Sevoflurane has been extensively employed for induction and maintenance of general anesthesia. The effect of sevoflurane-induced apoptosis in developmental neurotoxicity has been appreciated for some time now, but the underlying mechanism of developmental neurotoxicity has not been established. The aim of our study is to evaluate the role of autophagy in sevoflurane-induced neurotoxicity through observing changes in the levels of autophagy in hippocampal neurons after exposure to sevoflurane.
Primary cultured hippocampus neuronal cells were exposed to either 3.4% sevoflurane for 1 h (S1h group), 3 h (S3h group), 5 h (S5h group), or air (control group). We observed changes in autophagy proteins Beclin-1, LC3-II, p62, and Beclin-1mRNA, LC3mRNA and SQSTM1mRNA using Western Blot and QRT-PCR. We also determined the expression of LC3 using immunofluorescence staining, monitored the occurrence of autophagy using RFP-GFP-LC3 expression plasmid transient transfected hippocampal neuronal cells, detected the expression of LC3-II using siRNA Knockdown Beclin-1 and Atg5, and determined changes in cell apoptosis using Annexin V/PI staining and flow cytometry.
After primary cultured hippocampal neuronal cells were exposed to 3.4% sevoflurane for 5 h, the expression level of Beclin-1 and LC3-II increased and p62 decreased in Western blotting. The expression of Beclin-1mRNA, LC3mRNA increased and SQSTM1mRNA decreased in QRT-PCR. LC3 increased with cell immunofluorescence staining, LC3 expression plasmid increased after mRFP-GFP-LC3 expression plasmid transient transfection and LC3-II decreased after transfection with siRNA Beclin-1 and siRNA Atg5. The apoptosis rate of primary cultured hippocampal neuronal cells increased in Annexin V/PI staining and flow cytometry analysis.
This study demonstrates that sevoflurane may induce hippocampal neuron autophagy in primary cultured hippocampal neuronal cell and that Beclin-1 and Atg5 are involved in the process of sevoflurane-induced autophagy. Exposure of sevoflurane may not only induce autophagy of hippocampal neurons but also activate the apoptosis of hippocampal neurons. Autophagy may play an important role in sevoflurane-induced neurotoxicity in primary cultured hippocampal neuronal cells.