Lipoxin A₄ Methyl Ester Inhibits Endoplasmic Reticulum Stress And Ameliorates Apoptosis Of SH-SY5Y Neurons Induced By Ketamine Through Activating Leptin Signaling Pathway In Vitro

Part one Effects of different concentrations of ketamine on endoplasmic reticulum stress and apoptosis rate in SH-SY5 Y neuronsObjevtive: To evaluate the changes of apoptosis and ERS levels in SH-SY5 Y cells after ketamine exposure.Methods:Cell culture: SH-SY5 Y cells were cultured in DMEM:Ham's F12?1:1?with 10% fetal bovine serum,penicillin?100?g/ml?and streptomycin?100?g/ml?in a 37 °C,5%CO2 incubator.Cells were subcultured every 2-4days.Cell culture medium was changed every 1-2 days,cell growth and survival were observed by microscope.Ketamine cell model preparation: SH-SY5 Y cells in logarithmic growth phase were treated with 0 ?M,100 ?M,300 ?M,500 ?M,1000 ?M,2000 ?M ketamine respectively,and then incubated in a CO2 incubator for 24 hours.Experimental group: control group?0 ?M ketamine?,K1 group?100 ?M ketamine?,K2 group?300 ?M ketamine?,K3 group?500 ?M ketamine?,K4group?1000 ?M ketamine?,K5 group?2000 ?M ketamine?.Inhibition ratio was detected by CCK8,apoptosis rate was detected by flow cytometry,WB:Leptin,Lep Rb,GRP78.Observing whether the increase of ketamine concentration can increase the ERS level and apoptosis rate in SH-SY5 Y cells,and determine the appropriate ketamine concentration to expose the cells,which lays a foundation for subsequent experiments.The effects of ketamine on Leptin and Lep Rb were observed to determine whether ketamine inhibited Leptin and Lep Rb.Results:1.CCK8 results showed that the inhibition ratio of SH-SY5 Y cells increased with the increase of ketamine concentration.2.Flow cytometry results showed that the apoptosis rate increased with the increase of ketamine concentration.3.Western blot results showed that ketamine increased the expression of GRP78 protein and decreased the expression of Leptin protein and Lep Rb protein.Conclusions:With the increase of ketamine concentration,the endoplasmic reticulum stress level and apoptosis rate in SH-SY5 Y cells were increased,and Leptin,Lep Rb were inhibited.Part two Effects of LXA₄ ME treatment on ketamine-induced apoptosis and Leptin inhibition in SH-SY5 Y cellsObjective: To observe whether LXA₄ ME treatment can reduce the apoptosis rate of SH-SY5 Y cells induced by ketamine,and the inhibitory effect of ketamine on Leptin.Methods:SH-SY5 Y cells exposed to ketamine?the appropriate concentration of ketamine in the first part?were treated with 0,1,10,100 nmol/LLXA₄ ME?the appropriate concentration was selected for the gradient experiment?for 24 hours.Experimental group: control group,K group,K+LXA₄ ME group,LXA₄ ME group.Detection methods:Inhibition ratio was detected by CCK8,apoptosis rate was detected by flow cytometry,transmission electron microscopy observation of expanded endoplasmic reticulum,WB detection related proteins: including ERS marker proteins CHOP,GRP78.Apoptotic proteins: cleaved caspase-3,Bcl-2,Leptin,and Lep Rb protein.Results:1.CCK8 results showed that LXA₄ ME can reduce the decrease of inhibition ratio caused by ketamine.2.Flow cytometry results showed that LXA₄ ME can reduce the apoptosis rate caused by ketamine.3.Western blot results showed that ketamine caused an increase in the expression of CHOP,GRP78,cleaved caspase-3,and decreased Bcl-2,Leptin,Lep Rb proteins.LXA₄ ME reversed the effects of ketamine on these proteins.4.Transmission electron microscopy showed a clearly dilated endoplasmic reticulum in the ketamine group,and the endoplasmic reticulum expansion was observed in the LXA₄ ME+K group.The endoplasmic reticulum was normal in the control group and the LXA₄ ME group.Conclusion:LXA₄ ME can alleviate neurotoxicity by reducing endoplasmic reticulum stress caused by ketamine.Part three Mechanism of Leptin signaling pathway in reducing ketamine neurotoxicity in LXA₄ MEObjective: To analyze the role of Leptin in relieving of ketamineinduced neuronal apoptosis by LXA₄ ME.Methods:100 nmol/L Leptin inhibitors were added on the basis of the second step.Experimental group: control group,K group,K + LXA₄ ME group,K+LXA₄ME+Leptin TA group,Leptin TA group.Methods: flow cytometry detection of apoptosis rate,transmission electron microscopy observation of the expanded endoplasmic reticulum,WB detection related proteins including CHOP,GRP78.Apoptotic proteins: cleaved caspase-3,Bcl-2.Results:1.Flow cytometry results showed that the addition of leptin inhibitor reversed LXA₄ ME to attenuate the neuronal apoptosis of ketamine.2.Western blot analysis showed that leptin inhibitor can reverse the expression of ketamine-induced CHOP,GRP78,cleaved caspase-3 and Bcl-2proteins by LXA₄ ME.3.Transmission electron microscopy showed that the K+LXA₄ ME+Leptin TA group reversed the protective effect of the K+LXA₄ ME group on the endoplasmic reticulum.Conclusion:Blocking the leptin pathway reverses LXA₄ ME and attenuates ketamine-induced neuronal apoptosis.