Effects of diazoxide preconditioning on the expression of C-Jun N-terminal kinase In rat hippocampal neurons after anoxiareoxygenation In vitro

Abstract

Objective To investigate the effects of diazoxide preconditioning on the expression of C-Jun N-terminal kinase (JNK) in primary cultured rat hippocampal neurons after anoxia-reoxygenation (A/R). Methods One hundred SD rats (<24 h after birth) weighing 5-6 g were decapitated. Their hippocampi were removed and cut into chunks. Hippocampal neurons were enzymatically isolated. After being cultured for 9-10 d, the hippocampal neurons were randomly divided into 4 groups: group Ⅰ control (group C);group Ⅱ A/R;group Ⅲ, Ⅳ preconditioning with diazoxide 30 and 100 μmol/L (group D1, D2). In group A/R, D1 and D2 the neurons were exposed to 95 % N2-5% CO2 for 4 h followed by 24 hreoxygenation. In group D1 and D2 the neurons were pretreated with diazoxide 30 and 100 μmol/L respectively for 1 h before A/R for 3 consecutive days. The viability of neurons was measured by MTT assay. The expression of Bcl-2, Bax and JNK proteins was detected by Western blot analysis. Results The viability of neurons was significant lower in group A/R, D, and D2 than in control group and was significantly higher in group D2 than in group A/R. The expression of Bcl-2, Bax and JNK proteins was significantly up-regulated in group A/R, D1 and D2 as compared with control group. The expression of Bcl-2was significantly higher while the Bax and JNK protein expression was significantly lower in group D2 than in group A/R. Conclusion Preconditioning with diazoxide 100 μmol/L can ameliorate A/R injury to hippocampal neurons by down-regulatlng Bax expression and up-regulating Bcl-2 expression through inhibition of JNK signaling transduction pathway. Key words: Diazoxide; Ischemic preconditioning; JNK mitogen-activated protein kinases; Hippocampus; Neurons; Cell hypoxia; Oxygen