Effects Of IL-10on The Apoptosis And Expression Of PI3K/AKT/Caspase-3in Rat Cortical Neurons Followed OGD Injury

Object: To investigate the effects of IL‐10on the apoptosis and expression ofPI3K/AKT/Caspase-3signaling pathway, we employed oxygen and glucosedeprivation model of rat primary cortical neurons culture within adding exogenousIL-10. It provided the theory basis for IL-10to treat the ischemic cerebrovasculardisease.Methods:1. Primary cortical neurons from rats were cultured and identified in vitro.2. To establish OGD-injured model, after cultured5days, primarycortical neurons were cultured in anaerobic chamber for90min. After48h later,morphology of OGD-injured cortical neurons were observed by light microscope and laserscanning confocal microscope.3. OGD-injured neurons were cocultured with drugs. Then experimentswere randomly divided into5groups: Normal group (Control group), Co-culturedgroup (OGD+IL-10group), LY294002and IL-10treated group (OGD+IL-10+LY294002group), LY294002treated group (OGD+LY294002group), OGD-treatedgroup (OGD group).4. Following experiments were carried out after24h or48h culturing. Flowcytometry identified neuronal apoptosis. MTT identified neuronal vitality. Proteinexpression level of AKT、pAKT and Caspase-3were detected by Western-bolt.Immunofluorescent staining identified cell bodies and neurites. Images were capturedby ZEISS LSM710confocal microscope and the apoptotic rate was analyzed withZEN2009software.Results:1．The result of viability of neurons measured by MTT: IL-10increased cellssurvival induced by OGD in a concentration-dependent manner. The OGD group compared with normal control group, the average of viability was significantlydecreased （p <0.01）, There were no significant difference between IL-10+OGD andnormal control group （p＞0.05）. Compared with OGD group, the average ofviability was increased in OGD+IL-10（p <0.01）. Compared with OGD group, theaverage of viability was decreased in OGD+LY294002（p＜0.05）2．Neuron axon was damage to short and part of the nucleus was fragmentation,pyknosis and dissolution after OGD.3. The result of neuronal apoptosis by immunofluorescence: The OGD groupcompared with normal control group, the average of apoptotic rate was significantlyincreased （p <0.01）. There were no significant difference between IL-10+OGD andnormal control group （p＞0.05）. Compared with OGD+IL-10group, the averageof apoptotic rate was increased in OGD group and OGD+IL-10+LY294002（p＜0.05）. Compared with OGD group, the average of apoptotic rate was increased inOGD+LY294002（p <0.01）.4. The result of neuronal apoptosis by flow cytometry: The OGD group comparedwith normal control group, the average of apoptotic rate was significantly increased（p<0.01）. There were no significant difference between IL-10+OGD and normalcontrol group （p＞0.05）. Compared with OGD+IL-10group, the average ofapoptotic rate was increased in OGD group（p<0.01）. Compared with OGD group, theaverage of apoptotic rate was increased in LY294002（p＜0.05）.5．The result of western blot: compared with OGD group and OGD+IL-10+LY294002group, the expression of p-AKT was increased in OGD+IL-10group（p＜0.05）, but the expression of Caspase-3was decreased（p＜0.05）. There were nosignificant difference between IL-10+OGD and normal control group （p＞0.05）. Conclusion: Neuronal apoptosis could be induced by OGD. IL-10might promoterepair, which may be related to activation of PI3K/AKT/Caspase-3signalingpathway.