التأثيرات الوقائية لمستخلص القرنفل على الأضرار المؤكسدة والحمض النووي في كبد الفئران المعرضة للإيثانول

Protective Effects of Clove Extract on Oxidative and DNA Damage in liver of rats Exposed to Ethanol

Summary
The current study was conducted in the animal house of the College of Veterinary Medicine – University of Karbala to investigate the protective role of clove extract on oxidative stress and changes in gene expression and DNA resulting from exposure to ethanol alcohol,Alcohol consumption causes comprehensive liver disorders, designated as alcoholic liver disease (ALD). Cytochrome P450 2E1 (CYP2E1) in MEOS is one of the major ROS generators in liver and is recognized as a risk factor for ALD, Forty mature wister male rats weighting (140 – 190g) were used and divided randomly into four groups with ten males per group. All groups were dosed by oral intubation daily during the experiment period (four weeks), where the first group (the control group) was dosed with distilled water at an amount (1 ml per 1 kg) for a period of four weeks, while the second group (the clove group) was dosed with clove extract at a concentration (1 mg per kg) for four weeks and the third group (Ethanol group) ethanol was dosed at a concentration of (1 g per kg) and the last group was dosed with ethanol and then clove extract and at a concentration (1 g per kg) for ethanol and (1 mg per kg) for clove extract for a period of four weeks as well. Body weight of all rats (40 rats) has been monitored throughout the experiment. At the end of each treatment and control groups period, blood samples were obtained from heart for assessment of the following enzymes (catalase , glutathione peroxidase , Liver function test , TBARS and protein carbonyl) qRT-PCR technique was used for the quantification of CYP 2E1 gene expression levels relative to Housekeeping gene B actinin gene expression levels in rat liver have ethanol toxicity and treated with clove for estimation of fold changes in gene expression levels of target gene (CYP2E1) and housekeeping gene (B- actinin), Blood samples were collected from all rats, and samples were coded to avoid the possibility of bias. one ml of s blood was drawn once from the heart of animals by heparin injection. Samples were immediately transferred on ice to the central scientific laboratory in the veterinary medicine college, Ten microliter of tissue grinding cells were mixed with 75 μl of 0.5% lower melting point agarose in order to determine DNA damage analysis for all animals in this study (experimental and control) was done using the comet assay, the results were found in all of each the experimental periods (10-d, 20-d and 30- d), there was no significant difference (P>0.05) in body weight between male rats, the result also showed the effect of ethanol and clove on the catalase concentration in serum rats (U/ml), the treated male rats (T2) registered low significant (P<0.05) level of catalase throughout the experimental groups (C,T1 and T3), the oral intubation of (1g/kg) from ethanol and (100mg/kg) clove extract (T3) group rats caused significant (p<0.01) decrease in serum GPx concentration as compared to ethanol group(T2), it also found a significant elevation (P<0.05) in the liver enzymes (AST, ALT, ALP and Total bilirubin) in the second group (T2) and third (T3) groups when compared with male rats of control and first treatment group, on the other hand treated male rats (T2) registered highest significant (P<0.05) level of TBARS throughout the experimental groups (C,T1 and T3) male rats, the result showed a significant (P<0.01) increase in the Serum protein carbonyl after four weeks in T2 as compared to control group. On the other hand, among comet assay techniques the result found significant decrease of DNA in head in ethanol groups (30.829 ±5.82) rather than other groups as (90.104± 0.628, 91.057 ±0.727and 73.17 ± 8.13) for Control, cloves, and ethanol plus cloves, respectively.
Reverse transcriptase real-time PCR results have shown that CYP450E2 mRNA expression levels increased more than 10-folds in Treatment group 2 (ethanol treatment) male rats treated compared with corresponding of control male rats (slightly more than 2-fold) as well as in the treated male rats (around 1-fold).
In conclusion;the current study indicates that clove extract worked as antioxidant substance and the present study suggest that the ethanol exposure causes oxidative stress and liver disease and we recommended to avoiding exposure through treatment with clove extract .