| ObjectivesOn the basis of successful rat cortical neuron isolation, purification, culture and identification, to establish the model of ischemia/reperfusion (I/R), to clarify the mechanism of neuronal apoptosis during I/R induced injury and to explore the effect of polydatin (PD)on I/R induced cortical neuron injury in vitro.MethodsThe 24-hours-old newborn Sprague Dawley rats were sacrified by disconnecting the neck under aseptic conditions and the brain was taken out. The meninges and blood vessels on the surface of the brain were removed. The cortical nerons were dissociated from the cerebra, cultured for 7 days and the purity of the cortical nerons was identified. Then were randomly divided into three groups, the control group, the I/R injury group and the PD treatment group. The cortical neurons were cultuered with DMEM/F12 medium in control group. The neurons were treated with Sodium dithionite for 15 minutes and then cultuered with DMEM/F12 medium in I/R injury group. The PD was added before, at the same time with and after Sodium dithionite treatment in PD treatment group. The morphological features of the neurons were observed under fluorescence microscope with AO/EB and DAPI immunofluorescence staining. Apoptosis were measured through TUNEL method. The neuronal expressions of Bcl-2, Bax and NF-κBp65 were determined by immunohistochemical method.Results(1)At the 7th day, the primary cortical neurons grown well, there are plump and solid bodies, nervous processus contacted with eath other and a dense network formed under inverted phase contrast microscope.(2)Morphological changes of neuronal apoptosis can be observed under fluorescence microscope (AO/EB and DAPI) in I/R injury group.(3)The number of apoptotic cells in I/R injury group kept no change at 0h, increased at 6h, peaked at 24h, and decreased at 48h. The rate of neuronal apoptosis was increased in I/R injury group compared with that in control group at any corresponding time point of 6h, 12h, 24h and 48h, with statistical significance(P<0.05).The rate of neuronal apoptosis was lower in PD treatment group than that in I/R injury group at any corresponding time point of 6h, 12h, 24h and 48h, with statistical significance (P<0.05). (4)The expressions of neuron NF-κBp65 increased with time after reperfusion, peaked at 24h and then decreased in I/R injury group. Marked translocation of NF-κBp65 from cytolymph into nucleus were found from 24h to 48h in I/R injury group. There exists a strong negative correlation between NF-κBp65 and Bcl-2/Bax, with statistical significance (P<0.05). The expressions of neuron NF-κBp65 were decreased in PD treatment group, and compared with those in control group at any corresponding time point of 6h, 12h, 24h and 48h, with statistical significance (P<0.05). The expression of Bax increased significantly with time after reperfusion, peaked at 24h and then decreased in I/R injury group. The expressions of Bcl-2 and Bcl-2/Bax ratio increased with time after reperfusion, peak at 6h and then decreased. There was significantly negative correlation between Bcl-2/Bax and apoptosis (P<0.05). Increased expression of Bcl-2 but decreased Bax expresion were found in PD treatment group, and compared with those in I/R group at any corresponding time point of 6h, 12h, 24h and 48h, with statistical significances (P<0.05).Conclusions(1)On the basis of newborn rat primary cortical neuron isolation, purification and culture, we successfully established the model of I/R induced cortical neuron injury in vitro.(2)The Polydatin may have protective effect by decreasing the rate of neuron apoptosis during I/R insult in vitro. The possible mechanism of decreased neuron apoptosis may be through the suppression of NF-κBp65 activiation and up-regulation the ratio between Bcl-2 and Bax. |
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