慢性不可预见温和应激致抑郁通过糖皮质激素受体及肾上腺素能通路扰动药物代谢及机制研究

发布时间：2018-02-09 07:15

  本文关键词： CUMS 抑郁 瑞格列奈 糖皮质激素信号通路 肾上腺素能通路 代谢组学　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景与目的本实验室既往研究发现慢性不可预见性温和应激(Chronic Unexpected Mild Stress,CUMS)致抑郁扰动大鼠体内药物代谢过程,但机制未明。本课题将研究CUMS致抑郁大鼠瑞格列奈体内过程,对肝脏进行基因表达谱研究和验证,检测血清和尿液代谢谱,在细胞水平探索调控药物代谢酶表达的信号通路。方法1.建立CUMS致抑郁模型:将SD大鼠抑郁组孤养,予8种不可预见性应激造模8周。造模前后,进行旷场试验评分、糖水偏好实验,测定血浆皮质醇及肾上腺素浓度,共同测定抑郁建模是否成功。2.药代动力学及药物代谢酶表达的检测:造模成功后,用LC-MS/MS测定SD大鼠瑞格列奈血浆浓度,用qRT-PCR检测SD大鼠肝脏Cyp2c11等药物代谢酶的表达。3.GK大鼠肝脏mRNA表达及验证:GK大鼠发病后建立抑郁模型,用基因表达谱芯片检测大鼠肝脏mRNA表达水平,并进行生物信息学分析。qRT-PCR及Western blot验证所关注的差异基因表达水平。4.GK大鼠血清和尿液代谢谱研究:LC-Q/TOF-MS检测GK大鼠血清及尿液中代谢产物,Simca-P软件进行多元统计分析,寻找差异代谢产物及KEGG代谢通路。5.信号通路对BRL3A的影响:CCK8挑选无毒性药物浓度,单用地塞米松(Dexamethasone,DEX)、右美托咪定(Dexmedetomidine,DEXT)、异丙肾上腺素(Isoprenaline,ISO)、去氧肾上腺素(Phenylephrine,PE),或 DEX 分别联用8-Br-cAMP、RU486,干预 BRL3A48hr,qRT-PCR 及 Western blot 检测药物代谢酶的表达水平。结果1.SD大鼠经过8周造模后,抑郁组水平运动能力、垂直探索能力较对照组显著降低,糖水偏好减少,血浆皮质醇及肾上腺素浓度较对照组升高,说明建模成功。2.抑郁模型组与对照组相比,瑞格列奈血浆药物的半衰期(T1/2)、达峰时间(Tmax)、峰浓度(Cmax)、曲线下面积(AUC0-∞)、平均停留时间(MRT0-∞)均降低。抑郁组肝脏Cyp2c11表达上调,Cyp2c13、Cyp2c8/9表达无差异。3.抑郁组GK大鼠肝脏差异表达基因共49个。表达上调基因以Nr1i3为中心节点,调控其他基因的表达。表达上调基因在药物代谢、类固醇激素合成等KEGG通路富集。qRT-PCR验证所关注的基因,如Nr1i3、Cyp2b1/2、Ugt1a1、Ugt2b1、Cyp17a1、Cyp3a18等表达上调,CAR、Ugt1a1蛋白表达也显著上调。4.抑郁组GK大鼠血清及尿液代谢谱分别有16、28种代谢物显著改变,在22及17个KEGG代谢通路富集,其中也包括药物代谢、糖皮质激素信号通路。5.1umol/L DEX 及 8-Br-cAMP 可显著促进 BRL 3A Ugta1及 Nrli2 表达上调,PXR表达也显著上调,RU486可逆转这种上调效应。DEXT及PE可轻度上调Cyp3a18mRNA表达,ISO对上述基因及蛋白表达无影响。结论CUMS致抑郁可通过糖皮质激素受体及肾上腺素能通路调控多个药物代谢酶表达,体内多种内源性物质代谢谱改变,最终扰动瑞格列奈药物体内代谢过程。
[Abstract]:Background & AIM previous studies in our laboratory have found that chronic unpredictable mild stress (chronic Unexpected Mild StressCUMS) has disturbed the drug metabolism in rats with depression, but the mechanism is not clear. This study will study the in vivo process of repaglinide in depressive rats induced by CUMS. The gene expression profile of liver was studied and verified, the metabolic spectrum of serum and urine was detected, and the signal pathway of regulating the expression of drug metabolic enzymes was explored at the cell level. 1. The depression model induced by CUMS was established: SD rats were isolated in depression group. Eight unpredictable stress models were made for 8 weeks. The open field test scores, sugar water preference tests, plasma cortisol and epinephrine concentrations were measured before and after modeling. Detection of pharmacokinetics and expression of drug metabolic enzymes: after the model was successfully established, the plasma concentration of reaglinide in SD rats was measured by LC-MS/MS. The expression of Cyp2c11 and other drug metabolizing enzymes in SD rat liver was detected by qRT-PCR. 3. The expression of mRNA in liver of SD rats and the establishment of depression model after the onset of disease were verified. The expression of mRNA in rat liver was detected by gene expression microarray. Bioinformatics analysis. QRT-PCR and Western blot were used to verify the differentially expressed genes. 4. Metabolic spectrum of serum and urine of GK rats; multivariate statistical analysis of metabolite Simca-P in serum and urine of GK rats was carried out by means of 10% LC-QR TOF-MS. Search for differential metabolites and KEGG metabolic pathway. 5. The effect of signal pathway on BRL3A. Selection of nontoxic drug concentration by 1: CCK8, Dexamethasone alone, dexamethasone (Dexamethasone), dexmetomidine (Dexmedetomidine), isoprenaline ISO (isoproterenol), Phenylephrine DEX (8-Br-cAMPRU486), respectively, were used to detect the expression of drug metabolizing enzymes in SD rats. Results 1. After 8 weeks of modeling, SD rats were treated with 8-Br-cAMPRU486 to detect the expression of drug metabolizing enzymes. The horizontal motor ability, vertical exploration ability, sugar water preference and plasma cortisol and epinephrine concentrations in depression group were significantly lower than those in control group, which indicated that the model group was successful. 2. The depression model group was compared with the control group. The half-life of repaglinide plasma T1 / 2, peak time (Tmax), peak concentration (Cmax), area under curve (AUC0- 鈭，

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