Inhibitory effects of Allicin on apoptosis injury in hippocampus of young rats with lead poisoning

Abstract

Objective To investigate the inhibitory effect of Allicin on the apoptosis of hippocampal neurons induced by lead in rats. Methods Sixty male Sprague-Dawley rats aged 3 weeks were randomly divided into 6 groups, 10 rats in each group, which were low dose group(A-L), medium-dose group(A-M) and high dose (A-H) Allicin group and lead exposure group(Pb group), dimercaptosuccinic acid (DMSA) group and blank control group.The blank control group animals were treated with ultrapure water, and the other 5 groups received 1.0 g/L lead acetate aqueous solution instead of ultrapure water after 20 days and they were treated them with compounds by oral gavage.The doses of Allicin in group A-L, A-M group and A-H group were 2.7 mg/kg and 5.4 mg/kg, and 10.8 mg/kg, respectively.The DMSA dose was 10.8 mg/kg, and the Pb group was given 9 g/L saline.After the model was established, the rats were sacrificed to collect whole blood and hippocampus.Blood lead and tissue lead concentrations were measured, and the level of apoptosis in hippocampus was observed by TUNEL staining.The levels of cysteine-containing aspartate-specific proteases(caspase)-3, caspase-9, poly adenosine diphosphate-ribose polymerase (PARP) mRNA and caspase-3, caspase-9, PARP activated protein and cytochromes C distribution in the hippocampus cells were detected by using real-time quantitative PCR (qPCR), Western blot, and immunofluorescence staining. Results (1)Lead levels in the blood lead and hippocampus of rats in A-L group, A-M group and A-H group [(190.54±11.33) μg/L, (0.28±0.03) μg/L; (159.55±16.94) μg/L, (0.22±0.06) μg/L; (116.62±8.85) μg/L, (0.19±0.01) μg/L] were lower than those in Pb group [(271.34±21.23) μg/L, (0.31±0.04) μg/L], and there were significant differences (all P<0.05). The blood lead and hippocampal lead levels in the DMSA group [(50.12±7.44) μg/L, (0.15±0.03) μg/L] were lower than those in the A-L group, A-M group and A-H group.(2) The results of TUNEL staining showed that the apoptosis levels of hippocampus in A-L group, A-M group and A-H group were lower than that in Pb group [(2.81±0.17)%, (2.08±0.28)%, (1.33±0.08)% vs. (4.23±0.17)%], and there were significant differences (all P<0.05); the apoptosis level of hippocampus in the DMSA group [(2.63±0.32)%] was higher than that in the A-M group and the A-H group, which was lower than that in the Pb group.(3) qPCR results showed that the levels of caspase-3, caspase-9 and PARP mRNA in A-H group were down-regulated compared with Pb group (1.07±0.05, 1.02±0.02, 1.11±0.02 vs.1.34±0.02, 1.26±0.05, 1.93±0.07). The differences were statistically significant (P<0.05). The expression levels of caspase-3 and PARP mRNA in A-L group and A-M group were down-regulated (1.21±0.05, 1.43±0.12, 1.16±0.02, 1.20±0.06 vs.1.34±0.02, 1.93±0.07), and there were significant differences (all P<0.05), and there was no significant change in caspase-9 mRNA; the mRNA levels of caspase-3, caspase-9 and PARP in A-H group (1.07±0.05, 1.02±0.02, 1.11±0.02) were lower than those in DMSA group (1.14±0.02, 1.15±0.08, 1.32±0.05). (4) Western blot results: compared with Pb group, the expression levels of activated caspase-3, caspase-9 and PARP protein in A-H group were down-regulated (A-H group: 0.44±0.15, 0.58±0.25 and 0.31±0.19, 0.23±0.07 vs. Pb group: 0.69±0.13, 0.72±0.22 and 0.55±0.21, 0.43±0.10), the expression of activated caspase-9 protein in A-M group was lower than that in Pb group (A-M group: 0.59±0.18 vs. Pb group: 0.72±0.22), and there were significant differences (all P<0.05); the expression of activated caspase-3 and RARP protein in A-H group was lower than that in DMSA group.(5) Fluorescence staining showed that the expression of cytochrome C in cytoplasm of A-L group, A-M group and A-H group were significantly lower than that of Pb group and DMSA group. Conclusion Allicin can inhibit the apoptosis of hippocampus cells in rats with lead poisoning through mitochondrial pathway.The effect of Allicin on apoptosis inhibition may be better than DMSA. Key words: Allicin; Lead poisoning; Apoptosis; Mitochondrial pathway; Cysteine-containing aspartate-specific proteases family