| BackgroundOlanzapine is a second-generation antipsychotic drug that can significantly reduce side effects such as extrapyramidal diseases caused by a generation of antipsychotic drugs,but it causes metabolic disorders such as weight gain and elevated blood sugar.The mechanism of the olanzapine-induced metabolic disorder has not yet been explored.Previous studies have shown that adipose cell apoptosis can cause obesity and insulin resistance,and the relationship between olanzapine-induced metabolic disorder and adipocyte apoptosis has not been reported.From the perspective of olanzapine causing apoptosis of adipocytes in clinical drug concentration,and further feedback to increase the level of cell proliferation,we investigate the molecular mechanism of olanzapine-induced metabolic disorders.ObjectivesIn this study,we established a rat model of metabolic disorder induced by olanzapine to explore the mechanism of metabolic disturbance caused by olanzapine-induced abnormal proliferation of adipocytes.We verify how apoptosis induces cell proliferation through model animal tissues and fat cell line levels.This current study aimed to explore the specific mechanism of the olanzapine-induced obesity caused by lipid metabolism disorder.Methods1.Rat modeling and grouping: 210-230 g SD(Sprague-Dawley)rats were randomly divided into 2 groups: usual control group(Control group)and olanzapine administration group(Olanzapine group)(n=15/group).Rats in the Olanzapine group were given olanzapine(1.5 mg/kg),and rats in the Control group were given normal saline.Weigh once a week,measure blood glucose every week,and feed for 8 weeks.Insulin levels were measured at the 2nd,4th,6th,and 8th weekends,and after 8 weeks of feeding,the rat OGTT levels were measured.Animals were anesthetized at 8 weeks,and their white adipose tissue and ser?M were taken.Western-blotting determined the protein levels of Klotho/Egr-1/MnSOD,P-Pi3k/P-Akt and Cleaved/Caspase3 in rat adipose tissue.RT-PCR determined the mRNA levels of Klotho and Caspase3 in rat adipose tissue.2.Cell culture and experiment: Western-blotting was used to determine the protein levels of Klotho/Egr-1/MnSOD,P-Pi3k/P-Akt and Cleaved/Caspase3 in 3T3-L1 cells after olanzapine administration.After administration of different concentrations of olanzapine(0 ?m,1 ?M,2 ?M,5 ?M,10 ?M,20 ?M),the cell proliferation level was measured using CCK-8,and the level of apoptosis was measured by flow and confocal microscopy.3T3-L1 cells were dosed with 5 ?M(clinical drug concentration)and then treated with rKlotho to measure changes in cell proliferation and apoptosis levels.RT-PCR determined the mRNA levels of Klotho and Caspase3 in 3T3-L1 cells,and the levels of Klotho after olanzapine administration were observed by immunofluorescence technique.3.Data analysis: Statistical analysis was performed using SPSS13.0 statistical software.The data are expressed as the mean±standard error(S.E.)of at least three independent experiments.The t-test was used to compare the mean between the two groups.The repeated measures variance analysis was used in each index group.The difference was statistically significant at P<0.05.Results1.Long-term olanzapine administration can cause lipid metabolism disorder in rats.Compared to the control group,the olanzapine-administered group significantly increased in the body weight,and the drug-administered group grew faster.The weight of the drug-administered group was significantly higher than the control group from the first week to the eighth.The fasting blood glucose in the olanzapine group was significantly higher than that in the control group from the third week to the eighth week.The insulin level in the second week,the fourth week,the sixth week and the eighth week of the olanzapine administration group was significantly higher than that of the control group.The area under the OGTT curve in the olanzapine-administered group was significantly higher than that in the control group.The above results indicated that olanzapine could cause metabolic disorders such as impaired glucose tolerance and weight increase,indicating that the olanzapine rat metabolic disorder model was successfully established.2.Long-term olanzapine administration activates adipose cell apoptosis and proliferation-related pathways.In the Olanzapine group,the anti-aging factor Klotho protein and mRNA levels were significantly decreased,the mitochondrial anti-apoptotic factor MnSOD level was significantly decreased,and the inflammation-activated Egr-1 protein level was significantly increased.The level of Cleaved-Caspase3/Caspase3 apoptosis protein is significantly higher.The results showed that the apoptosis level of white adipose tissue in rats was significantly increased after olanzapine administration.Interestingly,the level of proliferation factor protein P-Pi3K/P-AKT was significantly increased,indicating that adipose tissue cell proliferation was simultaneously initiated.3.Olanzapine can induce drug-dependent apoptosis of adipocytes Apoptosis levels were observed in 3T3-L1 cells at different concentrations of olanzapine(1?M,2?M,5?M,10?M,20?M).Pearson correlation Analysis showed a significant positive correlation between olanzapine concentration and apoptotic levels(Pearson correlation test,r = 0.775,P = 0.000).The results showed that the clinical concentration of olanzapine(>=10 ?M)caused a large number of cell apoptosis.4.The clinical concentration of olanzapine fat cells was mainly caused by abnormal proliferation in 3T3-L1 cells.The cell proliferation level was detected by CCK-8 kit at different concentrations of olanzapine(1?M,2?M,5?M,10?M,20?M).At a concentration of 5 ?M(clinical concentration),cell proliferation levels were dominant.It was further confirmed that olanzapine caused an irreversible apoptosis trend at a concentration of 10 ?M(more than the clinical drug concentration).5.Recombinant Klotho could successfully suppress apoptosis induced by olanzapine of the clinical dosage and recover the proliferation of adipocytes into normal condition.3T3-L1 cells were co-cultured with recombinant Klotho protein at the concentration of olanzapine at a concentration of 5 ?M.CCK-8 results showed that the cell proliferation level returned to normal.Confocal microscopy showed that the level of apoptosis also recovered significantly.ConclusionThe clinical dose of olanzapine can induce cell apoptosis by activating the Klotho/Egr-1/MnSOD pathway.Due to the protective mechanism of the organism,Olanzapine feedback increases the level of adipocyte proliferation,which leads to metabolic disorders such as obesity.After the addition of rKlotho,the level of apoptosis was significantly decreased,and the abnormal proliferation level of the cells was also significantly inhibited to normal.This study observed that olanzapine caused cell apoptosis in a dose-dependent manner for the first time,which led us to understand the metabolic syndrome caused by olanzapine from a new perspective.The results of this study indicate that olanzapine-induced obesity-related metabolic complications can be treated by targeting the target of the apoptotic pathway,further improving the therapeutic effect of olanzapine. |
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