Chinese Journal of Natural Medicines 2015, 13(4): 02500256
Chinese Journal of Natural Medicines
Ethanol promotes saturated fatty acid-induced hepatoxicity through endoplasmic reticulum (ER) stress response YI Hong-Wei1, 2*, MA Yu-Xiang2, WANG Xiao-Ning2, WANG Cui-Fen3, LU Jian1, CAO Wei1, WU Xu-Dong2* 1
Department of Pharmacology, Medical School, Southeast University, Nanjing 210009, China;
2
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China;
3
Center for Diagnostic Nanosystems, Marshall University, Huntington, WV 25755, USA Available online 20 Apr. 2015
[ABSTRACT] Serum palmitic acid (PA), a type of saturated fatty acid, causes lipid accumulation and induces toxicity in hepatocytes. Ethanol (EtOH) is metabolized by the liver and induces hepatic injury and inflammation. Herein, we analyzed the effects of EtOH on PA-induced lipotoxicity in the liver. Our results indicated that EtOH aggravated PA-induced apoptosis and lipid accumulation in primary rat hepatocytes in dose-dependent manner. EtOH intensified PA-caused endoplasmic reticulum (ER) stress response in vitro and in vivo, and the expressions of CHOP, ATF4, and XBP-1 in nucleus were significantly increased. EtOH also increased PA-caused cleaved caspase-3 in cytoplasm. In wild type and CHOP–/– mice treated with EtOH and high fat diet (HFD), EtOH worsened the HFD-induced liver injury and dyslipidemia, while CHOP knockout blocked toxic effects of EtOH and PA. Our study suggested that targeting UPR-signaling pathways is a promising, novel approach to reducing EtOH and saturated fatty acid-induced metabolic complications. [KEY WORDS] Ethanol (EtOH); Palmitic acid (PA); ER stress; CHOP knockout; Liver toxicity
[CLC Number] R965
[Document code] A
[Article ID] 2095-6975(2015)04-0250-07
Introduction Non-alcoholic fatty liver disease (NAFLD) is a pathophysiological condition characterized by fat deposition in the liver in patients without a history of ethanol (EtOH) abuse [1]. NAFLD is an overarching term for liver histopathology that encompasses a spectrum from simple steatosis to non-alcoholic steatohepatitis (NASH). NAFLD poses a major public health problem, due to its high prevalence worldwide and potentially serious sequelae [2].
[Received on] 01-May-2014 [Research funding] This work was supported by National Natural Science Foundation of China (Nos. 81273569, 81001465), Natural Science Foundation of Jiangsu Province, China (No. BK2012726), the Ph.D. Programs Foundation of Ministry of Education of China (No. 20100091120028). [*Corresponding author] Tel/Fax: 86-25-83686552, E-mail: [email protected] (WU Xu-Dong); E-mail: [email protected] (YI Hong-Wei) All the authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
Overconsumption of fatty acids-containing foods is one of the major contributors to NAFLD. Saturated fatty acids csn be found in many kinds of food with high concentration, such as liver tissue and butter. Dietary saturated fatty acids such as palmitic acid (PA) in the liver may provoke endoplasmic reticulum (ER) stress and apoptosis in liver cells [3-4]. Apart from weight loss, there are currently no effective therapies for NAFLD [5]. Alcoholic liver disease (ALD) caused by chronic EtOH consumption, presents as a broad spectrum of disorders, ranging from simple fatty liver to more severe forms of liver injury, including alcoholic hepatitis (AH), cirrhosis, and superimposed hepatocellular carcinoma (HCC) [6]. The fact that only about 35% of heavy drinkers develop advanced ALD indicates that other factors are involved [6], including obesity, drinking patterns, dietary factors, non–sex-linked genetic factors, and cigarette smoking. Some people with obesity also have chronic EtOH consumption, and are more vulnerable to liver injury [7]. Moreover, experimental studies indicate that the synergistic effects of obesity and EtOH abuse involve the ER response to cell stress, type I macrophage activation, and adiponectin resistance [8], but the detailed mechanism remains
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unclear. ER is a specialized organelle that is integral in many cellular functions, particularly disulfide bond formation, proper protein folding, and synthesis and secretion of several critical biomolecules including steroids, cholesterol, and lipids [9]. Changes in cellular redox state or abnormal accumulation of toxic lipid species can result in activation of the unfolded protein response (UPR) and ER stress [10]. ER stress has been observed in liver and adipose tissue of patients with NAFLD and/or obesity [10]. Importantly, the signaling pathway activated by disruption of endoplasmic reticulum homeostasis, the unfolded protein response, has been linked to lipid and membrane biosynthesis, insulin action, inflammation, and apoptosis [11]. In the present study, we attempted to investigate effects of EtOH on PA-induced hepatocyte injury and the underlying mechanism(s). Our results indicated that EtOH promoted PA-induced hepatic lipid accumulation and inflammation by activating ER stress response, and CHOP knockout significantly increased the aggravation of EtOH on PA-caused toxic effects in liver. These results would help develop novel approach to preventing NAFLD and ALD, especially in people with obesity.
Methods and Materials Materials Palmitic acid (PA), bovine serum albumin (BSA) (fatty acid-free), and EtOH (200 proof, absolute) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Anti-CHOP, anti-ATF4, anti-XBP-1, anti-Lamin B, anti-cleaved caspase 3, and anti-Actin antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Fetal bovine serum (FBS) and Dulbecco's Modified Eagle Medium (DMEM) were purchased from Life Technology (Carlsbad, CA, USA). Normal chow diet (NCD) and high fat diet (HFD) were purchased from Research Diet (New Brunswick, NL, USA). All other chemicals and reagents used in the present study were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Primary rat hepatocytes culture Primary hepatocytes were isolated from Sprague Dawley (SD) rats (male, 8 weeks old) and cultured as described previously [12]. The hepatocytes were cultured in serum-free Williams’ E medium containing dexamethasone (0.1 μmol·L−1), penicillin (100 U·mL−1), and thyroxine (1 μmol·L−1), in a 5% CO2 environment at 37 °C, before additions to culture medium. Cells treatment PA was conjugated with BSA and the conjugation was added to medium to a final concentration of 200 or 400 μmol·L−1. BSA was used to increase cell uptake of PA. EtOH was added to medium to a final concentration of 1 or 5 μg·mL−1. The cultured primary rat hepatocytes were treated by different concentration of PA (0, 200, 400 μmol·L−1) with or without different concentration of EtOH (0, 1.0, 5.0
μg·mL−1) respectively for 24 h. Apoptosis analysis Annexin-V-FITC/PI double staining assay was used to detect apoptosis. Hepatocytes were treated by PA with or without EtOH respectively for 24 h. The cells were harvested and resuspended in Annexin-V binding buffer. The suspension was incubated with 2.5 μL of Annexin V-FITC and 2 μL of PI for 10 min at room temperature in the dark, followed by flow cytometric analysis (EPICS XL, Beckman Coulter, Brea city, CA, USA) within 30 min of staining. All experiments were performed in triplicate. Nile red staining Nile red staining was used to specifically stain the intracellular fat. The cells were fixed and stained with Nile red (100 ng·mL−1) in PBS for 30 min. After being washed thrice with PBS, the cells were examined under a fluorescent microscope at an excitation wavelength of 488 nm. Western blotting analysis Nucleus and cytoplasm protein lysates were separated by 10% SDS-PAGE and subsequently electrotransferred into a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). Then, the membrane was blocked with 5% nonfat milk for 2 h at room temperature. The blocked membrane was incubated with primary antibodies at 4 °C overnight. After incubated with a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature, protein bands were visualized using the Western blotting detection system according to the manufacturer’s instructions (Cell Signaling Technology, Danvers XX, MA, USA). Real-time PCR Total RNA was extracted from liver tissues, reverse transcribed to cDNA, and subjected to quantitative PCR, which was performed with the BioRad CFX96 ouch™ Real-Time PCR Detection System (BioRad, Hercules city, CA, USA) using iQ™ SYBR® Green permix (BioRad), and threshold cycle numbers were obtained using BioRad CFX manager software. The program for amplification was 1 cycle of 95 °C for 2 min followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. The primer sequences used in this study were as follows: mouse Chop, 5'-CAC ATC CCA AAG CCC TCG CTC TC-3' (forward) and 5'-TCA TGC TTG GTG CAG GCT GAC CAT-3' (reverse); mouse Atf4, 5'-GGA ATG GCC GGC TAT GG-3' (forward) and 5'-TCC CGG AAA AGG CAT CCT-3’ (reverse); mouse Xbp-1s, 5'-AAA CAG AGT AGC AGC TCA GAC TGC-3' (forward) and 5'-TCC TTC TGG GTA GAC CTC TGG GAG-3' (reverse); mouse Gapdh, 5'-GTC TAC TGG TGT CTT CAC CA-3 (forward) and 5'–GTG GCA GTG ATG GCA TGG AC-3' (reverse). Animal studies C57BL/6J mice and CHOP knockout C57BL/6J mice (CHOP–/–; back-crossed at least 12 generations to the C57BL/6J background) were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). All experiments and procedures involving mice were approved by the Ethics Re-
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view Comittes of Nanjing University and were conducted in accord with the Declaration of Helsinki, the Guide for the Care and Use of Animals (National Academies Press, Washington, DC, 1996). All efforts were made to minimize animals’ suffering and to reduce the number of animals used. To examine the role of CHOP in EtOH-promoted PA’s toxic effects in vivo, wild type mice and CHOP–/– mice (6–8 weeks old) were randomly assigned to four groups (n = 8): (1) NCD; (2) EtOH; (3) HFD; (4) HFD with EtOH. Mice were fed with HFD with or without intragastric administration of EtOH at 2 g·kg−1·d−1 for 4 weeks. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), free cholesterol, total cholesterol and total triglycerides were measured using commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Histology analysis Liver tissues were collected and fixed in 4% paraformaldehyde in 0.1 mol·L−1 of PBS at room temperature overnight. The regions of specimens collected were standardized for all the mice. The samples The sample were stained with Hematoxylin and Eosin (H&E) and examined in a blind manner to evaluate the presence of steatosis, inflammation, and fibrosis.
Statistical analysis The experimental data were expressed as mean ± SEM. The experiments were repeated three times. One-way ANOVA, followed by Student’s t-test, was used for the statistical analysis. The differences were considered statistically significant when P < 0.05.
Results EtOH aggravated PA-induced apoptosis in primary rat hepatocytes Apoptosis in primary rat hepatocytes was first examined after the cells were treated with PA with or without EtOH. As shown in Fig. 1A, PA at 200 μmol·L−1 induced apoptosis significantly. EtOH at 1 or 5 μg·mL−1 also induced hepatocytes apoptosis, as shown in Fig. 1A and Fig. 1B. Moreover, EtOH dose-dependently aggravated PA’s effects on apoptosis, with the percentage of apoptotic cells being from 14% (PA only) to 20% when combined with 1 μg·mL−1 of EtOH, and to nearly 27% when combined with 5 μg·mL−1 of EtOH. As shown in Fig. 1B, PA at 400 μmol·L−1 was more toxic than at 200 μmol·L−1, and the similar pattern was found for the EtOHincreased PA-induced apoptosis at two EtOH levels.
Fig. 1 EtOH aggravates the PA-induced apoptosis in primary rat hepatocytes. Hepatocytes were treated by PA (200 μmol·L−1 (A), 400 μmol·L−1 (B) with or without EtOH (1.0, 5.0 μg·mL−1) for 24 h, then stained with annexin V-FITC/propidium iodide. The apoptotic cells were detected by FACSCalibur Flow Cytometer
EtOH increased PA-enhanced lipid accumulation PA and EtOH both can cause disorder of lipid metabolism in liver. Our results demonstrated that, EtOH increased PA-enhanced lipid accumulation in primary rat hepatocytes (Fig. 2). EtOH greatly increased PA-upraised fluorescent intensity (fluorescent pictures), and more lipid plaques (bright field pictures) were seen in the cells treated with PA plus EtOH.
EtOH intensified PA-caused ER stress in vitro and in vivo ER stress is an important factor for lipid metabolism and cell apoptosis. Next, we detected ER stress response by PA and EtOH treatment. As shown in Fig. 3A, PA induced moderate ER stress in hepatocytes; the expressions of CHOP, XBP-1, and ATF4 in nucleus extracts were increased. Co-treatment PA with EtOH, the ER stress response was significantly intensified, and the expression
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levels of CHOP, XBP-1 and ATF4 were significantly higher than that of treatment of PA alone. In cytoplasm extracts, cleaved caspase 3 was raised by EtOH plus PA. EtOH alone also induced the ER stress response to some extent. In animal liver tissues EtOH promoted HFD-induced Chop, Xbp-1 and Atf4 mRNA expressions (Fig. 3B).
CHOP knockout blocked EtOH and HFD-induced liver injury and dyslipidemia CHOP is the core regulator of ER stress. In the present study, we further used wild type and CHOP–/– mice to access effects of EtOH and HFD in vivo. Histologic analysis (Figs. 4A and 4B) showed that EtOH at 2 g·kg−1·d−1 induced
Fig. 2 EtOH increases PA-induced lipid accumulation in primary rat hepatocytes. Hepatocytes were treated by PA (200 μmol·L−1 (A), and 400 μmol·L−1 (B) with or without various amount of EtOH (1.0 and 5.0 μg·mL−1) for 24 h. Cellular lipid were stained by nile red and detected under a fluorescent microscope
Fig. 3 EtOH intensifies PA-caused ER stress and caspase activation in vitro and in vivo. (A) Representative immunoblots of CHOP, XBP-1, and ATF-4 in the nucleus, and cleaved caspase 3 in cytoplasm of primary rat hepatocytes, treated by PA with or without EtOH (5 μg·mL−1) for 24 h. Lamin B and Actin were used as loading control. (B) Real-time quantitative PCR of ER stress factors in liver. C57/BL6 mice were fed with HFD with or without EtOH (2 g·kg−1·d−1) for 4 weeks. Total liver mRNA was isolated using Trizol and the first-strand cDNA was synthesized using the high-capacity cDNA archive kit. The mRNA levels of Chop, Atf4, and Xbp-1 were determined using SYBR Green Supermix
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inflammatory cells infiltration in the liver of wild type mice and HFD increased lipid accumulation. What’s more, the co-treatment of EtOH greatly worsened the HFD-induced inflammation and steatosis, and caused hepatocellular ballooning. However, CHOP knockout significantly reduced the EtOH and HFD-induced liver injury. The results of serum transaminase assay indicated that 2 g·kg−1·d−1 EtOH
and HFD greatly increased ALT and AST levels in wild type mice, which was remarkably improved in CHOP–/– mice (Figs. 4C and 4D). The results of serum lipid assay (Fig. 5) indicated that EtOH and HFD-induced dyslipidemia was blocked in CHOP–/– mice, compared with the wild type mice. Moreover, the ratio of liver/weight also was reversed by CHOP knockout.
Fig. 4 CHOP knockout reduceds the effects of liver injury induced by HFD and EtOH. H&E staining of wild type (A) and CHOP–/– (B) mice liver sections treated by HFD with or without EtOH (2 g·kg−1·d−1) for 4 weeks. Those mice serum ALT (C) and AST (D) were measured as described in methods. **P < 0.01 vs wild type mice (Student Two-Tailed t-test)
Discussion Cell death in the liver occurs mainly by apoptosis or necrosis, although other forms of cell death may occasionally occur. Liver apoptosis can be devided into extrinsic apoptosis and intrinsic apoptosis. The extrinsic apoptosis refers to a signaling pathway triggered by the binding of death receptors to their cognate ligands. Death receptors include Fas, TNF-αreceptor 1, TRAIL-R1, and TRAIL-R2, all of which are ubiquitously expressed in the liver to various extents. Their ligands (FasL/CD95L, TNF-α, and TRAIL) are mainly expressed by cells of the immune system and play a fundamental role in the elimination of virally infected, transformed, or damaged liver cells. In the present study, we found that PA and EtOH induced the primary rat liver apoptosis; the number of apoptotic cells was increased (Fig. 1), andmoreover, the levels of cleaved caspase 3 in PA or EtOH treated hepatocyte plasm were also increased (Fig. 3A). The treatment with PA
or EtOH resulted in lipid accumulation (Fig. 2). PA and EtOH could respectively cause hepatocytes apoptosis and lipid accumulation; while the effect of PA was greatly enhanced by EtOH; the content of lipid in the liver cells were increased after the co-treated with PA and EtOH, and the number of apoptotic cells were increased too (Figs. 1 and 2). We found that PA or EtOH induced liver cells apoptosis was not through the extrinsic pathway (data not show). PA can induce liver cell apoptosis through mitochondrial and ER pathway [3, 13-14]. EtOH could also induce liver cell apoptosis through the intrinsic pathway [15]. The intrinsic apoptosis can be triggered by a variety of intracellular stress inducers, including DNA damage, oxidative stress, UV and γ-irradiation, toxins, growth factor deprivation, and endoplasmic reticulum (ER) stress. PA induced liver cells mitochondrial membrane potential collapse (data not shown), and cause ER stress (Fig. 3), and its effect on ER stress was enhanced by EtOH. The ER stress signaling pathway is initiated
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Fig. 5 Plasma lipid levels and liver index are increased by HFD and EtOH co-treatment in wild type mice, which is reversed by CHOP knockout. Wild type and CHOP–/– mice were fed by HFD with or without EtOH (2 g·kg−1·d−1) for 4 weeks. (A) Free cholesterol, (B) Total cholesterol, (C) Total Triglyceride, and (D) Liver/Weight (g·g−1)
by three main ER transmembrane proteins, PERK, IRE-1, and ATF6, which together promote transcription of genes designed to increase protein folding and degradation [16]. IRE-1 splices XBP-1 mRNA to generate a transcription factor which leads to ER adaptation by activating a large number of unfolded protein response genes. PERK phosphorylates eIF2α, leading to an influence of ATF4 and CHOP. The ER stress in rat primary liver cells was induced by EtOH or PA, and the expressions of XBP-1, ATF4 and CHOP in nucleus were increased (Fig. 3A). The effect of EtOH was less than that of PA in liver cells. But when the cells were treated with PA and EtOH together, the ER stress was more serious. CHOP is a factor that mediates liver damage following diverse types of stress responses. CHOP transcription is primarily activated by ATF4 although ATF6a may also contribute. CHOP is the most well characterized proapoptotic pathway that emanates from the stressed ER. CHOP can induce the expression of proapoptotic BH3-only protein Bim, the cell surface death receptor TRAIL receptor 2, and other downstream of CHOP mRNAs, and inhibit Bcl-2 transcription [17] . Cholestasis induces CHOP-mediated ER stress and triggers hepatocyte cell death, and CHOP deficiency attenuates this cell death and subsequent liver fibrosis [18]. HFD induced liver injury through lipid accumulation and ER stress, and the mRNA levels of XBP-1, ATF4 and CHOP were increased (Fig. 3B). EtOH could increase the expression of ATF4 and CHOP, but had no effect on the expression of XBP-1. EtOH could
enhance the effect of PA, promoting ER stress in mice and increasing the mRNA levels of XBP-1, ATF4 and CHOP in the liver (Fig. 3B). In addition, CHOP deletion reduces oxidative stress, improves beta-cell function, and promotes cell survival in mouse with diabetes [19]. Our study showed that in wild type mice, HFD with high concentration of PA caused hepatic apoptosis, lipid accumulation and increases in serum ALT and AST in the liver, and EtOH, the possible risk factor, dose dependently aggravated lipotoxicity of PA via activating ER stress. However, in CHOP–/– mice, hepatic steatosis, serum aminotransferase increase and serum lipid accumulation were obviously attenuated, and the promoting effects of EtOH on HFD-induced liver injury was significantly blocked (Figs. 4–5). These finding suggested that promoting effects of EtOH on PA action were dependent on CHOP activation. Considering of these results, target CHOP will be a feasible treatment strategy for NAFLD patients with EtOH abuse. Hepatic steatosis is the result of dysregulation of lipid metabolism caused by increased lipid uptake, increased lipid synthesis, and reduced lipid oxidation and metabolism. Effect of EtOH on HFD-induced lipid accumulation indicated that CHOP also plays an important role in regulating the lipid metabolism. Inflammation is an important contributor to hepatic lipotoxicity [20–21]. Histological analysis showed that EtOH-induced inflammatory cells infiltration was also significantly reduced in CHOP–/– mice. Our studies suggested that the ER
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stress-mediated signaling might be as a potential connection for inflammation and metabolic disease [22]. In summary, our results demonstrated that EtOH promoted PA–induced activation of UPR, especially the upregulation of CHOP, representing an important molecular mechanism underlying EtOH and PA–associated dyslipidemia, inflammation, and hepatic lipotoxicity. Our studies suggest that targeting UPR-signaling pathways is a promising, novel approach to reducing EtOH and saturated fatty acid-induced metabolic complications.
References [1] [2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Sass DA, Chang P, Chopra KB. Nonalcoholic fatty liver disease: a clinical review [J]. Dig Dis Sci, 2005, 50(1): 171-180. Berlanga A, Guiu-Jurado E, Porras JA, et al. Molecular pathways in non-alcoholic fatty liver disease [J]. Clin Exp Gastroenterol, 2014, 7: 221-239. Cao J, Dai DL, Yao L, et al. Saturated fatty acid induction of endoplasmic reticulum stress and apoptosis in human liver cells via the PERK/ATF4/CHOP signaling pathway [J]. Mol Cell Biochem, 2012, 364(1-2): 115-129. Akazawa Y, Cazanave S, Mott JL, et al. Palmitoleate attenuates palmitate-induced Bim and PUMA up-regulation and hepatocyte lipoapoptosis [J]. J Hepatol, 2010, 52(4): 586-593. Kawano Y, Cohen DE. Mechanisms of hepatic triglyceride accumulation in non-alcoholic fatty liver disease [J]. J Gastroenterol, 2013, 48(4): 434-441. Gao B, Bataller R. Alcoholic liver disease: pathogenesis and new therapeutic targets [J]. Gastroenterology, 2011, 141(5): 1572-1585. Tsai J, Ford ES, Zhao G, et al. Co-occurrence of obesity and patterns of alcohol use associated with elevated serum hepatic enzymes in US adults [J]. J Behav Med, 2012, 35(2): 200-210. Xu J, Lai KK, Verlinsky A, et al. Synergistic steatohepatitis by moderate obesity and alcohol in mice despite increased adiponectin and p-AMPK [J]. J Hepatol, 2011, 55(3): 673-682. Leamy AK, Egnatchik RA, Young JD. Molecular mechanisms and the role of saturated fatty acids in the progression of nonalcoholic fatty liver disease [J]. Prog Lipid Res, 2013, 52(1): 165-174.
[10] Dara L, Ji C, Kaplowitz N. The contribution of endoplasmic reticulum stress to liver diseases [J]. Hepatology, 2011, 53(5): 1752-1763. [11] Pagliassotti MJ. Endoplasmic reticulum stress in nonalcoholic fatty liver disease [J]. Annu Rev Nutr, 2012, 32: 17-33. [12] Zhou H, Gurley EC, Jarujaron S, et al. HIV protease inhibitors activate the unfolded protein response and disrupt lipid metabolism in primary hepatocytes [J]. Am J Physiol Gastrointest Liver Physiol, 2006, 291(6): G1071-1080. [13] Wu X, Zhang L, Gurley E, et al. Prevention of free fatty acid-induced hepatic lipotoxicity by 18beta-glycyrrhetinic acid through lysosomal and mitochondrial pathways [J]. Hepatology, 2008, 47(6): 1905-1915. [14] Ji J, Zhang L, Wang P, et al. Saturated free fatty acid, palmitic acid, induces apoptosis in fetal hepatocytes in culture [J]. Exp Toxicol Pathol, 2005, 56(6): 369-376. [15] Ishii H, Adachi M, Fernandez-Checa JC, et al. Role of apoptosis in alcoholic liver injury [J]. Alcohol Clin Exp Res, 2003, 27(7): 1207-1212. [16] Leamy AK, Egnatchik RA, Young JD. Molecular mechanisms and the role of saturated fatty acids in the progression of non-alcoholic fatty liver disease [J]. Prog Lipid Res, 2013, 52(1): 165-174. [17] Malhi H, Kaufman RJ. Endoplasmic reticulum stress in liver disease [J]. J Hepatol, 2011, 54(4): 795-809. [18] Tamaki N, Hatano E, Taura K, et al. CHOP deficiency attenuates cholestasis-induced liver fibrosis by reduction of hepatocyte injury [J]. Am J Physiol Gastrointest Liver Physiol, 2008, 294(2): G498-505. [19] Song B, Scheuner D, Ron D, et al. Chop deletion reduces oxidative stress, improves beta cell function, and promotes cell survival in multiple mouse models of diabetes [J]. J Clin Invest, 2008, 118(10): 3378-3389. [20] Navab M, Gharavi N, Watson AD. Inflammation and metabolic disorders [J]. Curr Opin Clin Nutr Metab Care, 2008, 11(4): 459-464. [21] Harmon RC, Tiniakos DG, Argo CK. Inflammation in nonalcoholic steatohepatitis [J]. Expert Rev Gastroenterol Hepatol, 2011, 5(2): 189-200. [22] Hotamisligil GS. Endoplasmic reticulum stress and the inflammatory basis of metabolic disease [J]. Cell, 2010, 140(6): 900-917.
Cite this article as: YI Hong-Wei, MA Yu-Xiang, WANG Xiao-Ning, WANG Cui-Fen, LU Jian, CAO Wei, WU Xu-Dong. Ethanol promotes saturated fatty acid-induced hepatoxicity through endoplasmic reticulum (ER) stress response [J]. Chinese Journal of Natural Medicines, 2015, 13(4): 250-256
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Chinese Journal of Natural Medicines
Ethanol promotes saturated fatty acid-induced hepatoxicity through endoplasmic reticulum (ER) stress response YI Hong-Wei1, 2*, MA Yu-Xiang2, WANG Xiao-Ning2, WANG Cui-Fen3, LU Jian1, CAO Wei1, WU Xu-Dong2* 1
Department of Pharmacology, Medical School, Southeast University, Nanjing 210009, China;
2
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China;
3
Center for Diagnostic Nanosystems, Marshall University, Huntington, WV 25755, USA Available online 20 Apr. 2015
[ABSTRACT] Serum palmitic acid (PA), a type of saturated fatty acid, causes lipid accumulation and induces toxicity in hepatocytes. Ethanol (EtOH) is metabolized by the liver and induces hepatic injury and inflammation. Herein, we analyzed the effects of EtOH on PA-induced lipotoxicity in the liver. Our results indicated that EtOH aggravated PA-induced apoptosis and lipid accumulation in primary rat hepatocytes in dose-dependent manner. EtOH intensified PA-caused endoplasmic reticulum (ER) stress response in vitro and in vivo, and the expressions of CHOP, ATF4, and XBP-1 in nucleus were significantly increased. EtOH also increased PA-caused cleaved caspase-3 in cytoplasm. In wild type and CHOP–/– mice treated with EtOH and high fat diet (HFD), EtOH worsened the HFD-induced liver injury and dyslipidemia, while CHOP knockout blocked toxic effects of EtOH and PA. Our study suggested that targeting UPR-signaling pathways is a promising, novel approach to reducing EtOH and saturated fatty acid-induced metabolic complications. [KEY WORDS] Ethanol (EtOH); Palmitic acid (PA); ER stress; CHOP knockout; Liver toxicity
[CLC Number] R965
[Document code] A
[Article ID] 2095-6975(2015)04-0250-07
Introduction Non-alcoholic fatty liver disease (NAFLD) is a pathophysiological condition characterized by fat deposition in the liver in patients without a history of ethanol (EtOH) abuse [1]. NAFLD is an overarching term for liver histopathology that encompasses a spectrum from simple steatosis to non-alcoholic steatohepatitis (NASH). NAFLD poses a major public health problem, due to its high prevalence worldwide and potentially serious sequelae [2].
[Received on] 01-May-2014 [Research funding] This work was supported by National Natural Science Foundation of China (Nos. 81273569, 81001465), Natural Science Foundation of Jiangsu Province, China (No. BK2012726), the Ph.D. Programs Foundation of Ministry of Education of China (No. 20100091120028). [*Corresponding author] Tel/Fax: 86-25-83686552, E-mail: [email protected] (WU Xu-Dong); E-mail: [email protected] (YI Hong-Wei) All the authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
Overconsumption of fatty acids-containing foods is one of the major contributors to NAFLD. Saturated fatty acids csn be found in many kinds of food with high concentration, such as liver tissue and butter. Dietary saturated fatty acids such as palmitic acid (PA) in the liver may provoke endoplasmic reticulum (ER) stress and apoptosis in liver cells [3-4]. Apart from weight loss, there are currently no effective therapies for NAFLD [5]. Alcoholic liver disease (ALD) caused by chronic EtOH consumption, presents as a broad spectrum of disorders, ranging from simple fatty liver to more severe forms of liver injury, including alcoholic hepatitis (AH), cirrhosis, and superimposed hepatocellular carcinoma (HCC) [6]. The fact that only about 35% of heavy drinkers develop advanced ALD indicates that other factors are involved [6], including obesity, drinking patterns, dietary factors, non–sex-linked genetic factors, and cigarette smoking. Some people with obesity also have chronic EtOH consumption, and are more vulnerable to liver injury [7]. Moreover, experimental studies indicate that the synergistic effects of obesity and EtOH abuse involve the ER response to cell stress, type I macrophage activation, and adiponectin resistance [8], but the detailed mechanism remains
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unclear. ER is a specialized organelle that is integral in many cellular functions, particularly disulfide bond formation, proper protein folding, and synthesis and secretion of several critical biomolecules including steroids, cholesterol, and lipids [9]. Changes in cellular redox state or abnormal accumulation of toxic lipid species can result in activation of the unfolded protein response (UPR) and ER stress [10]. ER stress has been observed in liver and adipose tissue of patients with NAFLD and/or obesity [10]. Importantly, the signaling pathway activated by disruption of endoplasmic reticulum homeostasis, the unfolded protein response, has been linked to lipid and membrane biosynthesis, insulin action, inflammation, and apoptosis [11]. In the present study, we attempted to investigate effects of EtOH on PA-induced hepatocyte injury and the underlying mechanism(s). Our results indicated that EtOH promoted PA-induced hepatic lipid accumulation and inflammation by activating ER stress response, and CHOP knockout significantly increased the aggravation of EtOH on PA-caused toxic effects in liver. These results would help develop novel approach to preventing NAFLD and ALD, especially in people with obesity.
Methods and Materials Materials Palmitic acid (PA), bovine serum albumin (BSA) (fatty acid-free), and EtOH (200 proof, absolute) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Anti-CHOP, anti-ATF4, anti-XBP-1, anti-Lamin B, anti-cleaved caspase 3, and anti-Actin antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Fetal bovine serum (FBS) and Dulbecco's Modified Eagle Medium (DMEM) were purchased from Life Technology (Carlsbad, CA, USA). Normal chow diet (NCD) and high fat diet (HFD) were purchased from Research Diet (New Brunswick, NL, USA). All other chemicals and reagents used in the present study were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Primary rat hepatocytes culture Primary hepatocytes were isolated from Sprague Dawley (SD) rats (male, 8 weeks old) and cultured as described previously [12]. The hepatocytes were cultured in serum-free Williams’ E medium containing dexamethasone (0.1 μmol·L−1), penicillin (100 U·mL−1), and thyroxine (1 μmol·L−1), in a 5% CO2 environment at 37 °C, before additions to culture medium. Cells treatment PA was conjugated with BSA and the conjugation was added to medium to a final concentration of 200 or 400 μmol·L−1. BSA was used to increase cell uptake of PA. EtOH was added to medium to a final concentration of 1 or 5 μg·mL−1. The cultured primary rat hepatocytes were treated by different concentration of PA (0, 200, 400 μmol·L−1) with or without different concentration of EtOH (0, 1.0, 5.0
μg·mL−1) respectively for 24 h. Apoptosis analysis Annexin-V-FITC/PI double staining assay was used to detect apoptosis. Hepatocytes were treated by PA with or without EtOH respectively for 24 h. The cells were harvested and resuspended in Annexin-V binding buffer. The suspension was incubated with 2.5 μL of Annexin V-FITC and 2 μL of PI for 10 min at room temperature in the dark, followed by flow cytometric analysis (EPICS XL, Beckman Coulter, Brea city, CA, USA) within 30 min of staining. All experiments were performed in triplicate. Nile red staining Nile red staining was used to specifically stain the intracellular fat. The cells were fixed and stained with Nile red (100 ng·mL−1) in PBS for 30 min. After being washed thrice with PBS, the cells were examined under a fluorescent microscope at an excitation wavelength of 488 nm. Western blotting analysis Nucleus and cytoplasm protein lysates were separated by 10% SDS-PAGE and subsequently electrotransferred into a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). Then, the membrane was blocked with 5% nonfat milk for 2 h at room temperature. The blocked membrane was incubated with primary antibodies at 4 °C overnight. After incubated with a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature, protein bands were visualized using the Western blotting detection system according to the manufacturer’s instructions (Cell Signaling Technology, Danvers XX, MA, USA). Real-time PCR Total RNA was extracted from liver tissues, reverse transcribed to cDNA, and subjected to quantitative PCR, which was performed with the BioRad CFX96 ouch™ Real-Time PCR Detection System (BioRad, Hercules city, CA, USA) using iQ™ SYBR® Green permix (BioRad), and threshold cycle numbers were obtained using BioRad CFX manager software. The program for amplification was 1 cycle of 95 °C for 2 min followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. The primer sequences used in this study were as follows: mouse Chop, 5'-CAC ATC CCA AAG CCC TCG CTC TC-3' (forward) and 5'-TCA TGC TTG GTG CAG GCT GAC CAT-3' (reverse); mouse Atf4, 5'-GGA ATG GCC GGC TAT GG-3' (forward) and 5'-TCC CGG AAA AGG CAT CCT-3’ (reverse); mouse Xbp-1s, 5'-AAA CAG AGT AGC AGC TCA GAC TGC-3' (forward) and 5'-TCC TTC TGG GTA GAC CTC TGG GAG-3' (reverse); mouse Gapdh, 5'-GTC TAC TGG TGT CTT CAC CA-3 (forward) and 5'–GTG GCA GTG ATG GCA TGG AC-3' (reverse). Animal studies C57BL/6J mice and CHOP knockout C57BL/6J mice (CHOP–/–; back-crossed at least 12 generations to the C57BL/6J background) were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). All experiments and procedures involving mice were approved by the Ethics Re-
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view Comittes of Nanjing University and were conducted in accord with the Declaration of Helsinki, the Guide for the Care and Use of Animals (National Academies Press, Washington, DC, 1996). All efforts were made to minimize animals’ suffering and to reduce the number of animals used. To examine the role of CHOP in EtOH-promoted PA’s toxic effects in vivo, wild type mice and CHOP–/– mice (6–8 weeks old) were randomly assigned to four groups (n = 8): (1) NCD; (2) EtOH; (3) HFD; (4) HFD with EtOH. Mice were fed with HFD with or without intragastric administration of EtOH at 2 g·kg−1·d−1 for 4 weeks. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), free cholesterol, total cholesterol and total triglycerides were measured using commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Histology analysis Liver tissues were collected and fixed in 4% paraformaldehyde in 0.1 mol·L−1 of PBS at room temperature overnight. The regions of specimens collected were standardized for all the mice. The samples The sample were stained with Hematoxylin and Eosin (H&E) and examined in a blind manner to evaluate the presence of steatosis, inflammation, and fibrosis.
Statistical analysis The experimental data were expressed as mean ± SEM. The experiments were repeated three times. One-way ANOVA, followed by Student’s t-test, was used for the statistical analysis. The differences were considered statistically significant when P < 0.05.
Results EtOH aggravated PA-induced apoptosis in primary rat hepatocytes Apoptosis in primary rat hepatocytes was first examined after the cells were treated with PA with or without EtOH. As shown in Fig. 1A, PA at 200 μmol·L−1 induced apoptosis significantly. EtOH at 1 or 5 μg·mL−1 also induced hepatocytes apoptosis, as shown in Fig. 1A and Fig. 1B. Moreover, EtOH dose-dependently aggravated PA’s effects on apoptosis, with the percentage of apoptotic cells being from 14% (PA only) to 20% when combined with 1 μg·mL−1 of EtOH, and to nearly 27% when combined with 5 μg·mL−1 of EtOH. As shown in Fig. 1B, PA at 400 μmol·L−1 was more toxic than at 200 μmol·L−1, and the similar pattern was found for the EtOHincreased PA-induced apoptosis at two EtOH levels.
Fig. 1 EtOH aggravates the PA-induced apoptosis in primary rat hepatocytes. Hepatocytes were treated by PA (200 μmol·L−1 (A), 400 μmol·L−1 (B) with or without EtOH (1.0, 5.0 μg·mL−1) for 24 h, then stained with annexin V-FITC/propidium iodide. The apoptotic cells were detected by FACSCalibur Flow Cytometer
EtOH increased PA-enhanced lipid accumulation PA and EtOH both can cause disorder of lipid metabolism in liver. Our results demonstrated that, EtOH increased PA-enhanced lipid accumulation in primary rat hepatocytes (Fig. 2). EtOH greatly increased PA-upraised fluorescent intensity (fluorescent pictures), and more lipid plaques (bright field pictures) were seen in the cells treated with PA plus EtOH.
EtOH intensified PA-caused ER stress in vitro and in vivo ER stress is an important factor for lipid metabolism and cell apoptosis. Next, we detected ER stress response by PA and EtOH treatment. As shown in Fig. 3A, PA induced moderate ER stress in hepatocytes; the expressions of CHOP, XBP-1, and ATF4 in nucleus extracts were increased. Co-treatment PA with EtOH, the ER stress response was significantly intensified, and the expression
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levels of CHOP, XBP-1 and ATF4 were significantly higher than that of treatment of PA alone. In cytoplasm extracts, cleaved caspase 3 was raised by EtOH plus PA. EtOH alone also induced the ER stress response to some extent. In animal liver tissues EtOH promoted HFD-induced Chop, Xbp-1 and Atf4 mRNA expressions (Fig. 3B).
CHOP knockout blocked EtOH and HFD-induced liver injury and dyslipidemia CHOP is the core regulator of ER stress. In the present study, we further used wild type and CHOP–/– mice to access effects of EtOH and HFD in vivo. Histologic analysis (Figs. 4A and 4B) showed that EtOH at 2 g·kg−1·d−1 induced
Fig. 2 EtOH increases PA-induced lipid accumulation in primary rat hepatocytes. Hepatocytes were treated by PA (200 μmol·L−1 (A), and 400 μmol·L−1 (B) with or without various amount of EtOH (1.0 and 5.0 μg·mL−1) for 24 h. Cellular lipid were stained by nile red and detected under a fluorescent microscope
Fig. 3 EtOH intensifies PA-caused ER stress and caspase activation in vitro and in vivo. (A) Representative immunoblots of CHOP, XBP-1, and ATF-4 in the nucleus, and cleaved caspase 3 in cytoplasm of primary rat hepatocytes, treated by PA with or without EtOH (5 μg·mL−1) for 24 h. Lamin B and Actin were used as loading control. (B) Real-time quantitative PCR of ER stress factors in liver. C57/BL6 mice were fed with HFD with or without EtOH (2 g·kg−1·d−1) for 4 weeks. Total liver mRNA was isolated using Trizol and the first-strand cDNA was synthesized using the high-capacity cDNA archive kit. The mRNA levels of Chop, Atf4, and Xbp-1 were determined using SYBR Green Supermix
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inflammatory cells infiltration in the liver of wild type mice and HFD increased lipid accumulation. What’s more, the co-treatment of EtOH greatly worsened the HFD-induced inflammation and steatosis, and caused hepatocellular ballooning. However, CHOP knockout significantly reduced the EtOH and HFD-induced liver injury. The results of serum transaminase assay indicated that 2 g·kg−1·d−1 EtOH
and HFD greatly increased ALT and AST levels in wild type mice, which was remarkably improved in CHOP–/– mice (Figs. 4C and 4D). The results of serum lipid assay (Fig. 5) indicated that EtOH and HFD-induced dyslipidemia was blocked in CHOP–/– mice, compared with the wild type mice. Moreover, the ratio of liver/weight also was reversed by CHOP knockout.
Fig. 4 CHOP knockout reduceds the effects of liver injury induced by HFD and EtOH. H&E staining of wild type (A) and CHOP–/– (B) mice liver sections treated by HFD with or without EtOH (2 g·kg−1·d−1) for 4 weeks. Those mice serum ALT (C) and AST (D) were measured as described in methods. **P < 0.01 vs wild type mice (Student Two-Tailed t-test)
Discussion Cell death in the liver occurs mainly by apoptosis or necrosis, although other forms of cell death may occasionally occur. Liver apoptosis can be devided into extrinsic apoptosis and intrinsic apoptosis. The extrinsic apoptosis refers to a signaling pathway triggered by the binding of death receptors to their cognate ligands. Death receptors include Fas, TNF-αreceptor 1, TRAIL-R1, and TRAIL-R2, all of which are ubiquitously expressed in the liver to various extents. Their ligands (FasL/CD95L, TNF-α, and TRAIL) are mainly expressed by cells of the immune system and play a fundamental role in the elimination of virally infected, transformed, or damaged liver cells. In the present study, we found that PA and EtOH induced the primary rat liver apoptosis; the number of apoptotic cells was increased (Fig. 1), andmoreover, the levels of cleaved caspase 3 in PA or EtOH treated hepatocyte plasm were also increased (Fig. 3A). The treatment with PA
or EtOH resulted in lipid accumulation (Fig. 2). PA and EtOH could respectively cause hepatocytes apoptosis and lipid accumulation; while the effect of PA was greatly enhanced by EtOH; the content of lipid in the liver cells were increased after the co-treated with PA and EtOH, and the number of apoptotic cells were increased too (Figs. 1 and 2). We found that PA or EtOH induced liver cells apoptosis was not through the extrinsic pathway (data not show). PA can induce liver cell apoptosis through mitochondrial and ER pathway [3, 13-14]. EtOH could also induce liver cell apoptosis through the intrinsic pathway [15]. The intrinsic apoptosis can be triggered by a variety of intracellular stress inducers, including DNA damage, oxidative stress, UV and γ-irradiation, toxins, growth factor deprivation, and endoplasmic reticulum (ER) stress. PA induced liver cells mitochondrial membrane potential collapse (data not shown), and cause ER stress (Fig. 3), and its effect on ER stress was enhanced by EtOH. The ER stress signaling pathway is initiated
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Fig. 5 Plasma lipid levels and liver index are increased by HFD and EtOH co-treatment in wild type mice, which is reversed by CHOP knockout. Wild type and CHOP–/– mice were fed by HFD with or without EtOH (2 g·kg−1·d−1) for 4 weeks. (A) Free cholesterol, (B) Total cholesterol, (C) Total Triglyceride, and (D) Liver/Weight (g·g−1)
by three main ER transmembrane proteins, PERK, IRE-1, and ATF6, which together promote transcription of genes designed to increase protein folding and degradation [16]. IRE-1 splices XBP-1 mRNA to generate a transcription factor which leads to ER adaptation by activating a large number of unfolded protein response genes. PERK phosphorylates eIF2α, leading to an influence of ATF4 and CHOP. The ER stress in rat primary liver cells was induced by EtOH or PA, and the expressions of XBP-1, ATF4 and CHOP in nucleus were increased (Fig. 3A). The effect of EtOH was less than that of PA in liver cells. But when the cells were treated with PA and EtOH together, the ER stress was more serious. CHOP is a factor that mediates liver damage following diverse types of stress responses. CHOP transcription is primarily activated by ATF4 although ATF6a may also contribute. CHOP is the most well characterized proapoptotic pathway that emanates from the stressed ER. CHOP can induce the expression of proapoptotic BH3-only protein Bim, the cell surface death receptor TRAIL receptor 2, and other downstream of CHOP mRNAs, and inhibit Bcl-2 transcription [17] . Cholestasis induces CHOP-mediated ER stress and triggers hepatocyte cell death, and CHOP deficiency attenuates this cell death and subsequent liver fibrosis [18]. HFD induced liver injury through lipid accumulation and ER stress, and the mRNA levels of XBP-1, ATF4 and CHOP were increased (Fig. 3B). EtOH could increase the expression of ATF4 and CHOP, but had no effect on the expression of XBP-1. EtOH could
enhance the effect of PA, promoting ER stress in mice and increasing the mRNA levels of XBP-1, ATF4 and CHOP in the liver (Fig. 3B). In addition, CHOP deletion reduces oxidative stress, improves beta-cell function, and promotes cell survival in mouse with diabetes [19]. Our study showed that in wild type mice, HFD with high concentration of PA caused hepatic apoptosis, lipid accumulation and increases in serum ALT and AST in the liver, and EtOH, the possible risk factor, dose dependently aggravated lipotoxicity of PA via activating ER stress. However, in CHOP–/– mice, hepatic steatosis, serum aminotransferase increase and serum lipid accumulation were obviously attenuated, and the promoting effects of EtOH on HFD-induced liver injury was significantly blocked (Figs. 4–5). These finding suggested that promoting effects of EtOH on PA action were dependent on CHOP activation. Considering of these results, target CHOP will be a feasible treatment strategy for NAFLD patients with EtOH abuse. Hepatic steatosis is the result of dysregulation of lipid metabolism caused by increased lipid uptake, increased lipid synthesis, and reduced lipid oxidation and metabolism. Effect of EtOH on HFD-induced lipid accumulation indicated that CHOP also plays an important role in regulating the lipid metabolism. Inflammation is an important contributor to hepatic lipotoxicity [20–21]. Histological analysis showed that EtOH-induced inflammatory cells infiltration was also significantly reduced in CHOP–/– mice. Our studies suggested that the ER
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stress-mediated signaling might be as a potential connection for inflammation and metabolic disease [22]. In summary, our results demonstrated that EtOH promoted PA–induced activation of UPR, especially the upregulation of CHOP, representing an important molecular mechanism underlying EtOH and PA–associated dyslipidemia, inflammation, and hepatic lipotoxicity. Our studies suggest that targeting UPR-signaling pathways is a promising, novel approach to reducing EtOH and saturated fatty acid-induced metabolic complications.
References [1] [2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Sass DA, Chang P, Chopra KB. Nonalcoholic fatty liver disease: a clinical review [J]. Dig Dis Sci, 2005, 50(1): 171-180. Berlanga A, Guiu-Jurado E, Porras JA, et al. Molecular pathways in non-alcoholic fatty liver disease [J]. Clin Exp Gastroenterol, 2014, 7: 221-239. Cao J, Dai DL, Yao L, et al. Saturated fatty acid induction of endoplasmic reticulum stress and apoptosis in human liver cells via the PERK/ATF4/CHOP signaling pathway [J]. Mol Cell Biochem, 2012, 364(1-2): 115-129. Akazawa Y, Cazanave S, Mott JL, et al. Palmitoleate attenuates palmitate-induced Bim and PUMA up-regulation and hepatocyte lipoapoptosis [J]. J Hepatol, 2010, 52(4): 586-593. Kawano Y, Cohen DE. Mechanisms of hepatic triglyceride accumulation in non-alcoholic fatty liver disease [J]. J Gastroenterol, 2013, 48(4): 434-441. Gao B, Bataller R. Alcoholic liver disease: pathogenesis and new therapeutic targets [J]. Gastroenterology, 2011, 141(5): 1572-1585. Tsai J, Ford ES, Zhao G, et al. Co-occurrence of obesity and patterns of alcohol use associated with elevated serum hepatic enzymes in US adults [J]. J Behav Med, 2012, 35(2): 200-210. Xu J, Lai KK, Verlinsky A, et al. Synergistic steatohepatitis by moderate obesity and alcohol in mice despite increased adiponectin and p-AMPK [J]. J Hepatol, 2011, 55(3): 673-682. Leamy AK, Egnatchik RA, Young JD. Molecular mechanisms and the role of saturated fatty acids in the progression of nonalcoholic fatty liver disease [J]. Prog Lipid Res, 2013, 52(1): 165-174.
[10] Dara L, Ji C, Kaplowitz N. The contribution of endoplasmic reticulum stress to liver diseases [J]. Hepatology, 2011, 53(5): 1752-1763. [11] Pagliassotti MJ. Endoplasmic reticulum stress in nonalcoholic fatty liver disease [J]. Annu Rev Nutr, 2012, 32: 17-33. [12] Zhou H, Gurley EC, Jarujaron S, et al. HIV protease inhibitors activate the unfolded protein response and disrupt lipid metabolism in primary hepatocytes [J]. Am J Physiol Gastrointest Liver Physiol, 2006, 291(6): G1071-1080. [13] Wu X, Zhang L, Gurley E, et al. Prevention of free fatty acid-induced hepatic lipotoxicity by 18beta-glycyrrhetinic acid through lysosomal and mitochondrial pathways [J]. Hepatology, 2008, 47(6): 1905-1915. [14] Ji J, Zhang L, Wang P, et al. Saturated free fatty acid, palmitic acid, induces apoptosis in fetal hepatocytes in culture [J]. Exp Toxicol Pathol, 2005, 56(6): 369-376. [15] Ishii H, Adachi M, Fernandez-Checa JC, et al. Role of apoptosis in alcoholic liver injury [J]. Alcohol Clin Exp Res, 2003, 27(7): 1207-1212. [16] Leamy AK, Egnatchik RA, Young JD. Molecular mechanisms and the role of saturated fatty acids in the progression of non-alcoholic fatty liver disease [J]. Prog Lipid Res, 2013, 52(1): 165-174. [17] Malhi H, Kaufman RJ. Endoplasmic reticulum stress in liver disease [J]. J Hepatol, 2011, 54(4): 795-809. [18] Tamaki N, Hatano E, Taura K, et al. CHOP deficiency attenuates cholestasis-induced liver fibrosis by reduction of hepatocyte injury [J]. Am J Physiol Gastrointest Liver Physiol, 2008, 294(2): G498-505. [19] Song B, Scheuner D, Ron D, et al. Chop deletion reduces oxidative stress, improves beta cell function, and promotes cell survival in multiple mouse models of diabetes [J]. J Clin Invest, 2008, 118(10): 3378-3389. [20] Navab M, Gharavi N, Watson AD. Inflammation and metabolic disorders [J]. Curr Opin Clin Nutr Metab Care, 2008, 11(4): 459-464. [21] Harmon RC, Tiniakos DG, Argo CK. Inflammation in nonalcoholic steatohepatitis [J]. Expert Rev Gastroenterol Hepatol, 2011, 5(2): 189-200. [22] Hotamisligil GS. Endoplasmic reticulum stress and the inflammatory basis of metabolic disease [J]. Cell, 2010, 140(6): 900-917.
Cite this article as: YI Hong-Wei, MA Yu-Xiang, WANG Xiao-Ning, WANG Cui-Fen, LU Jian, CAO Wei, WU Xu-Dong. Ethanol promotes saturated fatty acid-induced hepatoxicity through endoplasmic reticulum (ER) stress response [J]. Chinese Journal of Natural Medicines, 2015, 13(4): 250-256
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