Protective Effect Of Progesterone On Sevoflurane-induced Neuronal Injury In Primary Hippocampal Neurons

Objective:Sevoflurane,one of the most commonly used inhalation anesthetics,is widely used in obstetrics and pediatric surgeries due to its unique pharmacological characteristics.However,increasing evidences showed that sevoflurane had neural damage to the developing brain,and early exposure to sevoflurane might affect brain development and long-term cognitive function.On the other hand,progesterone had gradually attracted attention for its neuroprotective effect.This study attempts to prove that progesterone has a protective effect on sevoflurane-induced neurotoxicity with isolated primary hippocampal neurons,and try to clarify some related molecular mechanisms and the role of progesterone receptor in this process,so as to provide a new idea to protect neurons from sevoflurane-induced neuronal injury.Methods:1、Primary culture of hippocampal neurons: hippocampal neurons were isolated from SD neonatal rats and were cultured in vitro for 7 days.Immunofluorescence assay was used to access the purity of neurons by Neu N staining.2、Establishment of sevoflurane injured model: Primary cultured hippocampal neurons were exposed to either 2%-8% sevoflurane for 6-12 h or air,and the most appropriate intensity of sevoflurane treatment was selected for subsequent experiments.3 、 Groups and treatments: the newly cultured primary hippocampal neurons were randomly divided into four groups: vehicle group(group C),sevoflurane group(group S),progesterone + sevoflurane group(group PS)and ulipristal acetate + progesterone +sevoflurane group(group UPS).Cells in group C were cultured without any treatment but vehicles,while cells in group PS were pretreated with progesterone 1 h before exposure to sevoflurane,and cells in group UPS were pretreated with ulipristal acetate 1 h before administration of progesterone,cells in group S were treated with vehicles at the same time point and then exposed to sevoflurane.4、Observation and measurement: The morphological and ultrastructural changes of neurons were observed under optical microscope and transmission electron microscope.Cell viability and apoptosis were respectively assessed by CCK-8 and TUNEL staining.Western blot analysis was performed to detect the expression of p-Akt,Akt and caspase-3.Results:1、Cell viability was not reduced significantly after exposure to sevoflurane at low concentration for a short time(2%/4%,6h)(P > 0.05),but significantly reduced at high concentration for comparatively long time(8%,6h;4%/8%,12h)(P<0.05).2、When exposed to 4% sevoflurane for 6 h,no obvious change in apoptotic rate or the expressions of caspase-3 was detected in neurons,while cell atrophy and lysis was observed with shorter and thinner neurites,and weakened intercellular connections under optical microscope.Compared to neurons exposed to air whose mitochondrial structure was clear,the mitochondria of neurons treated with 4% sevoflurane for 6 h displayed pyknosis and their integrity of bilayer membrane was destroyed.Sevoflurane also induced the appearance of autophagosomes in the neurons.3、Compared with group C,the cell viability of group S was significantly lower,the apoptotic rate was significantly higher,and the expression of p-Akt protein and p-Akt / Akt were significantly lower(P <0.01).Compared with group S,the cell viability of group P was significantly higher,the apoptotic rate was significantly lower,and the expression of p-Akt protein and p-Akt / Akt were significantly higher(P <0.01).Compared with group P,the cell viability of group U was significantly lower,the apoptotic rate was significantly higher,and the expression of p-Akt protein and p-Akt / Akt were significantly lower(P<0.01).Cells in group C and group P had normal or basically normal morphology,whilecells in group S and U manifested typical apoptosis character.Conclusion:1、The neurotoxicity of sevoflurane is concentration dependent.Exposure to 4%sevoflurane for 6h(usually used in clinic)can alter the morphology and ultrastructure of primary cultured hippocampal neurons,but has relatively slight effect on cell apoptosis.2、Progesterone plays a neuroprotective role in sevoflurane-induced neurotoxicity to hippocampal neurons by binding to progesterone receptors and up-regulating the expression of p-Akt.