药学学报  2017, Vol. 52 Issue (1): 44-50   PDF    
佐米曲坦诱导雄性大鼠肝CYP3A2的作用机制
韩坤, 陆思洁, 曾苏, 余露山     
浙江大学药学院药物代谢和药物分析研究所, 浙江省抗肿瘤药物研究重点实验室, 浙江 杭州 310058
摘要: 作者前期研究发现佐米曲坦(ZOL)对雄性大鼠肝中CYP3A2具有诱导作用,而对雌性大鼠没有诱导作用。阐明其机制对于研究ZOL的药物相互作用和个体化用药具有积极意义。由于生长激素(GH)与一些蛋白的性别差异性表达密切相关,因此,本研究探究了ZOL对大鼠GH分泌的影响,以及其对脑垂体化学损伤大鼠模型肝中CYP3A2的诱导作用。结果发现,ZOL可以抑制正常大鼠体内血浆GH水平。与正常组大鼠的诱导结果不同,ZOL抑制了损伤组雄性大鼠肝中CYP3A2的表达。另外,ZOL对于雌雄原代肝细胞中的CYP3A1/2无明显诱导作用。进一步研究发现,给予ZOL后正常雄性大鼠肝中肝细胞核因子4α(HNF4α)的入核水平明显增加,而正常雌性大鼠、脑垂体化学损伤大鼠和大鼠原代肝细胞中HNF4α的入核水平无明显变化。结果表明,GH和HNF4α在ZOL性别差异性诱导CYP3A2的过程中发挥了重要的作用。
关键词: 佐米曲坦     CYP3A2     性别选择性诱导     生长激素     肝细胞核因子4α    
Induction study of CYP3A2 in male rats by zolmitriptan
HAN Kun, LU Si-jie, ZENG Su, YU Lu-shan     
Institute of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
Abstract: In our preliminary studies, we observed zolmitriptan (ZOL) treatment led to induction of CYP3A2 in male not female rats. To figure out the reason is of great significance for drug-drug interactions and personalized administration. Since growth hormone (GH) is known as the major mechanistic determinant of sexually-dimorphic gene expression like CYP3A2 in rat liver, the impacts of ZOL on both plasma GH levels in non monosodium glutamate (MSG)-treated rats and CYP3A2 expression in GH depleted MSG-treated rats were studied. ZOL was shown to partially suppress GH levels in both genders. Furthermore, CYP3A2 protein and mRNA level declined in male not female MSG-treated rats. In order to study the possible molecular events involved in the depression of GH and gender-selective induction on rat CYP3A2 by ZOL, the mRNA and protein level (whole protein and nuclear protein) of hepatocyte nuclear factor 4α (HNF4α) was investigated. Nuclear accumulation of HNF4α was observed in the normal male not female rat liver tissue following ZOL treatment. However, this kind of nuclear translocation did not occur in rat hepatocytes and MSG-treated rats. These findings demonstrated CYP3A2 inducibility by ZOL was gender-selective. GH and HNF4α may play an important role in CYP3A2 induction.
Key words: zolmitriptan     CYP3A2     gender-dependent induction     growth hormone     hepatocyte nuclear factor 4α    

佐米曲坦 (zolmitriptan, ZOL) 是一种高选择性5-HTIB/ID受体激动剂, 临床上用于偏头痛的治疗[1, 2]。体内研究发现氟西汀、普萘洛尔[3]、地西泮[4, 5]、甲氧氯普胺、西咪替丁等都会导致佐米曲坦药物动力学的改变。因此, 若能获得更多的药物对细胞色素P450酶 (cytochrome P450, CYP450) 的诱导或抑制作用信息, 则可预测其在体内发生相互作用的可能性, 对临床安全、合理、有效用药, 避免不良反应的发生具有重要的指导意义。

实验室前期研究发现: 佐米曲坦能性别选择性的诱导雄性大鼠肝中CYP3A2, 而对雌性大鼠肝CYP3A没有明显诱导作用[6, 7]。生长激素 (growth hormone, GH)、肝细胞核因子 (hepatocyte nuclear factors, HNFs) 等均被报道和性别差异诱导相关。GH分泌过程受性腺激素的调节, 使得动物和人血浆中GH的浓度存在明显的性别差异[8]。大鼠肝脏中已知含有十几种性别相关的P450亚型, 雄性大鼠肝细胞含有CYP2A2、2C11和3A2等, 雌性大鼠肝细胞表达CYP2C12和CYP2C7等[9, 10]。GH在雄性大鼠中以脉冲式分泌, 在雌性大鼠中则不规则持续性分泌。据报道, GH是大鼠中许多性别差异性表达的CYP450的关键调控子[11-15], 而口服佐米曲坦能提高人血浆中GH的浓度[16], 这对进一步研究导致ZOL诱导大鼠CYP3A2具有性别差异的机制提供了思路。HNFs包括HNF1α、HNF4α、HNF3和HNF6等。许多肝CYP基因能被HNF4α活化[17, 18], HNF4α对人肝中孕烷X受体 (pregnane X receptor, PXR) 和组成型雄甾烷受体 (constitutive androstane receptor, CAR) 核受体调节CYP3A4的表达也具有重要作用。HNF4α对部分肝基因包括CYP基因的转录调节作用存在明显的性别差异, 能正向调节某些雄性动物中的特异性CYPs, 并且抑制雌性动物中某些特异性CYPs[19]

因此, 本文采用放射免疫 (RIA) 方法检测经佐米曲坦诱导后雌雄大鼠体内GH的变化, 同时采用2个GH缺陷模型即脑垂体损伤大鼠和原代大鼠肝细胞重复佐米曲坦的诱导实验, 从而观察当GH不存在的情况下佐米曲坦对CYP3A的性别诱导现象是否仍旧存在, 并用荧光定量PCR和Western blot方法观察佐米曲坦对HNF4α的mRNA、蛋白及入核水平的影响, 以探索佐米曲坦性别差异诱导肝CYP3A的分子机制。性别差异诱导机制的研究对预测活性化合物或药物候选物药代动力学性别差异以及个体化给药具有十分重要的意义。

材料与方法

试剂与仪器 Mastercycler ep Realplex荧光定量PCR仪 (Eppendorf), 核酸蛋白浓度测定仪Gene Quant pro (Amersham Biosciences), FDG-101伽马射线流式探头 (日本Aloka公司)。佐米曲坦 (ZOL, 浙江大学理学院化学系), 地塞米松 (DEX, Sigma), 谷氨酸单钠盐 (MSG, 康杰生物科技有限公司), Trizol试剂盒 (生工生物工程股份有限公司), M-MLV逆转录酶 (Fermentas), 荧光定量PCR试剂盒 (天根生化科技), 生长激素放射免疫试剂盒 (Millipore Linco), CYP3A1兔多克隆抗体 (Abcam), CYP3A2绵羊多克隆抗体 (Abcam), β-actin兔多克隆抗体 (Santa Cruz), HNF4α山羊多克隆抗体 (Santa Cruz)。

佐米曲坦对大鼠的诱导作用 Sprague-Dawley大鼠, 动物合格证号为: SCXK (沪) 2006-0016, 雌雄各半, 体重190 ± 10 g, 从中国科学院上海实验动物中心引进。雌雄动物按体重各均匀分为5组, 分别为空白, 佐米曲坦低剂量、中剂量、高剂量组及阳性对照组。每组共3批, 每批3或4只。低、中、高剂量组灌胃给药佐米曲坦分别为0.5、2.5和12.5 mg·kg-1, 3天; 空白组灌胃生理盐水, 3天; 阳性对照组地塞米松100 mg·kg-1, 3天。

微粒体制备 动物末次给药24 h后, 将动物断头处死, 剖腹, 用经冰浴冷却的生理盐水灌流, 除去肝中血液。取出肝脏 (以下均在4 ℃以下操作), 用生理盐水洗净, 滤纸吸干外部水分。取部分肝脏制备肝微粒体, 于-80 ℃下保存备用, 以Lowry法测定微粒体中的蛋白质浓度[20]。微粒体由Western blot检测。剩余的肝脏用于Real-time PCR和Western blot全蛋白及核蛋白的分析, 存储于-80 ℃。

放射免疫法测定血浆GH浓度 正常组大鼠经佐米曲坦诱导3天后, 尾静脉取血。每隔15 min取血100 μL, 共取5 h, 3000 r·min-1离心10 min后, 取上清液。血浆样品处理方法参照Linco生长激素RIA试剂盒的说明。

大鼠原代肝细胞培养及佐米曲坦的诱导处理 采用两步灌注消化法分离大鼠肝细胞[21]。将活力在85% 以上的肝细胞以1.25×105/cm2的密度接种于先铺有0.2% 明胶层的孔板上, 培养液为含10% 胎牛血清的RPMI 1640。新鲜分离的肝细胞贴壁后进行佐米曲坦的诱导实验, 含10 μmol·L-1地塞米松 (阳性对照组) 或含10、50和100 μmol·L-1 3个浓度佐米曲坦的培养液 (样品组), 分别替换六孔板中的培养液, 地塞米松和佐米曲坦均溶解于DMSO中, DMSO在培养液中的终浓度低于0.1%, 空白对照组在培养液中加入等量的DMSO。每组平行3孔, 培养3批原代肝细胞进行同样实验。

脑垂体损伤大鼠造模及佐米曲坦的诱导处理 脑垂体损伤大鼠造模方法如下[22]: 新生大鼠在出生24 h内皮下第一次注射谷氨酸单钠盐 (MSG , 4 mg·g-1体重), 此后每隔一天注射一次, 连续注射5次。新生大鼠与母鼠一起饲养。一个月后, 雌雄分开饲养。到3个月, 开始实验。造模大鼠与正常组大鼠相比主要差别在于脑垂体损伤大鼠血浆中检测不到GH。动物分组与佐米曲坦的给药处理同正常组大鼠, 见“佐米曲坦对大鼠的诱导作用”。

微粒体代谢实验和原代肝细胞代谢实验 取空白、佐米曲坦低中高剂量诱导、地塞米松诱导阳性对照的雌性或雄性鼠肝微粒体适量 (终质量浓度1.0 mg·mL-1), 分别加探针底物睾酮, 使其终浓度为30 μmol·L-1。37 ℃预孵育5 min, 加烟酰胺腺嘌呤二核苷酸磷酸 (NADP, 终浓度为0.25 mmol·L-1) 与还原型辅酶Ⅱ (NADPH, 终浓度为0.1 mmol·L-1) 启动反应, 再孵育一定时间后, 终止反应。按照“大鼠原代肝细胞培养及佐米曲坦的诱导处理”得到空白、佐米曲坦低中高剂量诱导、地塞米松诱导阳性对照的雌性或雄性鼠原代肝细胞, 进行同样实验。采用HPLC法[7]测定剩余底物浓度, 所得结果与空白对照组比较, 分析是否有显著性差异。

荧光定量PCR法测定CYP3A1CYP3A2HNF4α的mRNA表达 按Trizol一步法提取大鼠肝组织 (正常组大鼠和损伤组大鼠) 和原代肝细胞的总RNA, A260/A280为1.8~2.0。逆转录操作按照试剂盒说明书进行, 样品保存在-80 ℃。PCR反应体系参照荧光定量试剂盒说明, 实验结果采用2-ΔΔCt相对定量法。引物序列见表 1

Table 1 Primer sequences for amplification of specific rat genes

Western blot法测定CYP3A1、CYP3A2和HNF4α蛋白表达 大鼠原代肝细胞和肝组织 (正常组和损伤组) 均按照Abcam说明书, 用RIPA裂解液裂解获得总蛋白样本。核蛋白提取按照说明书操作。样品保存在-80 ℃。SDS-PAGE浓缩胶为5%, 分离胶为10%。电泳完成后, 将蛋白转至PVDF膜, 转膜条件为200 mA, 1 h (HNF4α为3 h)。CYP3A1和HNF4α封闭条件为含5% 脱脂牛奶的PBST (含0.1% Tween 20的磷酸缓冲液) 室温封闭1 h, CYP3A2封闭条件为含5% 脱脂牛奶的PBST 4 ℃封闭过夜。一抗条件为: 兔抗CYP3A1 (1:4 000) 和山羊抗HNF4α (1:100) 4 ℃过夜, 绵羊抗CYP3A2 (1:1 000) 和兔抗β-actin (1:3 000) 室温1 h。二抗条件: HRP标记的山羊抗兔 (1:3 000) , 兔抗绵羊 (1:2 000) 和兔抗山羊 (1:3 000) IgG室温孵1 h。最后蛋白用ECL显影, 胶片曝光后用扫描仪扫描。

数据处理 以上数据均用平均值及SD表示, 个体差异用Dunn’s检验表示, 非参数检验采用Kruskal-Wallis检验。所有计算均采用SPSS软件。

结果 1 佐米曲坦对原代肝细胞CYP3A1/2的酶活性、蛋白和mRNA水平均无显著影响

图 1表 2可见, 经佐米曲坦诱导后雄性和雌性大鼠肝细胞中CYP3A1和CYP3A2蛋白表达水平、酶活性水平和mRNA水平都没有明显升高。说明体外诱导实验中, 佐米曲坦对肝细胞CYP3A1/2并没有产生体内给药时所表现的性别差异诱导作用。

Figure 1 Hepatocytes were induced by media supplemented with zolmitriptan (ZOL, 10, 50 and 100 μmol·L-1), dexmethasone (DEX, 10 μmol·L-1) or control (DMSO) for 48 h after plating. (A) ZOL induction of CYP3A1 and 3A2 proteins in rat hepato-cytes (Western blot analysis). (B-C) ZOL induction of CYP3A activities (reduced amount of testosterone, a specific probe substrate of CYP3A), in male (B) and female (C) rat hepatocytes. Testosterone relative values are means ± SD; n ≥ 3. CYP3A proteins were monitored by Western blot in at least three animals/ group. **P < 0.01 vs Control

Table 2 Effects of ZOL on the expression of CYP3A in the liver of non-MSG (monosodium glutamate) or MSG-pretreated rats and in hepatocytes. CYP3A2 gene in rats treated with dexamethasone (DEX) is detectable in light of Ct and the melting curve. However, CYP3A2 gene in female control rats can’t be detected. Thus, we can’t calculate the normalized gene expression level. Gene expression relative level values are means ± SD; n ≥ 3. *P < 0.05, **P < 0.01 vs control group. ND: Not detected; NC: Not be calculated
2 佐米曲坦抑制正常组大鼠生长激素的分泌

图 2所示, 给药组、对照组雌雄大鼠的GH分泌模式和文献报道相符。雄性大鼠生长激素呈“开-关”式脉冲分泌, 在脉冲中间间隔时大鼠体内血液中几乎测不到GH。而雌性大鼠分泌GH呈“持续性分泌”, 虽有一定的脉动, 但是很微弱。从分泌模式上来看, 佐米曲坦并未改变GH的分泌形式。

Figure 2 Plasma levels of circulating growth hormone (GH) in non MSG-treated rats exposing to ZOL. (A: Male rats; B: Female rats). Plasma was obtained at 15-min intervals for 5 continuous hr. Rats were treated by ig with saline (control) or ZOL (0.5, 2.5 and 12.5 mg·kg-1, respectively) dissolved in saline for 3 consecutive days. GH profiles are representative of two collections from at least three rats/group

结果表明, 佐米曲坦显著抑制了血浆生长激素的峰值。给予低 (0.5 mg·kg-1)、中 (2.5 mg·kg-1)、高 (12.5 mg·kg-1) 剂量的佐米曲坦, 雄性大鼠的平均血浆GH浓度分别降低为原来的80%、30% 和10%。雌性大鼠的血浆GH峰值同样也显著降低, 为对照组的92%、60% 和34%。结果说明, 佐米曲坦显著抑制了正常组雌雄大鼠的血浆GH分泌。

3 佐米曲坦抑制损伤组雄性大鼠肝脏CYP3A酶活性、CYP3A2蛋白和mRNA表达

放射免疫法检测损伤组大鼠造模是否成功。结果显示, 损伤组大鼠GH皆在检测限以下。有趣的是, 佐米曲坦对损伤组大鼠CYP3A2的作用与正常组大鼠结果恰好相反。如图 3表 2, 高中低剂量的佐米曲坦显著抑制CYP3A2蛋白和mRNA表达水平。和正常组大鼠类似, 雌性大鼠CYP3A2的蛋白和mRNA也低于检测水平。酶活性结果与CYP3A2蛋白和mRNA基本一致, 雄性大鼠CYP3A酶活性被佐米曲坦抑制但雌性大鼠CYP3A酶活性基本不受影响。

Figure 3 MSG-treated rats were treated by ig with saline or ZOL (0.5, 2.5 and 12.5 mg·kg-1) or DEX (100 mg·kg-1) dis-solved in saline for 3 consecutive days. (A) ZOL induction of CYP3A1 and 3A2 proteins in MSG-treated rats (4 mg·g-1, neo-natally, Western blot analysis). (B-C) ZOL induction of testos-terone (reduced amount of testosterone, a specific probe substrate of CYP3A), in male (B) and female (C) MSG-treated rats (4 mg·g-1, neonatally). Testosterone relative values are means ± SD; n ≥ 3. CYP3A proteins were monitored by Western blot in at least three animals/group. *P < 0.05, **P < 0.01 vs Control
4 佐米曲坦对正常组大鼠, 原代培养大鼠肝细胞及损伤组大鼠HNF4α蛋白和mRNA水平的影响

为探究HNF4α是否参与佐米曲坦诱导雄性大鼠CYP3A2, 分别观察了正常组、损伤组和肝细胞中HNF4α的蛋白和mRNA的水平变化, 由于HNF4α入核蛋白水平的变化更直接、更准确地反映佐米曲坦可能引起的HNF4α介导下的CYP3A活性变化, 还观察了3个模型中HNF4α的蛋白入核水平变化。

在蛋白水平上, 由图 4可知, 佐米曲坦可以诱导正常组雌雄大鼠HNF4α蛋白, 但雄性的诱导倍数大于雌性, 且佐米曲坦对正常组雌雄大鼠HNF4α入核蛋白水平的影响呈性别选择性, 即对雄性有诱导但对雌性无明显诱导 (图 4A4B)。佐米曲坦对大鼠原代肝细胞HNF4α蛋白及入核蛋白水平均无显著影响 (图 4C4D)。佐米曲坦诱导损伤组雄性大鼠HNF4α蛋白但对雌性大鼠无显著影响, 核蛋白结果与总蛋白结果不同, 佐米曲坦对损伤组雌雄大鼠核蛋白中的HNF4α均无显著影响 (图 4E4F)。

Figure 4 Effects of ZOL on the expression of HNF4α whole cell proteins and nuclear proteins (Western blot), in non MSG-treated rats (A: Tissue lysate; B: Nuclear protein), rat hepatocytes (C: Cell lysate; D: Nuclear protein), and MSG-treated rats (E: Tissue lysate; F: Nuclear protein). Rats were treated by ig with saline or ZOL (0.5, 2.5 and 12.5 mg·kg-1) suspended in saline for 3 consecutive days. Hepatocytes were induced by media supplemented with ZOL (10, 50 and 100 μmol·L-1) or control (0.1% of DMSO) for 48 h after plating. HNF4α proteins were monitored by Western blot in at least three animals/group

在mRNA水平上, 如表 3所示, 佐米曲坦对正常组大鼠HNF4α有调控作用, 且这种调控作用具有性别差异, 即对雄性有诱导但对雌性无明显诱导。佐米曲坦对于原代培养肝细胞和损伤组大鼠的HNF4α mRNA有微弱诱导作用, 且这种诱导作用不呈雌雄差异。从整体趋势上看, HNF4α的mRNA实验结果和Western blot结果保持一致。

Table 3 Effects of ZOL on the expression of HNF4α in the liver of non-MSG or MSG-pretreated rats and in hepatocytes. Gene expression relative level values are means ± SD; n ≥ 3. *P < 0.05, **P < 0.01 vs Control
讨论

在原代培养大鼠肝细胞实验中, 佐米曲坦对肝细胞中CYP3A1/2并没有产生类似体内给药时所表现的性别差异诱导作用。原代培养大鼠肝细胞与正常组大鼠最大的区别在于大鼠肝细胞不受激素的调控, 因此推测这种性别特异性的产生是与体内各种激素调节相关的。在大鼠肝模型中, GH是性别二态性基因如CYP3A2的主要决定因素[23-27]。而且, 佐米曲坦相对于其他曲坦类药物有更好的渗透性[28], 神经内分泌的作用可能因此会更显著。因此, GH可能作为佐米曲坦对CYP3A2性别差异诱导的中间物。原代大鼠肝细胞上的实验结果支持了作者的假设。

据报道, GH峰值的高低会影响雄性大鼠CYP450的表达, 而雌性大鼠肝中CYP450对于“持续式”分泌的GH峰值变化响应不是十分敏感。对于雄性大鼠, CYP3A2在GH峰值降低时, 其活性、蛋白表达水平和mRNA水平反而上升。当GH浓度为0时, CYP3A2表达最高。当其接近生理浓度时, 表达才下降[29] 。而对于雌性大鼠来说, 只要GH分泌是持续性的, 峰值对CYP3A2影响很小, CYP3A2基本检测不到。他们的实验结果恰好可以解释实验当中两个结果的关联性。因为佐米曲坦可以抑制GH的分泌, 所以导致了佐米曲坦诱导雄性大鼠CYP3A2这个结果。因此, 佐米曲坦抑制GH的分泌可能是导致佐米曲坦诱导雄性大鼠CYP3A2的原因之一。

损伤组大鼠的实验结果表明, 佐米曲坦抑制损伤组雄性大鼠肝脏CYP3A酶活性、CYP3A2蛋白和mRNA表达。即在去除GH影响后, 佐米曲坦的诱导效应被逆转, 这说明佐米曲坦对CYP3A2的影响与GH存在一定关联。但是, 该结果提示GH不是佐米曲坦诱导正常组大鼠肝CYP3A2的唯一靶点。可能同时存在抑制途径, 只是诱导的效应强于抑制效应, 因此在正常大鼠中, 综合效应仍然显示为诱导作用。根据文献报道, 众多的五羟色胺再摄取抑制剂 (SSRIs) 因为有五羟色胺能的激活作用, 所以可以抑制CYP450s的表达[30-32], 而佐米曲坦是5-HT1B/1D的激动剂, 它的药理作用类似于SSRIs。因此认为佐米曲坦对损伤组大鼠的抑制作用可能是通过类似的机制引起。但是这种抑制作用在正常组大鼠中小于诱导作用。

HNF4α是肝中含量丰富的核受体, 对调节很多肝内基因, 包括许多CYP450家族成员有十分重要的作用, 其中还有的显示出性别依赖性[33-36]。而且据报道, HNF4α是由GH脉冲控制的[37]。综合上述结果, 佐米曲坦性别差异诱导正常组大鼠HNF4α的mRNA、总蛋白、核蛋白水平, 但对原代肝细胞和脑垂体损伤组大鼠这两个不受GH影响的模型并无明显影响。通常认为核受体和转录因子入核水平的变化和它们的功能存在关联。因此认为HNF4α可能介导了佐米曲坦性别差异诱导CYP3A2这一过程。

本文实验结果显示, 佐米曲坦可以抑制正常大鼠体内血浆GH水平。与正常组的诱导结果不同的是, 佐米曲坦不能诱导, 相反的是抑制了损伤组雄性大鼠肝中的CYP3A2。另外, 佐米曲坦对于雌雄原代肝细胞中的CYP3A1/2无明显诱导作用。因此, 认为GH可能介导了佐米曲坦诱导雄性大鼠肝中的CYP3A2。此外, 佐米曲坦性别差异诱导正常组大鼠肝HNF4α的入核水平, 但对于GH被化学敲除的损伤组大鼠, 这种性别差异诱导作用消失。这个结果说明HNF4α可能参与了佐米曲坦性别差异诱导CYP3A2, 且它的作用模式也和GH有关系。综上所述, 佐米曲坦可以性别选择性诱导雄性大鼠肝CYP3A2, GH和HNF4α可能参与了这个性别选择性诱导过程。值得注意的是, HNF4α发挥作用的分子机制以及这种性别选择性诱导过程对人体是否也有效仍需要更多研究, 如HNF4α基因敲除实验以及EMSA或免疫沉淀实验的进一步确证。

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