本文關鍵詞： CUMS 抑郁 瑞格列奈 糖皮質(zhì)激素信號通路 腎上腺素能通路 代謝組學　出處：《南方醫(yī)科大學》2017年博士論文　論文類型：學位論文

【摘要】：研究背景與目的本實驗室既往研究發(fā)現(xiàn)慢性不可預見性溫和應激(Chronic Unexpected Mild Stress,CUMS)致抑郁擾動大鼠體內(nèi)藥物代謝過程,但機制未明。本課題將研究CUMS致抑郁大鼠瑞格列奈體內(nèi)過程,對肝臟進行基因表達譜研究和驗證,檢測血清和尿液代謝譜,在細胞水平探索調(diào)控藥物代謝酶表達的信號通路。方法1.建立CUMS致抑郁模型:將SD大鼠抑郁組孤養(yǎng),予8種不可預見性應激造模8周。造模前后,進行曠場試驗評分、糖水偏好實驗,測定血漿皮質(zhì)醇及腎上腺素濃度,共同測定抑郁建模是否成功。2.藥代動力學及藥物代謝酶表達的檢測:造模成功后,用LC-MS/MS測定SD大鼠瑞格列奈血漿濃度,用qRT-PCR檢測SD大鼠肝臟Cyp2c11等藥物代謝酶的表達。3.GK大鼠肝臟mRNA表達及驗證:GK大鼠發(fā)病后建立抑郁模型,用基因表達譜芯片檢測大鼠肝臟mRNA表達水平,并進行生物信息學分析。qRT-PCR及Western blot驗證所關注的差異基因表達水平。4.GK大鼠血清和尿液代謝譜研究:LC-Q/TOF-MS檢測GK大鼠血清及尿液中代謝產(chǎn)物,Simca-P軟件進行多元統(tǒng)計分析,尋找差異代謝產(chǎn)物及KEGG代謝通路。5.信號通路對BRL3A的影響:CCK8挑選無毒性藥物濃度,單用地塞米松(Dexamethasone,DEX)、右美托咪定(Dexmedetomidine,DEXT)、異丙腎上腺素(Isoprenaline,ISO)、去氧腎上腺素(Phenylephrine,PE),或 DEX 分別聯(lián)用8-Br-cAMP、RU486,干預 BRL3A48hr,qRT-PCR 及 Western blot 檢測藥物代謝酶的表達水平。結(jié)果1.SD大鼠經(jīng)過8周造模后,抑郁組水平運動能力、垂直探索能力較對照組顯著降低,糖水偏好減少,血漿皮質(zhì)醇及腎上腺素濃度較對照組升高,說明建模成功。2.抑郁模型組與對照組相比,瑞格列奈血漿藥物的半衰期(T1/2)、達峰時間(Tmax)、峰濃度(Cmax)、曲線下面積(AUC0-∞)、平均停留時間(MRT0-∞)均降低。抑郁組肝臟Cyp2c11表達上調(diào),Cyp2c13、Cyp2c8/9表達無差異。3.抑郁組GK大鼠肝臟差異表達基因共49個。表達上調(diào)基因以Nr1i3為中心節(jié)點,調(diào)控其他基因的表達。表達上調(diào)基因在藥物代謝、類固醇激素合成等KEGG通路富集。qRT-PCR驗證所關注的基因,如Nr1i3、Cyp2b1/2、Ugt1a1、Ugt2b1、Cyp17a1、Cyp3a18等表達上調(diào),CAR、Ugt1a1蛋白表達也顯著上調(diào)。4.抑郁組GK大鼠血清及尿液代謝譜分別有16、28種代謝物顯著改變,在22及17個KEGG代謝通路富集,其中也包括藥物代謝、糖皮質(zhì)激素信號通路。5.1umol/L DEX 及 8-Br-cAMP 可顯著促進 BRL 3A Ugta1及 Nrli2 表達上調(diào),PXR表達也顯著上調(diào),RU486可逆轉(zhuǎn)這種上調(diào)效應。DEXT及PE可輕度上調(diào)Cyp3a18mRNA表達,ISO對上述基因及蛋白表達無影響。結(jié)論CUMS致抑郁可通過糖皮質(zhì)激素受體及腎上腺素能通路調(diào)控多個藥物代謝酶表達,體內(nèi)多種內(nèi)源性物質(zhì)代謝譜改變,最終擾動瑞格列奈藥物體內(nèi)代謝過程。
[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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