药学学报  2016, Vol. 51 Issue (1): 153-156   PDF    
槟榔碱对大鼠肝脏CYP2E1的体内诱导作用
黄祥涛, 肖润梅, 王明凤, 王俊俊, 陈勇     
湖北大学中药生物技术省重点实验室, 湖北大学生物资源绿色转化协同创新中心, 湖北 武汉 430062
关键词: 槟榔碱     CYP2E1     诱导作用    
Induction of rat hepatic CYP2E1 expression by arecoline in vivo
HUANG Xiang-tao, XIAO Run-mei, WANG Ming-feng, WANG Jun-jun, CHEN Yong     
Hubei Province Key Laboratory of Biotechnology of Chinese Traditional Medicine, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei University, Wuhan 430062, China
Abstract: The regulation mechanism of arecoline on rat hepatic CYP2E1 was studied in vivo. After oral administration of arecoline hydrobromide (AH; 4, 20 and 100 mg·kg-1·d-1) to rats for one week, the hepatic CYP2E1 mRNA level remained unchanged, but the hepatic CYP2E1 protein content was dose-dependently increased. Additionally, although the hepatic CYP2E1 activity was induced by AH treatment, the induction was attenuated with the increase in dosage. The results indicate that the effect of arecoline on rat hepatic CYP2El does not involve transcriptional activation of the gene, but largely involves the stabilization of CYP2E1 protein against degradation or increased efficiency of CYP2E1 mRNA translation, and additionally involve the post- translational modification of CYP2E1 protein. Furthermore, the CYP2E1 response is fairly equal among the different species, the induction of rat hepatic CYP2E1 by arecoline suggests that there is a risk of metabolic interaction among the substrate drugs of CYP2E1 in betel-quid use human.
Key words: arecoline     CYP2E1     induction    

槟榔或槟榔果实,是广泛使用的嗜好品。研究表明,槟榔咀嚼物的使用与口腔癌、肥胖、糖尿病、高血压、高脂血症、肝硬化和肝细胞癌等相关[1, 2]。槟榔碱 (图 1) 是槟榔所含的主要生物碱,对肝细胞有毒性[3, 4, 5],且巯基消耗及活性氧 (reactive oxygen species,ROS) 攻击是槟榔碱所致肝损伤的主要原因[5, 6]。此外,CYP450酶在槟榔诱导的N-亚硝胺诱变激活中也扮演了关键角色[7]

Figure 1 The chemical structure of arecoline

肝脏CYP2E1过表达或活性异常增高,是肝脏ROS增生的重要原因。由ROS引发的氧化应激是各种肝病常见的病理生理机制[8]。作者曾研究了槟榔碱灌胃给药一周对大鼠肝脏氧化应激及几种重要CYP450酶活性的影响,结果提示槟榔碱对大鼠肝脏CYP2E1活性的诱导作用,可能是槟榔碱诱导大鼠肝脏氧化应激损伤的重要机制[9]。本文进一步研究了槟榔碱体内对大鼠肝脏CYP2E1的调控机制。

材料与方法 实验动物

雄性Wistar大鼠(6周龄,体重200~250 g) 购自湖北省实验动物研究中心 [许可证号: SCXK (鄂) 2008-0005]。

主要试剂及仪器

氢溴酸槟榔碱 (AH,美国Sigma公司); RIPA裂解液,超敏ECL化学发光试剂盒 (中国碧云天生物技术研究所); PageRuler 预染蛋白 Ladder ( Thermo Scientific,美国 ) ; 小鼠抗β-actin 单克隆抗体 (Santa Cruz,美国); 兔抗大鼠CYP2E1抗体 (上海Abcam公司); HRP-山羊抗兔IgG、HRP-山羊抗小鼠IgG (KPL公司,美国); TRIzol Reagent (Invitrogen,美国); PCR引物 (上海桑尼生物); ReverTra Ace qPCR逆转录试剂盒、THUNDERBIRDTM SYBR qPCR Mix (Toyobo Co.,Ltd.,日本); 氯唑沙宗,6-羟基氯唑沙宗 (TRC,加拿大)。BCA蛋白测定试剂盒 (Rockford,美国)。

组织匀浆器 (IKA,德国); 核酸蛋白测定仪 (Eppendorf公司,德国); CFX ConnectTM实时荧光定量PCR仪(Bio-Rad,美国); DYY-6C型电泳仪、DYC-40A型垂直电泳槽及DYC-40A型电转仪 (北京市六一仪器厂)。

实验动物分组与给药方法

雄性Wistar大鼠饲养于SPF级动物房 (温度在20~25 ℃,相对湿度在40%~70%),自由取食、进水,昼夜交替时间为12/ 12 h。给药前禁食12 h,并随机分为低、中、高剂量 (4、20和100 mg·kg-1·d-1) AH(溶于生理盐水) 给药组和对照组 (灌胃等体积生理盐水),每组6只。每天于同一时间点灌胃给药 (灌胃体积小于2.0 mL),连续给药7天。末次给药后1 h,处死大鼠并取肝组织,迅速用4 ℃生理盐水冲洗、擦干后,保存于液氮中备用。

定量PCR检测CYP2E1 mRNA的表达

每只 大鼠取肝组织约100 mg,按TRIzol试剂盒说明书提取总RNA。用0.7% 琼脂糖凝胶电泳检测总RNA的完整性,用分光光度法检测260和280 nm处吸收度值检测总RNA的纯度及浓度。取总RNA 2 μg按逆转录试剂盒说明书逆转录成cDNA。cDNA再经实时荧光定量PCR扩增检测mRNA的表达量。定量PCR的引物序列如下: 大鼠GAPDH (GenBank No.: NC_ 005103.4): Forward Primer AGGGCTGCCTTCTCTT GTGAC,Reverse Primer TGGGTAGAATCATACTG GAACATGTAG; 大鼠CYP2E1 (GenBank No.: XM_ 006230520.1): Forward Primer CCTACATGGATGCT GTGGTG,Reverse Primer CTGGAAACTCATGGCT GTCA。定量PCR实验条件为: 94 ℃变性15 s,58.4 ℃退火30 s,72 ℃延伸30 s,40个循环。基因表达水平的比较采用2-ΔΔCt法计算[10]

Western blotting 检测 CYP2E1 蛋白表达量

取适量肝组织在RIPA裂解液中冰浴匀浆,离心取上清液,用BCA蛋白测定试剂盒检测蛋白含量。取蛋白样品 (60 µg) 经SDS-PAGE (5% 的浓缩胶-10% 分离胶) 分离后转移到PVDF膜上。膜用5% 脱脂奶粉封闭1.5 h后,于4 ℃与一抗 (小鼠抗β-actin单克隆抗体,1∶1 000稀释; 兔抗大鼠CYP2E1抗体,1∶1 000稀释) 温孵过夜,TBST洗涤后再于37 ℃与二抗 (HRP-山羊抗小鼠IgG ,1∶5 000稀释; HRP-山羊抗兔IgG ,1∶5 000稀释) 温孵1.5 h,TBST洗涤后用超敏ECL化学发光试剂盒分析蛋白的表达量。

HPLC 测定 CYP2E1 活性

采用CaCl2沉淀低速离心法分别制备每只大鼠的肝微粒体 (RLM),并用BCA法测定RLM中蛋白含量。以氯唑沙宗的6-羟基化作用表示RLM中CYP2E1的活性 (即ng·min-1·mg-1 protein)。RLM温孵反应体系及代谢物6-羟基氯唑沙宗含量测定方法参考文献 [8] 。

统计学方法

采用t检验,以P < 0.05为具有统计学意义。

结果 1 槟榔碱对大鼠肝脏 CYP2E1 mRNA 表达的影响

连续给大鼠灌胃低、中、高剂量氢溴酸槟榔碱 7天后,经实时荧光定量PCR检测大鼠肝脏CYP2E1 的mRNA表达量见图 2。结果表明,给药后CYP2E1 mRNA的表达量有一定上调,但与对照组相比无显著性差异 (P > 0.05),且无剂量依赖性。

Figure 2 Effect of arecoline on hepatic CYP2E1 mRNA expression after oral administration of arecoline hydrobromide (AH; 4, 20 and 100 mg·kg−1·d−1) to rats for one week. n = 6, ± s
2 槟榔碱对大鼠肝脏 CYP2E1 蛋白表达的影响

连续给大鼠灌胃低、中、高剂量氢溴酸槟榔碱7天后,Western blotting检测大鼠肝脏CYP2E1的蛋白表达量见图 3。结果表明,槟榔碱对大鼠肝脏CYP2E1的蛋白表达量有明显上调作用 (P < 0.05),且具有剂量依赖性。

Figure 3 Effect of arecoline on hepatic CYP2E1 protein content after oral administration of AH (4, 20 and 100 mg·kg−1·d−1) to rats for one week
3 槟榔碱对大鼠肝脏 CYP2E1 活性的影响

给大鼠连续灌胃低、中、高剂量氢溴酸槟榔碱7天后,对照组及低、中、高剂量给药组大鼠RLM中CYP2E1活性分别为 (16 ± 1.7)、(26 ± 2.6)、(19 ± 2.4) 和 (17 ± 1.7) ng·min-1·mg-1蛋白。与对照组相比,低剂量组有极显著性差异 (P < 0.01),中剂量组有显著性差异 (P < 0.05),高剂量组无显著性差异 (P > 0.05)。结果表明,低剂量槟榔碱对大鼠肝脏CYP2E1活性具有较显著诱导作用,且随着给药剂量的增大,诱导作用反而减弱。

讨论

在众多负责外源化合物代谢活化的CYP450酶中,对肝脏CYP2El的诱导 (如四氯化碳、对乙酰氨基酚等) 因其能激活一些重要的肝毒素而显得特别重要。研究表明,肝脏中CYP2E1的mRNA与其蛋白和酶活间没有明显相关性,如其蛋白表达占CYP总量的7%,但mRNA超过CYP总量的50%[11]。 CYP2E1的mRNA分布有两种形式,约30%不参与翻译蛋白,约70% 可活化翻译蛋白[12]。已知CYP2E1的诱导因素包括异烟肼、乙醇、丙酮、苯、二甲亚砜以及饥饿和糖尿病[13],它们能封闭一个对CYP2E1蛋白降解很关键的磷酸化位点,从而提高CYP2E1的蛋白含 量[14, 15]。CYP2E1的磷酸化状态也影响其活性,一方面磷酸化调节CYP2E1的蛋白酶体降解途径,进而影响其活性; 另一方面,磷酸化也可以在不影响其蛋白表达水平的同时,降低其酶活性[16, 17, 18]。与其他大多 数CYP450酶不同,底物对CYP2E1的诱导一般不涉及转录激活[19],而主要是稳定其蛋白或提高翻译效率[20, 21]。本文研究表明,槟榔碱对大鼠肝脏CYP2E1 mRNA的表达无明显影响,但对大鼠肝脏CYP2E1的蛋白含量及活性却有较显著的诱导作用,说明槟榔碱体内对大鼠肝脏CYP2E1的调控不在其mRNA的转录阶段,而主要是通过增加其基因的翻译效率或蛋白的稳定性,这与文献[20, 21]报道的结果基本一致。本研究还发现槟榔碱对CYP2E1蛋白含量及活性的诱导作用存在相反的剂量关系,即蛋白含量随槟榔碱的给药剂量增加而增加,而活性虽被诱导,但却随着给药剂量的增加而减弱,提示槟榔碱对CYP2E1的调控可能还存在翻译后修饰。

此外,研究表明外源化合物对大鼠、小鼠和人肝脏CYP2E1表达与活性的调控机制基本一致[22]。本文研究表明,低剂量 (4 mg·kg-1·d-1) 氢溴酸槟榔碱是大鼠肝脏CYP2E1活性的诱导剂,提示槟榔咀嚼嗜好者在接受CYP2E1底物药物治疗时,存在一定的代谢相互作用风险。

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