Study On Endoplasmic Reticulum Stress-Related Apoptosis Of Rat Cerebral Cortical Neurons

PART Ⅰ Study on Endoplasmic Reticulum Stress related apoptosis of rat cerebral cortical neurons injuryed by Oxygen Glucose Deprivation in vitro.Objective To observe the apoptosis and Endoplasmic Reticulum Stress related changes of rat cerebral cortical neurons injuryed by Oxygen Glucose Deprivation in vitro. Methods Neonatal SD rat cerebral cortical neurons were cultured in vitro, identified with neuron-specific enolase by immunofluorescene staining. The neurons were randomly divided into Control group and OGD groups, and the simulated ischemia-reperfusion models were established by oxygen glucose deprivation on the neurons. Nerve cells’ morphology were observed by inverted phase contrast microscope, apotosis rates were determined by Annexin V-FITC-. PI double staining with flow cytometry. Nuclear morphology of neurons were observed with hoechst33258staining by fluorescense microscopy. The protein of GRP78and active caspase12were detected by Western-Blot method. Results SD neonatal rat cortical neurons can be cultured in vitro, the neurons purity was above90%. With the prolongation of reperfusion, the apoptosis rate of model groups increased (P<0.05. The apoptotic nuclei were dense stained, with fluorescence enhancement. Moreover, With the prolongation of reperfusion, GRP78protein levels and active caspasel2protein levels in model groups were gradually increased. However, GRP78protein levels dropped at48h. And the differences were statistically significant (P<0.05). Conclusions Simulated ischemia and reperfusion models were successfully built through oxygen-glucose deprivation in vitro. Neuronal apoptosis can be induced by the OGD models.Endoplasmic reticulum stress was involved in the apoptosis. PART Ⅱ The effect of low molecular weight heparin on ER-related apoptosis in rat cortical neurons injured by oxygen-glucose deprivation in vitro.Objective To observe the effect of low molecular weight heparin on cell apoptosis in rat cortical neurons injured by oxygen-glucose deprivation.Methods Neonatal rat cerebral cortical neurons were cultured in vitro,identified by Neuron-specific endolase immunofluorescene staining. The neurons were randomly divided into three groups:Control group, OGD group(Is6h/Re24h),and LMWH group(LMWH were gived two hours before OGD). The optical density value were calculated and neurons’ viabilities were detected by the MTT method. Flow cytometry was used to detect cell apotosis.Nuclear morphology of neurons were observed with hoechst33258staining by fluorescense microscopy. Results SD neonatal rat cortical neurons can be cultured in vitro,the neurons purity was above90%.Compared with normal control group, neuron activity decreased significantly and cell apoptosis rate increased drastically in OGD group.The difference is statistically significant (P<0.01).The neurons activity increased but apoptosis rate decreased significantly in LMWH group,and the difference is statistically significant (P<0.01). Conclusions Low molecular weight heparin can improve cell viability and reduce apoptosis rate of neurons injured by oxygen-glucose deprivation in vitro.Low molecular weight heparin has protective effects on rat cerebral cortical neurons. PART Ⅲ Research on the neuroprotective effects of low molecular weight heparin on the apoptosis induced by endoplasmic reticulum stress on rat cortical neuronsObjective To investigate the neuroprotective mechanisms of low molecular weight heparin on rat cortical neurons injured by oxygen-glucose deprivation.Methods Neonatal rat cerebral cortical neurons were cultured in vitro,identified by Neuron-specific endolase immunofluorescene staining. The neurons were randomly divided into three groups: Control group, OGD group,and LMWH group. The proteins of bcl-2,bax,GRP78and active caspase12in each group were detected by Western-Blot method.Intracellular cytoplasmic calcium concentration was detected by dual-wave length fluorescence spectrophotometer.Results SD neonatal rat cortical neurons can be cultured in vitro,the neurons purity was above90%.Compared with control group, bcl-2protein levels and the Bcl-2/Bax ratio in model group were significantly decreased,but bax protein levels was significantly increased(P<0.01);Compared with model group, bcl-2protein levels and the Bcl-2/Bax ratio in LMWH group were significantly increased, bax protein levels was decreased significantly(P<0.01);(P<0.01).Compared with control group,GRP78protein expression levels in Model group increased, GRP78protein levels of low molecular weight heparin group was highest (P<0.05).Compared with the control group, the active casase12protein levels in model group increased, but it decreased in LMWH group (P<0.05).The calcium assay Show:cytoplasmic [Ca2+] i in model group increased significantly compared with the control group (P<0.01),and it decreased in model group (P <0.01).Conclusions Low molecular weight heparin has neuroprotective effects on rat cortical neurons injuryed by ischemia-reperfusion in vitro.The mechanisms may be associated with the increase of bcl-2/bax ratio and GRP78expression, the decrease of active caspase12protein expression and cytoplasmic calcium concentration.