Effect Of EGR3 Gene On Cognitive Impairment In Patients With Schizophrenia Induced By Clozapine Combined With RTMS

Objective(s):Schizophrenia is a serious mental illness whose symptoms include positive symptoms,negative symptoms and cognitive impairment,and the antipsychotic drug Clozapine can significantly improve positive symptoms in patients,but there are serious adverse effectss.Repetitive transcranial magnetic stimulation(rTMS)is more effective and efficient than drug therapy.EGR3 gene plays an important role in memory and cognitive function,and is also a susceptibility gene for schizophrenia,which may be related to cognitive impairment in schizophrenia.This study explores whether the improvement effect of Clozapine combined with rTMS on cognitive impairment in schizophrenia is related to EGR3 gene,And the relationship between EGR3 and apoptosis.Methods: Both clozapine and rTMS can improve the clinical symptoms of patients with schizophrenia.The cognitive function of patients was evaluated during the treatment,the expression of EGR3 was detected by Reverse Transcription-Polymerase Chain Reaction(RT-q PCR),and its efficacy was investigated.Meanwhile,the correlation between EGR3 and cognitive function was studied.Schizophrenia model mice were induced by didroxepine maleate(MK-801)and treated with clozapine and rTMS.Eighty 6-week-old male BALB/C mice were randomly divided into control group(CT,n=20),schizophrenia group(MK,n=30),and MK-801+ clozapine group(CLO,n=30).CLO group was injected 2mg/kg clozapine per day according to the body weight of the mice.Mk-801(0.6mg/kg)was intraperitoneally injected with the MK group at a interval of at least 20 minutes,and the CT group was injected with equal volume of normal saline for 2 weeks.Water maze behavior experiment was carried out.After successful modeling,half of the mice in each group were randomly selected for rTMS intervention(the parameters were 5 Hz,1.26 T stimulation intensity,15 s stimulation interval,1000 pulses in total,14 days of continuous stimulation).The group of transcranial magnetic therapy was divided into rTMS group(r T),MK-801+rTMS group(MK+r T),MK-801+ Clozapine+rTMS group(CLO+r T).Meanwhile,the prefrontal cortex tissue of the mouse brain was taken for protein Western blot and immunohistochemistry(IHC)determine EGR3 expression in the prefrontal cortex;impact of clozapine combined with rTMS on EGR3,Bcl-2 and Caspase-3 expression is explored.The expressions of EGR3 gene,Bcl-2 protein and Caspase-3 protein in prefrontal cortex of mice were detected by Western blot.The expressions of Bcl-2 protein and Caspase-3 protein in prefrontal cortex were determined by immunohistochemistry.Results:1.Compared with healthy normal persons,the expression of EGR3 gene in serum q PCR of schizophrenia patients showed no significant difference;Serum EGR3 gene expression was decreased in patients with schizophrenia treated with clozapine compared to patients with schizophrenia(P<0.05).2.The detection index of Morris water maze experiment was escape latency and residence time ratio of target quadrant,which could indicate the learning and memory ability of experimental mice.There was no significant difference in swimming speed in the platform stage.Compared with CT group,the escape latency of MK model group was significantly increased,and the rate of residence time in the peripheral target quadrant was significantly increased.Compared with MK group,the escape latency of CLO group and transcranial magnetic stimulation group was significantly shortened(P<0.001),the rate of residence time in the peripheral target quadrant was significantly lower(P<0.001);The escape latency was shorter in the transcranial magnetic stimulation group(CLO+r T)than in the rTMS alone group(MK+r T)(P<0.05),there was no significant difference in escape latency compared with CLO group,only the target quadrant time ratio was lower(P<0.05);The motor trajectories of mice in the MK group were the most disorderly,and the motor trajectories of mice in the CLO group and the transcranial magnetic stimulation group were more converging than those in the rTMS only group(MK+r T)and CLO group.3.(1)Western blot assay: Compared with CT group,the expression of EGR3 gene in prefrontal cortex of MK group was significantly increased(P<0.05),the expression of Bcl-2 protein decreased significantly(P<0.05),the expression of Caspase-3 protein was significantly increased(P<0.05);Compared with MK group,the expression of EGR3 gene and Caspase-3 protein in prefrontal cortex of CLO group was decreased,with no statistical significance(P<0.05),but increased the expression of Bcl-2 protein(P<0.05),EGR3 gene expression in prefrontal cortex of mice in MK+r and CLO+r T groups was significantly decreased(P<0.01),the expression of Bcl-2 protein was significantly increased(P<0.01),the expression of Caspase-3 protein decreased significantly(P<0.05);Compared with the CLO group,the expression of EGR3 in the prefrontal cortex was significantly decreased in the CLO+r T group(P<0.01),the expression of Bcl-2 protein increased significantly(P<0.05),the expression of Caspase-3 protein decreased slightly,and the difference was not statistically significant.(2)Immunohistochemical experiment: Compared with CT group,the number of Bcl-2 positive cells decreased(P<0.01)and the number of Caspase-3 positive cells increased in the prefrontal cortex of mice in MK group(P<0.01).Compared with MK group,the number of Bcl-2 positive cells in the prefrontal cortex of mice in MK+r and CLO+r T groups was significantly increased(P<0.001),the number of Caspase-3positive cells decreased significantly(P<0.01),the number of Bcl-2 positive cells increased significantly in CLO group(P<0.01),the number of Caspase-3 positive cells decreased significantly(P<0.05);Compared with the CLO group,the number of Bcl-2 positive cells increased in the CLO+r T group(P<0.05),the number of Caspase-3 positive cells decreased,but the difference was not statistically significant.Conclusion(s): 1.Behavioral results further confirm that clozapine combined with rTMS can significantly improve cognitive impairment in mice.2.Clozapine combined with rTMS may regulate apoptosis by affecting EGR3 gene in prefrontal cortex of schizophrenic mice,thereby improving cognitive impairment in mice.3.rTMS treatment can enhance the efficacy of clozapine.