Neuroprotective Effects Of GM1 On Ketamine Anesthesia In Young Rats Via PI3k/AKT/GSK3? Signaling Pathway

Anesthesia plays an important role in the diagnosis and treatment of animals.However,the use of general anesthetics may cause short-term or long-term adverse effects on anesthetized patients,such as damage to learning and memory,neurodegenerative diseases,etc.,which are particularly prominent in the anesthesia of young mammals.Neonatal period of animals is an important period of central nervous system development,and also the fastest period of development of animal nervous system.During this period,the intervention of anesthetic may cause neuronal apoptosis and neurotoxicity in animal brain area.As a commonly used anesthetic in clinical practice,ketamine has good analgesic and sedative effects.It is an indispensable drug for animals.It is widely used in anesthesia,pain management,antidepression and other aspects,and is often used in perinatal period and young animal anesthesia practice.However,whether ketamine anesthesia will cause neurotoxicity and long-term adverse effects,there is no systematic research in veterinary clinic.As a neuroprotective agent,GM1 is widely used in the treatment of nerve damage and neurodegenerative diseases.However,it is unclear whether it can protect the neuronal apoptosis caused by ketamine.Therefore,this experiment selected 7-day-old rats for ketamine anesthesia to explore its possible toxic effects,and selected GM1 as a protective agent to explore whether GM1 has protective effects and its mechanism,for the clinical application and reasonable protection method of ketamine in the future.Young rats were randomly divided into four groups.The control group(group C)was saline group.Ketamine group(group K)received intraperitoneal injection of 20 mg/kg of ketamine,every 1.5 h one time for 7.5 h.GM1 group received 20 mg/kg of GM1,and GM1+K group received two drugs,and the GM1 was injected 0.5 h before and administered in the same manner as ketamine.Then the cerebrum and hippocampus of rats were collected.Nissl staining was used to detect the number of neurons in cerebrum and hippocampus of rats.The protein expression levels of Caspase-3,BAX and Bcl-2 were detected by immunohistochemistry.The m RNA expression levels of Caspase-3,BAX,Bcl-2 and AKT,GSK3? were detected by RT-PCR.Western blot was used to detect the protein expression changes of AKT,p-AKT,GSK3? and p-GSK3?.PC12 cell model was selected for the validation experiment in vitro,and the optimal concentration of ketamine and GM1 on PC12 cells was determined by CCK-8 method.PI3K inhibitor LY294002 was introduced.Cell flow assay was used to detect the change of apoptosis rate in each group.Then RT-PCR and Western Blot were used to detect the changes of the above apoptosis-related genes,pathway-related genes and protein expressions.Results:(1)GM1 do not affect the anesthetic effect of ketamine,and there is no significant change in cell apoptosis in GM1 group compared with group C in the in vivo model and in vitro model.(2)In vivo experiment,the number of neurons in hippocampal CA1 region,CA3 region and cerebrum of K group decreased,and in GM1+K group increased significantly compared with K group(P<0.01);In vitro experiment,the cell activity of K group decreased and the cell apoptosis rate increased,while the activity of GM1+K group was not significantly different from that of normal group(P>0.05).(3)Except that there was no significant difference between the BAX groups in cerebral cortex,the m RNA and protein expressions of Caspase-3 and BAX in K group were significantly up-regulated(P<0.01 or 0.05),and the m RNA and protein expressions of Bcl-2 were significantly down-regulated(P<0.01 or 0.05)in vivo and in vitro models.There was no significant difference between GM1+K group and C group.(4)Except that the expression of AKT m RNA in hippocampus was not significantly different among groups(P>0.05),the expression of AKT m RNA in K group was significantly decreased(P<0.01 or 0.05)and the expression of GSK3? m RNA was significantly increased(P<0.01 or 0.05)in vivo and in vitro experiments,but the expression of AKT and GSK3? total protein in each group was not significantly different,while the expressions of p-AKT and p-GSK3? in K group were significantly decreased(P<0.01).The expression of p-AKT and p-GSK3? in GM1+K group was significantly higher than that in K group(P<0.01).(5)After adding PI3K inhibitor LY294002,the apoptosis rate of GM1+K+LY group was not significantly different from that of K group,and the expression trend of signaling pathway genes and proteins was similar to that of K group.The results show that ketamine prolonged anesthesia or exposure led to increased apoptosis in young rats and PC12 cells.GM1 has a neuroprotective effect by regulating the PI3K/AKT/GSK3?signaling pathway and downstream apoptosis-related genes and proteins,improving the neurotoxicity of ketamine in young rats.