ObjectiveTo investigate the influence of sepsis on the expression of apoptotic protease caspase-3 in hippocampus neurons of rats. MethodsModels of rats with sepsis were established by the cecal ligation and puncture (CLP) method. Eighty 30-day-old male Wistar rats were randomly divided into CLP group (n=50) and control group (n=30). In the CLP group, only CLP was performed on the rats. Ten rats in the CLP group and another 10 in the control group were taken at 6, 12, and 24 hours after operation, respectively. Five of them in each group were taken for neurobehavioral score, and the other five were killed and their brains were removed. Then the Western blot and immunohistochemistry staining were used to detect the expression changes of apoptosis protein caspase-3. ResultsIn the control group, there were very low expression of apoptotic protease caspase-3 and high scores of neurological behavior. In the CLP group, the expression of apoptotic protease caspase-3 started to increase at the 6th hour, and reached the peak at the 24th hour after CLP, both of which were significantly higher than the control group (P<0.05). The scores of neurological behavior of the CLP group began to decline at 6h after CLP, and decreased gradually along with the time, and the scores were significantly lower at various time points after CLP in the CLP group than those in the control group (P<0.05). ConclusionThe scores of neurological behavior decrease and the expression of apoptosis protease caspase-3 increase in the rat hippocampus with sepsis, and fluctuate with time change.
ObjectiveTo observe the effect of integrin β8 on the neuronal apoptosis after hypoxia ischemia (HI) in astrocyte/neuron co-culture system. MethodsAstrocytes 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 β8 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 β8 small interering RNA (siRNA) system was established to specifically block astrocyte β8 expression, the efficiency of integrin β8 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 β8 siRNA for 2 days/normal neurons group (β8 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). ResultsGlial fibrillary acidic protein and neuronal nuclei staining suggested a purity of more than 90% in cultured cells. HI resulted in an increase of integrin β8 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 β8 siRNA (P<0.05). The neuronal apoptosis was significantly increased in HI group, co-cultured HI group, and β8 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 β8 RNA interference group when compared with HI group (P<0.05). The significant difference of AI was found between co-cultured HI group and β8 RNA interference group (P<0.05). ConclusionIntegrin β8 expression can be induced with hypoxic-ischemic brain damage, leading to decreased AI of neurons and obvious protective effect.