ABSTRACT
Objective To investigate the effect of transduction of heme oxygenase-1 (HO-1) protein on oxygen-glucose deprivation and restoration (OGD/R)-induced injury to hippocampal neurons in rats.Methods Plasmid 11R-HO-1 was constructed using plasmid pET-21a(+)-p53-11R (plasmid 11R) and 11R-HO-1 fusion protein was identified and collected.Hippocampal neurons obtained from newborn Wistar rats (＜ 48 h) were cultured for 7 days in vitro and then the neurons were randomly divided into 5 groups (n =171 each) using a random number table:OGD/R group,normal saline group (group NS),plasmid 11R group (11R group),300 nmol/L 11R-HO-1 group (H1 group),and 1 500 nmol/L 11R-HO-1 group (H2 group).In NS,11R,H1 and H2 groups,the neurons were incubated for 2 h with 300 nmol/L normal saline,300 nmol/L plasmid 11R,300 nmol/L 11R-HO-1 fusion protein,and 1 500 nmol/L 11R-HO-1 fusion protein,respectively,and then OGD/R was performed.The neurons were incubated in deoxygenated glucose-free DMEM medium and sealed under 5 ％ CO2-95 ％ N2 in an anaerobic chamber equilibrated to 37 ℃ for 45 min.OGD was terminated by replacement of the medium with high glucose DMEM medium and by returning the cultures to a standard incubator maintained at 37 ℃ in 5 ％ CO2-95 ％ air and the neurons were then incubated for 24 h.Immediately after OGD/R was established,the cell survival rate (by MTT assay),apoptosis rate (using TUNEL),and expression of HO-1 and caspase-3 protein (by using Western blot) were measured.Results Compared with group OGD/R,the cell survival rate was significantly increased,the apoptosis rate was decreased,the caspase-3 expression was down-regulated,HO-1 protein expression was up-regulated in H1 and H2 groups (P ＜ 0.05),and no significant change was found in the parameters mentioned above in NS and 11R groups (P ＞ 0.05).Compared with group H1,the cell survival rate was significantly increased,the apoptosis rate was decreased,the caspase-3 expression was down-regulated,and HO-1 protein expression was up-regulated in group H2 (P ＜ 0.05).Conclusion Transduction of HO-1 protein can reduce OGD/R-induced injury to hippocampal neurons of rats.
ABSTRACT
ObjectiveTo investigate the effect of heme oxygenase-1 (HO-1) on cyclin-dependent kinase 5 (CDK5)-ataxia telangiectasia mutated (ATM)-P53 signal transduction pathway in rat hippocampal neurons subjected to oxygen-glucose deprivation (OGD) injury.MethodsHippocampal neurons of newborn Wistar rats ( ＜ 48 h) were cultured for 7 days in vitro.The primary cultured neurons were randomly divided into 4 groups with 10 wells in each group:control group (group C),OGD (group D),OGD + hemin (HO-1 inducer) group (group D + H ) and OGD + hemin + zinc protoporphyrin ( HO-1 inhibitor) group ( group D + H + T).For OGD experiments,cultures were washed three times in a glucose-free balanced salt solution (BSS).They were then placed in deoxygenated glucose-free medium and sealed under 95％ N2-5％ CO2 in an anaerobic chamber equilibrated to 37°C and 100％ humidity for 45 min.OGD was terminated by replacement of stored medium and by returning the cultures to a standard incubator maintained at 37 ℃ in 95％ air-5％ CO2.The OGD model was established after the neurons were preconditioned with hemin 10 μmol/L for 24 h in group D + H.The OGD model was established after the neurons were preconditioned with hemin 10 μmol/L and zinc protoporphyrin 10 μmol/L for 24 h in group D + H + T.After 24 h of culture,the neuronal viability,apoptosis rate,and expression of HO-1 mRNA and protein,and CDK5,ATM and P53 protein were detected.ResultsCompared with group C,the expression of HO-1 mRNA,and HO-1,CDK5,ATM and P53 protein was up-regulated,the neuronal viability was significantly decreased,and the apoptosis rate was significantly increased in group D (P ＜ 0.01 ).Compared with group D,the expression of HO-1 mRNA and protein was up-regulated,the expression of CDK5,ATM and P53 protein was down-regulated,the neuronal viability was significantly increased,and the apoptosis rate was significanlly decreased in group D + H ( P ＜ 0.01 ).Compared with group D + H,the expression of HO-1 mRNA and protein was down-regulated,the expression of CDK5,ATM and P53 protein was up-regulated,the neuronal viability was significantly decreased,and the apoptosis rate was significantly increased in group D + H + T ( P ＜ 0.01 ).ConclusionHO-1 can inhibit neuronal apoptosis through blocking CDK5-ATM-P53 signal transduction pathway in rat hippocampal neurons subjected to OGD injury.