In our study, while liver function tests and an oxidative stress product MDA increased significantly; an antioxidant enzyme GSH and DDAH which plays a role in NO metabolism decreased significantly with high cholesterol diet. Sinusoidal congestion, cloudy bloating, periportal cell infiltration, periasiner lubrication and apoptosis were observed in the liver tissue by hypercholesterolemia in the histopathological analysis. Both melatonin treatments protected liver tissue by normalizing these parameters. However, the effect of prophylactic (HCT-MEL) melatonin was more successful than therapeutic (HCT-2MEL) use in the histopathological analysis.
Serum transaminase levels are considered a sensitive marker in liver injury. Amin et al. 17 showed that liver transaminases (ALT, AST and GGT) increased in hypercholesterolemic rats at 3 and 6 weeks of age compared to controls. In hepatic steatosis in non-alcoholic fatty liver disease, ALT, AST and oxidative stress parameters have been shown to decrease serum levels in melatonin doses of 10 mg/kg 18. On thioacetamide-induced liver fibrosis, melatonin administered for 4 weeks was reported to have a positive effect on ALT and AST 19. In our study, increased ALT, AST and GGT levels in the hypercholesterolemia group were decreased in melatonin groups but changes in ALT levels did not reach statistical significance. Similarly, in another study, ALT values of fibrosis stage 1 and 4 were not significantly increased compared to stage 0 20.
Oxidative stress plays a critical role in the inflammatory process in the liver by increasing the migration and activation of inflammatory cytokines and mediators. In an experimental study, a high cholesterol diet has been shown to trigger oxidative stress in plasma, liver and aortic tissue 21. Some animal studies have shown that melatonin inhibits hypocholesterolemia and lipid peroxidation 22. GSH and MDA effectively reduce the level of lipid peroxidation, therefore it is considered as an indicator of oxidative stress in the liver 23. MDA, the final product of lipid peroxidation, has been shown to activate inflammation 24. It has been observed that melatonin treatment increased the antioxidant enzymes SOD, catalase and glutathione peroxidase activity and decreased GSH levels in the liver 25. In a study, it was shown that melatonin administration for seven days did not change liver MDA levels in a mouse model of carbon tetrachloride-induced liver damage 26. But in another study, liver MDA levels were reduced when treatment with melatonin administrated for 84 days in a rat model of carbon tetrachloride injury 27.
NO, which is synthesized from L-arginine by NOS in endothelial cells, plays a pivotal role in maintainance of vascular structure and function, and it is generally described as an ‘endogenous anti-atherosclerotic molecul’. It was reported that ADMA, a major endogenous inhibitor of NOS, could reduce NO production and decrease acetylcholine-induced vasodilator responses 28. It was studied that the elevation of circulating ADMA level is involved in endothelial dysfunction in some cardiovascular abnormalities 29. Decrease in activity of DDAH, a major hydrolase of ADMA, causes accumulation of ADMA under cardiovascular abnormalities 30. DDAH is known to be highly susceptible to oxidation. In our study, melatonin treatment significantly increased the DDAH level compared to the hypercholesterolemia group. Since the ADMA level will decrease due to the increase in DDAH, melatonin may increase the level of DDAH and protect the tissue from endothelial damage.
DDAH activity is accepted as the main determinant of endogenous ADMA concentration 31. In an experimental study, the hypercholesterolemic diet has been shown to increase ADMA levels and decrease DDAH activity and arginine/ ADMA ratio 32. In a rat study, it has been shown that melatonin treatment increased DDAH activity thus, reduce the level of ADMA and thereby inhibit kidney damage caused by bile duct ligation 33. Similarly, atherogenic lipid profile and serum ADMA levels were increased in rats exposed to high fructose diet, and oral melatonin treatment was reported to have positive effects on these parameters 34. In a clinical study, high ADMA level has been shown to reduce the drop in blood pressure due to increased endogenous melatonin release at night 35.
The accumulated evidence shows that the parameters counted as inflammation and oxidative stress indicator in liver tissue and plasma are associated to clinical and histological findings. Observations regarding the most significant histopathological changes in the HCT groups support the finding that cholesterol may damage the liver 36. In our study, significant changes such as sinusoidal congestion, cloudy bloating, periportal cell infiltration, periasiner lubrication and apoptosis were observed in the liver tissue by hypercholesterolemia. Melatonin prevented all these changes. In a recent study, Hong and colleagues 27 showed protective antifibrotic effects of melatonin on hepatic fibrosis induced by carbon tetrachloride in experimental rats. It has been reported that liver damage observed in ApoE -/- hypercholesterolemic mouse model was significantly reduced by oral support of melatonin 21. Melatonin has a free radical scavenger (receptor-independent) effect directly against reactive oxygen and nitrogen types, while indirectly (receptor-dependent) upregulates antioxidant enzymes and downregulates pro-oxidant enzymes 35. Besides that, it protects cellular function by showing antiapoptotic and anti-inflammatory effects with local receptor-mediated functions 37.
As a result, melatonin may influence NO bioavailability by modulating DDAH levels. The therapeutic administration of melatonin may also have effective consequences for liver protection. Melatonin may be a hepatoprotective and/or therapeutic agent as an antioxidant and regulator of NO metabolism to prevent histopathological changes in liver pathogenesis in diet-induced hypercholesterolemia.