Protective Effect Of Genistein On The Apoptosis Of Rat Cerebellar Granule Neurons Induced By Acrylamide

Objective:To established the apoptosis model of rat cerebellar granule neurons induced byacrylamide in vitro and investigated the protective effect and mechanism of genistein onapoptosis of cerebellar granule neurons.Methods:Cerebellar granule neurons were prepared from the cerebellar cortex cells of5-7day-old Spragule-Dawley rats.The neurons were identified by neuron-specific enolaseimmunofluorescence staining.After culturing for8days, the cells were passaged andwere divided randomly into different groups:①control group;②acrylamide modelgroup:cerebellar granule neurons were exposed to acrylamide with different dose of160μmol/L、640μmol/L、3.2mmol/L、16mmol/L、160mmo/L for24h,The IC50wereevaluated by cell inhibition rate in different groups;③genistein pretreatmentgroup:cerebellar granule neurons were pretreated with different concentration of10、25、50、100μmol/L of genistein for12h,then the culture medium was get rid of and thefresh solution with acrylamide IC50was added to the above mentioned volume andcultured for another24h.The neuronal viability was measured by MTT.Themorphology of neurons and their nuclei were observed by phase-contrast andHochest33342staining,respectively.The neuronal apoptosis index was observed byTdT-mediated dUTP nick end labeling (TUNEL).The mRNA expression of Bax andBcl-2mRNA were detected with Semi-quantitative RT-PCR.Results:1.After culturing for8d,the proportion of rat cerebellar granule neurons was about90%detected by neuron-specific enolase immunofluorescence staining.2.The results of MTT showed that the damage of rat cerebellar granule neurons inducedby acrylamide with the relationships of dosage-effect and time-effect. IC50ofacrylamide is10mmol/L and the dosage pulse for24h is proved to be the best model foracrylamide induced neurons apoptosis.The time and concentration of genisteinpretreatment had different effect on cerebellar granule neurons,The10μmol/L and 25μmol/L of genistein pretreatment for12h could significantly block the apoptosis ofcerebellar granule neurons induced by acrylamide.3.The inverted phase-contrast microscope and TUNEL assay showed that the shrink andfragmentation of neuronal cells nuclei and apoptosis index of cells induced byacrylamide were markedly inhibited.4.The results of Semi-quantitative RT-PCR showed that there were a higher levelexpression of Bax mRNA and a lower level expression of Bcl-2mRNA in acrylamidegroup,but the ratio of Bcl-2/Bax decreased in cerebellar granule neurons.In the10μmol/L and25μmol/L genistein pretreatment group there were a higher levelexpression of Bcl-2mRNA and a lower level expression of Bax mRNA, the ratio ofBcl-2/Bax increased in cerebellar granule neurons（P＜0.05）。.Conclusions:1. The apoptosis of cerebellar granule neurons induced by acrylamide in vitro wasrelated to decreasing the expression of Bcl-2mRNA and increasing the expression ofBax mRNA and decreasing the ratio of Bcl-2/Bax.2.Protective effect of genistein was related to its concentration, The concentration of10μmol/L and25μmol/L of genistein blocked significantly apoptosis of cerebellar granuleneurons induced by acrylamide, while no protection for apoptotic cells induced byacrylamide in the concentration of50μmol/L and100μmol/L.3.Protective effect of genistein against apoptosis induced by acrylamide in culturedcerebellar granule neurons was related to its role of increasing the expression of Bcl-2mRNA and decreasing the expression of Bax mRNA and increasing the ratio ofBcl-2/Bax.