Hepaprotective Effect of Standardized Ecklonia stolonifera Formulation on CCl4-Induced Liver Injury in Sprague-Dawley Rats

Abstract

The liver is an essential organ for the detoxification of exogenous xenobiotics, drugs and toxic substances. The incidence rate of non-alcoholic liver injury increases due to dietary habit change and drug use increase. Our previous study demonstrated that Ecklonia stolonifera (ES) formulation has hepatoprotective effect against alcohol-induced liver injury in rat and tacrine-induced hepatotoxicity in HepG2 cells. This present study was designated to elucidate hepatoprotective effects of ES formulation against carbon tetrachloride (CCl₄)-induced liver injury in Sprague Dawley rat. Sixty rats were randomly divided into six groups. The rats were treated orally with ES formulation and silymarin (served as positive control, only 100 mg/kg/day) at a dose of 50, 100, or 200 mg/kg/day for 21 days. Seven days after treatment, liver injury was induced by intraperitoneal injection of CCl₄ (1.5 ml/kg, twice a week for 14 days). The administration of CCl₄ exhibited significant elevation of hepatic enzymes (like AST and ALT), and decrease of antioxidant related enzymes (superoxide dismutase, glutathione peroxidase and catalase) and glutathione. Then, it leaded to DNA damages (8-oxo-2'-deoxyguanosine) and lipid peroxidation (malondialdehyde). Administration of ES formulation inhibited imbalance of above factors compared to CCl₄ induced rat in a dose dependent manner. Real time PCR analysis indicates that CYP2E1 was upregulated in CCl₄ induced rat. However, increased gene expression was compromised by ES formulation treatment. These findings suggests that ES formulation could protect hepatotoxicity caused by CCl₄ via two pathways: elevation of antioxidant enzymes and normalization of CYP2E1 enzyme.

Keywords: Antioxidant enzymes; CYP2E1; Carbon tetrachloride; Ecklonia stolonifera; Hepatoprotective effect; Non-alcoholic liver injury.

Fig. 1.

Effect of ES formulation on serum hepatic aminotransferases. To evaluate the effects of ES formulation on hepatic functions, we measured the (A) serum AST and (B) ALT levels. The results are presented as the mean ± SD of three independent experiments (n=8). ^###p<0.001 vs. the normal control, ^*p<0.05, ^**p<0.01, ^***p<0.001 vs. the CCl₄ induced control. ALT, alanine aminotransaminase; AST, aspartate aminotransferase.

Fig. 2.

Effects of ES formulation on triglyceride, total cholesterol, HDL cholesterol in serum and liver tissue. To evaluate (A) triglyceride, (B) hepatic TG, (C) total cholesterol, (D) hepatic TC, (E) HDL cholesterol, we investigated above factors in both serum and liver tissues. Then, we calculated (F) HDL cholesterol to total cholesterol. The results are presented as the mean ± SD of three independent experiments (n=8). ^#p<0.05, ^##p<0.01, ^###p<0.001 vs. the normal control, ^*p<0.05, ^**p<0.01, ^***p<0.001 vs. the CCl₄ induced control. HDL, high density lipoprotein; TG, triglyceride; TC, total cholesterol.

Fig. 3.

Effect of ES formulation on the SOD, CAT, GPx and GSH levels in liver tissue. Activities of (A) SOD, (B) CAT and (C) GPx, and level of (D) GSH were estimated in the liver tissue. The results are presented as the mean ± SD of three independent experiments (n=8). ^##p<0.01, ^###p<0.001 vs. the normal control, ^*p<0.05, ^**p<0.01, ^***p<0.001 vs. the CCl₄ induced control. GSH, glutathione; SOD, superoxide dismutase; GPx, glutathione peroxidase; CAT, catalase.

Fig. 4.

Effect of ES formulation on MDA and 8-OHdG levels in liver tissue. Hepatic oxidative substances ((A) MDA and (B) 8-OHdG) were measured to verify DNA and lipid oxidation in liver tissue. The results are presented as the mean ± SD of three independent experiments (n=8). ^##p<0.01, ^###p<0.001 vs. the normal control, ^*p<0.05, ^**p<0.01 vs. the CCl₄ induced control. MDA, malondialdehyde; 8-OHdG, 8-hydroxy-2′-deoxyguanosine.

Fig. 5.

Effect of ES formulation on the gene expression levels of CYP2E1 in liver tissue. CYP2E1 gene expression was evaluated in the liver tissue of rats, then, analyzed by comparative real-time PCR and normalized to GAPDH. The primer sequence was as follows: CYP2E1, 5′-TCCAACCTACCCCATGAAGC-3′ (forward) and 5′-CCAACACAC AC ACGCTTTCC-3′ (reverse); GAPDH, 5′-GCCAGCCTCGTCTCATAGACA-3′ (forward) and 5′-AGAGA-AGGCAGCCCTGGTAAC-3′ (reverse). The results are presented as the mean ± SD of three independent experiments (n=8). ^##p<0.01 vs. the normal control, ^*p<0.05, vs. the CCl₄ induced control.