| Background:Olanzapine,as the widely used second-generation antipsychotic drug,shows good therapeutic effects on various types of psychotic symptoms.However,despite its beneficial neuropsychiatric effects in patients,Olanzapine exhibits some potential adverse metabolic effects such as weight gain/obesity,diabetes,dyslipidemia and cardiovascular.At present,due to the new clinical antipsychotic drugs that can replace Olanzapine has not yet appear,the side-effects of Olanzapine are still of great attention.However,the mechanism by which Olanzapine causes side effects is not completely clear.In addition to regulating the central nervous system,Olanzapine but also act on the lipid metabolism of adipose tissue,liver and other peripheral tissue through inhibiting lipolytic enzyme activity and activating of SREBPs related lipid synthesis pathways.Simvastatin,an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A(HMG-Co A)reductase,is a commonly used hypolipidemic drug.Except controlling blood cholesterol level,high cholesterol and preventing cardiovascular diseases,numerous studies in vitro and in vivo have suggested that Simvastatin can also decrease lipid accumulation in liver and hepatocyte.Moreover,Simvastatin also show neuroprotective effect among neurodegenerative diseases like cerebral ischemic stroke,Alzheimer’s disease,and Parkinson’s diseases.In recent years,Simvastatin is also found to inhibit m TOR signaling pathway,then inhibiting cell growth and inducing cell cycle arrest in a variety of tumors such as glioma,melanoma,breast cancer,colon cancer and so on.Mammalian rapamycin target protein(m TOR)is a class of atypical serine / threonine protein kinase,in addition to regulating cell growth and energy metabolism,m TOR also mediate the regulation of fat metabolism,involved in the development of obesity and diabetes.Studies have shown that Olanzapine can activate the m TOR pathway,triggering abnormal lipid metabolism.This provides a new thinking for the study of antipsychotic drug induced lipid metabolism.Therefore,we conclude that whether Simvastatin can inhibit the activation of mTOR pathway induced by Olanzapine and improve Olanzapine-induced abnormal lipid metabolism,thus providing a theoretical basis for clinical combination therapy.Objective:The purpose of our study was to explore whether Simvastatin could prevent Olanzapine-induced heptic lipid metabolism and to explore the mechanism,then proving references for searching drugs that improve Olanzapine-induced side effects of lipid metabolism.The study was carried out on rat model and Hep G2,LO2 cells.Methods:1.The Olanzapine-induced lipid metabolism model was established as the voluntary oral administration rat model established in our previous study.45 ~ 55 g rats were randomly divided into blank administration group and Olanzapine group(1.0 mg/kg,t.i.d).After two weeks of continuous treatment,the blank administration group was divided into control group and Simvastatin group(10 mg/kg/day).The Olanzapine group was divided into Olanzapine group(1.0 mg/kg,t.i.d)and Olanzapine(1.0 mg/kg,t.i.d)+ Simvastatin group(10 mg/kg/day)for 5 weeks.The body weight of the rats was measured weekly and the dietary intake was assessed.2.After treatment,the changes of plasma TG and TC contents were measured,and the liver slices were stained with oil red O to detect the changes of lipid accumulation in the liver.3.The toxicity of Olanzapine/Simvastatin to Hep G2 and LO2 cells was determined by MTT assay.Different concentrations of Olanzapine/Simvastatin were used to observe the effects of different concentrations of Olanzapine/Simvastatin on Hep G2 and LO2 cells in the accumulation of lipid droplets.4.The effect of Olanzapine/Simvastatin on the transcription level of lipid synthesisrelated genes was analyzed by RT-qPCR technique.5.Western Blot was used to analyze the expression of m TOR and its phosphorylation in the liver and Hep G2 and LO2 cells.6.Olanzapine / Simvastatin on the transcription level of lipid synthesis related genes were investigated.Results:1.Effects of Olanzapine or/ and Simvastatin on Serum TG and TC Contents in RatsAfter treating with Olanzapine for 2 weeks,TG and TC were measured.Compared with the control group,Olanzapine significantly increased the contents of TG and TC by about 39.72%,5.26%.And the content of TG and TC in plasma were 1.02 ± 0.06 mmol/L,2.40 ± 0.04 mmol/L;the content of TG and TC in the blank administration group were 0.73 ± 0.02 mmol/L and 2.28 ± 0.03 mmol/L,respectively.After completion of the administration,TG and TC were measured again.Compared with the control group,Olanzapine significantly increased the contents of TG and TC by about 49.41% and 11.84%,Simvastatin significantly decreased the contents by about 30.59% and 9.39%.Compared with Olanzapine group,the content of TG and TC in combination group decreased by 33.86% and 11.31%,respectively.2.The effect of Olanzapine or/and Simvastatin on lipid accumulation in liverCompared with the control group,the accumulation of lipid in the Olanzapine group was significantly increased by 49.82%,and that of the Simvastatin group was about 10.11%,but there was no significant difference.Compared with the Olanzapine group,the O+S co-treatment significantly decreased the lipid accumulation by 26.72%.3.Effects of Olanzapine or/and Simvastatin on m RNA expression of hepatic lipogenesisCompared with the control group,Olanzapine significantly inrcreased the transcriptional levels of Srebp1,Fasn,Acc,Acly and Scd-1,which were respectively 2.77 fold,3.84 fold,1.84 fold,3.53 fold and 11.47 fold of the control.While,Simvastatin significantly decreased by about 57.32%,11.74%,18.31%,53.01%,36.40%.Compared to the Olanzapine-only treatment group,the transcription level of the above genes were significantly decreased in the combined group,which decreased by about 73.35%,47.98%,56.95%,62.84% and 94.23%,respectively.4.Effects of Olanzapine or/ and Simvastatin on m TOR and phosphorylation of protein in rat liverCompared with the control,the relative expression of m TOR in the Olanzapine-treated group was 1.26± 0.10,and the relative expression of Simvastatin treatment was 0.91±0.11;the relative expression of p-m TOR in the Olanzapine-treated group was 1.54±0.12,the relative expression p-m TOR in Simvastatin-treeated group was 0.78±0.10.Compared with the Olanzapine group,the expression of m TOR protein was significantly decreased by about 23.03%,the expression of p-m TOR protein was also significantly decreased by about 40.14%.5.Effects of Simvastatin on the accumulation of lipid droplets in Hep G2 and LO2 cells induced by OlanzapineHep G2 and LO2 cells were treated with 1 μmol/L,5 μmol/L and 10 μmol/L Olanzapine,respectively.Olanzapine promoted lipid droplets accumulation in both kinds of cells,and had a concentration-dependent effect.When treated with 1 μmol/L,5 μmol/L and 25 μmol/L Simvastatin combining with 10 μmol/L Olanzapine,Simvastatin could interfere with Olanzapine-induced lipid accumulation in a dose-dependent manner.Moreover,50 nmol/L Rapamycin could decrease the accumulation of lipid droplets in HepG2 and LO2 cells.6.Effects of Olanzapine /Simvastatin on on m RNA expression of hepatic lipogenesis in HepG2 and LO2 Cells10μmol/L Olanzapine and 25μmol/L Simvastatin were used alone or in combination to treat cells and DMSO used as control.Compared with the control,10 μmol/L Olanzapine could significantly upregulate the transcription level of Srebp1,Fasn,Acc,Acly and Scd-1.25μmol/L Simvastatin could reduce the transcription level of obove genes,in which Fasn and Acly were significantly lower than the control group in Hep G2,and Srebp1,Fasn,Acly were significantly lower than the control group in LO2.Compared with the Olanzapine alone group,the transcription level of each gene in O+S co-treatment group was also significantly decreased.7.Effects of Olanzapine/Simvastatin on protein expression of m TOR,p-m TOR and SREBP1 and S6K1 in Hep G2 and LO2 cells.Compared with the control,the relative protein expression level of m TOR,p-m TOR,S6K1 and SREBP1 in Hep G2 cells were 1.80 ± 0.13,1.40 ± 0.20,1.16± 0.18,1.16 ± 0.18;the relative expression level of Simvastatin group were 0.82 ± 0.22,0.75 ± 0.15,0.74 ± 0.20 and 0.86±0.20,respectively;relative protein expression level of the co-treatment group were 1.38±0.09,1.04±0.17,0.84±0.22 and 1.07±0.20.Compared with Olanzapine-only group,the co-treatment group decreased the protein level by about 23.37%,26.06%,27.51% and 7.63%,respectively.Compared with the control,the protein expression of m TOR,p-m TOR,S6K1,SREBP1 in LO2 cells was significantly increased by Olanzapine,the relative expression were 1.56±0.14,1.36±0.11,1.26±0.12 and 1.37±0.14;Simvastatin significantly decreased the protein expression,and the relative expression were 0.85±0.19,0.89±0.11,0.91±0.11 and 0.93±0.15,respectively;the relative protein expression level of the cotreatment group were1.32±0.15,1.12±0.10,1.10±0.10 and 1.22±0.12,respectively.Compared with Olanzapine group,the expression of these proteins in the combination group decreased by about 15.28%,17.21%,12.90% and 10.91%,respectively.11.When the cells were pretreated with rapamycin for 24 h,the protein expression of m TOR,p-m TOR,SREBP1 and S6K1 in both kinds of cells were significantly inhibited.Conclusion:Simvastatin exerted beneficial effect on Olanzapine-induced lipid metabolism disorder in voluntary oral administration SD rat model and Hep G2,LO2 cells,and may partly through m TOR pathway.Our study provide a basis for the study of the mechanism of Olanzapine-induced lipid metabolism side effects. |
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