Effect Of Combination Treatment With Isoflurane And Propofol On BiP-GABA_A Receptor ?1 Subunit Proteostasis In Hippocampal Neurons Injured By Hypoxia

Objective:Neurons are vulnerable to damage and dysfunction in hypoxic conditions.In patients with ischemic brain damage,cognitive dysfunction is very common.For patients with preoperative cognitive decline,inappropriate selection of anesthetic drugs will aggravate cognitive impairment.Isoflurane can induce neuronal damage with neurotoxicity in a dose-dependent manner.Subantasone anesthetic dose(20 mg·kg^-1·h^-1)propofol exerts brain protection through activation of the KCC2-GABA_A receptor?GABA_AR?pathway.The combined use of anesthetic drugs can reduce the dosage of single agents and reduce single drug-induced adverse reactions.The effect of combined anesthesia on cognitive function in these patients remains to be studied.In this study,we cultured rat hippocampal neurons and established a hypoxic injury model to simulate the mechanism of cognitive impairment caused by hypoxic injury.To study the effects of different combinations of isoflurane and propofol on hypoxic injury of rat hippocampal neurons and its possible mechanism.Method:The study is divided into two parts.In the first part,we investigated the effects of different doses of isoflurane/propofol combined use on the viability,the GABA_AR?1 proteostasis,and the level of endoplasmic reticulum stressof rat hippocampal neurons injured by hypoxia.Primary cultured hippocampal neurons of Wistar rat were divided into 6 groups by random number table?n=12?:normal control group?C group?,hypoxic group?H group?,different doses of isoflurane/propofol combined use groups?IP₁ group and IP₂ group?,isoflurane group?I group?,propofol group?P group?.Group C was cultured under normal conditions;Group H was cultured under hypoxic conditions for 3 h;Group I and P were cultured under hypoxic conditions for 3 h,then incubated with 1.9%isoflurane and 22.4?mol/L?Corresponds to 4?g/ml?propofol respectively.IP₁ group and IP₂ group were cultured under hypoxic conditions for 3 h,then incubated with 1.0%isoflurane+6.7?mol/L?Corresponds to 1.2?g/ml?propofol and 1.4%isoflurane+3.4?mol/L?Corresponds to 0.6?g/ml?propofol for 3 h.Finally,change to normal culture medium to continue cultivation.The cell viability was detected by CCK-8 after cultured for 24 h in each group.The expression of GABA_AR?1 subunit mRNA was detected by qRT-PCR.The expression of GABA_AR?1 subunit,BiP and the activation level of Caspase 12 were detected by Western blot.The expression of endoplasmic reticulum-associated degradation?ERAD?of GABA_AR?1 subunit was detected by immunoprecipitationandWesternblot,andtheexpressionof CCAAT/enhancer-binding protein homologous protein?CHOP?was detected by western blot immunofluorescence.In the second part,we investigated the effect of BiP-GABAAR?1 subunit pathway on hypoxic injury of primary rat hippocampal neurons incubated with isoflurane/propofol in hypoxia.We randomly divided the hippocampal neurons into 6 groups?n=12?:?1?Control-transfected cells?Con-siRNA+C?group;?2?Hypoxia-treated control-transfected cells?Con-siRNA+H?group;?3?Hypoxia-treated control-transfected cells treated with 1%isoflurane and6.7?M propofol?Con-siRNA+IP₁?group;?4?Control BiP-transfected cells?BiP-siRNA+C?group;?5?Hypoxia-treated BiP-transfected cells?BiP-siRNA+H?group;and?6?Hypoxia-treated BiP-transfected cells treated with 1%isoflurane and6.7?M propofol?BiP-siRNA+IP₁?group.After incubation with the corresponding drugs,cell viability was measured using the CCK-8 method.The expression of GABA_AR?1 subunit mRNA was detected by qRT-PCR.The expression of GABA_AR?1 subunit and the activation level of Caspase 12 were detected by Western blot.Immunoprecipitation and Western blot were used to detect the ERAD of GABA_AR?1subunit.Western blot and immunofluorescence was used to detect the expression of CHOP.Results:In the first part of the experiment,compared with group C,the viability of neurons in the remaining 5 groups were decreased,GABA_AR?1 subunit mRNA and GABA_AR?1 protein were down-regulated,ERAD levels were increased,BiP and CHOP expressions were up-regulated,the activation levels of Caspase 12 were increased?P<0.05?.Compared with group H,the viability of neurons in group I,group P,and group IP₂ were decreased,the expression of GABA_AR?1 subunit mRNA and the surface GABA_AR?1 protein were down-regulated,the ERAD level was elevated,and the expression of CHOP was up-regulated,the activation level of Caspase 12 was increased?P<0.05?.There was no significant difference in the above indicators in group IP₁,but BiP expression was significantly increased?P<0.05?.Compared with group I or group P,the viability of neurons in IP₁ group and IP₂ group were increased,GABA_AR?1 subunit mRNA and surface GABA_AR?1 protein were up-regulated,ERAD levels were decreased.CHOP expression and Caspase 12activation level were down-regulated,but BiP expression were elevated?P<0.05?.Compared with IP₁ group,the neuronal viability in IP₂ group was decreased,GABA_AR?1 subunit mRNA and surface GABA_AR?1 protein were down-regulated,ERAD level was increased,BiP expression was down-regulated and CHOP expression and Caspase 12 activation level were up-regulated?P<0.05?.The second part of the experimental results:compared with Con-siRNA+C group,neuronal viability,GABA_AR?1 subunit mRNA,membrane GABA_AR?1 protein expression and ERAD levels,CHOP expression and and Caspase 12 activation levels of neurons in the BiP-siRNA+C group were not obviously changed.Compared with Con-siRNA+H group,the activity of neurons in BiP-siRNA+H group was decreased,GABA_AR?1 mRNA and GABA_AR?1 protein were down-regulated,ERAD level was increased,CHOP expression and Caspase 12 activation levels were increased?P<0.05?.Compared with Con-siRNA+IP₁ group or BiP-siRNA+H group,the activity of neurons in BiP-siRNA+IP₁ group was decreased,GABA_AR?1 subunit mRNA,cell GABA_AR?1 subunit expression was down-regulated,ERAD level,CHOP expression and Caspase 12 activation level were decreased?P<0.05?.Conclusion:The combination use of isoflurane and propofol,especially 1%isoflurane and 6.7?mol/L propofol,can reduce endoplasmic reticulum stress and maintain GABA_AR?1 subunit proteostasis through BiP-GABA_AR?1 pathway,in turn,maintains neuronal viability.