The Role Of Autophagy In Sevoflurane-induced Neurotoxicity In Developing Rats Hippocamp And Mechanism Research

Sevoflurane is one of the most commonly used drugs of general anesthesia and has been used for induction and maintenance of pediatric anesthesia for many years.A large number of animal studies have shown that the early exposure to common anesthetic agents such as sevoflurane can lead to widespread hippocampal neuron apoptosis and persistent spatial learning ability deficits in the developing rat brain.However,at present,the specific mechanism of sevoflurane neurotoxicity has not been fully elucidated and the effective clinical prevention and treatment measures has not been found.Sanchez et al.have found that inhaled anesthetics caused long-term abnormal mitochondria morphology and synaptic transmission,accompanied by autophagy activity enhancement in the developing rat.It is speculated that autophagy may play an important role in sevoflurane neurotoxicity,but now the research on the relationship between autophagy and sevoflurane neurotoxicity is not thorough.In order to investigate the changes of autophagy levels in the hippocampus of neonatal rats induced by sevoflurane and pinpoint the role of autophagy in sevoflurane-induced neurotoxicity in developmental rats hippocamp and the related mechanism as well as the relationship between autophagy and apoptosis of neurons so as to further find its corresponding therapeutic targets and the safest clinical exposure time,we carried out our study as follows.Part one To investigate neuron autophagy phenomenon induced by sevoflurane and its specific regulation signal passway by using animal modelIn this part of work,newborn Sprague-Dawley(SD)rats were exposed to 3.4%for different time,we observed the changes in the levels of autophagy in hippocampal tissue and discussed the relationship between autophagy activity and sevoflurane the exposure time by Western blotting detecting autophagy proteins Beclin-1,LC3-Ⅱ and p62,QRT-PCR detecting Beclin-1 mRNA,LC3mRNA and p62mRNA,immunohistochemical and immunofluorescence staining detecting the expression of Beclin-1.LC3 and p62.Aiming to is classic signaling pathways Class Ⅰ PI3K/Akt/mTOR/p70S6K and Class Ⅲ PI3K of regulating autophagy,autophagy inducer(Rapamycin)and autophagy inhibitor(3-methyl adenine,3-MA)were used respectively to observe autophagy.At the same time the levels of the apoptotic protein Bax protein and Bcl-2 protein were detected by Western blotting,and the TUNEL method was used to detect the apoptosis of neurons.The results found that after newborn SD rats exposed to 3.4%sevoflurane for 4h x 3d,Western blotting tests showed Beclin-1 and LC3-Ⅱ raised and autophagy substrate p62 decreased in Western blotting.At the same time,Bax protein significantly increased while Bcl-2 protein decreased significantly.Beclin-1mRNA,LC3mRNA raised and p62mRNA decreased in QRT-PCR,Beclinl and LC3 raised and p62 decreased in immunohistochemical and immunofluorescence staining.Rapamycin activated autophagy,3-MA inhibited autophagy.TUNEL detection results showed a significant increase in the number of tunnel positive neurons.The study showed that sevoflurane may induce autophagy in hippocampal neurons in the developing SD rat brain and promote the formation of autophagy and the fusion of lysosomes and autophagy.Sevoflurane may initiate autophagy by activating the Class Ⅲ PI3K pathway or inhibiting mTOR.Sevoflurane promotes autophagy in neurons while promoting the formation of neuronal apoptosis.Autophagy may play an important role in sevoflurane neurotoxicity in the developing SD rat hippocampal neurons.Part two To investigate the formation characteristics and signal transduction pathways of neuronal autophagy induced by sevoflurane through using the hippocampal neuron modelIn this part of work,primary cultured hippocampal neuron cells were exposed to 3.4%for different time.We observed the changes in the level of autophagy by Western blotting detecting the levels of autophagy proteins Beclin-1,LC3-Ⅱ and p62,and apoptotic protein Bax protein and Bcl-2 protein,QRT-PCR detecting Beclin-1mRNA,LC3mRNA and p62mRNA,cells immunofluorescence staining detecting the expression of LC3,monitored the occurrence of autophagy by using RFP-GFP-LC3 expression plasmid transient transfection hippocampal neuron cells,and detected the expression of LC3-Ⅱ by using siRNA Knockdown Beclin 1 and Atg5 and the change of cell apoptosis with Annexin V/PI.The results found that after primary cultured hippocampal neuron cells exposed to 3.4%sevoflurane for 5h,Beclin-1 and LC3-Ⅱ raised and p62 decreased,at the same time,the level of apoptotic protein Bax protein significantly increased,and Bcl-2 protein decreased significantly in Western blotting,Beclin-1mRNA,LC3mRNA raised and p62mRNA decreased in QRT-PCR,LC3 raised in cell immunofluorescence staining,LC3 expression raised after RFP-GFP-LC3 expression plasmid transient transfection and LC3-Ⅱ decreased after transfection siRNA Beclin-1 and siRNA Atg5,early and late apoptotic cells in Annexin V/PI.The study showed that sevoflurane may induce hippocampal neuron autophagy in primary cultured hippocampal neuron cells and the signaling pathway regulated by Beclin 1 and Atg5 is involved in the regulation process of sevoflurane induced autophagy.The 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 neurotoxicity in primary cultured hippocampal neuron cells.In conclusion,the study showed that autophagy may play an important role in sevoflurane neurotoxicity in the developing SD rats hippocamp neuron and primary cultured hippocampal neuron cells.Enhanced protective autophagy can be used as a potential way to prevent the neurotoxicity induced by sevoflurane,but more experimental studies are needed in the future.The study investigated the relationship between autophagy activity and sevoflurane exposure time and provided some experimental basis for deeply understanding the pathogenesis of inhalation anesthetics neurotoxicity so as to find the clinical corresponding therapeutic targets and the safest dose and concentration of sevoflurane exposure.