Effects Of IL-10 On Neurites Outgrowth And Apoptosis Of Cultured Cortical Neurons After Oxygen-Glucose Deprived Via NF-κB Pathway

Object: To investigate the effects of IL‐10 on the apoptosis and the length of longest neurites,the expression of NF- κ B,NAIP and PSD-95, we employed oxygen and glucose deprivation model of rat primary cortical neurons culture within adding exogenous IL-10. It provided the theory basis for IL-10 to treat the ischemic cerebrovascular disease.Methods:1. Primary cortical neurons from rats were cultured and identified in vitro.2.To establish OGD-injured model, after cultured 5 days, primary cortical neurons were cultured in anaerobic chamber for 90 min. After 48 h later, morphology of OGD-injured cortical neurons were observed by light microscope and laser scanning confocal microscope.3.OGD-injured neurons were cocultured with drugs. Then experiments were randomly divided into 4 groups: Normal group(Control group),OGD-treated group(OGD group), PDTC and IL-10 treated group(OGD+IL-10+ PDTC group), Co-cultured group(OGD+IL-10 group).4.Following experiments were carried out after 24 h or 48 h culturing. Flow cytometry identified neuronal apoptosis. MTT identified neuronal vitality. Protein expression level of NF- κ B,NAIP and PSD-95 were detected by Western-bolt.Immunofluorescent staining identified cell bodies and neurites. Images were captured by ZEISS LSM 710 confocal microscope and the average length of the longest neurite was analyzed with ZEN2009 software.Results:1.The result of western blot: compared with normal control group, the expression of NF-κB was increased in OGD group(p<0.05), and the expression of NAIP and PSD-95 was decreased(p<0.01). The OGD+IL-10 group compared with OGD group,the expression of PSD-95 was increased(p<0.01),and the expression of NF-κB,NAIP was increased too(p<0.05). Compared with the OGD+IL-10 group,the expression of NF- κ B, NAIP and PSD-95 of the OGD+IL-10+PDTC group is decreased(p<0.01).2. The result of viability of neurons measured by flow cytometry: IL-10 increased cells survival induced by OGD in a concentration-dependent manner. The OGD group compared with normal control group, the average of viability was significantly decreased(21.333±0.788 vs. 6.767±0.185,p <0.01).Compared with OGD group, the average of viability was increased in OGD+IL-10( 12.000 ± 0.650 vs.21.333±0.78,p<0.01).Compared with OGD+IL-10 group, the average of viability was decreased in OGD+IL-10+PDTC group( 21.167 ± 2.974 vs. 12.000 ± 0.650, p<0.05).3.The result of average length of the longest neurite by immunofluorescence: The OGD group compared with normal control group, the average length of the longest neurites was significantly decreased(74.160±3.967 vs. 130.632±3.563,p <0.01).Compared with OGD group, the average length of the longest neurites was increased in OGD+IL-10 group(94.066±7.449 vs.74.160±3.967,p<0.01). Compared with OGD+IL-10 group, the average length was decreased in OGD+IL-10+PDTC(73.126±4.066 vs.94.066±7.449,p<0.01).Conclusion: IL-10 might promote anti-apoptosis and faciliates neurites outgrowth in neurons after OGD, which may be related to activation of NF- κ B signaling pathway and its down-stream targets NAIP and PSD-95.