| Object: To explore the effects of Neuritin on Endoplasmic Reticulum Stress(ERS)related apoptosis induced by oxygen–glucose deprivation and reperfusion in rat cerebral cortical neurons.Methods:(1)The cortical brain tissue of neonatal SD suckling mice(within 24 h)was digested and then inoculated into a six-well plate with DMEM-HG medium containing 10% fetal calf serum.After 2–4 h,the medium was changed to neurobasal growth medium containing 2% B27 to cultivate.(2)The growth medium was changed every 2–3 days and observed under an inverted phase contrast microscope.Morphological changes of neurons were recorded.NSE/PI double–staining immunofluorescence was used to identify the purity of neurons.(3)The primary cortical neurons of mature rat cortical neurons cultured for 7–8 days were randomly divided into the blank control group,the oxygen–glucose deprivation/reperfusion(OGD/R)group and the Neuritin–treatment group.(4)The expressions levels of GRP78,Caspase-12,and CHOP protein were detected by Western blot.The expression levels of GRP78,caspase-12,and CHOP mRNA were detected by quantitative real time polymerase chain reaction(qRT-PCR).(5)The neuronal apoptosis rate was detected by flow cytometry after Annexin V–FITC/PI double–staining.(6)MTT method was used to calculate the relative survival rate of neurons under different concentrations of Neuritin and the optimal concentration of Neutritin was screened.(7)Ultrastructural changes of neuronal rough ER were observed under a transmission electron microscope.Results:(1)Cortical neurons were successfully isolated and cultured,and the cytoplasm,dendrites,and axonal patterns were found to be typical.(2)Neuron purity was examined by immunofluorescence assay and found to be >90%.(3)In the OGD/R group,the protein and mRNA expression levels of GRP78 were increased at 6,12,24,and 48 h and peaked at 24 h,and Caspase-12 were increased at 12,24,and 48 h and peaked at 48 h,and CHOP were increased at 6,12,24 and 48 h and peaked at 48 h,the difference were statistically significant compared with the blank control group(P<0.05).(3)There was no statistically significant difference of neuronal activity in the 50ng/mL and 100ng/mL Neutritin groups compared with the blank control group(P > 0.05).Neuronal viability was significantly different in the 150ng/mL,200ng/mL,and 250ng/mL Neuritin groups compared with the blank control group(P<0.05).There was a statistically significant difference of neuronal activity in 200ng/mL compared with the 150ng/m L and250ng/mL Neuritin groups(P<0.05).(4)The apoptotic rates of neurons in the OGD/R group at 6,12,24,and 48 h were significantly higher than those of the blank control group,and the difference was statistically significant(P<0.05).Compared with the OGD/R group,Neuritin–treatment group can significantly reduce the expression of GRP78,Caspase-12,and CHOP and the rate of neuronal apoptosis,and the difference was statistically significant(P<0.05).(5)Compared with the blank control group,the rough ER volume of the OGD/R group were increased and with severely edematous.Compared with the OGD/R group,the Neuritin–treatment group showed a significantly restored regular shape,reduced number of vesicles,improved edema,and increased neuron content of in ER.Conclusion: OGD/R can lead to the apoptosis–induced by ER stress as reflected by the expressionchanges of GRP78,Caspase-12,and CHOP in rat cerebral cortical neurons.200ng/mL Neuritin can significantly inhibit the apoptosis and improve the neuronal activity of cortical neurons after OGD/R.Neuritin can inhibit the expression of GRP78,caspase-12,and CHOP,then reduce the apoptosis rate of neurons,and alleviate the ultrastructural changes of neurons in rough ER... |
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