Hepatosteatosis or simple fatty liver is characterized by accumulation of fat in liver cells. There are several different causes of hepatosteatosis, including chronic alcohol consumption, B and C viral hepatitis, type 2 diabetes, obesity and some metabolic aberrations [1
]. Actually, nonalcoholic fatty liver disease (NAFLD) is considered the most prevalent form of hepatosteatosis associated with obesity and metabolic syndrome [3
]. During the last 20 years, NAFLD has reached worrying proportion involving 20–30% of adults and 3–10% of children in Western countries [5
]. NAFLD genesis is multifactorial and comprises different patterns of liver injuries including simple hepatosteatosis alone or in combination with nonalcoholic steatohepatitis (NASH), with or without fibrosis [6
In recent years, many studies have provided new insights explaining potential mechanisms responsible for the switch from hepatosteatosis to NASH. So far it has been established that NAFLD pathogenesis and progression depends on different “hits” and it has been shown that the genetic makeup and dietary intake play key roles as leading factors [7
]. A working model [9
] has been proposed consisting of two sequential “hits”, the first conducting to the hepatic steatosis and the second towards the hepatic necro-inflammation determining the NASH condition and possibly fibrosis. Firstly, the insulin resistance (IR), and/or the derangement of fatty acid metabolism (de novo
lipogenesis, lower beta oxidation, impairment of triglyceride clearance and the diminished export of very-low-density lipoprotein), leads to hepatic fat accumulation and increased liver sensitivity to other possible subsequent hits [3
]. Followed by a still largely unknown mechanism, multifactorial complex interactions have been described as responsible for the “second hit” leading to the more advanced form of NASH which can possibly predispose to cirrhosis [11
]. This further hit includes oxidative stress, lipid peroxidation, imbalance of inflammatory cytokines and adipokines and augmentation of pathogen- or damage-associated molecular patterns [13
Nowadays the intervention against the NAFLD status encompasses two different and complementary directions: lifestyle changes and/or pharmacological treatment against specific hits potentially involved in NAFLD pathogenesis (i.e.
, insulin resistance and oxidative stress) [16
]. In the last decade many noteworthy efforts have been made for ameliorating the hepatic damage in NAFLD. It has been extensively demonstrated that metformin, vitamin E or placebo treatments do not have positive effects on liver injury although vitamin E is able to improve the hepatocellular ballooning degeneration [17
The current known targets for treatment of NAFLD are limited in number and are not even sufficiently defined and a breakthrough for new tolerated and efficient compounds is needed [16
]. In fact, many studies are aimed at testing the effect of natural agents on NAFLD evolution [18
]. It has been proven that Silibinin (silybin), a polyphenolic molecule constituent of silymarin (a flavonolignan extracted from Silibum marianum),
has anti-oxidant and hepatoprotective effects [20
]. Moreover, it protects against cirrhosis, decreases fibrosis if complemented to vitamin E and phospholipids and decreases both insulin resistance and plasma markers of liver fibrosis in NAFLD patients [21
]. Curcumin, a polyphenol and an active component of turmeric (Curcuma longa
), is another natural compound investigated by several laboratories. Clinical studies showed a protective action against fructose-induced hepatic steatosis by improving inflammation, hyperlipidemia, reducing insulin resistance and interrupting leptin signaling [22
]. Interestingly, it has been demonstrated that emodin (1,3,8-trihydroxy-6-methylanthraquinone), which is an active herbal component traditionally used in China for treating a variety of diseases, might have a role in the disease regression in NAFLD-induced rats. In fact, emodin significantly decreased the body weight, liver index, serum activities of ALT, blood lipids, hepatic triglyceride and considerably improved the hepatic histology features [25
]. Despite these encouraging results, to date no further thoughtful studies have been made to understand mechanisms and reliability of emodin in NAFLD models.
The wide diffusion of NAFLD in developed countries and its close correlation with cirrhosis, place the study of both prevention and therapeutic approaches, based on natural safe and efficient agents, in a central position of interest. Thus, in this study we attempted to investigate the potential preventive properties of emodin in a diet-induced hepatosteatosis in rats.
3. Experimental Section
3.1. Animals and Primary Hepatocytes
Twenty-four male Sprague–Dawley rats (120–140 g) were obtained from Harlan Italy (San Pietro al Natisone, UD, Italy). The animals received treatment in agreement with the European guidelines of the local committee for animal care and welfare. The animals used in this study were part of a large experimental protocol approved by Italian Ministry of Health. They were located in plastic cages under standard conditions with free access to water and food, at the Certified Animal Facility of the University of Rome, “La Sapienza”. The animals were fed with standard rat chow for 5 days then equally grouped based on two different dietetic regimens: a standard diet (SD) and a high-fat/high-fructose diet (HFD/HF). SD contained 5% of energy derived from fat, 18% from proteins, and 77% from carbohydrates (3.3 kcal/g), while HFD/HF contained 58% of energy derived from fat, 18% from protein, and 24% from carbohydrates (5.6 kcal/g; Laboratorio Dottori Piccioni, Gessate Milano, Italy); plusfructose (30%) that was added to the drinking water. After 5 weeks half of SD and HFD/HF were treated with emodin (40 mg/kg/day) from Sigma-Aldrich, Milan, Italy. Fluid and food intake were assessed every two-days at the replacement. We found no significant differences between consumption of food and water among the groups.
Randomly after 6 h fasting, from each group of animal, liver tissues were taken for biochemistry and histology. Further, from each group primary hepatocytes have been isolated using a perfusive method as previously described [52
]. Briefly, the rats were anesthetized by intraperitoneal administration of sodium pentobarbital (5 mg/100 g body weight). The liver was perfused firstly with a calcium-free Hank’s balanced salt solution containing 2% BSA and 0.6 mM ethyleneglycotetraacetic acid, and secondly with Hank’s solution containing 4 mM calcium chloride and 0.04% collagenase. Liver cells were released into a Krebs–Henseleit buffer with 2% BSA. The hepatocytes were seeded on collagen-coated plates at density between 1.5 × 104
and 3 × 104
. After 24 h from plating hepatocytes from SD, HFD/FD, SD + Emodin, HFD/HF + Emodin animals were subjected to following treatments: 10 μL PBS (NT) or N
-acetylcysteine 1mM (NAC) or H2
). Hepatocytes were harvested 24 h later, centrifuged and collected for the experiments.
3.2. Biochemical Determinations and Inflammatory Markers
Blood samples obtained from caudal vein after 6 h fasting were collected in sterile glass tubes containing 0.15% EDTA. Blood samples were centrifuged at 3000 for 15 min to obtain plasma. Plasma samples were immediately used to perform enzymatic and photocolorimetric assay to determine the levels of alanine aminotransferase (ALT), triglycerides, total cholesterol, glucose and insulin. Enzymatic and colorimetric assays were performed using standard procedures as indicated by kits purchased from different companies: ALT assay kit from Randox Laboratories Ltd (Antrim, UK), triglycerides and cholesterol assay kits from Cayman Chemical (Ann Arbor, MI, USA), glucose assay kit from Abcam Inc (Cambridge, MA, USA), and rat insulin enzyme immunoassay kit from SPI-BIO (France). At 14 weeks, insulin resistance was calculated according to the homeostasis model assessment of insulin resistance (HOMA-IR) calculation: fasting plasma insulin (μU/mL) × fasting plasma glucose (mmol/L)/22.5. ELISA-based kits were used to assay the circulating levels TNF-α (Peprotech, Rocky Hill, New Jersey, USA) and IL6 (R&D Systems, Abingdon, UK).
Liver was fixed in 4% buffered formalin and embedded in paraffin. A measure of 3–5 μm sections were stained with haematoxylin and eosin (Bio-Optica, Milan, Italy). Then the specimens were evaluated under 10 × 20 light microscopic fields.
3.4. High-Performance Liquid Chromatography of GSH
The tissues and primary hepatocytes from liver mouse model NASH diet-induced or NASH diet-induced treated with Emodin were sonicated (Sonics Vibra Cell, Sonics & Material Inc., Newtown, CT, USA), three times for 2 s in 0.1 mL of 0.1 M potassium phosphate buffer (pH 7.2). Following the sonication levels of total (GSH Tot), reduced (GSH), oxidized (GSSG) and protein-bound (ProSSG) glutathione were analyzed by HPLC. HPLC equipment and conditions for analyzing the several forms of glutathione have been reported [53
3.5. Immunoprecipitation and Western Blotting
Liver tissues were lysed in ice-cold Ripa buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 1% sodium deoxycholate and phosphatases 10% cocktail protease inhibitors. For the immunoprecipitation protocol of the glutathionylated proteins see previous published work [46
]. Then protein extracts were resolved on 10–12.5% SDS-PAGE, transferred and immobilized onto nitrocellulose membrane (Amersham, Germany), blocked with 5% nonfat dry milk and incubated with appropriate primary and secondary antibodies. The anti-PTEN, anti-pPTEN, primary antibodies were purchased from Santa Cruz Biotech (CA, USA). Immunoblots were detected with the ECL system (Amersham) and the relative intensities of the specific bands were determined by densitometric analysis and referring to beta-actin protein expression.
3.6. Cell Viability
Cell viability was determined by a simple vital stain method that evaluates the accumulation of the neutral red dye in the lysosomes of viable, uninjured cells [54
]. The simple vital Neutral red (Sigma-Aldrich) was dissolved in culture medium and added to cells for 1 h. The pH of the neutral red solution was adjusted in all the experiments to 6.35 with the addition of 1 M KH2
. Then, cells were washed thrice with PBS, and 1 mL of elution medium (EtOH/AcCOOH, 50%/1%) was added followed by gentle shaking for about 10 minutes to obtain the complete dissolution. Measures were acquired with spectrophotometer at 540-nm of absorbance.
3.7. Statistical Analysis
The results are reported as means ± SD. for at least four independent experiments. Statistical differences were determined by Student’s t test considering P < 0.05 as statistically significant.
In summary, in this study we reported for the first time the preventive effect of emodin on hepatosteatosis-dependent metabolic derangement and liver cell injury. In particular, our results demonstrated that emodin was able to protect rats, treated with high fat/high fructose diet, from insulin resistance, hypertriglyceridaemia, histological damage, systemic necro-inflammation, and oxidative stress. Furthermore, interestingly, we demonstrated that emodin treatment conferred to HFD/HF hepatocytes an important defense from additional oxidative stress, and an improved ability to react to classical anti-oxidant agents. Our data suggested that PTEN could be a target of emodin, but the full comprehension of the existing molecular mechanisms of this natural agent requires further study.
In conclusion, all these findings suggest the use of emodin, not only as a potential preventive agent in NAFLD diet-induced and a promising agent for hampering the progression to NASH, but also as a natural coadjuvant of the more classical antioxidant therapy.