Objective: To observe the effect of integrin beta8 on the neuronal apoptosis after hypoxia ischemia (HI) in astrocyte/neuron co-culture system.
Methods: Astrocytes and neurons were cultured in vitro from cerebral cortex of the P1-3 days Sprague Dawley rats and E16 days fetal rats, respectively. Immunocytochemistry staining was used to identify the purity of cells. Integrin beta8 mRNA expression was qualified in the astrocytes at 12 hours, 1 day, and 2 days after HI and reoxygenation (experimental group) and in normal astrocytes (control group) by RT-PCR. Integrin beta8 small interering RNA (siRNA) system was established to specifically block astrocyte beta8 expression, the efficiency of integrin beta8 inhibition was detected by real-time fluorescent PCR. The astrocytes and neurons were co-cultured to established the astrocyte/neuron co-culture system. The neuronal apoptosis was detected with TUNEL in the normal neurons/astrocytes group (co-cultured HI group), the astrocytes infected by integrin beta8 siRNA for 2 days/normal neurons group (beta8 RNA interference group), and normal neurons in vitro with HI treatment group (HI group) at 1 day after HI and reoxygenation. The normal neurons without treatment as control (control group).
Results: Glial fibrillary acidic protein and neuronal nuclei staining suggested a purity of more than 90% in cultured cells. HI resulted in an increase of integrin beta8 mRNA expression at 12 hours after reoxygenation in astrocytes, which peaked at 1 day after reoxygenation, then slowly decreased and remained higher at 2 days, showing significant differences between control group and experimental group and among different time points in experimental group (P < 0.05). RNA interference efficiency was most significant at 2 days after astrocytes infected with integrin beta8 siRNA (P < 0.05). The neuronal apoptosis was significantly increased in HI group, co-cultured HI group, and beta8 RNA interference group when compared with control group (P < 0.05). But neuronal apoptosis index (AI) was significantly decreased in co-cultured HI group and beta8 RNA interference group when compared with HI group (P < 0.05). The significant difference of AI was found between co-cultured HI group and beta8 RNA interference group (P < 0.05).
Conclusion: Integrin beta8 expression can be induced with hypoxic-ischemic brain damage, leading to decreased AI of neurons and obvious protective effect.