[Effects of arsenic poisoning on neuronal cell apoptosis and mRNA and protein expression of calpain 1, calpain 2, and cdk5/p25].

OBJECTIVE

To study the effect of arsenic on neuronal cell apoptosis and the mRNA and protein expression of calpain 1, calpain 2, and cyclin-dependent kinases 5 (cdk5)/p25 and to provide a scientific basis for the research on neurotoxic mechanism of arsenic trioxide (As2O3).

METHODS

Primary cultured rat neurons were divided into untreated control group, dimethyl sulfoxide (DMSO) solvent control group, and 1, 5, and 10 µmol/L As2O3 treated groups. Eight hours after being treated with As2O3, cell apoptosis rate was determined by flow cytometry, the mRNA expression of calpain 1, calpain 2, cdk5, and p35 was measured by real-time fluorescence quantitative PCR, and the protein expression of calpain 1, calpain 2, cdk5, p35, and p25 was measured by Western blot.

RESULTS

Compared with those in the untreated control group and DMSO solvent control group, the cell apoptosis rates in the 5 and 10 µmol/L As2O3 treated groups were significantly increased (P < 0.05). The mRNA expression levels of calpain 1 were 6.36±3.26, 7.11±5.13, and 7.47±2.59 in the 1, 5, and 10 µmol/L As2O3 treated groups, respectively, and the mRNA expression levels of cdk5 were 1.27±0.19, 1.54±0.04, and 1.79±0.21 in the 1, 5, and 10 µmol/L As2O3 treated groups, respectively, which were significantly higher than those in the untreated group (0.72±0.15 and 1.77±0.87) and those in the DMSO solvent control group (0.96±1.23 and 1.18±0.09) (P < 0.05). The mRNA expression levels of p35 in the 1 and 5 µmol/L As2O3 treated groups were 2.17±0.59 and 2.51±0.51, respectively, which were significantly higher than that in the untreated control group (1.26±0.37) (P < 0.05). The protein expression levels of calpain 1 were 0.37±0.10, 0.42±0.13, and 0.51±0.18 in the 1, 5, and 10 µmol/L As2O3 treated groups, respectively, which were significantly higher than those in the untreated control group (0.11±0.08) and DMSO solvent control group (0.13±0.07) (P < 0.05). In the 5 and 10 µmol/L As2O3 treated groups, the protein expression levels of cdk5 were 0.34±0.12 and 0.37±0.21, while the protein expression levels of p25 were 0.31±0.23 and 0.55±0.16, all of which were significantly higher than those in the untreated control group and DMSO solvent control group (P < 0.05). The protein expression levels of p35 were reduced in the 5 µmol/L As2O3 treated group (0.31±0.23) and 10 µmol/L As2O3 treated group (0.26±0.16), as compared with those in the untreated control group and DMSO solvent control group (P < 0.05). The mRNA and protein expression of calpain 2 showed no significant differences between all groups (P > 0.05).

CONCLUSION

The calpain 1-cdk5/p25 pathway may be involved in the process of As2O3-induced neuronal cell apoptosis.