Short-term exposure to stavudine results in neuron apoptosis, neurite shrink and down-regulated expression of thymidine kinase 2 / 中华传染病杂志

ABSTRACT

Objective To investigate the central neurotoxicity induced by nucleoside analog reverse transcriptase inhibitors (NRTIs)-stavudine (D4T).Methods Mouse primary cortical neurons were cultured and treated with different concentrations of stavudine.Neuron apoptosis was analyzed by calcein/acetomethoxy/propidium iodide (AM/PI) staining. Morphological change of neuron was confirmed by immunofluorescence.Mitochondrial DNA copies which were usually evaluated through Cycloxygenase 2 (COX-2) and thymidine kinase2 (TK2) mRNA were determined by real-time quantitative polymerase chain reaction.Chi-square test,student t test and Wilcoxon nonparameter test were used to analyze the data.Results Neuronal apoptosis observed in 50 μmol/L D4T treatment group was more significant than that in 0μmol/L D4T treatment group and 25 μmol/L D4T treatment group (51.3％±12.4％ vs 24.9％±8.2％ and 26.5％±10.6％,respectively; x2 =7.25 and 6.93,respectively; both P＜0.01).The average neurite numbers of each neuron were 11.2±3.6 in 0μmol/L D4T treatment group,8.6±2.8 in 25 μmol/L D4T treatment group and 4.3±2.4 in 50 μmol/L D4T treatment group.The difference was statistically significant between 25 μmol/L D4T treatment group and 0 μmol/L D4T treatment group (t=4.06,P＜0.01) and between 50 μmol/L D4T treatment group and 25 μmol/L D4T treatment group (t =4.35,P＜ 0.01). Furthermore,the average lengths of neuritis were (319.9±100.2) μm in 0 μmol/L D4T treatment group,(298.3±83.9) μm in 25 μmol/L D4T treatment group and (258.4±82.2) μm in 50 μmol/L D4T treatment group.The difference was statistically significant between 25 μmol/L D4T treatment group and 0 μmol/L D4T treatment group (t=4.58,P＜0.01) and between 50 μmol/L D4T treatment group and 25 μmol/L D4T treatment group (t=4.65,P＜0.01).TK2 mRNA expression dramatically decreased along with the increasing D4T concentration.The fold changes were 0.34 in 25 μmol/L D4T treatment group and 0.08 in 50 μmol/L D4T treatment group. The difference was statistically significant between 25 μmol/L D4T treatment group and 0μmol/L D4T treatment group (Z=- 3.28,P＜0.01) and between 50 μmol/L D4T treatment group and 25 μmol/L D4T treatment group (Z=-4.25,P＜0.01).Compared with 0μmol/L D4T treatment group,the relative fold changes of COX-2 copies were 1.01 in 25 μmol/L D4T treatment group and 1.12 in 50 μmol/L D4T treatment group.The differences were not significant among the three groups (Z=0.98 and 1.24,respectively; both P＞0.05).Conclsion It suggests that short-term exposure to D4T may result in neuron apoptosis,neurite shrink and down-regulated expression of TK2,but the level of mitochondrial DNA copies keeps stable.