【作者】 李杰；

【导师】 乐江；

【作者基本信息】 武汉大学 ， 病理与病理生理学， 2015， 博士

【摘要】 细胞色素P450 (Cytochrome P450, CYP)是一类富含血红素的药物代谢酶超家族,参与内源性物质和外源性物质代谢,具有重要的药理学和毒理学作用。目前,已知人CYPs共有57个家族成员,分为18个亚家族。CYPs催化功能受到基因、内源性物质(如激素)、外源性物质(如药物和环境毒物)等多种因素影响,存在明显的个体差异。CYPs的活性水平差异是临床药物的药效和毒性存在明显个体差异的重要原因之一。CYPs酶系在肝脏表达丰富,其中CYP3A为含量最多的亚家族。CYP3A参与代谢约50%的临床药物(如硝苯地平、环孢菌素、红霉素等)。研究表明,肝脏CYP3A的表达量和催化功能存在性别差异,女性均高于男性。性激素是否直接影响了肝脏CYP3A的表达,或通过影响其它内源性物质(如生长激素)间接引发CYP3A的性别差异表达,尚不十分清楚。研究证实,CYPs超家族酶系亦表达于肝外器官,如脑。由于血脑屏障的存在,脑组织与血液之间的物质交换种类、交换量和速度受到制约,从而形成了相对独立于外周组织的脑内环境。近年,国外实验室证实脑CYPs具有原位代谢功能。CYP2D广泛表达于脑内,在大脑皮层、丘脑、海马、黑质纹状体、小脑等脑区均能检测到CYP2D mRNA和蛋白表达,为脑内表达的主要CYP亚型。文献报道,性激素如雌激素、雄性激素可调控脑CYP2D表达,但缺乏机制认识。本文利用多种动物模型和体外细胞实验,采用分子生物学技术结合分析化学手段,深入研究雄性激素对肝脏、脑主要表达CYP亚型(CYP3A和CYP2D)的表达调控机制,在分子水平阐明机体代谢功能差异的原因。研究将揭示性激素调控CYPs表达进而影响外源性物质代谢的机制,有利于认识内源性物质和外源性物质之间的相互作用,以及丰富对干预CYPs表达靶点的认识。第一部分：雄激素通过影响生长激素分泌模式调控肝脏CYP3A表达的分子机制研究目的：文献报道,女性肝脏CYP3A4蛋白表达量和酶催化活性约为男性2倍。啮齿类动物肝脏CYP3A表达亦有明显性别差异：小鼠CYP3A亚族包括11、13、16、25、41和44等亚型,其中CYP3A41为雌性小鼠特异性表达亚型；大鼠CYP3A亚族包括1、2、9、18、23和62等亚型,其中CYP3A2为雄性大鼠特异性表达亚型。课题组前期建立大鼠去势,去垂合并去势动物模型,实验数据提示雄性动物去势可明显抑制肝脏CYP3A2表达,但切除垂体后去势则无法观察到雄性激素对肝脏CYP3A2的调控作用。已知,生长激素为垂体分泌的重要激素,且其分泌模式具有性别差异。男性分泌模式表现为间断性脉冲分泌,女性则表现为连续性脉冲分泌。前期数据表明,去势并不影响调控肝脏CYP3A的重要转录因子HNF4和PXR的水平,但HNF6变化明显。本文拟利用HepG2细胞,去势及去垂大鼠模型,以及生长激素受体敲除(GHR-KO)小鼠模型,研究雄性激素是否对肝脏CYP3A4, CYP3A2和CYP3A41具有直接调控作用,或通过影响生长激素分泌模式间接调控CYP3A表达水平的分子调控机制。方法：体外实验,生理剂量(30 nM)和高剂量睾酮(300 nM),以及低剂量(0.2 ng/ml)和生理剂量(2 ng/ml)重组生长激素分别处理HepG2细胞。在体实验,采用去势、去垂、去势合并去垂大鼠模型,以及利用生长激素受体敲除小鼠模型。利用real-time RT-PCR, Western blotting分别检测大鼠CYP3A2,小鼠CYP3A41,人CYP3A4, HNF6, C/EBPa和RXRa mRNA和蛋白水平；利用Co-IP, ChIP,构建高表达质粒和荧光素酶基因报告系统,检测HNF6, C/EBPa, RXRa重要转录因子之间的相互作用,及其对CYP3A4, CYP3A2以及(Cyp3a41启动子的影响。结果：与对照组相比,持续型生长激素处理模式(雌性)可明显诱导CYP3A4的mRNA和蛋白水平,而脉冲型模式(雄性)无明显影响。同时,相比脉冲型生长激素分泌模式,持续型模式对HNF6 mRNA和蛋白水平的诱导作用更为明显,而对RXRa mRNA和蛋白水平的诱导效应低。两种生长激素模式均剂量依赖性抑制C/EBPa表达,但无明显程度差异。动物实验发现,雌性野生型小鼠肝脏CYP3A41和HNF6 mRNA和蛋白水平均明显高于雄鼠。与野生型小鼠相比,雌性和雄性GHR-KO小鼠肝脏CYP3A41, HNF6及RXRa的mRNA和蛋白水平均明显降低,而C/EBPa表达上升,但上述基因在雌性和雄性GHR-KO小鼠间并无明显差异。大鼠实验发现,与假手术组比较,垂体摘除可以导致HNF6和RXRa显著降低,而C/EBPa明显升高。细胞实验与动物实验均证实,生长激素分泌模式对肝脏CYP3A, HNF6及RXRa的表达调控存在差异。与假手术组比较,去势组大鼠肝脏CYP3A2, RXRα和C/EBPαmRNA和蛋白水平明显降低,HNF6升高；然而,在去垂组、去垂+去势组以及去垂+去势+睾酮组间,CYP3A2, HNF6, RXRα以及C/EBPα的mRNA和蛋白水平并无差异。生理剂量睾酮处理HepG2细胞后发现,细胞CYP3A4, HNF6, RXRα和C/EBPα的表达亦无明显变化。上述结果提示,睾酮对肝脏CYP3A并无直接调控作用。HepG2细胞和GHR-KO小鼠肝脏ChIP实验发现,两种生长激素分泌模式均可增加HNF6、C/EBPα与CYP3A4启动子结合,且持续型模式的诱导效应更强；两种模式生长激素均抑制RXRα与CYP3A4启动子结合,而持续型模式的抑制效应更为显著。Co-IP实验发现,HNF6可与RXRα和C/EBPα结合形成异源多聚体,而RXRα和C/EBPα之间并无结合。持续型生长激素分泌模式可明显诱导HNF6和C/EBPα形成异源多聚体,而脉冲型生长激素模式诱导效应低；持续型生长激素分泌模式可明显抑制HNF6与RXRα形成异源多聚体,而脉冲型抑制效应低。荧光报基因检测发现,HNF6可与CYP3A2启动子-140 bp到-106 bp之间发生结合且活化启动子；C/EBPα与CYP3A2启动子-163 bp到-140 bp之间发生结合且能增强其活性；RXRα与CYP3A2启动子-163 bp到-87 bp之间发生结合,具有较弱的活化作用。HNF6与C/EBPα对CYP3A2, CYP3A4, CyP3a41启动子具有协同激活效应,而当共转染RXRα后,HNF6与C/EBPα对启动子的激动作用被明显抑制。结论：雄性激素通过影响生长激素分泌模式间接调控肝脏CYP3A表达。性激素依赖性生长激素分泌模式差异是引发肝脏CYP3A表达出现性别差异的重要原因。不同生长激素分泌模式可影响RXRα与HNF6与C/EBPα结合,以及RXRα, HNF6, C/EBPα与CYP3A启动子的结合,并进而影响肝脏CYP3A的表达水平。生长激素雌性分泌模式可以诱导HNF6与C/EBPα结合,以及HNF6, C/EBPα与人CYP3A4禾口小鼠Cyp3α41启动子的结合,增强其表达；雄性分泌模式使得RXRα与HNF6结合增加,以及RXRα与HNF6与人CYP3A4和小鼠Cyp3α41启动子的结合,进而拮抗了HNF6和C/EBPα的协同激活作用,使得表达量下降。RXRα具有介导转录因子间相互作用的重要功能,在生长激素调控CYP3A表达过程中发挥重要作用。第二部分：睾酮器官选择性调控脑CYP2D表达的分子机制研究目的：研究报道,人成年后脑CYP2D表达水平随年龄增加而升高。动物实验发现,雌性大鼠去卵巢后给予孕激素和雌激素后,脑CYP2D mRNA均明显下降。研究提示,内源性物质可能影响脑CYP2D表达。已知肝脏CYP2D基因不可被诱导而增加表达,且目前尚未发现肝脏CYP2D的诱导剂。研究报道,外源性物质尼古丁可在转录后水平诱导脑CYP2D表达,但不影响肝脏CYP2D水平,提示脑CYP2D的表达调控机制可能区别于肝脏。大鼠CYP2D表达亚型包括CYP2D1, CYP2D2, CYP2D3, CYP2D4, CYP2D5和CYP2D18,其表达量存在器官差异：肝脏主要表达CYP2D2,脑内主要为CYP2D4,而CYP2D18仅表达于脑组织。小鼠CYP2D表达亚型包括CYP2D9, CYP2D10, CYP2D11, CYP2D12, CYP2D13, CYP2D22, CYP2D26, CYP2D34和CYP2D40,其中CYP2D22与人CYP2D6具有高同源性。本文拟利用SH-SY5Y, U251和HepG2细胞系,大鼠和生长激素受体敲除(GHR-KO)小鼠模型,深入探讨睾酮对脑CYP2D的表达调控机制。方法：人神经母细胞瘤细胞系SH-SY5Y,神经胶质瘤细胞U251和肝癌细胞HepG2细胞,经生理剂量(30 nM)和高剂量(300 nM)睾酮处理后检测CYP2D6 mRNA表达和催化功能活性,以及脑内特异性表达microRNA (miRNA)的表达量；转染miRNAs后,检测CYP2D6 mRNA水平；利用荧光素酶基因报告系统,检测miRNAs与CYP2D63’-UTR的结合情况。利用GHR-KO小鼠的去势模型,在体观察睾酮对海马和肝脏CYP2D22和miRNAs表达的影响。生理剂量睾酮处理大鼠,分别检测脑和肝脏CYP2D表达情况；利用HPLC-DAD和HPLC-MS/MS分别检测血浆和脑脊液中曲马多及其活性代谢物M1的水平；利用甩尾镇痛实验检测曲马多镇痛效应的变化。结果：睾酮可剂量依赖性抑制SH-SY5Y和U251细胞CYP2D6 mRNA表达水平及其催化活性,而对HepG2细胞CYP2D6 mRNA和功能无明显影响。生理剂量睾酮可明显诱导SH-SY5Y和U251神经细胞中miR-101, miR-124a, miR-125b和miR-128-2的表达水平。HepG2细胞中miR-101, miR-124a, miR-125b和miR-128-2表达量明显低于SH-SY5Y和U251神经细胞,且仅大剂量睾酮可诱导miR-125b表达量。生长激素受体敲除小鼠去势后,脑内海马区CYP2D22 mRNA水平明显升高,而肝脏表达水平无变化；同时,海马miR-101和miR-128-2表达水平下降,肝脏miR-125b表达量减少。通过构建表达质粒并转染细胞系,发现miR-101和miR-128-2高表达可使得SH-SY5Y,U251和]HepG2细胞中CYP2D6 mRNA水平下降,但转染miR-124a和miR-125b对CYP2D6 mRNA水平无影响。荧光素酶基因报告实验,发现miR-101和miR-128-2可与CYP2D6的3’UTR结合并明显抑制其活性。生理剂量睾酮处理大鼠,发现大脑额叶皮层、海马、小脑和脑干CYP2D蛋白表达量降低,而丘脑CYP2D蛋白表达不受影响；实时定量RT-PCR检测证实,海马CYP2D1/5, CYP2D2, CYP2D3和CYP2D4/18 mRNA表达水平均下降；脑脊液中曲马多半衰期延长,活性代谢物M1的AUC0-180明显减少,而血浆中曲马多及M1药代动力学参数无变化。甩尾实验发现,睾酮处理使得曲马多镇痛效应减弱。已知,曲马多对甩尾实验的镇痛作用主要经μ型阿片受体介导,可完全被纳洛酮拮抗,而活性代谢物M1与μ型阿片受体的亲和力较曲马多高300倍。因而,睾酮处理组脑内M1生成量减少是曲马多镇痛效应下降的重要原因。结论：睾酮对脑CYP2D表达调控作用具有器官选择性,可通过改变脑内特异性表达的miRNAs水平,直接调控脑CYP2D表达,而不影响肝脏CYP2D水平。睾酮通过影响经CYP2D代谢的中枢活性物质的靶器官浓度,而改变CYP2D底物的药效学。随年龄变化的雄性激素水平改变,或使用激素治疗,亦或通过手术影响雄性激素分泌,均可能影响脑内CYPs表达,进而改变中枢活性物质的药理毒理学效应,以及改变环境污染物所致神经系统疾病的易患性。更多还原

【Abstract】 Cytochrome P450 (CYP) superfamily is principally responsible for the biotransformation of exogenous and endogenous compounds and plays the important pharmacological and toxicological roles. There are at least 18 subfamilies comprising 57 isoforms. The catalytic function of CYPs can be affected by multiple factors including gene, endogenous substances (such as hormones), exogenous substances (such as drugs and environmental toxins) and so on. The variation of the activity of drug-metabolizing enzymes among the population is one of the primary causes for the differences in the pharmacological and toxic profiles of clinical drugs.CYPs are mainly expressed in liver, and CYP3A subfamily is the most abundant subfamily among hepatic isoforms. Approximately 50% of clinical drugs (e.g. nifedipine, cyclosporine, erythromycin) has been shown to be metabolized by CYP3A. Previous studies have shown the gender differences in the expression and catalytic activity of hepatic CYP3A. The mechanism of gender difference in CYP3A expression is still unclear, which could be due to the direct regulation of hepatic CYP3A by sex hormones or due to indirect regulation via the alteration of other endogenous substances (e.g. growth hormone) of by sex hormones.Previous studies have revealed that CYPs superfamily also exist in the extrahepatic organ, such as brain. Because of the blood-brain barrier, the capacity and speed of compound exchange between brain tissue and blood are restricted. Thus, brain is a relatively independent environment from the peripheral tissues. In recent years, researchers have confirmed that brain CYPs can be involved in the situ metabolism. The mRNA and protein of CYP2D are widely expressed in the brain including the cerebral cortex, thalamus, hippocampus, substantia nigra, cerebellum, which has been shown to be one of the major CYP isoforms in brain. Sex hormones (e.g. estrogens and androgens) have been reported to affect the expression of brain CYP2D; however, the mechanism was largely unknown.The present study intends to investigate the regulation mechanism of the major CYP isoform in the liver (CYP3A) and brain (CYP2D) by androgen in diverse animal models and cell lines by using molecular biology and analytical chemistry techniques. The data will reveal the regulation mechanism of CYPs by sex hormones, which will be helpful to understand the interaction between the endogenous and exogenous compounds and the regulation target of CYP isoforms.Part I:Regulation mechanism of hepatic CYP3A via the alteration of growth hormone secretory pattern by androgenObjective: Previous study has shown that the levels of hepatic CYP3A4 expression and catalytic activity in female were 2-fold higher than those in male. The significant sex differences in hepatic CYP3A in rodents have been observed. Mouse CYP3A subfamily includes CYP3A11,13,16,25,41, and 44. Among them, the expression of CYP3A41 is female-specific. Rat CYP3A subfamily consists of CYP3A1,2,9,18,23 and 62, and CYP3A2 is male-specific. Our previously data have shown that orchidectomy in male rat markedly reduced hepatic CYP3A2 expression; however, no change in CYP3A2 levels was observed in rats in hypophysectomy-orchidectomy group compared with the orchidectomized rats. It is known that the secretory of growth hormone (GH), an important pituitary hormone, is sex-dependent. Masculine secretion pattern was an intermittent pulse secretion, but feminine pattern was a continuous secretion. Previous data showed orchidectomy affected the levels of transcription factor HNF6, but not HNF4 and PXR. In the present study, the regulation of hepatic CYP3A4, CYP3A2 and CYP3A41 by androgen was investigated in orchidectomy, hypophysectomy, and orchidectomy-hypophysectomy rat models, GH knock out (GHR-KO) mouse models, and HepG2 cell line. Methods:HepG2 cells were treated with physiological (30 nM) and supraphysiological (300 nM) doses of testosterone, or subphysiological (0.2 ng/ml) and physiological (2 ng/ml) doses of recombinant human growth hormone. Orchidectomy, hypophysectomy, orchidectomy-hypophysectomy rat models, and GH knock out (GHR-KO) mouse model were used in the present study. The mRNA and protein levels of rat CYP3A2, mouse CYP3A41, human CYP3A4, HNF6, C/EBPa and RXRa were respectively detected by real-time RT-PCR and Western blotting. The interactions between HNF6, C/EBPa and RXRa proteins and the effect of transcriptional factors on CYP3A4, CYP3A2 and Cyp3a41 promoters were respectively analyzed by using Co-IP, ChIP and luciferase assays. Results: The mRNA and protein levels of CYP3A4 were increased following the constant (feminine) GH administration compared with control group, but not by the pulsatile (masculine) GH administration. The constant GH secretion increased HNF6 mRNA and protein levels more efficiently compared with the pulsatile GH secretion, but the induction of RXRα mRNA and protein following the constant (feminine) GH administration was inefficiently. Both the constant and pulsatile GH secretion dose-dependently inhibited C/EBPα expression without the differences between the two patterns. The basal levels of CYP3A41 and HNF6 mRNA and protein in the liver were higher in the wild-type female mice compared with the male mice. CYP3A41, HNF6, and RXRα mRNA and protein levels were significantly lower in both male and female GHR-KO mice compared with wild-type mice, while C/EBPα mRNA and protein levels were higher in GHR-KO mice. There were no difference in the mRNA and protein levels of these genes between male and female GHR-KO mice. Hypophysectomy significantly decreased HNF6 and RXRa levels in rats, but increased C/EBPα levels. The differences in the regulation of CYP3A, HNF6 and RXRα between two GH secretion patterns were observed in vivo and in vitro. Orchiectomy significantly decreased CYP3A2, RXRα and C/EBPα mRNA and protein levels and increased HNF6 expression in rats. However, no differences in the mRNA and protein levels of CYP3A2, HNF6, RXRα, and C/EBPα were observed between the hypophysectomized rats and the hypophysectomized-orchiectomized rats treated with or without testosterone. No changes in CYP3A4, HNF6, RXRα, and C/EBPα mRNA levels in HepG2 cells were observed in response to testosterone at a physiologic concentration. These data suggest that there is no direct regulation of CYP3A by testosterone.The ChIP data showed the increases in the binding of nuclear HNF6 and C/EBPα proteins to a region of the CYP3A4 promoter under the control of two GH secretion patterns, especially the constant secretion. In contrast, a greater inhibition of the binding of RXRα to the CYP3A4 promoter was observed in response to the constant GH administration compared with the pulsatile pattern. HNF6 can respectively bind to RXRα and C/EBPα, but RXRα cannot bind to C/EBPα. A greater induction of HNF6/C/EBPα complex levels was observed by the constant GH administration compared with the pulsatile GH administration; meanwhile, a greater inhibition of HNF6/RXRα complex levels was observed in response to the constant GH administration compared with the pulsatile GH administration. HNF6 activated the CYP3A2 promoter located at -140 to -106 bp, and C/EBPa activated the CYP3A2 promoter located at -163 to -140 bp. Weak activation of the CYP3A2 promoter located at -163 and -87 bp was observed by RXRa. HNF6 and C/EBPa can active the CYP3A2, CYP3A4, and Cyp3a41 promoters synergistically; however, the activation of the CYP3A2 promoter by HNF6 was completely inhibited following cotransfection with RXRa. Conclusion: The data suggest that androgen indirectly regulated hepatic CYP3A expression via the alteration of growth hormone secretion. Sex-dependent GH secretion pattern can be an important cause for the gender difference in hepatic CYP3A expression. GH secretion patterns can affect the binding of RXRa, HNF6, and C/EBPa proteins, and also affect the binding of RXRa, HNF6, and C/EBPa binding to CYP3A promoter. Feminine GH secretion may induce HNF6/C/EBPa complex levels and the binding of HNF6 and C/EBPa to the CYP3A4 and Cyp3a41 promoters leading to the high level of hepatic CYP3A expression. Masculine GH secretion may increase the binding of RXRa to HNF6, and the binding of RXRa/HNF6 to the CYP3A4 and Cyp3a41 promoters leading to the attenuation of the synergistic activation by HNF6 and C/EBPa and the low level of hepatic CYP3A expression. RXRa may be an important signalling molecule that mediates interactions between transcription factors, and play a critical role in the regulation of CYP3A expression by GH.Part Ⅱ: Testosterone selectively regulates cerebral CYP2D via brain-enriched miRNAsObjective:Previous study has shown that brain CYP2D levels were increased with age. It has been reported that brain CYP2D mRNA was decreased in ovariectomized female rats treated with progesterone and estrogen. It provides a hint that endogenous substances can influence brain CYP2D expression. It is known that hepatic CYP2D transcription cannot be induced and no CYP2D inducer has been reported so far. Previous study has shown that xenobiotics nicotine can induce brain CYP2D expression at post-transcriptional level but not hepatic CYP2D, suggesting the different regulation mechanism of brain CYP2D. Rat CYP2D subfamily includes CYP2D1, CYP2D2, CYP2D3, CYP2D4, CYP2D5, and CYP2D18. The expression of rat CYP2D exists organ difference. CYP2D2 is the major hepatic isoform, and CYP2D4 was the most abundant isoform in brain. CYP2D18 is only expressed in brain. Mouse CYP2D subfamily consists of CYP2D9, CYP2D10, CYP2D11, CYP2D12, CYP2D13, CYP2D22, CYP2D26, CYP2D34, and CYP2D40. CYP2D22 shares high sequence identity of amino acid with human CYP2D6. In the present study, the regulation mechanism of brain CYP2D by testosterone was investigated in SH-SY5Y, U251, HepG2 cell lines and rats, GHR-KO mice. Methods:Human neuroblastoma SH-SY5Y cell line, Glioma U251 cell line and HepG2 cell line were respectively treated with physiological (30 nM) and supraphysiological (300 nM) doses of testosterone. The levels of CYP2D6 mRNA and catalytic activity were detected by real-time RT-PCR and AMMC probe. The expression of brain-enriched microRNA (miRNA) was also analyzed. The CYP2D6 mRNA level was also determined after miRNAs transfection. The binding of miRNAs to CYP2D6 3’-UTR was assayed using luciferase assay. The changes in CYP2D22 and miRNAs in hippocampus and liver were detected in orchidectomized GHR-KO mice. CYP2D expression in brain and liver were respectively detected in rats treated with or without physiological dose of testosterone. Tramadol and its active metabolite M1 in plasma and cerebrospinal fluid were determined by HPLC-DAD and HPLC-MS/MS. Analgesic action of tramadol was assayed by tail flick test. Results:Testosterone does-dependently decreased CYP2D6 mRNA expression and catalytic activity in SH-SY5Y and U251 cells, but did not affect CYP2D6 mRNA expression and catalytic activity in HepG2 cells. Testosterone at a physiological dose induced miR-101, miR-124a, miR-125b and miR-128-2 in SH-SY5Y and U251 cells. The basal levels of miR-101, miR-124a, miR-125b, and miR-128-2 were significantly higher in SH-SY5Y and U251 cells than those in HepG2 cells. Only supraphysiological dose of testosterone can increase miR-125b. CYP2D22 mRNA in hippocampus was enhanced in orchidectomized GHR-KO mice compared with sham operation, but no change in hepatic CYP2D22 mRNA. Meanwhile, miR-101 and miR-128-2 in hippocampus were reduced in orchidectomized GHR-KO mice compared with sham operation, and miR-125b in liver was decreased. Overexpression of miR-101 and miR-128-2 decreased CYP2D6 mRNA levels in SH-SY5Y, U251 and HepG2 cells; however, no change in CYP2D6 mRNA levels observed following the transfection of miR-124a and miR-125b. MiR-101 and miR-128-2 inhibited the luciferase activity of CYP2D6 3-’UTR. CYP2D protein levels in frontal cortex, hippocampus, cerebellum and brain stem were decreased in rats treated with testosterone compared with the control, but no change in thalamus. Compared with the control, CYP2D1/5, CYP2D2, CYP2D3 and CYP2D4/18 mRNA levels were reduced in hippocampus from the rats treated with testosterone detected by real-time RT-PCR; half-life of tramadol in cerebrospinal fluid (CSF) was prolonged and CSF AUC0-180 of active metabolite Ml was reduced in the rats treated with testosterone, but no change in the pharmacokinetic parameters of tramadol or M1 in plasma. Analgesic action of tramadol was decreased by testosterone treatment. Analgesic effects of tramadol in tail flick test were mediated by μ-opioid receptor which can be completely antagonized by naloxone. The affinity of M1 for μ-opioid is more than 300-fold higher than that of tramadol. Thus, the decrease in CYP2D-mediated production of M1 by testosterone resulted in the reduction of tramadol-induced analgesia. Conclusion: The data suggest that testosterone organ-selectively regulates brain CYP2D via the alteration of brain-enriched miRNAs, but not hepatic CYP2D. Testosterone altered the pharmacokinetic profile of centrally active substances in the biological phase leading to the changes in pharmacodynamics. Brain CYPs can be affected by the fluctuation of androgen levels with age, hormone therapy, or sex organ operation, which may result in the changes in the pharmacological and toxic effects of centrally active substances and the risk to neurological disorders in individuals.更多还原