Screening Of Susceptibility Genes For Olanzapine-induced Metabolic Syndrome And Phenotypic Mechanism Research

Objective: Psychiatric disorders including schizophrenia are highly disabling mental illnesses characterized by emotional,cognitive,and behavioral dysregulation.Due to their efficacy,Atypical antipsychotics(APs)are widely used in psychiatry.However,a significant clinical problem associated with antipsychotic therapy is APs-induced metabolic abnormalities,such as dyslipidemia.The IRSk of APs-induced Metabolic Syndrome(APs-induced Mets)is significantly higher than that of the general population,especially olanzapine and clozapine-induced Mets.It is believed that the cause of olanzapine-induced Mets is a combination of genetic and epigenetic variables that result in aberrant changes in the neuroendocrine and substance metabolic pathways of cells.Based on this perspective,our research identified 12 prospective susceptibility genes for olanzapine-induced Mets to elucidate the molecular process behind olanzapine-induced Mets and identify appropriate therapeutic intervention targets.Methods: In the first part of the project,functional validation was conducted at both the systemic and cellular levels based on multi-omics data.We constructed the in vitro and in vivo model of Mets caused by chronic treatment with olanzapine.Validated the model’s reliability by evaluating fasting glucose,triglycerides,serum insulin,glucose tolerance tests,and the insulinresistance index(HOMA-IR).The Quantitative real-time polymerase chain reaction(q RT-PCR)and Western Blot were then used to screen for candidate susceptible genes in the model.In the second part,focusing on the functional changes of the ACSBG 1 up-regulation phenotype,we changed the ACSBG 1 gene level by Lipo 3000 transfection to create an ACSBG 1overexpression in vitro model.q RT-PCR was used to assess the effectiveness of the model.A link between ACSBG 1 expression and the pathogenic phenotype of Mets was estimated by using the glucose uptake and lipid accumulation that followed ACSBG 1 overexpression.In the third section,we administered intravenous injections of ACSBG 1 siRNA targeted therapy to the animal model of olanzapine-induced Mets action in order to further clarify whether inhibiting ACSBG 1 expression can improve AP-induced Mets.The effectiveness of targeting was evaluated using Western Blot and q RT-PCR.The therapeutic effect was judged by the detection of fasting glucose,triglycerides,serum insulin,oral glucose tolerance tests,HOMA-IR,and other indicators.Results: In the first part,this study screened the susceptibility genes of olanzapine-induced Mets at multiple levels in vivo and in vitro.Compared with the control group,C 57 BL/6 male mice in the olanzapine injection group showed changes in metabolic phenotypes such as blood glucose,blood lipids,and insulin resistance index from the 6 th week of administration(P < 0.05).Compared with the control group,HEPG 2 cells also showed changes in metabolic phenotypes such as decreased glucose consumption and increased triglyceride content(P < 0.05).Real-time fluorescence quantitative PCR was used to verify the 12 significantly different genes(KCNJ 2,ACSBG 1,PTPN 11,PLTP,ABCG 1,CDK 5,EHHADH,GSK 3β,CD 36,PTPN 11,IKBKB,PRKCE)obtained in the previous experiment.In vivo and in vitro experiments,compared with the control group,the expression changes of the five genes were consistent,among which KCNJ 2,ACSBG 1,and PTPN 11 were up-regulated,ABCG 1 and GSK 3β were down-regulated.Compared with other gene changes,the change of ACSBG 1 was the most significant(P < 0.005).In the second part,the ACSBG 1 gene level was changed by Lipo 3000 to study the correlation between the change of ACSBG 1 expression level and the functional phenotype of Mets induced by olanzapine.The overexpression efficiency of ACSBG 1 was detected by real-time fluorescence q RT-PCR.Compared with the control group,the expression of ACSBG 1 in the Lipo 3000 transfection group was significantly increased(P < 0.005).The glucose consumption,triglyceride content and lipid accumulation of ACSBG 1 overexpressed cells were analyzed.Compared with the control group,the results showed that the glucose consumption of ACSBG 1 overexpressed cells decreased(P < 0.05),the triglyceride content increased(P < 0.05),the expression of p-PI 3 K and p-IRS-1 was inhibited(P < 0.05),p-Gsk 3β was activated(P < 0.05),and the abnormal pathological phenotype of lipid accumulation was produced.In addition,JC-1 staining results showed that compared with the control group,the mitochondrial membrane potential of the ACSBG 1 overexpression group was decreased and the mitochondrial function was impaired.This phenomenon is basically consistent with the phenotype of mitochondrial dysfunction in HEPG 2cells induced by olanzapine.In the third part,the mice induced by olanzapine were treated with targeted injection of ACSBG 1 siRNA to determine whether ACSBG 1 was involved in olanzapine-induced Mets key molecular targets,and to verify the feasibility of precise targeted intervention of ACSBG 1expression up-regulation in the treatment of olanzapine-derived Mets.Real-time fluorescence quantitative PCR was used to detect the liver knockdown efficiency of ACSBG 1 siRNA.Compared with the control group,tail vein injection of si-h-ACSBG 1 significantly down-regulated the level of ACSBG 1 in the liver of mice(P < 0.05).The glucose and lipid metabolism of mice were detected to determine whether reducing the expression of ACSBG 1 in the liver could alleviate or improve the pathological phenotype of olanzapine-induced glucose and lipid metabolism disorders.The results of blood biochemical analysis showed that compared with the control group,the increase of blood glucose,blood lipid and serum insulin in ACSBG 1 siRNA group gradually returned to normal level(P < 0.05).Compared with the control group,Western blot results also showed that the expression of p-PI 3 K and p-IRS-1 in the liver tissue of Mets mice in the ACSBG 1 siRNA group was activated(P < 0.05),and the expression of p-Gsk 3β was inhibited.The liver mitochondrial function of mice was detected by a reactive oxygen species(DCFH-DA)detection kit.Compared with the control group,the liver ROS level of mice in the ACSBG 1 siRNA group was significantly decreased(P < 0.05).At the same time,Western blotting results showed that liver TNF-α and NF-κB were inhibited during this process,and liver inflammation was improved(P < 0.05).The above results indicate that olanzapine induces the production of Mets and high expression of ACSBG 1 in the liver.Conclusion: Mitochondrial dysfunction caused by up-regulation of ACSBG 1 gene is one of the key mechanisms involved in olanzapine-derived Mets.This study further expands the understanding of the molecular mechanism of APs-induced Mets,and provides a new target for the individualized prevention and precise treatment of Mets in patients with schizophrenia.