Oxidative Stress Injuries Induced By Lead And The Expression Of JWA MRNA In Primary Cultured Rat Cerebral Neurons

Lead,as a toxic heavy metal with steady chemical property,could cause severe damage to the nervous system which is important target organ of lead toxicity.Exposure to lead could cause cellular oxidative damage by reducing the activities of antioxidant enzymes and the contents of some antioxidants such as superoxide dismutase,catalase,glutathione,etc.Therefore,lead neurotoxicity has close correlation with oxidative stress and oxidative injury.As an active environmental response gene,JWA widely participated in the regulation of oxidative stress induced by a variety of environmental factors so as to protect cells against oxidative injury.Up to now,the effect of JWA in lead toxicity has not been reported.In this research,primarily cultured cortical neurons of newborn rats in vitro will be used to study the expression of JWA and its possible mechanism in lead neurotoxicity.Objective: To study the effects of lead on the oxidative stress and the expression of JWA in cerebral neurons of rats so as to explore the possible mechanism of JWA in neuronal oxidative stress induced by lead and provide new clues and basis for the further investigate on the neurotoxic mechanism of lead from the molecular level.Methods: After separated from the newborn Sprague-Dawley rats within 24 hours,cortical neurons were cultured primarily and identified by immunocytochemistry method.The well-grown cortical neurons in vitro were treated with neural feeding medium containing lead acetate with final concentration of 0 μmol/L,31 μmol/L,54 μmol/L,92 μmol/L,150 μmol/L respectively for a definite time.Then the rates of cerebral neurons’ survival were detected by MTT method and the activity of superoxide dismutase,the content of malondialdehyde,the activity of catalase and the content of glutathione in cerebral neurons were measured by the related kits respectively.The expression of JWA mRNA in cortical neurons was detected with QRT-PCR technique.Results:(1)The results of morphological analysis and immunocytochemistry showed that the cortical neurons formed an integrated neural network on day 7 of culturing and developed satisfactorily with a high purity,which could be used for the subsequent research.(2)With increasing of concentrations of lead acetate,the number of neurons gradually reduced.After exposed to lead for 24 hours or 48 hours,the growth status of cortical neurons in 92 μmol/L and 150 μmol/L lead acetate groups had statistically significant difference with other dose groups(P<0.05),and the average optical density of cortical neurons were 0.218 ± 0.008 and 0.181 ± 0.006 after exposed to 92 μmol/L lead,while they were 0.199 ± 0.008 and 0.166 ± 0.009 after exposed to 150 μmol/L lead respectively.From the above,the appropriate concentration of lead acetate was 92 μmol/L and the appropriate exposure time was 24 hours.(3)After the cortical neurons were exposed to different concentrations of lead and incubated for 6 hours,12 hours,24 hours and 48 hours,the activity of SOD,the activity of CAT and the content of GSH decreased significantly(P<0.05),while the content of MDA increased significantly(P<0.05)compared with control group.Correlation analysis results revealed that the activities of SOD and CAT and the content of GSH in cerebral neurons of rats were negatively correlated with lead dose,while MDA contents were positively correlated(P<0.01).(4)With increasing of concentrations of lead acetate,the expression of JWA mRNA decreased gradually(P<0.05).Appropriate superoxide dismutase addition to cerebral neurons after exposed to lead,the expression of JWA mRNA had no statistical significance(P>0.05).While appropriate catalase addition to cerebral neurons after exposed to lead,the expression of JWA mRNA increased significantly(P<0.05).Correlation analysis results revealed that the activities of SOD and CAT and the content of GSH in cerebral neurons of rats were positively correlated with the expression of JWA mRNA,while the content of MDA were negatively correlated(P<0.01).Conclusion: Exposure to lead could cause oxidative stress injury to cortical neurons of rats and affect the expression of JWA.