RESUMO
Objective:To explore the effect of rapamycin on 1-methyl-4-phenylpyridinium iodide (MPP+ )-induced activation of Nod-like receptor protein 3 (NLRP3) inflammasome in microglia.Methods:The BV2 microglia cells were divided into control group, model group and rapamycin group.The model group and rapamycin group were treated by MPP+ to activate NLRP3 inflammasome, and rapamycin group was pretreated with rapamycin.Quantitative real-time PCR (RT-qPCR) was used to detect the mRNA levels of NLRP3, apoptosis-associated speck-like protein (ASC) and caspase-1.Immunofluorescence was used to detect the protein expression of NLRP3 and interleukin-1β (IL-1β). Western blot was carried out to assess the protein expression of NLRP3, ASC, caspase-1, beclin1 and microtubule-associated protein 1 light chain 3 (LC3).Results:The mRNA levels of NLRP3, ASC and caspase-1 in model group were higher than those in control group ( t=4.825, 3.015, 5.853, all P<0.05). The mRNA levels of NLRP3 and caspase-1 in rapamycin group were lower than those in model group ( t=2.75, 2.89, both P<0.05). In model group, the protein expressions of NLRP3 (1.54±0.22), ASC (1.02±0.13) and caspase-1 (1.42±0.30) were higher than NLRP3 (0.66±0.15), ASC (0.41±0.14) and caspase-1 (0.70±0.10) in control group ( t=5.653, 5.602, 3.964, all P<0.01), while the protein expression of beclin1 (0.28±0.09) and LC3II/LC3I ratio(0.69±0.14) were lower than beclin1 (0.60±0.11) and LC3II/LC3I (1.29±0.23) in control group ( t=4.010, 3.982, both P<0.01). The protein expressions of NLRP3 (0.80±0.18) and ASC (0.68±0.14) in rapamycin group were lower than those in model group ( t=4.413, 3.077, both P<0.05), while the protein expression of beclin1 (0.65±0.20) and LC3II/LC3I ratio(1.42±0.36) were higher than those in model group ( t=2.965, 3.278, both P<0.05). Conclusion:MPP+ activates NLRP3 inflammasome and impairs autophagic function in microglia.Rapamycin inhibits MPP+ -induced activation of NLRP3 inflammasome by restoring autophagic impairment in microglia.
RESUMO
Objective To establish the cell model of intractable epilepsy and to observe its neuronal damage and morphologic change of neurites.Methods The model was established by exposing hippocampal neurons to Mg2+ -free media for 3 hours on days 10 of culture.Expression of lactic acid dehydrogenase (LDH) in supernatant was measured as an index of neuronal damage.The morphologic change of neurons and neurites was observed by optical microscope and scanning electron microscope (SEM).Results Compared to the control group, level of LDH (U/L) was significantly increased in the model group at different time points (3 hours: 4.26 ± 1.28, 6 hours: 6.56 ±2.34 and 24 hours: 16.67 ±3.57, P <0.05).With time prolonging, release of LDH in the model group was gradually increased (F = 39.316,P <0.05).Under optical microscope, neurons of model group migrated closely to each other and neurite connections appeared to be gradually "reticulated" after Mg2+ -free media treatment for 24 hours; and the "reticulated" neurites connections become more obvious after 72 hours.Under SEM, neuronal membrane was rough and had several small depressions, neurites were interlaced in cluster.Conclusions Neuronal damage and morphologic change of neurites are verified in the cell model of intractable epilepsy.