Effect Of GPx4 On Ketamine Anesthesia Induced Nerve Injury In Young Rats

Ketamine,an NMDA receptor blocker with sedative and analgesic effects,which is widely used for anaesthesia and pain management in perinatal and young animals.Studies have shown that long-term or repeated exposure to ketamine in young animals may cause brain damage.However,the underlying mechanism of ketamine-induced neurotoxicity,and the measures of treatment are still unclear,which is an urgent problem to be solved in current study.Pyroptosis and ferroptosis are new forms of programmed cell death in recent years.Pyroptosis is mainly characterized by inflammasome assembly,massive release of pro-inflammatory factors and cell lysis.Several studies have confirmed that NLRP3 inflammasome-induced pyroptosis is involved in the pathological process of various neurological diseases.Ferroptosis is caused by an imbalance between oxidative and antioxidant systems,characterized by lipid peroxidation and cell membrane damage.Glutathione Peroxidase 4(GPx4),a lipid repair enzyme,is a negative regulator of ferroptosis.GPx4 activity is decreased during ferroptosis,resulting in ROS accumulation and mitochondrial damage.In addition,mitochondrial damage and ROS accumulation are also necessary for NLRP3/caspase-1 pathway activation.This suggests that GPx4 may also be involved in the regulation of pyroptosis.However,it remains unknown whether pyroptosis and ferroptosis are involved in ketamine-induced hippocampal injury in young animals,and the role of GPx4 in both types of cell death.N-acetylcysteine(Nac),the main synthetic precursor of glutathione(GSH),is currently clinically used:(1)as a liver-protective drug for the treatment of liver damage caused by acetaminophen overdose;(2)as a mucolytic agent for mucolytic therapy.At present,Nac is widely used in the adjuvant therapy of neurological diseases.It remains to be further explored whether Nac has a protective role in ketamine-induced nerve damage in developing animals.Based on the above problems,this paper explores the regulatory effect of GPx4 on pyroptosis and ferroptosis in ketamine-induced neuronal injury in young rats,and further explores the potential mechanism of Nacs neuroprotective effect.The purpose of this experiment is to provide theoretical reference for the neurotoxicity mechanism of ketamine and the screening of protective drugs in veterinary clinical practice.Research includes:(1)To explore the effect of NLRP3/caspase-1 pathway on ketamine-induced nerve damage in young rats: 7-day-old SD rats,PC12 cells and HAPI cells were used to establish ketamine neurotoxicity models in vivo and in vitro,respectively.After using MCC950 and VX765 to inhibit the NLRP3/caspase-1 pathway,the histological changes of hippocampus,the water maze test in adult rats,NLRP3/caspase-1-related pyroptosis pathway,and neuroinflammation related moleculars were detected.(2)Based on ketamine-induced nerve injury model in young rats and cell model,the role of GPx4 on ferroptosis and NLRP3/caspase-1 pathway-related pyroptosis was explored.In vivo and in vitro models of ketamine neurotoxicity were established,respectively,using RSL3(GPx4 specific inhibitor)to inhibit GPx4 protein expression,and using Nac(GPx4 substrate GSH precursor)to activate GPx4,the groups were divided into: control group(Con group),Ketamine group(Ket group),RSL3 group,Ket+RSL3 group and Ket+RSL3+Nac group.Histological damage of hippocampus,water maze test in adult,NLRP3/caspase-1-related pyroptosis pathway,neuroinflammatory indexes,iron concentration,mitochondrial damage,lipid peroxidation and ROS levels were detected.The above experiments were performed to explored the regulatory effect of GPx4 on ketamine-induced NLRP3/caspase-1 pathway,ferroptosis and cognitive dysfunction.(3)To investigate whether Nac can regulate the passage of NLRP3/caspase-1 and ferroptosis through GPx4,and play a neuroprotective role.Nac was treated for ketamine neurotoxicity model in vitro and in vivo,and the histopathology of the hippocampus,the water maze test in adult,GPx4/GSH activity,NLRP3/caspase-1-related pyroptosis indicators,neuroinflammatory factors,iron concentrations,mitochondrial morphology and function,lipid peroxidation levels,and ROS content were detected.The results are shown as follows:(1)The effect of NLRP3/Caspase-1 pathway on ketamine-induced cognitive impairment in young ratsIn the hippocampus of young rats,ketamine leads to activation of the NLRP3/caspase-1pathway,and increases in GSDMD-N expression,neuroinflammatory cytokines levels,and the number of positive cells in hippocampal TUNEL fluorescence staining;in PC12 and HAPI cells,ketamine up-regulates the expression of NLRP3,caspase-1p20 and GSDMD-N,increased the release of LDH,the secretion of inflammatory cytokines,and the number of caspase-1/PI doublepositive cells in flow cytometry;after inhibiting the NLRP3/caspase-1 pathway with MCC950 and VX765,ketamine induced the histological damage of the hippocampus of young rats and the decline of learning and memory ability of young rats in adulthood were alleviated,and the number of positive cells in TUNEL staining was reduced.At the same time,after this pathway was inhibited,the expression of pyroptosis protein and the levels of inflammatory cytokines in the hippocampus and two cells were decreased,suggesting that NLRP3/caspase-1-induced pyroptosis is involved in ketamine-induced nerve damage.(2)The effect of GPx4 on ketamine-induced ferroptosis,NLRP3/caspase-1 induced pyroptosis and cognitive dysfunction in hippocampus of developing ratKetamine induces ferroptosis in hippocampus,PC12 and HAPI cells of young rats,manifested as down-regulation of SOD,GSH and GPx4 expression,and increase in Fe2+ level,intracellular ROS and lipid peroxide(MDA and 4-HNE)content,mitochondrial damage;GPx4 inactivation with RSL3 aggravates ketamine-induced hippocampal histological damage,cognitive impairment,ferroptosis(increased iron concentrations,ROS and lipid peroxides)and pyroptosis(increased levels of pyroptosis-related proteins and cytokines);whereas in the RSL3+Ket+Nac group,Nac counteracted the effect of RSL3 on ketamine.The above results suggest that GPx4 has a negative regulatory effect on ketamine-induced hippocampal pyroptosis,ferroptosis and nerve damage in developing rats,while Nac has a potential neuroprotective effect.(3)Protective effect of Nac on ketamine-induced nerve injury in young ratsIn hippocampal tissue,PC12 and HAPI cells,Nac pretreatment alleviated ketamine-induced decrease in GPx4/GSH activity,iron overload,ROS accumulation and lipid peroxidation,mitochondrial damage,and high expression of pyroptosis-related proteins and inflammatory cytokines;Nac pretreatment can effectively alleviate ketamine-induced neuronal loss in the CA1 and CA3 regions of the hippocampus of young rats,and improve the spatial learning and memory ability of rats in adulthood,suggesting that Nac can alleviate ketamine-induced ferroptosis and cell pyroptosis,and has a potential neuroprotective effect.In conclusion,in young rats,ketamine reduced GPx4 activity,caused ROS accumulation and mitochondrial dysfunction,induced lipid peroxidation and ferroptosis,and activated the NLRP3/Caspase-1 induced pyroptosis,resulting in hippocampal damage and cognitive dysfunction in adult rats.In addition,we found that the neuroprotective effect of Nac is mediated by upregulating the activity of GPx4 and increasing the content of GSH.