↓TNFα

[43] Losartan 10 µM

 

 

 

 

 

 

 

 

 

10 mg/kg/day

24 hours

 

 

 

2 days

 

 

 

 

 

3 days

Tunicamycin-induced ER stress

 

TGF-β-, AII-, high glucose-, and albumin-induced ER stress

 

 

 

 

Tunicamycin-induced ER stress

Human HK-2 cells

 

 

 

 

 

 

 

 

Rats/6

↓Tunicamycin-, AII-, high glucose-, and albumin-induced ER stress. ↓BiP expression

↓p-eIF2α expression

↑SIRT1 expression

↑HO-1 and thioredoxin expression

[44] Polyphenols

(Naringenin/ Hesperetin)

15 mg/kg/day 4 weeks 24-month-old Wistar rats Rats/30 ↑AOE activity

↑GSH levels

↑MUFAs content

↑n-3 PUFA content

↓n-6 PUFA content

↑CAT, GPx, SOD2, and GR gene expression
Note: AII, angiotensin II; ALP, alkaline phosphatase; ALT, alanine transferase; AOE, antioxidant   enzymes; AST, aspartate transferase; BiP, binding immunoglobulin protein; CAT, catalase; ER, endoplasmic reticulum; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; HFD, high fat diet; HO-1, heme oxygenase 1; IFNγ, interferon γ; IL-1β, interleukin 1β; IL-6, interleukin 6; JNK, c-Jun N-terminal kinases; LDL-c, low-density lipoprotein cholesterol; MUFAs, monounsaturated fatty acids; n-3 PUFA, n-3 polyunsaturated fatty acids; n-6 PUFA, n-6 polyunsaturated fatty acids; NAFLD, non-alcoholic fatty liver disease; p-eIF2α, phosphorylated eukaryotic initiation factor 2; ROS, reactive oxygen species; SIRT1, silent mating type information regulation 2 homolog 1; SOD, superoxide dismutase;  SOD2, superoxide dismutase 2; TG, triglycerides; TGFβ, transforming growth factor β; TNFα, tumor necrosis factor α; XBP-1, X-box binding protein 1.

6. CONCLUDING REMARKS

It should be noted that NAFLD remains as an unsolved problem in the clinical practice. Nevertheless, oxidative stress and ER stress are two key factors. Accordingly, evidence suggests that novel therapies, such as the use antioxidants, might be beneficial to slow down the progression of the disease. Therefore, it should be expected that targeting the pathophysiology of these factors could contribute to improving the clinical outcome of NAFLD patients. The administration of antioxidant vitamins and PJ could provide a low risk and economic alternative in the near future. It is important to investigate the unknown steps in the genesis and progression of the disease, expanding the knowledge that will not only be beneficial to patients with NAFLD/NASH, but also to those with MS and cardiovascular risk. Thus, it is necessary to enhance investigation about the pharmacokinetic and pharmacodynamic properties of the compounds used as novel therapies. It is necessary to know their therapeutic margins and possible adverse effects to run new clinical trials that may involve combined therapies with antioxidants and drugs, looking forward not only to treating the disease, but also to preventing it.

REFERENCES

  1. Angulo P. Nonalcoholic fatty liver disease. N Engl J Med 2002; 346(16):1221‒31. doi: 10.1056/NEJMra011775.
  2. Ludwig J, Viggiano TR, McGill DB, Oh BJ. Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease. Mayo Clin Proc 1980; 55(7):434‒8.
  3. Day CP. From fat to inflammation. Gastroenterology 2006; 130(1):207‒10. doi: 10.1053/j.gastro.2005.11.017.
  4. Feldstein AE, Werneburg NW, Canbay A, Guicciardi ME, Bronk SF, Rydzewski R, et al. Free fatty acids promote hepatic lipotoxicity by stimulating TNF-alpha expression via a lysosomal pathway. Hepatology 2004; 40(1):185‒94. doi: 10.1002/hep.20283.
  5. Videla LA, Rodrigo R, Orellana M, Fernandez V, Tapia G, Quinones L, et al. Oxidative stress-related parameters in the liver of non-alcoholic fatty liver disease patients. Clin Sci (Lond) 2004; 106(3):261‒8. doi: 10.1042/CS20030285.
  6. Casoinic F, Sampelean D, Buzoianu AD, Hancu N, Baston D. Serum levels of oxidative stress markers in patients with type 2 diabetes mellitus and non-alcoholic steatohepatitis. Rom J Intern Med 2016; 54(4):228‒36. doi: 10.1515/rjim-2016-0035.
  7. Magee N, Zou A, Zhang Y. Pathogenesis of nonalcoholic steatohepatitis: interactions between liver parenchymal and nonparenchymal cells. Biomed Res Int 2016; 2016:5170402. doi: 10.1155/2016/5170402.
  8. Shimomura Y, Takaki A, Wada N, Yasunaka T, Ikeda F, Maruyama T, et al. The serum oxidative/anti-oxidative stress balance becomes dysregulated in patients with non-alcoholic steatohepatitis associated with hepatocellular carcinoma. Intern Med 2017; 56(3):243‒51. doi: 10.2169/internalmedicine.56.7002.
  9. Puri P, Baillie RA, Wiest MM, Mirshahi F, Choudhury J, Cheung O, et al. A lipidomic analysis of nonalcoholic fatty liver disease. Hepatology 2007; 46(4):1081‒90. doi: 10.1002/hep.21763.
  10. Puri P, Wiest MM, Cheung O, Mirshahi F, Sargeant C, Min HK, et al. The plasma lipidomic signature of nonalcoholic steatohepatitis. Hepatology 2009; 50(6):1827‒38. doi: 10.1002/hep.23229.
  11. Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, et al. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the American Gastroenterological Association. Hepatology 2012; 55(6):2005‒23. doi: 10.1002/hep.25762.
  12. Rector RS, Thyfault JP, Wei Y, Ibdah JA. Non-alcoholic fatty liver disease and the metabolic syndrome: an update. World J Gastroenterol 2008; 14(2):185‒92.
  13. Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease: meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology 2016; 64(1):73‒84. doi: 10.1002/hep.28431.
  14. Day CP, James OF. Steatohepatitis: a tale of two “hits”? Gastroenterology 1998; 114(4):842‒5.
  15. Takaki A, Kawai D, Yamamoto K. Multiple hits, including oxidative stress, as pathogenesis and treatment target in non-alcoholic steatohepatitis (NASH). Int J Mol Sci 2013; 14(10):20704‒28. doi: 10.3390/ijms141020704.
  16. Jou J, Choi SS, Diehl AM. Mechanisms of disease progression in nonalcoholic fatty liver disease. Semin Liver Dis 2008; 28(4):370‒9. doi: 10.1055/s-0028-1091981.
  17. Friedman SL. Liver fibrosis in 2012: Convergent pathways that cause hepatic fibrosis in NASH. Nat Rev Gastroenterol Hepatol 2013; 10(2):71‒2. doi: 10.1038/nrgastro.2012.256.
  18. Kohen R, Nyska A. Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicol Pathol 2002; 30(6):620‒50. doi: 10.1080/01926230290166724.
  19. Dufour JF, Oneta CM, Gonvers JJ, Bihl F, Cerny A, Cereda JM, et al. Randomized placebo-controlled trial of ursodeoxycholic acid with vitamin e in nonalcoholic steatohepatitis. Clin Gastroenterol Hepatol 2006; 4(12):1537‒43. doi: 10.1016/j.cgh.2006.09.025.
  20. Liochev SI, Fridovich I. The relative importance of HO˙ and ONOOˉ in mediating the toxicity of O2˙ˉ. Free Radic Biol Med 1999; 26(5‒6):777‒8.
  21. Sozen E, Ozer NK. Impact of high cholesterol and endoplasmic reticulum stress on metabolic diseases: An updated mini-review. Redox Biol 2017; 12:456‒61. doi: 10.1016/j.redox.2017.02.025.
  22. Baiceanu A, Mesdom P, Lagouge M, Foufelle F. Endoplasmic reticulum proteostasis in hepatic steatosis. Nat Rev Endocrinol 2016; 12(12):710‒22. doi: 10.1038/nrendo.2016.124.
  23. Muraki Y, Makita Y, Yamasaki M, Amano Y, Matsuo T. Elevation of liver endoplasmic reticulum stress in a modified choline-deficient l-amino acid-defined diet-fed non-alcoholic steatohepatitis mouse model. Biochem Biophys Res Commun 2017; 486(3):632‒8. doi: 10.1016/j.bbrc.2017.03.072.
  24. Kandel-Kfir M, Almog T, Shaish A, Shlomai G, Anafi L, Avivi C, et al. Interleukin-1alpha deficiency attenuates endoplasmic reticulum stress-induced liver damage and CHOP expression in mice. J Hepatol 2015; 63(4):926‒33. doi: 10.1016/j.jhep.2015.05.012.
  25. Ozcan U, Cao Q, Yilmaz E, Lee AH, Iwakoshi NN, Ozdelen E, et al. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science 2004; 306(5695):457‒61. doi: 10.1126/science.1103160.
  26. Zhang XQ, Xu CF, Yu CH, Chen WX, Li YM. Role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease. World J Gastroenterol 2014; 20(7):1768‒76. doi: 10.3748/wjg.v20.i7.1768.
  27. Meakin PJ, Chowdhry S, Sharma RS, Ashford FB, Walsh SV, McCrimmon RJ, et al. Susceptibility of Nrf2-null mice to steatohepatitis and cirrhosis upon consumption of a high-fat diet is associated with oxidative stress, perturbation of the unfolded protein response, and disturbance in the expression of metabolic enzymes but not with insulin resistance. Mol Cell Biol 2014; 34(17):3305‒20. doi: 10.1128/MCB.00677-14.
  28. Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 2007; 8(7):519‒29. doi: 10.1038/nrm2199.
  29. Wang M, Kaufman RJ. Protein misfolding in the endoplasmic reticulum as a conduit to human disease. Nature 2016; 529(7586):326‒35. doi: 10.1038/nature17041.
  30. Yoshida H, Nadanaka S, Sato R, Mori K. XBP1 is critical to protect cells from endoplasmic reticulum stress: evidence from Site-2 protease-deficient Chinese hamster ovary cells. Cell Struct Funct 2006; 31(2):117‒25.
  31. Lee AH, Scapa EF, Cohen DE, Glimcher LH. Regulation of hepatic lipogenesis by the transcription factor XBP1. Science 2008; 320(5882):1492‒6. doi: 10.1126/science.1158042.
  32. Chapados NA, Lavoie JM. Exercise training increases hepatic endoplasmic reticulum (er) stress protein expression in MTP-inhibited high-fat fed rats. Cell Biochem Funct 2010; 28(3):202‒10. doi: 10.1002/cbf.1643.
  33. Koo JH, Lee HJ, Kim W, Kim SG. Endoplasmic reticulum stress in hepatic stellate cells promotes liver fibrosis via PERK-mediated degradation of HNRNPA1 and up-regulation of SMAD2. Gastroenterology 2016; 150(1):181‒93 e8. doi: 10.1053/j.gastro.2015.09.039.
  34. Sreenivasa Baba C, Alexander G, Kalyani B, Pandey R, Rastogi S, Pandey A, et al. Effect of exercise and dietary modification on serum aminotransferase levels in patients with nonalcoholic steatohepatitis. J Gastroenterol Hepatol 2006; 21(1 Pt 1):191‒8. doi: 10.1111/j.1440-1746.2005.04233.x.
  35. Lazo M, Solga SF, Horska A, Bonekamp S, Diehl AM, Brancati FL, et al. Effect of a 12-month intensive lifestyle intervention on hepatic steatosis in adults with type 2 diabetes. Diabetes Care 2010; 33(10):2156‒63. doi: 10.2337/dc10-0856.
  36. Zelber-Sagi S, Kessler A, Brazowsky E, Webb M, Lurie Y, Santo M, et al. A double-blind randomized placebo-controlled trial of orlistat for the treatment of nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol 2006; 4(5):639‒44. doi: 10.1016/j.cgh.2006.02.004.
  37. Spahis S, Delvin E, Borys JM, Levy E. Oxidative stress as a critical factor in nonalcoholic fatty liver disease pathogenesis. Antioxid Redox Signal 2017; 26(10):519‒41. doi: 10.1089/ars.2016.6776.
  38. Noori M, Jafari B, Hekmatdoost A. Pomegranate juice prevents development of non-alcoholic fatty liver disease in rats by attenuating oxidative stress and inflammation. J Sci Food Agric 2017; 97(8):2327‒32. doi: 10.1002/jsfa.8042.
  39. Ekhlasi G, Kolahdouz Mohammadi R, Agah S, Zarrati M, Hosseini AF, Arabshahi SS, et al. Do symbiotic and Vitamin E supplementation have favorite effects in nonalcoholic fatty liver disease? A randomized, double-blind, placebo-controlled trial. J Res Med Sci 2016; 21:106. doi: 10.4103/1735-1995.193178.
  40. Takahara I, Akazawa Y, Tabuchi M, Matsuda K, Miyaaki H, Kido Y, et al. Toyocamycin attenuates free fatty acid-induced hepatic steatosis and apoptosis in cultured hepatocytes and ameliorates nonalcoholic fatty liver disease in mice. PLoS One 2017; 12(3):e0170591. doi: 10.1371/journal.pone.0170591.
  41. Inzaugarat ME, De Matteo E, Baz P, Lucero D, Garcia CC, Gonzalez Ballerga E, et al. New evidence for the therapeutic potential of curcumin to treat nonalcoholic fatty liver disease in humans. PLoS One 2017; 12(3):e0172900. doi: 10.1371/journal.pone.0172900.
  42. Huang F, Zhao S, Yu F, Yang Z, Ding G. Protective effects and mechanism of meretrix meretrix oligopeptides against nonalcoholic fatty liver disease. Mar Drugs 2017; 15(2). doi: 10.3390/md15020031.
  43. Kim H, Baek CH, Lee RB, Chang JW, Yang WS, Lee SK. Anti-fibrotic effect of losartan, an angiotensin ii receptor blocker, is mediated through inhibition of ER stress via up-regulation of SIRT1, followed by induction of HO-1 and thioredoxin. Int J Mol Sci 2017; 18(2). doi: 10.3390/ijms18020305.
  44. Miler M, Zivanovic J, Ajdzanovic V, Orescanin-Dusic Z, Milenkovic D, Konic-Ristic A, et al. Citrus flavanones naringenin and hesperetin improve antioxidant status and membrane lipid compositions in the liver of old-aged Wistar rats. Exp Gerontol 2016; 84:49-60. doi: 10.1016/j.exger.2016.08.014.