Protective Effect And Mechanism Of Esketamine On LPS-induced HT22 Cell Injury

Background:Inflammation and oxidative stress in neurons and glial cells are regulators of neurodegenerative diseases.Neuroinflammation itself is a defense mechanism against acute central nervous system damage,but persistent neuroinflammation is harmful,inhibiting nerve regeneration and impairing brain development.With the aging of society,neurodegenerative diseases have more and more impact on the global economy.It is urgent to explore the pathogenesis and genetic susceptibility of neurodegenerative diseases.Ferroptosis is a newly discovered mode of nonapoptotic cell death,which is dependent on iron metabolism and lipid peroxidation.A growing body of evidence suggests that the underlying mechanism between neuroinflammatory and neurodegenerative diseases can be explained by ferroptosis.And inhibiting ferroptosis can alleviate neurological damage.Esketamine（ESK）is a dextrorotatory isomer of ketamine.It is not only a commonly used sedative and analgesic drug,but also has anti-inflammatory and neuroprotective effects.Therefore,we used lipopolysaccharide（LPS）to simulate neuronal inflammation and selected mouse hippocampal neurons（HT22 cells）to investigate the neurotoxicity induced by esketamine and its effect on ferroptosis induced by LPS.Methods:HT22 cells were selected and cultured in conventional cells,and different doses of LPS groups（1μg/mL,5μg/mL,10μg/mL）were used to observe cell viability and ferroptosis-related indicators,including ferroptosis marker proteins（ACSL4、PTGS2）,intracellular reactive oxygen species ROS levels,intracellular lipid peroxidation production,and changes in intracellular iron divalent ions.HT22 cells were randomly grouped and pretreated with esketamine for 2 hours or Fer-1 for 2 hours,and then pretreated with 10μg/mL LPS for 24 hours.The cells were grouped as follows:control group（Con group）,LPS group（LPS group）,LPS group after esketamine pretreatment（ESK+LPS group）,LPS group after Fer-1 pretreatment（Fer-1+LPS group）,and then observed cell morphology,cell viability,ferropptosis marker protein（ACSL4,PTGS2）,inflammatory factor（IL-1β,TNF-α）,iron homeostasis key protein（TFR1,FPN,Ferritin）,HMGB1-MAPK pathway protein（HMGB1,p-ERK,p-JNK,p-P38）,lipoxidation of ROS in cells and ultrastructural changes of Fe2+were detected.Transfected plasmid,constructed HMGB1 siRNA cell line,HMGB1 siRNA pretreated with esketamine for 2 h and then 10 μg/mL LPS treatment for 24 hours,grouped as follows:control group（Con group）,LPS treatment group（LPS group）,esketamine pretreated LPS group（esketamine+LPS group）,LPS group after HMGB1 siRNA pretreated with esketamine（HMGB1 siRNA+esketamine+LPS group）,NC siRNA plus esketamine pretreatment LPS group（NC siRNA+esketamine+LPS group）,comparing TLR4,RAGE protein levels,as well as IL-1β,TNF-α,IL-6,brain-derived nerve growth factor（BDNF）,nerve growth factor（NGF）,nerve growth factor-3（NT-3）,Expression of nerve growth factor-4（NT-4）.Results:LPS is dose-dependent in promoting cell mortality,ROS,lipid peroxidation,and Fe2+levels,as well as ACSL4 and PTGS2 protein expression;Compared with the LPS group,the cell mortality rate of the esketamine+LPS group decreased significantly;the levels of ROS,lipid peroxidation and Fe2+ were significantly lower;the mitochondrial crest swelling was reduced;esketamine significantly reduced the levels of ACSL4,PTGS2,TFR1,FPN proteins and the expression of HMGB1,p-ERK,p-JNK,p-P38,and reduced the degradation of ferritin.After knocking down HMGB1,TLR4,RAGE protein levels in the HMGB1 siRNA+esketamine+LPS group were reduced in the HMGB1 siRNA+esketamine+LPS group,as well as the levels of IL-1β,IL-6,NGF,NT-3,NT-4.Conclusion:Ferroptosis plays a key role in LPS-induced inflammation in HT22 cells.Esketamine inhibits ferroptosis and may inhibit inflammation by regulating HMGB1 expression,thereby protecting HT22 cells from LPS damage.