Objective: No effective drugs have been developed to date to prevent or treat non-alcoholic fatty liver disease (NAFLD), although diet modification and exercise to improve obesity have been attempted. Therefore, development of a novel drug/strategy to treat NAFLD is urgently needed. In the present study, a novel concept is proposed for the treatment of NAFLD.
Methods: Fisher 344 male rats were given a choline-deficient, l-amino acid-defined (CDAA) diet or a high-fat high-calorie (HF/HC) diet with or without the antiplatelet agents, aspirin, ticlopidine or cilostazol for 16 weeks. Liver steatosis, inflammation and fibrosis, and the possible mechanisms involved were investigated.
Results: All three antiplatelet drugs, namely aspirin, ticlopidine and cilostazol, significantly attenuated liver steatosis, inflammation and fibrosis in the CDAA diet group. Of the three agents, cilostazol was the most effective, and the drug also suppressed HF/HC diet-induced liver steatosis. Cilostazol appeared to exert its beneficial effect against NAFLD by suppressing mitogen-activated protein kinase activation induced by oxidative stress and platelet-derived growth factor via intercepting signal transduction from Akt to c-Raf.
Conclusion: Antiplatelet agents, especially cilostazol, offer the promise of becoming key agents for the treatment of NAFLD.
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Up to almost a quarter of adults in many industrialised countries are found to show excessive fat accumulation in the liver.1 Although the cause of fatty liver is not clearly known, in our ageing and overfed population obesity and diabetes appear to be common conditions frequently associated with non-alcoholic fatty liver disease (NAFLD), which includes non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH).2 While NASH is proposed as a fatty liver disease associated with diffuse fatty infiltration and inflammation,3 NAFL, in contrast, is characterised by only steatosis, indicating that NAFL might represent the state of first hit in NASH.2 NAFLD has been reported to be the most common cause of chronic liver injury, and represents a spectrum of conditions that are histologically characterised by macrovesicular hepatic steatosis.3 Liver damage gradually progresses over years in patients with fatty liver, eventually resulting in liver dysfunction.4 To date, no effective drugs have been found for the prevention or treatment of fatty liver disease, although dietary modification and exercise to improve obesity have been attempted.5 Therefore, development of a novel drug/strategy for the treatment of NAFLD is urgently needed.
Recently, we observed that several NAFLD patients treated with antiplatelet drugs showed a tendency towards a decrease in plasma levels of hepatic transaminase and triglyceride (unpublished data). However, no studies have been conducted to date to investigate the therapeutic efficacy of antiplatelet drugs against NAFLD or the possible mechanisms involved. Therefore, we investigated the effects of antiplatelet drugs on the development of NAFLD in animal models. We used two experimental NAFLD models, the high-fat/high-calorie (HF/HC) diet model as the NAFL model,6 and the choline-deficient/l-amino acid-defined (CDAA) diet model as the NASH model.7 Both are well-established animal models of NAFLD; the former used as the model for the investigation of liver steatosis, and the latter used as the model for the investigation of liver inflammation and fibrosis.
Reagents and special diet
The antiplatelet drugs aspirin, ticlopidine and cilostazol were kindly provided by Bayer Healthcare (Tokyo, Japan), Daiichi-Sankyo (Tokyo, Japan) and Otsuka Pharma (Tokushima, Japan), respectively. The CDAA and choline-sufficient, l-amino acid-defined (CSAA) diets were obtained in powdered form from Dyets (Bethlehem, Pennsylvania, USA; product nos 518753 and 518754).7 High-fat diet 32 was obtained in powdered form from Japan CLEA (Tokyo, Japan).
Animal treatment and experimental procedures
All animals were treated humanely according to the guidelines of the National Institutes of Health and the AERI-BBRI Animal Care and Use Committee. All animal experiments were approved by the Institutional Animal Care and Use Committee of Yokohama City University School of Medicine.
In brief, male Fischer 344 rats (6 weeks, body weight 150–160 g) were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan). They were quarantined for 1 week and then housed in stainless-steel mesh cages under controlled conditions of temperature (25±2°C), humidity (50±10%) and lighting (12 h light, 12 h dark). The animals were allowed free access to food and tap water throughout the acclimatisation and experimental periods.
After acclimatisation for 1 week, the rats were randomly divided into two experimental groups, and one of these experimental groups was further subdivided into five groups (G1–G5). The G1–G4 groups were given the CDAA diet throughout the experimental period, and the G5 group was given the CSAA diet. In addition to the diet, the G2 group was administered oral aspirin once a day at a dose of 150 mg/kg/day,8 the G3 group was administered oral ticlopidine once a day at a dose of 100 mg/kg/day,9 and the G4 group was administered oral cilostazol once a day at a dose of 100 mg/kg/day.10
The other experimental group was subdivided into four groups (g1–g4). The g1 and g2 groups were given the HF/HC diet, and the g3 and g4 groups were given normal diet throughout the experimental period. In addition to the diet, the g2 and g4 groups were administered oral cilostazol once a day at a dose of 100 mg/kg/day.10 After the experimental period, all the animals were sacrificed under ether anaesthesia and samples were collected.
Measurement of serum biochemical markers
Serum alanine aminotransferase (ALT) was measured using Spotchem SP-4410 (Arklay, Kyoto, Japan). Serum levels of leptin, adiponectin, total cholesterol, low-density lipoprotein (LDL)-cholesterol, high-density lipoprotein (HDL)-cholesterol, triglyceride (TG), fasting blood sugar and fasting insulin were determined by radioimmunoassay using commercially available kits (Linco Research, St. Charles, Missouri, USA). Hepatic tumour necrosis factor α (TNFα) was measured by ELISA using the Quantikine Rat ELISA kit (R&D Systems, Minneapolis, Minnesota, USA).
Measurement of the liver triglyceride content
Liver samples were homogenised in 50 mM Tris–HCl buffer, pH 7.4, containing 150 mM NaCl, 1 mM EDTA and 1 μM phenylmethylsulfonyl fluoride (PMSF). TGs were analysed enzymatically using a diagnostic kit11 (Infinity, Thermo DMA, Arlington, Texas, USA) and measured by spectrophotometry (Beckman Coulter, Fullerton, California, USA).
Histopathological and immunohistochemical evaluations
Liver samples were excised and embedded in Tissue-Tek OCT compound (Sakura Finetek USA, Torrance, California, USA) and paraffin for histological analysis. Formalin-fixed and paraffin-embedded sections (5 μm thick) were processed routinely for H&E staining. The presence of collagen, as an index of fibrosis in the lesions, was examined in Masson’s trichrome-stained preparations. The OCT-embedded samples were serially sectioned at 4 μm. For the evaluation of fat deposition, the liver sections were stained with Oil red O.
Liver histology and scoring systems used
All histopathological findings were scored by the same two pathologists, who were unaware of the treatment that the animals had received. The Committee reviewed the study set cases as a group and then proposed an evaluation method for each of the recognised features of NAFLD. The Committee agreed that only H&E and Masson’s trichrome stains should be necessary to perform the evaluation. The histological features were grouped into three broad categories: steatosis, inflammation and fibrosis.12 ,13 The system of evaluation is detailed in tables 1 and 2.
Western blot analysis for the expression of molecules of the mitogen-activated protein kinase (MAPK) pathway and procollagen type 1
Frozen liver tissue was isolated mechanically and extracted using T-PER tissue protein extraction reagent (Pierce, Rockford, Illinois, USA) with 1 mM Na3VO4, 25 mM NaF and one tablet of proteinase inhibitor cocktail (complete mini, Roche, Tokyo, Japan). Protein concentrations were determined using the Bio-Rad Protein Assay Reagent (Bio-Rad, Richmond, California, USA). Protein was separated by sodium dodecylsulfate–plyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene difluorene (PVDF) membrane (Amersham, London, UK). Thereafter, the membranes were incubated in blocking buffer (10 mM Tris, 100 mM NaCl, 0.1% Tween 20 and 5% non-fat milk) overnight at 4°C, followed by incubation with the primary antibodies specific for phospho-Akt, Akt, phospho-c-Raf, c-Raf, phospho-MEK1/2, MEK1/2, phospho-p42-MAPK, p42-MAPK, pro-collagen type 1 (Cell Signaling Technology, Danvers, Massachusetts, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Trevigen, Gaithersburg, Maryland, USA) diluted 1:1000 in the blocking buffer, for 90 min at room temperature. The membranes were washed three times for 10 min each in washing buffer (10 mM Tris, 100 mM NaCl and 0.1% Tween 20) and incubated with the secondary antibody diluted in blocking buffer (goat anti-rabbit peroxidase-conjugated antibody) for 60 min at room temperature. The membranes were then washed and developed using an enhanced chemiluminescence (ECL) detection kit as described by the manufacturer.14
Determinations of the oxidative stress levels
Liver portions were homogenised in ice-cold 0.15 M KCl. The degree of lipid peroxidation in the liver was assessed by measurement of the malondialdehyde (MDA) levels using the OxiSelect TBARS Assay Kit (Cell Biolabs, San Diego, California, USA) and the values were expressed as nmol/mg protein.15
RNA isolation and reverse transcription
Total RNA was isolated from the samples using an RNeasy Mini Kit (Qiagen, Hilden, Germany, Catalogue no. 74126) according to the manufacturer’s instructions. The protocol included a DNase treatment step to remove genomic DNA. The RNA quantity and quality were assessed by measurement of the relative absorbance at 260 and 280 nm, and ethidium bromide agarose gel electrophoresis. Reverse transcription to produce cDNA was performed using a TaqMan Gold RT-PCR Kit (Applied Biosystems, Foster City, California, USA), according to the manufacturer’s instructions. The reaction mixtures (100 μl) contained 2.5 μg of total RNA, and the reaction was carried out for 50 min at 48°C, and then the reverse transcriptase was inactivated by heating the samples to 95°C for 5 min.16
Quantification of the expression of angiogenic cytokine genes by TaqMan reverse transcription–PCR (RT–PCR)
The mRNA levels of the angiogenic cytokine genes, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), inducible nitric oxide synthase (iNOS), transforming growth factor β1 (TGFβ1), platelet-derived growth factor C (PDGF-C) and endothelial nitric oxide synthase (eNOS), as well as of the housekeeping gene, GAPDH, in the liver tissue were determined by a fluorescence-based RT–PCR on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The primer sequences were purchased from ABI. RT–PCR was performed using the TaqMan Universal PCR Master Mix reagent, according to the manufacturer’s instructions (Applied Biosystems).16 ,17 All TaqMan RT–PCR data were obtained and processed using Sequence Detection Software version 1.6.3 (Applied Biosystems). The values in all the samples were normalised to the expression level of the endogenous control, GAPDH.16 ,18
All results were expressed as means (SE). Statistical comparisons were made using Student t test or Scheffé multiple comparisons after analyses of variance. The results were considered significantly different at p<0.05.
Effects of the antiplatelet drugs on liver inflammation and fibrosis
Because the HF/HC diet model showed less liver inflammation and fibrosis, we examined the CDAA diet-fed animals to investigate the effects of the antiplatelet drugs on liver inflammation and fibrosis. First, examination of the biochemical parameters of inflammation, such as the serum ALT and TNFα levels, revealed a significant decrease in the levels of these parameters in the animals treated with aspirin, ticlopidine or cilostazol (table 3). On histological examination, reduced inflammatory cell infiltration was noted in the liver of the rats treated with aspirin, ticlopidine or cilostazol as compared with that in the liver of the animals in the control CDAA group. The scoring of the pathological findings revealed a tendency towards a reduced severity of inflammation in the animals treated with one of the antiplatelet agents, in particular, in those treated with cilostazol (see table 1).
In addition, liver fibrosis was also markedly suppressed by treatment with aspirin, ticlopidine or cilostazol as compared with that in the control CDAA group (data not shown). The index of fibrosis—that is, the increase in the procollagen 1 protein expression levels, was significantly suppressed by treatment with aspirin, ticlopidine or cilostazol (fig 1A,B). Among the three drugs, cilostazol appeared, based on the scores, to be the most effective at suppressing liver fibrosis (see table 1). These results indicate that antiplatelet drugs, especially cilostazol, possess the ability to suppress liver inflammation and fibrosis.
Effect of antiplatelet drugs on liver steatosis and metabolic disorder
Three antiplatelet drugs, aspirin, ticlopidine or cilostazol, were administered to rat models fed the CDAA diet. Liver steatosis and the TG content were found to be suppressed in the animals treated with the antiplatelet drugs (fig 2A,B). Among the three drugs, cilostazol was the most effective at suppressing liver steatosis and reducing the liver TG content. However, strictly speaking, this model was not the most suitable for the evaluation of liver steatosis. We therefore investigated the effects of cilostazol on the liver steatosis induced by the HF/HC diet. Liver steatosis was attenuated and the liver TG content decreased in the animals treated with cilostazol (fig 2C,D). In addition, several parameters, such as insulin resistance and hyperlipidaemia, also improved, despite the absence of any significant changes in the daily caloric intake, body weight, visceral and subcutaneous fat area or adipocytokine levels in the animals (table 4). The scoring of the pathological findings showed a tendency towards reduction of liver steatosis in the animals treated with cilostazol (tables 1 and 2). Cilostazol clearly improved the liver steatosis and metabolic disorder in both CDAA and HF/HC diet-fed NAFLD models.
Effect of cilostazol on the hepatic oxidative stress levels
A significant increase in the hepatic MDA levels was found in both CDAA and HF/HC diet-fed animals as compared with those in the animals fed the control normal diet (p<0.01); however, a significant decrease in the expression levels of MDA in the liver was observed with the antiplatelet agents, especially cilostazol-treated animals compared with non-treated animals (p<0.01) (tables 3 and 4).
Effect of cilostazol on the expression of angiogenesis-related genes
Chronic intermittent hypoxia is associated with NAFLD in obese subjects, and hypoxia is believed to play an important role in the pathogenesis of acute and chronic liver disease via inducing alterations of gene expression.19 ,20 We therefore investigated the effects of cilostazol on the expression of various angiogenic genes. As shown in fig 3A–C, we observed significant increases in the expression of VEGF, HGF and eNOS mRNA in the liver tissues of the cilostazol-treated rats compared with the non-cilostazol-treated animals of the HF/HC diet group; all of these genes have been reported to directly related to angiogenesis.
In contrast, we found significant decreases in the expression of TGFβ1, PDGF-C and iNOS mRNA in the liver tissues of cilostazol-treated rats compared with the non-cilostazol-treated animals of the HF/HC diet group (fig 3D–F). These results indicate that cilostazol upregulates the genes that encode direct angiogenic factors, but downregulates those that encode indirect angiogenic factors.
Effect of cilostazol on activation of the PDGF-induced MAPK pathway
As mentioned above, we found a significant decrease in the PDGF-C mRNA expression levels in the liver tissues of cilostazol-treated rats compared with those of the non-cilostazol-treated rats given the HF/HC diet. We therefore investigated the effects of cilostazol on the molecules in the pathway downstream of the PDGF receptor. It has been reported that PDGF interacts with its receptor on the cell membrane, which causes sequential phosphorylation of several enzymes in the MAPK cascade, such as MAPK kinase kinase (c-Raf), MAPK kinase (MEK) and MAPK, to induce cell proliferation.21 ,22 As shown in fig 4D, significant inhibition of the phosphorylated MAPK proteins was observed in the liver tissues of cilostazol-treated rats compared with those of the non-cilostazol-treated animals of the HF/HC diet group. In addition, significant inhibition of phosphorylation of other members of the MAPK pathway, such as c-Raf and MEK, was also observed in the liver tissues of the cilostazol-treated rats compared with those in the non-cilostazol-treated animals of the HF/HC diet group (fig 4B,C). In contrast, no significant effect of the drug was observed on the phosphorylation of Akt (fig 4A).
The majority of NAFLD patients present with complications, such as diabetes mellitus, hyperlipidaemia, hypertension and obesity. Therefore, most drug therapies—for example, insulin sensitisers, hepatoprotectants, antioxidants and antihyperlipidaemic drugs—are targeted against components of NAFLD.23–27 In the present study, we have proposed a novel concept for the treatment of NASH. We clearly showed that antiplatelet drugs dramatically improved the pathological features of NAFLD, such as liver steatosis in the HF/HC-fed NAFL model, and inflammation and fibrosis in the CDAA-fed NASH model, despite the absence of any significant alterations in the daily calorie consumption, body weight, visceral and subcutaneous fat volume and adipocytokine levels in the animals. In the past, Wanless et al demonstrated similar effects with dipyridamole on experimental steatohepatitis induced by cholesterol and stilbestrol exposure.28 However, one of the decisive differences between their report and our present study was the difference in the extent of the changes in the lipid content of the tissues. One of the possible reasons for the less pronounced alterations of the tissue lipid contents in their study is that their animal model was not strictly a liver steatosis model but a model of liver cirrhosis, because they showed only a few small lipid droplets in the hepatocytes, which accounted for <15% of the liver dry weight.
To clarify the mechanisms underlying the effectiveness of antiplatelet drugs against NAFLD, we examined several potential mechanisms. First, antiplatelet drugs are known to stimulate the expression of eNOS and VEGF.29 Both of these factors induce the synthesis of vasodilatory molecules, such as nitric oxide and prostacyclin, by endothelial cells, resulting in antioxidant activity. Secondly, antiplatelet drugs reduce the expression of iNOS, which is considered as a mediator of oxidative stress that generates TNFα and, thereby, causes cellular inflammation and fibrosis, and of the liver MDA levels.30 Thirdly, antiplatelet drugs reduce the expression of PDGF-C, a marker of fibrosis, and induce the expression of various profibrotic genes, such as procollagen type 1, α-smooth muscle actin and TGFβ, which cause liver fibrosis, inflammation and steatosis.31
Among the mechanisms, we focused on the PDGF-mediated signalling pathways. In fact, cilostazol, the most effective antiplatelet drug against NAFLD, has been shown to reduce the phosphorylation of the MAPKs downstream of PDGF receptor signalling.32 A recent study demonstrated that the inhibition of the MAPK cascade leads to reduced serine phosphorylation of insulin receptor substrate-1 (IRS-1) in the liver, with a reduction of the Jun N-terminal kinase (JNK) activity, which may cause stimulation of the insulin signalling pathway and improve insulin resistance, which, in turn, may prevent both the first and second hits in NASH.32 These results indicate that cilostazol may inhibit activation of the PDGF-induced MAPK pathway, which may be one of the potential mechanisms underlying the effectiveness of the drug against NAFLD.
The effects of aspirin, ticlopidine and cilostazol of suppressing NAFLD may also be observed with other antiplatelet drugs. However, in the present study, cilostazol was found to be the most effective agent. What is the difference between cilostazol and the other two antiplatelet drugs? One difference may be in the effects of the drugs on cAMP activation. Cilostazol, a selective cGMP-inhibited family (PDE-3) inhibitor, increases protein kinase A (PKA) activity via elevation of the cAMP level, which suppresses MAPK activation.33 The cAMP/PKA signalling pathway is known to play a major role in the activation of the cAMP response element-binding protein (CREB) in hepatocytes. Activation of the cAMP/PKA signalling pathway, in turn, is responsible for the enhanced phosphorylation of CREB. CREB is a transcription factor that has been reported to have diverse functions in various tissues,34 ,35 and activation of CREB along with other transcription factors in the hepatocytes has been demonstrated to contribute to increased gene expression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and cholesterol biosynthesis in the liver.36 In the liver, the cAMP/CREB signalling pathway regulates the expression of the key genes involved in glucose metabolism as well as lipid metabolism,35 and activation of CREB is necessary for HMG-CoA reductase expression and increased cholesterol biosynthesis in the liver,36 HMG-CoA reductase serves as a direct link between cAMP/PKA signalling and lipid metabolism, and activation of CREB might contribute to insulin resistance in individuals susceptible to diabetes. These results explain the superior activity of cilostazol against liver steatosis as compared with that of the other two antiplatelet agents examined in this study.
In conclusion, we have proposed a novel concept for the treatment of NAFLD. We clearly showed that antiplatelet drugs dramatically improved the pathological features of NAFLD, including liver steatosis, inflammation and fibrosis, despite the absence of any significant alterations in the daily caloric consumption, body weight, visceral and subcutaneous fat volume or adipocytokine levels in the animals.
The skillful technical assistance of Machiko Hiraga is gratefully acknowledged. Supported in part by a Grant-in-Aid from the Ministry of Health, Labour and Welfare, Japan, to AN, a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan Kiban-B, to AN, and a grant from the National Institute of Biomedical Innovation to AN.
Competing interests: None.
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