| Background:Schizophrenia is a serious chronic mental illness that has a huge impact on individuals,families and society.Olanzapine is the first-line clinical agent for the treatment of schizophrenia.However,long-term medication is easy to cause metabolic adverse reactions such as obesity,dyslipidemia and diabetes,and increases the risk of death in patients with schizophrenia.Olanzapine-mediated thermogenesis impairment leading to energy imbalance is one of the important causes of excessive obesity caused by this drug.Brown adipose tissue is the main thermogenic organ of the body,and epigenetics plays an important role in regulating the thermogenesis of brown adipose tissue.The previous study of our group found that histone methylation modification in brown adipose tissues of mice changed significantly after olanzapine action,and the expression of protein 2B in the jumonji domain of histone demethylase(jumonji domaincontaining protein 2B,JMJD2B)was also significantly downregulated by drug action.However,the specific mechanism of JMJD2 B in olanzapine mediated thermogenic injury of brown adipose tissue remains unclear.Therefore,elucidating the role of JMJD2 B in olanzapine-mediated energy metabolism abnormalities in brown adipose tissue is of great significance for understanding the molecular mechanism of epigenetic regulation in the development and progression of drug-induced metabolic diseases.Object:To explore the abnormal histone methylation modification mediated by olanzapine and its molecular mechanism,and provide a new strategy for clinical intervention in metabolic side reactions such as weight gain caused by olanzapine.Methods and results:1.Effects of olanzapine on body weight,thermogenesis and glycolipid metabolism in SD ratsTwenty female SD rats,8 weeks old,body weight 180-220 g,randomized: blank control group,olanzapine administration group(1 mg/kg,3 times a day),administered for 28 days,to monitor the body weight,water drink,food intake,body temperature,blood glucose and lipids of the rats.The results showed that olanzapine caused significant weight gain(p<0.05),a significant decrease in scapular temperature(p<0.01),a significant increase in serum triglyceride(TG)levels(p<0.05),and an extremely significant increase in total cholesterol(TC)levels compared with the control group(p<0.01).HE staining found that olanzapine can make fat droplets larger and more in brown adipose tissue and white adipose tissue.In addition,by immunohistochemistry,Western Blot and q PCR analysis,olanzapine significantly reduced the m RNA expression(p<0.01)and protein expression(p<0.01)of Fgf21,Pparγ,Prdm16,Rxrα and Ucp1.2.Olanzapine upregulates H3K9me3 levels in brown adipose tissueTo explore the effect of olanzapine on histone methylation in brown adipose tissue,the brown adipose tissue of rats was taken for Chi P-seq and transcriptome sequencing.The results showed that several pathways related to brown fat energy metabolism,including "adipose tissue development","white adipocytes differentiation" and "positive regulation of adipocyte differentiation",underwent significant changes.Further analysis revealed an increase in the enrichment of the promoter region H3K9me3 of genes associated with energy metabolism,such as Prdm16,and Rxrα.In addition,the transcriptome sequencing results were analyzed and further validated,and it was found that the transcription level of Jmjd2 b was significantly downregulated after olanzapine log2(multiple change)| ≥1.4,P value≤ 0.05.3.Olanzapine action JMJD2 B reduces the expression of thermogenic genes in brown adipose tissueTo further explore the role of JMJD2 B in olanzapine mediated thermogenesis imbalance in brown adipose tissue,we first analyzed Immunohistochemistry and Western Blot detect the expression of JMJD2 B in energy-metabolizing-related tissues such as liver,muscle,white fat and brown fat.The results showed that compared with the control group,the protein expression of JMJD2 B in brown adipose tissue showed significant(p<0.01).Subsequently,the knockdown and overexpression C3H10T1/2 stably transformed cell lines of JMJD2 B were constructed respectively.After being induced to differentiate into brown adipose tissue cells,they were treated with 5 μ M olanzapine for 24 hours.Compared with the control group,the global epigenetic markers of H3K9me3(p<0.05)were significantly increased after knocking down JMJD2 B,while the protein expression of RXRα(p<0.05)and PRDM16(p<0.05)decreased significantly,and these epigenetic markers increased more significantly after olanzapine compared with the knockdown group.compared with the control group,Overexpression of JMJD2 B led to a significant decrease in the global epigenetic markers of H3K9me2(p<0.05)and H3K9me3(p<0.01),and the protein expression of RXRα(p<0.01)and PPARγ(p<0.05)was significantly increased,and compared with the olanzapine group,the protein expression of H3K9me3 after overexpression of JMJD2 B decreased significantly(p<0.01)and the protein expression of RXRα increased significantly(p<0.01).4.JMJD2B-Ser478 phosphorylation abnormality is a key site of olanzapine-induced damage to brown fat thermogenesisIn order to explore whether olanzapine can reduce the activity of JMJD2 B by affecting the phosphorylation level of JMJD2 B,the expression of JMJD2 B and PKA in brown adipose tissue after olanzapine treatment was detected by Western Blot.The results showed that compared with the control group,olanzapine significantly reduced the protein expression of JMJD2 B in nucleus(p<0.01)and the protein expression of PKA(p<0.05).In order to confirm the effect of olanzapine on the phosphorylation of JMJD2 B and further explore its possible action sites,the two phosphorylation sites Ser452 and Ser478 that exert enzymatic activity in JMJD2 B were first predicted and screened through the Net Phos 3.1 Server website.Then these two sites were mutated into alanine(Ala),which were S478 A and S452 A,respectively.Western blot analysis showed that compared with the wild-type,the protein expression of JMJD2 B in the nucleus was significantly reduced after the combination of olanzapine and H89 treatment(p<0.01).Compared with the S478 A mutant group,the protein expression of JMJD2 B in the nucleus of the S478 A mutant group was significantly reduced after the combination of olanzapine and H89treatment(p<0.05).Analysis of the protein expression of heat related factors in cells after S478 A mutation revealed that compared to the wild-type,the mutant group after the combination of olanzapine and H89 treatment,the protein level of RXRα was significantly downregulated(p<0.05).Conclusion:Olanzapine inhibits PKA activity,affects the phosphorylation of JMJD2 B and reduces its activity,so that multiple transcription factors Rxra,Fgf21 and Prdm16 promoter regions of the thermogenic key protein UCP1 recruit more H3K9me3,thereby inhibiting its transcription,which in turn leads to thermogenic damage and energy metabolism disorders.This study can provide a basic theory for the treatment of metabolic adverse reactions caused by olanzapine antipsychotic drugs in the clinic. |
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