Testosterone Decreases Sevoflurane-induced Tau Phosphorylation And Cognitive Deficiency By Regulating GSK3?

Tau hyperphosphorylation and associated cognitive impairment induced by sevoflurane anesthesia is one of the key mechanisms studied for the neurotoxicity of sevoflurane.One of the possible pathways is by reducing the phosphorylation levels of GSK3? Ser9 and leading to enhanced GSK3?-Tau interaction.Testosterone levels have a positive correlation with men's cognitive performance.Studies have shown that testosterone has a regulatory effect on GSK3? phosphorylation,in addition,we speculate that testosterone may influence Tau-GSK3? interaction.Sevoflurane induced more serious Tau phosphorylation and cognitive impairment in young mice than in adult mice,we assume that inadequate level of testosterone protection from birth to adolescence may be one of the reasons why young mice are more sensitive to anesthetic damage.This study set GSK3? kinase activity and protein interaction as the breakthrough points of mechanism,using multiple sevoflurane anesthesia animal/cell models and testosterone injection,explore the effect of testosterone in protecting multiple sevoflurane anesthesia-induced Tau protein phosphorylation,neural function injury and cognitive function in young mice,and further explore the molecular mechanism of testosterone in regulating GSK3?.Experiment Part 1: The difference in the content of testosterone and the effects of sevoflurane anesthesia on Tau expression and cognitive function in young and adult miceObjective: To investigate the difference in the content of testosterone in young(Postnatal 6 day old,P6)and adult(Postnatal 60 day old,P60)male mice and the effects of sevoflurane anesthesia(2h × 3d)on Tau protein phosphorylation and cognitive function in the brains of young and adult mice.Methods: P6 and P60 adult male mice were randomly separated into 2 groups:Control and Sevoflurane.The mice in Sevoflurane group were given 3% sevoflurane anesthesia for 2 hours for 3 days,and the mice in Control group was given 40% oxygen for 2 hours for 3 days under the same conditions.Mice were sacrificed immediately after treatment on day 3(young: P8,adult: P62)and brain tissues were harvested.TauSer202/Thr205 and total Tau expression were analyzed using Western blot,testosterone levels were detected using ELISA,cognitive function were detected using Morris water maze.Also,in order to record the change trend of testosterone levels in C57BL/6 mice from birth to adulthood(P60),brains of mice were harvested at different time points from P0 to P60,and the levels of testosterone were detected using ELISA.Results: 1.Multiple sevoflurane anesthesia significantly increased the level of TauPS202 /PT205 in the brain tissues of P6 male mice but not in P60 male mice;2.Multiple sevoflurane anesthesia caused cognitive impairment in P6 but not P60 male mice in Morris water maze;3.The level of testosterone in the hippocampus of P6 male mice is much lower than that of P60 male mice;4.Three times of sevoflurane anesthesia had no effect on the testosterone level of the brain tissues;5.With the growth of age,the change trend of testosterone in the hippocampus of mice was as follows: the level was high on day 0-1 of newborn,then gradually decreased,the level was about 14% of that of P60 mice on P6-10,testosterone level significantly increased on P25-30,reached a peak around P40,then stayed stable.Conclusion: Repeated sevoflurane anesthesia caused Tau hyperphosphorylation and long-term cognitive impairment in P6 mice,but not in P60 mice.The level of testosterone in the brain of P6 mice was significantly lower than that of P60 mice.Therefore,the neurotoxic injury induced by sevoflurane anesthesia in young mice may be related to the insufficient testosterone level and associated protective effect.Experiment Part 2: The protective effect of testosterone against sevoflurane anesthesia induced Tau hyperphosphorylation and function damage on SH-Sy5 y cells and primary neuronsObjective: To investigate the effects of testosterone against sevoflurane anesthesia induced Tau protein phosphorylation,cell viability and function damage on SH-Sy5 y cells and primary neurons by regulating GSK3? kinase activity.Methods: 1.SH-Sy5 y cells: the concentration of testosterone was first determined,and then SH-Sy5 y cells were randomly divided into 4 groups: Control + Vehicle,Control + Testosterone,Sevoflurane + Vehicle,and Sevoflurane + Testosterone.The cells in Control + Testosterone group and Sevoflurane + Testosterone group were given 100 n M testosterone pretreatment for 1 hour,then Sevoflurane + Vehicle group and Sevoflurane + Testosterone group were anesthetized with 4% sevoflurane for 6 hours while cells in Control + Vehicle and Control + Testosterone groups remained in the incubator.After all experiments,the levels of Tau-Ser202/Thr205,Tau-Ser262,total Tau,GSK3? Ser 9,GSK3? Tyr 216 and total GSK3? Tau were detected using Western blot,the expression of Tau-Ser202/Thr205 was also detected using immunofluorescence,and the cell viability was detected by Live/dead cell viability kit.2.Primary neurons: neurons were randomly divided into 4 groups: Control + Vehicle Control + Testosterone,Sevoflurane + Vehicle and Sevoflurane + Testosterone.Control + Testosterone and Sevoflurane + Testosterone were given 100 n M testosterone for 1 hour,then Sevoflurane + Vehicle and Sevoflurane + Testosterone were given 3% sevoflurane anesthesia 2 hours daily for 3 days,while Control + Vehicle,Control + Testosterone were placed in the incubator.After all treatment,levels of TauSer202/Thr205,Tau-Ser262,total Tau,GSK3?-Ser9,GSK3?-Tyr216,total GSK3? and PSD-95 were detected using Western blot,the expression of Tau-Ser202/Thr205 was also detected using immunofluorescence,the cell viability was detected using Live/dead cell viability kit,and the activity of neural stimulatory excitatory postsynaptic currents(s EPSCs)was recorded by whole-cell patch clamp technique.Results: 1.4% sevoflurane anesthesia for 6 hours significantly increased the expression of Tau-Ser202/Thr205 in SH-Sy5 y cells and decreased the expression of GSK3?-Ser9.Testosterone treatment reduced the expression of Tau-Ser202/Thr205 and increased the expression of GSK3?-Ser9.On the other hand,two hours of 3% sevoflurane anesthesia for 3 days significantly increased the expression of TauSer202/Thr205 and Tau-Ser262 in neurons,decreased the expression of GSK3? Ser9 and PSD-95,and increased the expression of GSK3?-Tyr216.Testosterone treatment reduced the expression of Tau-Ser202/Thr205,Tau-Ser262 and GSK3?-Tyr216,and increase the expression of GSK3?-Ser9 and PSD-95;2.Sevoflurane anesthesia or testosterone pre-treatment did not change the cell viability of SH-Sy5 y cells or neurons;3.Multiple sevoflurane anesthesia inhibited the amplitude and frequency of neuronal s EPSCs,and testosterone treatment restored the amplitude and frequency of s EPSCs.Conclusion: Sevoflurane causes increased kinase activity of GSK3? in SH-Sy5 y cells and neurons and the hyperphosphorylation of Tau.Testosterone can reverse the neurotoxicity caused by sevoflurane,inhibit the kinase activity of GSK3?,reduce the phosphorylation level of Tau and protect synaptic function of neurons,therefore play a neuroprotective role.Experiment Part 3: The protective effect of testosterone on multiple sevoflurane anesthesia induced Tau hyperphosphorylation and cognitive dysfunction in young miceObjective: To investigate whether testosterone can regulate the kinase activity of GSK3?,therefore reduce the Tau hyperphosphorylation and reverse cognitive impairment induced by multiple sevoflurane anesthesia in young P6 mice.Methods: P6 male mice were randomly separated into 4 groups: Control + Vehicle,Control + Testosterone,Sevoflurane + Vehicle and Sevoflurane + Testosterone.Mice in Control + Testosterone and Sevoflurane + Testosterone received subcutaneous injection of 100?g of testosterone solution,while Control + Vehicle and Sevoflurane + Vehicle received subcutaneous injection of corn oil,after 1 hour of pre-treatment,mice in Sevoflurane + Vehicle and Sevoflurane + Testosterone received 3% sevoflurane anesthesia for 2 hours while Control + Vehicle,Control + Testosterone received 40% oxygen inhalation under the same conditions.After 3 days of anesthesia and testosterone administration,brain tissues of each group were harvested.The expression of Tau-Ser202/Thr205,Tau-Ser262,total Tau,GSK3? Ser9,GSK3? Tyr216 and total GSK3? were detected using Western blot.Testosterone levels were detected using ELISA.The changes of hippocampus structure were observed using HE stain.The changes of cognitive function in hippocampus zone were tested using Morris water maze.Results: 1.The testosterone level in the hippocampus of mice increased significantly 1 hour after the injection,and basically decreased to the normal level 24 hours after the third administration;2.Sevoflurane anesthesia and testosterone administration didn't cause morphological changes in hippocampus;3.Multiple sevoflurane anesthesia significantly increased the expression of Tau-Ser202/Thr205 in the cortex and hippocampus of P6 mice,while decreased the expression of GSK3?-Ser9.The expression of Tau-Ser262 and GSK3?-Tyr216 in the cortex remained unchanged,while the expression of Tau-Ser262 and GSK3?-Tyr216 in the hippocampus increased.Testosterone treatment decreased the expression of Tau-Ser202/Thr205,Tau-Ser262 and GSK3?-Tyr216,and increased the expression of GSK3?-Ser9;4.Testosterone pretreatment reversed multiple sevoflurane anesthesia induced cognitive impairment in the hippocampus in Morris water maze.Conclusion: Repeated administration of testosterone temporarily increased the level of testosterone in the hippocampus of mice without long-term effects or changes in brain structure.However,testosterone treatment inhibited the kinase activity of GSK3? caused by multiple sevoflurane anesthesia in the cortex and hippocampus of young mice,reduced the expression of Tau-Ser202/Thr205 and Tau-Ser262,thus showed a protective effect on the sevoflurane anesthesia induced hippocampus cognitive function impairments.Experiment Part 4: Adverse effects of sevoflurane and testosterone on interaction between Tau and GSK3?Objective: To investigate the different effects of sevoflurane and testosterone on TauGSK3? interaction.Methods: 1.The grouping and treatment of SH-Sy5 y cells were shown in Part 2.After the treatment,the binding between Tau and GSK3? in all groups were detected using immunoprecipitation.2.Brain tissues of P8 C57BL/6 mice were harvested to isolate and purify Tau protein.Interaction between Tau and GSK3? was detected using nanochip.Results: 1.Sevoflurane anesthesia increased the binding between Tau and GSK3?,while testosterone pre-treatment reduced the binding between Tau and GSK3?;2.Testosterone weakened the protein interaction between Tau and activated GSK3?.Conclusion: Sevoflurane enhances the interaction between Tau and GSK3?,making it easier for the phosphorylation of Tau protein.Testosterone,on the other hand,inhibits the strong interaction between Tau and activated GSK3?,making it difficult for the two to bind,thus reducing the possibility of Tau phosphorylation at various sites.