| Background Second generation antipsychotic drugs(SGA)are considered to possess superior efficacy in treating both positive and negative symptoms of schizophrenia compared to first-generation antipsychotics(FGA).These drugs are also increasingly applied to the treatment of other psychiatric disorders,such as mental disorders,autism and depression.However,in clinical applications,SGA main side effects are weight gain and metabolic abnormalities,including hyperglycemia,insulin resistance,type ? diabetes,dyslipidemia and cardiovascular disease.Bone marrow mesenchymal stem cells is a considerable potential of genetically engineered cells with multiple differentiation potential.Mesenchymal stem cells can be induced to differentiate in vitro into adipocytes,is adipocyte differentiation in vitro studies the most widely used cell lines.About SGA mouse bone marrow mesenchymal stem cells in vitro study less.Mechanism SGA cause weight gain can help guide the design of clinical medicine and new treatments to reduce the side effects of these drugs.Objectives1.To discuss the influence of olanzapine and clozapine to proliferation and adipogenic differentiation in mice BMSCs.2.To explore the mechanism olanzapine and clozapine to proliferation and adipogenic differentiation in mice BMSCs,in order to lay the foundation for understanding SGAs induced obesity and diabetes pathogenesis.Methods1.Cell culture : BMSCs using sugar DMEM + 10% FBS were cultured,when cell fusion reaches 80~90%,according to 1: 2 or 1: 3 ratio for passage.2.Cells induce :(1)Adipogenic induction : First,containing 10 mg/L insulin,0.5 mmol/L IBMX,1.0 ?mol/L dexamethasone,10% and fetal bovine serum DMEM low glucose medium(adipogenic medium)cultured cells for 2 days.Subsequently the medium was replaced by containing 10 mg/L insulin,10% DMEM low glucose medium fetal bovine serum,and changed for once every 2 day until maturation;(2)Add a series of concentrations of second generation antipsychotics into it to induce.(3)Both adipogenic medium and the drug was added to simultaneously induce BMSCs.3.In order to measure the influence caused by different concentrations of olanzapine and clozapine on cell proliferation,MTT assay and cell counting method were used,and select the appropriate concentration of olanzapine for subsequent experiments.4.Oil red O staining,Western blotting,q RT-PCR were used to detect effects of olanzapine on adipogenic differentiation and its mechanism.Results1.MTT assay and cell counting results showed that high concentration(80 ?M and 100?M)of olanzapine and clozapine inhibited the growth of cells and the minimal toxicity was 20 ?mol/L olanzapine and clozapine.2.The Oil Red O staining showed that the level of fat cells drops in the experimental group was significantly higher than control group after adding olanzapine;However the level of fat cells drops in the MDI+ olanzapine group and MDI+clozapine group was significantly reduced than MDI group.3.When the olanzapine was used alone,western blot showed that the expression level of fat differentiation marker gene and protein ?p2 and C/EBP increased 36%(P<0.01)and25%(P<0.05)respectively,which had the statistical significance;when the clozapine was used alone,the expression level of C/EBP? and ?P2 protein increased35%(P<0.01)and 25%(P > 0.05)respectively,so the former had the statistical significance,the later was not significant,and did not have the statistical significance;when both the MDI and olanzapine were used,the expression level of C/EBP? and ?P2protein in the MDI+20 ?M group was decreased than the MDI group,besides gray value analysis showed the difference was significant;when both the MDI and clozapine were used,the expression level of C/EBP? and ?P2 protein in the MDI+20?M group was decreased than the MDI group.4.When the olanzapine was used alone,the results of RT-q PCR showed that the expression levels of adipogenic genes Leptin,C/EBP? and Tnf-? significantly increased than the control group,increased about 68%(P<0.001)?79%(P<0.01)and 60%(P<0.01)respectively.When the clonzapine was used alone,the expression levels of adipogenic genes PPAR?,Leptin and Tnf-? significantly increased than the control group,increased about 52%(P<0.01)?66%(P<0.05)?65%(P<0.05)respectively,which had the statistical significance;When both the MDI and olanzapine were used,the expression levels of adipogenic genes Leptin,C/EBP? significantly decreased than the MDI group,decreased about 65%(P<0.01)?84%(P<0.05)respectively.however,he expression levels of Tnf-? increased than the MDI group.When both the MDI and clozapine were used,the expression levels of adipogenic genes Leptin,significantly decreased than the MDI group,decreased about 50%(P<0.001).The expression levels of Tnf-? and C/EBP? increased than the MDI group,and did not have the statistical significance.5.When the olanzapine was used alone,the results of western blot showed that the expression levels of p-Akt significantly higher than the control group,the expression of GSK-3? with the increase of p-Akt expression gradually reduced.The expression levels of ?P2 and C/EBP? were inhibited after BMSCs were pretreated with PI3K/Akt inhibitor(LY294002),Oil red O staining results also showed fat droplets in cells also decreased significantly.When MDI and clozapine together,,the expression levels of p-Akt lower than the MDI group,and the expression of its upstream and downstream p-IRS and p-GSK-3? also has changed.Where in,p-Akt and p-IRS expression than MDI group have come down,and with the increasing concentration gradually reduced.The expression of p-GSK-3? with the olanzapine concentration increased and increased.Conclusions1.Olanzapine and clozapine can promote BMSCs adipogenic differentiation,and the promoting effect of olanzapine is stronger than clozapine.Olanzapine can activate PI3K/Akt signaling pathway by increasing the level of Akt phosphorylation to promote adipogenic differentiation of BMSCs in mice.2.Compared with the single use MDI,MDI respectively with olanzapine and clozapine combined action,inhibits adipogenic differentiation of mouse BMSCs.When MDI and olanzapine together,insulin signaling pathway is inhibited,it may lead to insulin resistance,the specific mechanism needs further study. |
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