Hepatoprotective Effects of Liv.52 on Ethanol-induced Liver Damage in Rats
Rajat Sandhir* and Gill, K.D. Department of Biochemistry, Postgraduate Institute of Medical Education & Research Chandigarh, India
RESULTS
The activity of ϒ-glutamyl transpeptidase was used as an index of ethanol induced hepatic damage. It was observed that ethanol exposure (3g/kg body wt., intragastrically) for 4 weeks resulted in a 2-fold increase of ϒ-glutamyl transpeptidase activity, whereas in the animals given Liv.52 along with ethanol, the activity of ϒ-glutamyl transpeptidase was completely restored, indicating the in vivo protective effects of Liv.52 against ethanol induced damage (Fig.1).
In an attempt to understand the mechanism, by which Liv.52 prevents hepatic damage caused by ethanol, detailed investigations were carried out relating to lipid peroxidation and antioxidant enzymes. The results in Fig.2 indicate that ethanol in vitro (10 µmol) enhanced the amount of malondialdehyde formed, confirming that ethanol induced hepatotoxic effects are mediated through enhanced generation of free radicals. However, the effect of exogenously added Liv.52 on ethanol induced lipid peroxidation could not be studied, since the colour of Liv.52 extract interfered with the assay of lipid peroxidation. Therefore the in vivo effect of Liv.52 on ethanol induced lipid peroxidation was studied. The data in Fig. 3 indicate that ethanol could accentuate lipid peroxidation, a mediator of tissue damage, even after in vivo exposure. Whereas, when Liv.52 was given along with ethanol, the levels of lipid peroxidation were restored to that observed in control, indicating protective efficacy of Liv.52 against hepatotoxicity of ethanol.
The activity of antioxidant enzymes, superoxide dismutase and glutathione peroxidase was significantly inhibited in liver following ethanol exposure, whereas the activity of catalase increased markedly following ethanol exposure. The levels of reduced glutathione were observed to decrease in liver of ethanol exposed animals. Liv.52 treatment on the other hand was able to restore the activity of superoxide dismutase and the levels of glutathione in ethanol treated animals (Table 1). No significant effect was observed on the activity of catalase and glutathione peroxidase.
Table 1: Ethanol induced alterations in antioxidant enzymes and glutathione levels in rat liver (Values are mean ± SD of 6 animals/group) |
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|
Superoxide dismutase (Units/mg protein) |
Catalase (µ mol H2O2 decomposed/min/mg protein) |
Glutathione peroxidase (µ mol NADP oxidized /min/mg protein) |
Glutathione (µ mol GSH/mg protein) |
Control group |
16.47 ± 1.47 |
166.57 ± 7.93 |
297.85 ± 13.44 |
45.11 ± 2.13 |
Ethanol treated group |
10.05 ± 0.83a |
209.84 ± 8.97a |
200.34 ± 10.43a |
32.73 ± 1.62a |
Ethanol + Liv.52 treated group |
15.43 ± 1.29b |
201.58 ± 10.09NS |
213.34 ± 6.25NS |
43.13 ± 1.54b |
p values: a<0.001 compared to control group; b<0.001 compared to ethanol treated group; NS Not significant |
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DISCUSSION
The results obtained indicate that ethanol induced hepatotoxic damage in terms of the increase in ϒ-glutamyl transpeptidase activity, a known marker of ethanol induced hepatic damage21. The increase in ϒ-glutamyl transpeptidase activity was prevented by Liv.52 treatment, thereby confirming the efficacy of Liv.52 in counteracting the ethanokl induced liver damage. Liv.52 treatment also restored the levels of ethanol induced lipid peroxidation to that in control liver. An increase in lipid peroxidation has already been reported after both acute and chronic exposure22,23. The effect of ethanol has been suggested to be a result of the enhanced generation of oxyfree radicals during its oxidation in liver24. The peroxidation of membrane lipids, resulting in elevated levels of ϒ-glutamyl transpeptidase, a membrane bound enzyme in serum. Goel and Dhiman25 have reported protective effect of Liv.52 on carbon tetrachloride induced NADPH dependent lipid peroxidation and hepatic functions. These authors have further reported efficacy of Liv.52 in preserving the structural integrity of liver.
Our study demonstrates that ethanol exposure in peroxide dismutase, glutathione peroxidase and levels of glutathione in liver, whereas, Liv.52 treatment restored the activity of superoxide dismutase and the levels of glutathione to nearly those observed in control livers. Superoxide dismutase is an enzyme responsible for dismutation of highly reactive and potentially toxic superoxide radicals’ (O–2) to H2O2. A reduced activity of this enzyme may reduce its cellular efficacy to detoxify these potentially toxic oxyradicals, which will lead to an increase in the levels of lipid peroxidation26. Glutathione is an important naturally occurring antioxidant as it prevents the hydrogen of the sulfhydryl group to be abstracted instead of methylene hydrogen of unsaturated lipids27. Therefore, the levels of glutathione are of critical importance in tissue injury caused by toxic substances. The binding of acetaldehyde, a metabolite of ethanol with glutathione may contribute to reduction in the levels of glutathione29. The ability of Liv.52 to protect the liver from ethanol induced damage might be attributed to its direct antiperoxidative effect or may be due to its ability to restore the activity of antioxidants, superoxide dismutase and glutathione. The enzyme superoxide dismutase and glutathione. The enzyme superoxide dismutase and glutathione constitute the first line of defense against free radical induced damage and a restoration of the superoxide dismutase, activity and glutathione levels by Liv.52 may account for its protective effects. The decrease in the activity of antioxidant enzymes superoxide dismutase, glutathione peroxidase and glutathione are speculated to be due to the damaging effects of free radicals produced following ethanol exposure or alternatively could be due to a direct effect of acetaldehyde, formed from oxidation of ethanol, on these enzymes30,31. A decrease in the activity of certain metabolic enzymes induced by free radicals generated on the oxidation of ethanol has been reported following ethanol exposure32. The antioxidant effect and resultant hepatoprotective ability of Liv.52 may be attributed to flavinoids, ∝- and β-carotenes, vitamin A and C present in the multiherbal preparation33,34, which explains its ability to reduce the levels of lipid peroxidation and restore the antioxidant status. Chauhan et al.35, have demonstrated that Liv.52 enhances acetaldehyde elimination and also prevents binding of acetaldehyde to cellular proteins and thereby exerts its protective effects. The activity of glutathione peroxidase, an enzyme, which reduces the levels of peroxides in the cell and thus protects the cell from peroxidative damage was also inhibited on ethanol exposure. On the contrary, coexposure of ethanol and Liv.52 failed to restore the activity to that observed in the control animals. The reduced activity of glutathione peroxidase might not contribute towards peroxidative damage following ethanol exposure, since the critical antioxidants superoxide dismutase and glutathione, which are the first lines of defense offer protection against free radicals and thus maintain low levels of lipid peroxides. However, the increase in the activity of catalase, an important antioxidant enzyme responsible for detoxification of H2O2 dependent ethanol oxidation36, may be adaptive mechanism in response to ethanol exposure is considered to be harmful as it results in the formation of acetaldehyde, a very reactive compound.
The results of the present study thus demonstrate that Liv.52 protects liver from ethanol-induced damage by preventing the peroxidation of membrane lipids. Further studies are, however, needed to isolate the specific components responsible for the antioxidant action of this multiherbal drug and to establish its mechanism of action.
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