EUGENOL AMELIORATES PATHOGENIC FEATURES IN EXPERIMENTAL MODEL OF NAFLD IN RATS BY

Background: Nonalcoholic fatty liver disease (NAFLD) is emerging one of the major causes of chronic liver damage that includes a wide spectrum of liver injury ranging from steatosis to steatohepatitis evolving to fibrosis. Aim: This study was designed to evaluate the possible effect of EUG on NAFLD induced by high fat cholesterol diet. Methods: Rats were fed either normal rat chow diet (control) or high fat cholesterol diet and both received olive oil (10 mg/kg) for eight weeks to induce NAFLD model. Eugenol (10 mg/kg) was administrated to rats by oral intubation 3 times weekly for 8 weeks. Results: Our results showed that EUG significantly ameliorated the histopathological lesions induced by HFCD. Furthermore, HFCD induced a significant elevation in liver enzymes (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)). This was significantly attenuated by EUG co-treatment. Conclusion: These findings indicate that EUG possess es a marked role in modulation of NAFLD.


Introduction:
Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome.It represents a spectrum of disease, ranging from simple steatosis to steatohepatitis through to fibrosis and cirrhosis (Than and Newsome, 2015).Currently, NAFLD is recognized as the most common form of liver disease worldwide affecting between 25-30% of the general population.Frequently, it has been associated with obesity, type 2 diabetes mellitus, hyperlipidemia, and insulin resistance (Berlanga et al., 2014).
NAFLD hallmark is the excessive hepatic accumulation that results from an imbalance between lipid availability and lipid removal (Arguello et al., 2015).Once the energy expenditure is less than energy intake, lipid overload can be found in organs such as adipose tissue organs (Shulman, 2014) .As a consequence of excess lipid contents that spill over from the dysfunctional adipose tissue, ectopic lipid deposition in liver would occur (Stienstra et al., 2010).This flux in particular is a major determinant of accumulation of hepatic and lipoprotein fat in NAFLD that induce marked liver injury (Loria et al., 2008).
The effective therapy for NAFLD has not been established, but there are many proposed strategies and agents used for liver support

Drugs and chemicals:
Eugenol was purchased from Sigma Chemical Co.(USA) and dissolved in olive oil.Cholesterol, casein, and methionine were purchased from Medico pharm Co., for Pharmaceutical and Medicine (Cairo, Egypt).Vitamin mixture and mineral mixture were obtained from Misr feed additives company (Egypt).All other chemicals were of the highest available commercial grade.

Animals:
Forty male albino rats weighed 150-180 g were obtained from Nile Co. for pharmaceutical and chemical industries (Egypt).The normal chow was acquired from Meladco for Animal Food (Egypt).Pellets and tap water were provided ad libitum.Temperature was maintained at 25 °C.Rats were allowed at least 1 week to acclimatize to the lab conditions.The study protocol was approved by the Ethical Committee, Faculty of Pharmacy, Ain Shams University, Egypt.

Experimental design:
White albino rats were randomly divided into four groups (n = 10): group (1) fed normal rat chow diet and given olive oil (10mg/kg) orally 3 times weekly for 8 weeks and is considered as control group.Group (2) fed HFCD diet and given olive oil (10 mg/kg) orally 3 times weekly for 8 weeks and is considered as HFCD group.Group (3) fed high-HFCD diet and given EUG (10mg/kg) orally 3 times weekly for 8 weeks and is considered as HFCD+EUG group.Group (4) fed normal rat chow diet and given EUG (10mg/kg) orally 3 times weekly for 8 weeks and is considered as EUG only group.
After 8 weeks, fasting blood samples were collected from the retro orbital venous plexus, allowed to clot and serum was separated by centrifugation at 3000g for 15 min for biochemical assessment.Rats were then sacrificed; liver tissues were dissected out and washed with ice-cold saline.Then, parts from liver were fixed in 10% buffered formaldehyde for histopathological examination.

Biochemical analysis:
Serums alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were determined by spectrophotometric method using commercially available kits (Teco Diagnostics, C.A., USA), and the results were expressed as IU/L.

Histopathological examination
For light microscopy, liver specimens were taken from the different lobes and fixed in 10% formalin and processed for paraffin sections of 4 μm thickness.Sections were stained with hematoxylin and eosin (H & E) and evaluated under light microscope.

Statistical analysis
All data are expressed as mean ± SD.Statistical analysis differences among groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey Kramer test as a post hoc test.Differences were considered significant at p < 0.05.All analyses were performed using lnstat version 3 software package.Graphs were sketched using Graph Pad prism (ISI® software, USA) version 5 software.

Effect of Eugenol on liver index and liver enzymes
Body weight was monitored throughout the feeding period.After 8 weeks, there was significant increase in liver index ratio in HFCD group by 86% as compared to the control group.On the contrary, co-treatment with EUG significantly decreased the liver index by 30% as compared to HFCD group (Fig. 1).Similarly, ALT and AST significantly increased in HFCD group by 3 folds as compared to the control group.EUG co-treated rats showed significant decrease in ALT and AST by 65 and 62%, respectively, as compared to HFCD group (Fig. 2  Fig ( 1).Effect of EUG on liver index in rats fed HFCD.Rats were fed HFCD diet for 8 weeks, HFCD+ EUG, and EUG groups received EUG (10mg/kg) orally 3times weekly for 8 weeks .Data are represented as mean ± S.D. (n=10).a, or b : significant difference from control, or HFCD groups, respectively at p<0.05 using ANOVA followed by Tukey-Kramer as a post-hoc test.

Fig.(2): Effect of EUG on serum level of ALT in rats in rats fed HFCD.
Rats were fed HFCD diet for 8 weeks, HFCD+ EUG, and EUG groups received EUG (10mg/kg) orally 3times weekly for 8 weeks.Data are represented as mean ± S.D. (n=10).a, or b : significant difference from control, or HFCD groups, respectively at p<0.05 using ANOVA followed by Tukey-Kramer as a post-hoc test.

Fig.(3): Effect of EUG on serum level of AST in rats in rats fed HFCD.
Rats were fed HFCD diet for 8 weeks, HFCD+ EUG, and EUG groups received EUG (10mg/kg) orally 3times weekly for 8 weeks .Data are represented as mean ± S.D. (n=10).a, or b : significant difference from control, or HFCD groups, respectively at p<0.05 using ANOVA followed by Tukey-Kramer as a post-hoc test.

Liver histopathological assessment
Rats fed normal chow diet showed normal hepatic architecture.Rats fed with HFCD diet showed liver steatosis, lobular inflammation, hepatocellular ballooning in diffuse manner all over the hepatocytes as nuclear pyknosis in the hepatocytes surrounding and adjacent the dilated central vein.HFCD+EUG group showed little congestion in the central vein with mild degeneration in hepatocytes (Fig4).

Discussion
NAFLD is a major health public issue due to its high prevalence and it is considered one of the major causes of chronic liver damage (Tilg and Moschen, 2008) . .Recently, EUG is of interest for many recent researchers because of its broad range of pharmacological and biological activities (Jo et al., 2014).It has been used as anti-inflammatory agent (Huang et al., 2015) , anti-hyperglycemic agent (Jeong et al., 2014), and anti-oxidant agent (Gulcin, 2011).The present study was designed to assess the potential protective effect of EUG on IR in NAFLD model in rats.
In the present study, by feeding rats HFCD diet for 8 weeks, rat model of NASH, namely steatosis, was established in HFCD group, as indicated by significant elevation of plasma ALT and AST activities which reproduce marked liver injury.These results are in alignment with previous study (Yogalakshmi et al., 2010).On the contrary, co-treatment with EUG showed significant decrease in hepatic enzyme levels.Our results were supported by previous study (Srinivasan et al., 2014).
An ideal animal model should displays hepatic histopathologic and pathophysiologic features associated with NASH (Hebbard and George, 2011) .In our model, increase the fat intake showed liver steatosis, lobular inflammation , hepatocellular ballooning in diffuse manner all over the hepatocytes.These results are in alighment with previous studies (Xu et al., 2010; Takahashi et al., 2012) .On the other hand, EUG co-treatment ameliorated these changes.Our results are supported by previous study (Venkadeswaran et al., 2014).

Conclusion
In conclusion, EUG co-treatment significantly ameliorates NAFLD by modulating of liver enzymes (ALT&AST) levels.Furthermore, Histopathological results supported hepatoprotective role of EUG against HFCD.The results of this study make EUG a promising dietary supplement to improve NAFLD.

Figure 1 :
Figure 1: Effect of EUG on liver index in rats fed HFCD :

Figure 2 :
Figure 2: Effect of EUG on serum level of ALT in rats fed HFCD :

Figure 3 :
Figure 3: Effect of EUG on serum level of AST in rats fed HFCD :

(Yogalakshmi et al., 2010).
Nowadays, renewed attention to alternative medicines and natural therapies has stimulated new wave of research to look for more efficacious agents with lesser side effects.Eugenol (4-allyl-2-methoxyphenol) is a naturally occurring phenolic compound extracted from clove, basil, cinnamon and nutmeg (

Yogalakshmi et al., 2010).
It has been used as: a fragrant and favoring agent (

Nuchuchua et al., 2009), in
dentistry as a filling material and a pulp capping agent or as a neuroprotective (