We have previously demonstrated that ageing decreases the synthesis of phosphatidylcholine in isolated Wistar rats hepatocytes. Accumulating evidence indicates that free radicals (FR) play a major role in tissue and cell damage associated with ageing. FR generated within cells include the superoxide anion radical, the hydroxyl radical and nitric oxide (NO). Because of their high reactivity, these radicals can be devastatingly toxic to other molecules and cause cellular dysfunction and sometimes death of cells. Melatonin is an important antioxidant molecule. Besides its ability to scavenge highly reactive radicals, melatonin’s antioxidant activity is augmented by its ability to stimulate enzymes related to antioxidative defence systems. This study was designed to investigate a possible protective effect of melatonin against age-induced hepatocyte injury.
Hepatocytes were isolated from young (2 months old, n = 8) and old (24 months old) male Wistar rats. Old rats were randomly separated into three groups: non-treated rats (n = 8), rats treated with melatonin (1 mg kg-1 day-1) for 2.5 months (mel 2.5; n = 8), and rats treated with melatonin for 5 months (mel 5; n = 8). After humanely killing the animals by decapitation, cells were cultured in RPMI 1640 medium (supplemented with serum, glutamine, antibiotics and insulin) for 24 h. Then, media and cells were collected separately and CO and NO release to the medium, and cGMP, phosphatidylcholine (PC), and lipid peroxide (LPO) content of the cells were measured. Results are presented as means ± S.E.M. Mean comparison was done by Friedman’s analysis of variance followed by Wilcoxon’s two-tailed test for paired data; a confidence level of 95 % (P < 0.05) was considered significant. Experimental procedures employed are in accordance with the National rules of Spain (RD 223/1986).
Age increased LPO (5.6 ± 0.03 vs. 0.91 ± 0.05 fmol (µg protein)-1, P < 0.01). This increase was attenuated when animals were treated with melatonin (2.6 ± 0.03 and 2.19 ± 0.01 for mel 2.5 and mel 5, respectively). NO (1.99 ± 0.02 vs. 1.24 ± 0.005 nmol (µg protein)-1, P < 0.05), and CO (5.33 ± 0.1 vs. 1.93 ± 0.09 pmol (µg protein)-1, P < 0.05) release to the medium and cGMP (248 ± 2.1 vs. 46.6 ± 4.1 fmol (µg protein)-1, P < 0.01) content of the cells were increased in 24-month-old rats, and again this effect was diminished by melatonin treatment (1.05 ± 0.004, mel 2.5 and 1.04 ± 0.003, mel 5; 1.96 ± 0.1, mel 2.5 and 1.86 ± 0.2, mel 5; and 70.5 ± 3.7, mel 2.5 and 67.3 ± 2, mel 5 for NO, CO and cGMP, respectively). Additionally, melatonin was able to attenuate the decrease in PC synthesis induced by ageing.
In summary, these results suggest a possible protective effect for melatonin in age-induced liver injury. This effect could be mediated by reduction of FR generation.
This work was supported by a grant of C.A.M. 8.5/0062/2001.