Research On Diazoxide Preconditioning To Anti-apoptosis Of Hippocampal Neurons By Hypoxia-reoxygenation In Rats

Objective 1. To investigate if there is influence on expression of Akt, Bcl-2 and Bax proteins in cultured hippocampal neurons by hypoxia-reoxygenation after DZ Preconditioning duiring its anti-apoptosis in rats. 2. To investigate if Akt signal transduction passageway completely or partly mediated the expression of Bcl-2 and Bax proteins in cultured hippocampal neurons by hypoxia-reoxygenation in rats after DZ Preconditioning based on Part One Experiment. Methods 1. Cultured hippocampal neurons were assigned into the following five groups randomly, normal control group (Group A), oxygen deficiency group (Group B), oxygen deficiency with DZ preconditioning group (Group C), oxygen deficiency with DZ and 5-HD preconditioning group (Group D), oxygen deficiency with 5-HD preconditioning group (Group E). Adopting sixteen holes or two holes for every group in erery time of these experiments, and these experiments repeat three times. Two days before oxygen deficit, oxygen deficiency group (Group B), oxygen deficiency with DZ preconditioning group (Group C), oxygen deficiency with DZ and 5-HD preconditioning group (Group D), oxygen deficiency with 5-HD preconditioning group (Group E) add the final concentration of DZ 0μmol/L, DZ 100μmol/L, DZ 100μmol/L+5-HD 100μmol/L, 5-HD 100μmol/L for Preconditioning. Observe the morphology change of hippocampal neurons; Determin cell vitality by blue tetrazoline chromatometry; Apply flow cytometry to detect the early apoptosis rate of hippocampal neurons, and assay express level of Akt, Bcl-2 and Bax proteins in hippocampal neurons 24h after hypoxia-reoxygenation. 2. Cultured hippocampal neurons were assigned into the following four groups randomly, normal control group (Group A), oxygen deficiency group (Group B), oxygen deficiency with DZ preconditioning group (Group C), oxygen deficiency with DZ and LY294002 preconditioning group (Group D). Adopting sixteen holes or two holes for every group in erery time of these experiments, and these experiments repeat three times. Two days before oxygen deficit, oxygen deficiency group (Group B), oxygen deficiency with DZ preconditioning group (Group C), oxygen deficiency with DZ and LY294002 preconditioning group (Group D), add the final concentration of DZ 0μmol/L, DZ 100μmol/L, DZ 100μmol/L+LY294002 50μmol/L for Precondi- tioning. Observe the morphology change of hippocampal neurons; Determin cell vitality by blue tetrazoline chromatometry; Apply flow cytometry to detect the early apoptosis rate of hippocampal neurons, and assay express level of Akt, Bcl-2 and Bax proteins in hippocampal neurons 24h after hypoxia-reoxygenation. Results 1. There are more amount, better shape, less neorobiosis and more cell-cell junction in Group C compared to those in other three oxygen deficiency groups; The vitality of hippocampal neurons in Group C is significant lower than that in Group A (P<0.01), but obviously higher than those in other three oxygen deficiency groups (P<0.01); The early apoptotic rate of hippocampal neurons in Group C is significantly higher than that in Group A (P<0.01), but obviously lower than those in other three oxygen deficiency groups (P<0.01); Akt, Bcl-2 and Bax proteins in Group C expressed more intensively than that in Group A (P<0.01), compared to other three oxygen deficiency groups, the express of Akt and Bcl-2 proteins in Group C is more intensively (P<0.01), whereas Bax protein was opposite (P<0.01). 2. Compared with cells in Group C, there is a great amount of neorobiosis and drop-off in Group D, the remains become sparse, lose the normal shapes and decrease in junction between cells; The vitality of hippocampal neurons in Group D is significantly lower than that in Group C (P<0.01), there is no statistical significance between Group D and Group B (P>0.05); (3)The early apoptotic rate of hippocampal neurons in Group D is significantly higher than that in Group C (P<0.01), there is no statistical significance between Group D and Group B (P>0.05); (4) The expression of Akt, Bcl-2 proteins in Group D is weaker than that in Group C (P<0.01), whereas the expression of Bax protein is stronger (P<0.01). And there is no statistical significance between Group D and Group B in the expression of Akt, Bcl-2 and Bax proteins (P>0.05). Conclusions 1. DZ precondi- tioning could enhance the hippocampal neuronal vitality and cut down the hippocampal neuronal early apoptotic rate; DZ Preconditioning could up-regulating expression of Akt, Bcl-2 proteins and down-regulating expression of Bax protein in Cultured hippocampal neurons by hypoxia-reoxygenation. 2. Akt signal transduction passageway completely or partly mediated the up-regulating expression of Bcl-2 protein and the down-regulating expression of Bax protein in Cultured hippocampal neurons by hypoxia-reoxygenation in rats after Diazoxide preconditioning.