盐酸川芎嗪对小鼠肝细胞色素P酶活性及表达调控的影响

王文华¹^,², 张泽萍¹^,², 杨洋¹^,², 李勇军¹^,², 龚菲¹^,³, 李黎¹^,², 何彬¹^,³, 孙佳¹^,², 刘亭¹^,²

贵州医科大学1.药学院

2.贵州省药物制剂重点实验室

3.民族药与中药开发应用教育部工程研究中心,贵阳 550004

WANG Wenhua¹^,², ZHANG Zeping¹^,², YANG Yang¹^,², LI Yongjun¹^,², GONG Fei¹^,³, LI Li¹^,², HE Bin¹^,³, SUN Jia¹^,², LIU Ting¹^,²

1. School of Pharmacy

2.Guizhou Provincial Key Laboratory of Pharmaceutics

3.Engineering Research Center for the Development and Application of Ethnic Medicine and Traditional Chinese Medicine, Ministry of Education, Guizhou Medical University,Guiyang 550004, China

通信作者刘亭(1981-),贵州遵义人,男,副教授,博士,从事中药药理研究。电话:0851-86908468,E-mail: t-liu@163.com。

作者简介王文华(1989-),内蒙古人,女,在读硕士,从事中药药理研究。电话:0851-86908468,E-mail: 1026512104@qq.com。

基金资助: 国家自然科学基金资助项目(81603189)

中图分类号: R285.5;R543 文献标识码: A 文章编号: 1004-0781(2017)04-0390-06

摘要:

目的探讨盐酸川芎嗪对小鼠肝细胞色素P₄₅₀酶(CYPs)主要亚型及相关核受体的影响。方法将小鼠分为5组:空白对照组、苯巴比妥组、盐酸川芎嗪小剂量(13.0 mg·kg^-1·d^-1)组、盐酸川芎嗪中剂量(19.5 mg·kg^-1·d^-1)组、盐酸川芎嗪大剂量(26.0 mg·kg^-1·d^-1)组,连续7 d给药。取5组小鼠的肝脏组织,制备肝微粒体、提取RNA,检测5组小鼠CYP活性、mRNA水平和蛋白表达的变化。结果与空白对照组比较,盐酸川芎嗪大剂量组CYP1A2酶活性、 mRNA水平和蛋白表达分别上调1.43,1.44和1.40倍(P<0.05)。盐酸川芎嗪组CYP2E1、CYP2D22、CYP3A的酶活性、mRNA水平与空白对照组比较,均差异无统计学意义。与空白对照组比较,盐酸川芎嗪大剂量组AhR mRNA水平上调1.6倍(P<0.05)。盐酸川芎嗪对HNF-4α、PXR和PPARα的mRNA水平无显著影响。随着盐酸川芎嗪的剂量增高,CYP2E1蛋白表达有上升趋势,但无显著性影响。结论盐酸川芎嗪对小鼠体内肝CYP2E1、CYP2D22、CYP3A、HNF-4α、PXR和PPARα无显著影响,但对CYP1A2活性有诱导作用,该作用很可能与盐酸川芎嗪上调AhR水平从而促进CYP1A2表达有关。

关键词: 川芎嗪,盐酸 ; 细胞色素 P450酶 ; 细胞色素P450 mRNA表达

Abstract:

Objective To investigate the influence of ligustrazine hydrochloride on cytochrome P₄₅₀ and nuclear receptor in hepatocyte of mice. Methods Mice were randomly divided into blank control group, phenobarbital group, and ligustrazine hydrochloride low-, medium- and high-dose groups (13.0,19.5 and 26.0 mg·kg^-1·d^-1). Then the mice were sacrificed after were administered medicines once daily for consecutive 7 days. Liver microsomes were prepared to determine the enzyme activities. Quantitative real-time PCR and Western blotting was employed to examine the expression of these four CYP450 enzymes and nuclear receptor in liver tissue of mice. Results Compared with the blank control group, CYP1A2 activity, mRNA and protein expression were increased by 1.43,1.44 and 1.40 times (P<0.05) respectively in the ligustrazine hydrochloride dose group.Ligustrazine hydrochloride was found to have no impact on the activities of CYP2E1, CYP3A and CYP2D22.AhR mRNA was increased by 1.6 times (P<0.05) in the ligustrazine hydrochloride dose group.Ligustrazine hydrochloride was found to have no impact on the expression of HNF-4α,PXR and PPARα. Conclusion Ligustrazine hydrochloride is found to have no impact on the expression of CYP2E1,CYP2D22,CYP3A,HNF-4α,PXR and PPARα,but induces the activity of CYP1A2. This effect is likely to be related toin creasing AhR level to promote the expression of CYP1A2.

Key words: Ligustrazine,hydrochloride ; Cytochrome P450 enzymes ; Cytochrome P450 mRNA expression

中药成分复杂,具有较多的化学成分,不同配伍组成的复方机制也较多^[1]。中药的多种成分作用于机体形成复杂的相互作用体系,而复方整体效应的重要基础正是这些复杂的相互作用。产生相互作用的原因是中药的有效成分进入体内要经过CYP酶进行代谢,并有可能导致酶活性被诱导或抑制,从而影响其他物质的代谢,引起药物之间的相互作用^[2]。参芎葡萄糖注射液主要由丹参和盐酸川芎嗪组成,盐酸川芎嗪有扩张血管、改善组织微循环、抑制血小板粘附等作用^[3]。临床常用于治疗缺血性心脑血管疾病和缺血性肢体血管疾病^[4]。本课题组前期研究了参芎葡萄糖注射液及其组分丹参对小鼠CYP同工酶的影响。笔者在本研究中继续考察参芎葡萄糖注射液中的另一种主要成分盐酸川芎嗪对细胞色素P₄₅₀同工酶的影响,进而完善参芎葡萄糖注射液的代谢性相互作用的研究。

1 材料与方法

1.1 实验动物

清洁级昆明雄性小鼠,体质量22~25 g,由贵州医科大学实验动物房提供,动物生产许可证号:SCXK(黔)2012-0001。饲养条件:室温(22±1)℃,湿度:58%,昼夜规律饲养,标准饮食、饮水。

1.2 材料和试剂

盐酸川芎嗪(北京索莱宝科技有限公司,批号:YC-0720131020);苯巴比妥(北京索莱宝科技有限公司,批号:20150508);Eastep总RNA提取试剂盒(Promega公司,批号:20140801);SYBR^® Premix Ex Taq^TMⅡ(大连宝生物有限公司,批号:RR820A)、TransScript^TMOne-Step RT-PCR SuperMix(大连宝生物有限公司,批号:RR047A);CYP1A2、CYP2E1、CYP3A11、CYP2D22、肝细胞核因子4α(human liver cell nucleus factor-4α,HNF-4α)、孕甾烷X受体(pregnane X receptor,PXR)、过氧化物酶体增殖物激活受体α(proxisome proliferator activation receptors,PPARα)、芳香烃受体(aryl hydrocarbon receptor,AhR)及甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)的引物均由上海生工公司合成;氧化型辅酶Ⅱ二钠(Na₂NADP,批号:SM0313KB14)、6-磷酸葡萄糖(G-6-P,批号:WJ0717EA14)、G-6-PDH(批号:K09M6C1)均由上海海叶生物公司提供;右美沙芬(批号:A0516AS)、普萘洛尔(批号:F0315AS)、非那西丁(批号:A1116AS)均由大连美仑生物公司提供;β-actin抗体(批号:BS6007M)、CYP1A2抗体(批号:BS2188)、CYP2E1抗体(批号:BS6577)购自巴傲德生物有限公司。

1.3 仪器

CFX96实时荧光定量PCR仪(美国BIO-RAD公司),Biomate 3S核酸蛋白紫外测定仪(美国Thermo公司);Thermo fresco 17高速冷冻离心机(美国Thermo公司);Ultimate 3000 UPLC-PDA高效液相仪器(美国Thermo公司);电泳仪、转膜仪(美国BIO-RAD公司)。

1.4 小鼠肝微粒体的制备

将小鼠分为5组:空白对照组、苯巴比妥组、盐酸川芎嗪小剂量(13.0 mg·kg^-1·d^-1)组、盐酸川芎嗪中剂量(19.5 mg·kg^-1·d^-1)组、盐酸川芎嗪大剂量(26.0 mg·kg^-1·d^-1)组,连续7 d给药。5组小鼠末次给药后禁食16 h,处死,剪开腹腔取其肝脏,用装满冰冷0.9%氯化钠溶液的注射器,经肝门静脉灌洗法除去肝中残血,用冰冷的氯化钾溶液漂洗肝脏3次。把肝组织剪碎,匀浆后两层纱布过滤,将得到的组织匀浆液在10 000×g离心场力作用下离心20 min,采用氯化钙沉淀法制备肝微粒体,最后在15 000×g离心场力作用下离心20 min,弃去上清液,粉红色沉淀即为肝微粒体。用氯化钾磷酸盐缓冲液洗涤上述沉淀,在15 000×g离心场力作用下离心20 min,用20%甘油的磷酸盐缓冲液重新均匀混悬,即为微粒体混悬液。混悬液分装后-80 ℃冰箱中保存。采用BCA法测定微粒体蛋白浓度。

1.5 药物代谢酶的活性测定

1.5.1 CYP1A2酶活性的测定采用非那西丁体外代谢法来测定CYP1A2酶活性^[5]。将待测肝微粒体稀释到5 g·L^-1 ,取100 μL肝微粒体于1.5 mL离心管中,加入非那西丁(0.05 mol·L^-1)200 μL,37 ℃振荡孵育10 min。然后加入200 μL 还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)再生系统(2 mmol·L^-1 Na₂NADP,20 mmol·L^-1 G-6-P,4 U·L^-1 G-6-PDH,20 mmol·L^-1 MgCl₂),于37 ℃水浴中振荡30 min后,加入三氯乙酸200 μL终止反应。15 000×g离心20 min,取90 μL上清液加入普萘洛尔(1.07 g·L^-1)10 μL,混匀,用高效液相检测。色谱条件:上样量20 μL;Luna C₁₈色谱柱(4.6 mm×250 mm,5 μm);流动相:60%甲醇-40%水(0.04%三乙胺和0.04%乙酸乙酯),用冰醋酸调pH值至3.0;流速0.6 mL·min^-1;柱温40 ℃;检测波长280 nm。

1.5.2 CYP2D22酶活性的测定采用右美沙芬体外代谢高效液相色谱法测定,将待测肝微粒体稀释到5 g·L^-1 ,取100 μL肝微粒体于1.5 mL离心管中,加入右美沙芬(0.03 mol·L^-1)200 μL。37 ℃振荡孵育10 min后,加入NADPH再生系统200 μL,于37 ℃水浴中振荡孵育30 min,最后加入三氯乙酸200 μL终止反应。15 000×g离心20 min,取上清液90 μL加入普萘洛尔(1.07 g·L^-1)10 μL,混匀,用高效液相色谱法检测。色谱条件同“1.5.1”项^[5]。

1.5.3 CYP3A酶活性的测定采用氨基比林N-脱甲基酶ADM法,通过Nash比色法测定甲醛生成量来反映CYP3A的活性^[6]。将待测肝微粒体稀释到5 g·L^-1,取100 μL,加入磷酸盐缓冲液(PBS)0.15 mL,混匀后加入86.4 mmol·L^-1氨基比林0.05 mL,混匀,37 ℃水浴3 min后加入NADPH发生系统0.05 mL,37 ℃孵育60 min后取出,加入30%三氯醋酸0.25 mL终止反应。再加Nash试剂0.25 mL,混匀后60 ℃水浴10 min,室温放置20 min。以不含微粒体的空白体系调零,于412 nm波长处测定吸光度值(A值)。

1.5.4 CYP2E1酶活性的测定采用苯胺羟化酶ANH法,用分光光度法测定对氨基酚的生成量,从而反映CYP2E1的活性^[6]。将待测肝微粒体稀释到5 g·L^-1 ,取100 μL加入测定管,再加入苯胺溶液0.05 mL,对照管加入水0.05 mL。37 ℃水浴30 min,加入冰冷20%三氯乙酸溶液1.0 mL,冰浴5 min。取上清液1.0 mL,加入1%酚试剂1.0 mL,混匀,再加入碳酸钠1.0 mL混匀。室温下反应30 min,于630 nm波长处测定吸光度。

1.6 小鼠RNA的提取与纯化

末次给药后禁食16 h,断颈处死,剪开腹腔取其肝脏,用预冷0.9%氯化钠溶液将肝脏的血冲洗干净,然后取大约20 mg的肝脏于手动玻璃匀浆器中,并按Eastep RNA提取试剂盒说明书,提取纯化肝脏总RNA。核酸蛋白紫外测定仪检测RNA的A₂₆₀/A₂₈₀值均在1.8~2.1之间符合要求。采用1.0%琼脂糖凝胶电泳,比较28S和18S条带的灰度值。

1.7 小鼠肝脏mRNA水平测定

取cDNA 2.0 μg,按照TransScript^TMOne-Step RT-PCR SuperMix说明书进行逆转录反应。荧光定量聚合酶链反应体系20 μL包括:逆转录反应产物2.0 μL,上下游引物(10 μmol·L^-1)各0.8 μL,SYBR^® Premix Ex Taq^TMⅡ 10 μL。在CFX96荧光定量PCR仪上进行反应,PCR扩增程序为:95 ℃ 30 s,95 ℃ 5 s,60 ℃ 34 s,共40个循环,循环结束后绘制溶解曲线。每次扩增均设置GAPDH内参照,用2^(-△△Ct)方法分析数据。引物序列见表1。

基因	正向引物	反向引物
AhR	ACCAGAACTGTGAGGGTTGG	CTCCCATCGTATAGGGAGCA
HNF-4α	CGGAGCCCCTGCAAAGT	CCAGTCTCACAGCCCATTCC
PXR	CCCATCAACGTAGAGGAGGA	TCTGAAAAACCCCTTGCATC
PPARα	CCATACAGGAGAGCAGGGATTT	TTACCTACGCTCAGCCCTCTTC
CYP1A2	CATCCCCCACAGCACAACAA	TCCCACTTGGCCAGGACTTC
CYP3A11	ACAAACAAGCAGGGATGGAC	GGTAGAGGAGCACCAAGCTG
CYP2D22	CAGTGGTTGTACTAAATGGGCT	GCTAGGACTATACCTTGAGAGCG
CYP2E1	AGTGCAGAGCGCTTGTACACA	AAGAACAGGTCGGCCACAGT
GAPDH	AGTATGACTCCACTCACGGCAAAT	GTCTCGCTCCTGGAAGATGGT

表1 引物序列

Tab.1 Primer sequences

1.8 小鼠肝脏CYP蛋白表达的测定

小鼠肝微粒体采用BCA蛋白定量方法,用裂解液把肝微粒的浓度调节至4 mg·mL^-1。取蛋白50 μg,100 V电压电泳至溴酚蓝前缘移动至凝胶底部。电泳完成后,将蛋白从胶上转印到聚偏二氟乙烯膜(polyvinylidene fluoride,PVDF)上,将PVDF浸于含5%牛血清清蛋白(albumin from bovine serum,BSA)的1×TBST,室温震荡1.5 h,加适量以含5%BSA的1×TBST稀释的的一抗(兔抗CYP1A2的抗体浓度为1∶500,兔抗CYP2E1的抗体浓度为1∶400,鼠抗GAPDH的抗体浓度1∶5 000);在室温下孵育1 h,4 ℃过夜。用1×TBST洗膜3次,每次5 min,各加入1×TBST稀释的鼠、兔二抗(1∶20 000),室温孵育 1 h后用1×TBST洗膜3次,每次5 min。按照ECL发光试剂盒说明书进行显色。

1.9 统计学方法

采用SPSS18.0版统计软件进行One-Way ANOVA检验分析,组间数据进行Dunnett或Dunnett`s T₃检验;计量资料以均数±标准差(ヌ±s)表示,以P<0.05为差异有统计学意义。

2 结果

2.1 盐酸川芎嗪对小鼠的体质量和肝质量及肝指数的影响

与空白对照组比较,盐酸川芎嗪和苯巴比妥组的体质量都有增加,但差异无统计学意义;各给药组与空白对照组之间的肝指数差异无统计学意义,说明盐酸川芎嗪对小鼠肝脏质量无明显影响。肝脏指数(%)=(肝脏质量/体质量)×100%。见表2。

组别	体质量 g	肝质量	肝指数/ %
空白对照组	24.9±0.880	1.52±0.101	0.061±0.002
苯巴比妥组	26.5±1.003	1.52±0.111	0.057±0.005
盐酸川芎嗪
小剂量组	26.66±0.761	1.40±0.044	0.052±0.001
中剂量组	27.62±0.446	1.72±0.115	0.062±0.004
大剂量组	26.06±0.542	1.58±0.090	0.060±0.003

表2 5组小鼠体质量及肝脏质量和肝脏指数比较

Tab.2 Comparison of body weight, liver weight and liver index among five groups of mice ヌ±s,n=5

2.2 盐酸川芎嗪对CYP1A2、CYP2D22、CYP2E1和CYP3A代谢活性的影响

盐酸川芎嗪对小鼠肝脏4种亚酶酶活性的影响(表3)。与空白对照组比较,盐酸川芎嗪大剂量组的CYP1A2酶活性升高了1.43倍(P<0.05)。盐酸川芎嗪对CYP2E1、CYP2D22、CYP3A的酶活性与空白对照组比较,均差异无统计学意义。

组别	CYP1A2	CYP2E1	CYP3A	CYP2D22
空白对照组	0.433±0.076	0.042±0.292	0.543±0.008	1.390±0.162
苯巴比妥组	0.524±0.036	0.087±0.022^*1	0.852±0.057^*1	1.815±0.424
盐酸川芎嗪
小剂量组	0.498±0.008	0.035±0.320	0.550±0.005	1.511±0.133
中剂量组	0.493±0.021	0.048±0.092	0.565±0.009	1.712±0.115
大剂量组	0.620±0.017^*1	0.051±0.106	0.616±0.032	1.334±0.055

表3 5组小鼠肝CYP酶活性比较

Tab.3 Comparison of the activity of hepatic CYP enzyme among five groups of mice nmol·mg^-1·min^-1,ヌ±s,n=5

2.3 盐酸川芎嗪对CYP1A2、CYP2D22、CYP2E1和CYP3A11 mRNA水平的影响

本实验所提取的RNA条带清晰,没有模糊弥散的现象,且28S条带的亮度是18S条带的约2倍,说明提取的RNA完整性符合实验要求。紫外分光光度计检测结果表明提取的RNA的A₂₆₀/A₂₈₀比值在1.8~2.0之间,说明提取的RNA纯度满足实验要求。实时荧光定量PCR考察了盐酸川芎嗪对小鼠肝脏4种P₄₅₀同工酶mRNA表达的影响(表4)。与空白对照组比较,盐酸川芎嗪大剂量组上调了CYP1A2 mRNA水平(P<0.05);盐酸川芎嗪对CYP3A11 、CYP2D22和CYP2E1的mRNA的水平差异无统计学意义。

组别	CYP1A2 mRNA	CYP2E1 mRNA	CYP3A11 mRNA	CYP2D22 mRNA
空白对照组	1	1	1	1
苯巴比妥组	1.325±0.106	1.189±0.123	1.102±0.101	1.152±0.130
盐酸川芎嗪
小剂量组	0.980±0.150	1.300±0.055	0.933±0.202	0.633±0.080
中剂量组	1.170±0.040	1.076±0.106	0.980±0.055	1.176±0.088
大剂量组	1.435±0.234^*1	1.296±0.146	1.333±0.123	1.023±0.039

表4 5组小鼠肝CYP mRNA表达的比较

Tab.4 Comparison of the mRNA expression of hepatic CYP enzyme among five groups of mice ヌ±s,n=5

2.4 盐酸川芎嗪对AhR、HNF-4α、PXR和PPARα mRNA表达的影响

本实验用实时荧光定量聚合酶链反应考察了盐酸川芎嗪对小鼠肝脏相关核受体mRNA表达的影响。与空白对照组比较,盐酸川芎嗪大剂量组AhRmRNA水平上调1.56倍(P<0.05),对HNF-4α、PXR和PPARα的mRNA水平均差异无统计学意义。见表5。

组别	AhR mRNA	PPARα mRNA	PXR mRNA	HNF-4α mRNA
空白对照组	1	1	1	1
苯巴比妥组	1.760±0.231^*1	1.567±0.090^*1	2.067±0.099^*2	1.850±0.090^*1
盐酸川芎嗪
小剂量组	1.050±0.115	0.661±0.085	0.928±0.083	0.993±0.115
中剂量组	1.133±0.089	1.140±0.058	1.100±0.057	1.110±0.057
大剂量组	1.567±0.145^*1	1.333±0.088	1.237±0.058	1.266±0.145

表5 5组小鼠肝核受体mRNA表达的比较

Tab.5 Comparison of the mRNA expression of hepatic nuclear receptor among five groups of mice ヌ±s,n=5

2.5 盐酸川芎嗪对CYP1A2和CYP2E1蛋白表达的影响

Western-blotting测定肝脏微粒体中CYP1A2、CYP2E1的蛋白表达,结果见图1和表6。与空白对照组比较,盐酸川芎嗪大剂量组CYP1A2/GAPDH蛋白表达上调1.4倍(P<0.05);盐酸川芎嗪对小鼠肝脏中的CYP2E1/GAPDH蛋白表达无显著性影响。

图1 CYP1A2、CYP2E1蛋白表达相对水平

Fig.1 Relative protein expression of CYP1A2 and CYP2E1

组别	CYP1A2/GAPDH	CYP2E1/GAPDH
空白对照组	1.020±0.076	0.989±0.065
苯巴比妥组	1.310±0.087	1.188±0.120
盐酸川芎嗪
小剂量组	0.723±0.060	0.904±0.096
中剂量组	0.857±0.072	0.908±0.080
大剂量组	1.430±0.062^*1	1.217±0.110

表6 5组小鼠肝CYP蛋白表达的比较

Tab.6 Comparison of the protein expression of hepatic CYP enzyme among five groups of mice ヌ±s,n=5

3 讨论

细胞色素P₄₅₀是体内重要的Ⅰ相代谢酶之一。主要存在于动物肝脏,中药复方配伍使用时,中药有效成分与其他药物之间产生的代谢性相互作用,会对CYP₄₅₀酶的活性产生影响,从而影响治疗效果或产生药物毒性^[7]。参芎葡萄糖注射液是由丹参和盐酸川芎嗪配伍组成的中药注射剂,在临床上广泛应用。本实验是对参芎葡萄糖注射液的主要成分盐酸川芎嗪的研究。实验结果表明,盐酸川芎嗪对小鼠肝脏中的CYP3A11、CYP2D22和CYP2E1的活性及表达无显著影响,对CYP1A2的药物代谢酶的酶活性、mRNA以及蛋白表达具有诱导作用。

近年来,研究人员对细胞色素的调控因子进行了广泛研究,发现大部分CYP1A、2B、2D、2E、3A、4A家族成员的表达受到NR如PXR的调节^[8-9]。NR是一类脂溶性配体依赖的转录因子,此类因子在外源性物质对转录水平的基因表达调控中起着重要的作用^[10]。AHR调控CYP1A家族的表达,HNF-4α主要调节CYP2D22的水平;PXR主要激活CYP3A异构体;PPARα激活CYP2E亚型等^[11]。许多化合物对CYP酶活性的调节作用是通过NR介导的^[12]。因此,本文进一步研究了盐酸川芎嗪对CYP3A11、CYP2D22、CYP2E1和CYP1A2及调控因子表达的影响。给药7 d后,盐酸川芎嗪组对CYP3A11 、CYP2D22和CYP2E1的调控因子PXR、HNF-4α、和PPARα的表达无显著变化,但盐酸川芎嗪可以上调AhR表达。这一结果与盐酸川芎嗪对CYP3A11 、CYP2D22、CYP2E1和CYP1A2的作用一致。

CYP1A2在前致癌物的代谢活化中发挥了重要作用,它的活性与许多药物的疗效或毒性以及一些肿瘤的易感性密切相关,是人体参与药物的代谢的重要亚型。诱导剂与AhR相结合,形成复合物进入细胞核与转运因子结合成异二聚体,该异二聚体通过与CYP1A2启动子区上游的特异性结合位点结合,从而调节CYP1A2的转录,改变CYP1A2的表达,起到诱导作用^[13]。实验结果表明,盐酸川芎嗪对小鼠肝脏中CYP1A2活性和表达有诱导作用,很可能是由盐酸川芎嗪上调AhR的表达所致。

笔者在本实验中从酶活性、mRNA与蛋白水平检测盐酸川芎嗪对小鼠肝药物代谢酶的影响。结果显示盐酸川芎嗪对小鼠体内肝CYP2E1、CYP2D22、CYP3A、HNF-4α、PXR和PPARα没有显著性影响,但对CYP1A2的活性有诱导作用,而这种作用很可能与盐酸川芎嗪上调AhR水平从而促进CYP1A2表达有关。本课题组前期发现参芎葡萄糖注射液可以诱导CYP1A2及抑制CYP2E1的表达,其主要成分丹参对CYP2E1酶活性、mRNA及蛋白表达都具有显著抑制作用,结合本实验结果,推测参芎葡萄糖注射液的CYP1A2诱导作用主要由盐酸川芎嗪产生。

CYP1A2酶在肝中的含量次于CYP3A和CYP2C^[14],是乙酞氨基酚、非那西丁、华法林、芳香胺、丙咪嗓、乙酞苯胺、雌二醇、茶碱等的药物主要代谢酶^[15]。因此当盐酸川芎嗪与这些药物联合使用时,应考虑二者之间可能存在潜在的相互作用。但有文献报道,盐酸川芎嗪对人肝微粒体CYP2E1、CYP1A2等主要的药物代谢酶的活性没有显著影响^[16]。对大鼠体外肝细胞CYP1A2无诱导作用^[17]。但盐酸川芎嗪对大鼠肝微粒体CYP1A2,CYP2D6,CYP2E1的酶活性有诱导作用^[18]。盐酸川芎嗪可以诱导小鼠的CYP1A2的表达,但对其他代谢酶虽有上调趋势,但差异无统计学意义。这表明不同的动物模型、给药方式及给药周期等因素都会影响药物的代谢过程。因此研究中药对CYP酶的影响还需结合临床的试验来验证。

The authors have declared that no competing interests exist.

参考文献

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[15]	于盼盼,杭鹏洲,张波,等.稳心颗粒对大鼠细胞色素P₄₅₀酶的影响[J].医药导报,2012,31(7):834-838. [本文引用:1]
[16]	易飞,黄心一.人肝微粒体中川芎嗪对CYP酶活性的影响[C].成都:2008年成渝药学学术年会,2008,144-148. [本文引用:1]
[17]	谭妍,沈国林,庄笑梅,等.四物汤效应成分基于CYP酶的相互作用研究[J].中国药理学通报,2014,30(10):1456-1461. 目的：研究四物汤效应成分果糖、阿魏酸、芍药苷和川芎嗪及其配伍对CYP同工酶的抑制及诱导作用，为从代谢角度解释中药方剂的配伍规律提供依据。方法应用人肝微粒体体外孵育模型和探针底物代谢产物的LC-MS/MS定量方法，评价各单药及药对配伍组对 CYP 酶同工酶CYP1A2、2B6、2C9、2C19、2D6和3A4的抑制作用。应用“三明治”培养的大鼠原代肝细胞模型，评价各单药及其药对配伍组对CYP1A 和 CYP3A 的诱导作用。结果果糖、阿魏酸、芍药苷、川芎嗪及其配伍组(100μmol·L-1)，对人肝微粒体 CYP1A2、2B6、2C9和2C19的抑制率均阳性诱导剂的40%。结论四物汤各成分及其药对配伍对6个主要CYP同工酶均无明显的抑制作用，芍药苷单药对CYP1A2有一定诱导作用，与果糖、阿魏酸和川芎嗪配伍时，诱导活性明显增强。 DOI:10.3969/j.issn.1001-1978.2014.10.026 URL [本文引用:1]
[18]	尚芳红,俸珊,张飞燕,等.加味佛手散及其配伍对大鼠肝脏P₄₅₀酶活性及肝细胞形态的影响[J].中国中药杂志,2015,40(10):2030-2036. 考察加味佛手散及其不同药物组合对大鼠肝脏P450酶活性及肝细胞形态的影响。大鼠灌胃给药4周后处死,制备肝微粒体,采用肝微粒体体外孵育"鸡尾酒"法,HPLC-MS/MS法测定代谢产物,考察各受试组肝脏P450酶活性,HE染色观察用药前后肝组织病理变化。与对照组相比,加味佛手散组CYP1A2,CYP3A4的酶活性显著升高(P0.05);阿魏酸+川芎嗪组、川芎嗪+延胡索乙素组CYP1 A2,CYP2 C9,CYP2 D6,CYP2 E1,CYP3 A4的酶活性显著升高(P0.05);川芎嗪组CYP1A2,CYP2D6,CYP2E1的酶活性显著升高(P0.05);阿魏酸组CYP3A4的酶活性显著降低(P0.05)。用药后阿魏酸和川芎嗪组肝细胞形态正常,延胡索乙素组肝小叶界限不清,肝细胞排列紊乱,边缘模糊,细胞核大小不均,炎细胞浸润。阿魏酸+延胡索乙素、川芎嗪+延胡索乙素、加味佛手散组有炎细胞浸润,但病理变化程度明显较延胡索乙素组轻微,其中加味佛手散组最轻。研究结果表明加味佛手散对大鼠肝脏的CYP1A2,CYP3A4的酶活性具有诱导作用,川芎嗪可能是加味佛手散对CYP1A2产生诱导作用的效应物质。配伍阿魏酸、川芎嗪后可降低延胡索乙素对大鼠肝脏的毒性作用。 DOI:10.4268/cjcmm20151034 URL [本文引用:1]

基于药物代谢酶和转运蛋白的中药方剂配伍规律研究

2008

... 中药成分复杂,具有较多的化学成分,不同配伍组成的复方机制也较多^[1].中药的多种成分作用于机体形成复杂的相互作用体系,而复方整体效应的重要基础正是这些复杂的相互作用.产生相互作用的原因是中药的有效成分进入体内要经过CYP酶进行代谢,并有可能导致酶活性被诱导或抑制,从而影响其他物质的代谢,引起药物之间的相互作用^[2].参芎葡萄糖注射液主要由丹参和盐酸川芎嗪组成,盐酸川芎嗪有扩张血管、改善组织微循环、抑制血小板粘附等作用^[3].临床常用于治疗缺血性心脑血管疾病和缺血性肢体血管疾病^[4].本课题组前期研究了参芎葡萄糖注射液及其组分丹参对小鼠CYP同工酶的影响.笔者在本研究中继续考察参芎葡萄糖注射液中的另一种主要成分盐酸川芎嗪对细胞色素P₄₅₀同工酶的影响,进而完善参芎葡萄糖注射液的代谢性相互作用的研究. ...

CYP酶特性及其应用研究进展

2008

川芎的化学成分及药理研究进展

2006

中西医结合治疗急性脑梗死70例临床观察

2006

柴胡总皂苷对小鼠药物代谢酶及P-糖蛋白的影响

2012

... 1.5.1 CYP1A2酶活性的测定采用非那西丁体外代谢法来测定CYP1A2酶活性^[5].将待测肝微粒体稀释到5 g·L^-1 ,取100 μL肝微粒体于1.5 mL离心管中,加入非那西丁(0.05 mol·L^-1)200 μL,37 ℃振荡孵育10 min.然后加入200 μL 还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)再生系统(2 mmol·L^-1 Na₂NADP,20 mmol·L^-1 G-6-P,4 U·L^-1 G-6-PDH,20 mmol·L^-1 MgCl₂),于37 ℃水浴中振荡30 min后,加入三氯乙酸200 μL终止反应.15 000×g离心20 min,取90 μL上清液加入普萘洛尔(1.07 g·L^-1)10 μL,混匀,用高效液相检测.色谱条件:上样量20 μL;Luna C₁₈色谱柱(4.6 mm×250 mm,5 μm);流动相:60%甲醇-40%水(0.04%三乙胺和0.04%乙酸乙酯),用冰醋酸调pH值至3.0;流速0.6 mL·min^-1;柱温40 ℃;检测波长280 nm. ...

... 1.5.2 CYP2D22酶活性的测定采用右美沙芬体外代谢高效液相色谱法测定,将待测肝微粒体稀释到5 g·L^-1 ,取100 μL肝微粒体于1.5 mL离心管中,加入右美沙芬(0.03 mol·L^-1)200 μL.37 ℃振荡孵育10 min后,加入NADPH再生系统200 μL,于37 ℃水浴中振荡孵育30 min,最后加入三氯乙酸200 μL终止反应.15 000×g离心20 min,取上清液90 μL加入普萘洛尔(1.07 g·L^-1)10 μL,混匀,用高效液相色谱法检测.色谱条件同“1.5.1”项^[5]. ...

2010

... 1.5.3 CYP3A酶活性的测定采用氨基比林N-脱甲基酶ADM法,通过Nash比色法测定甲醛生成量来反映CYP3A的活性^[6].将待测肝微粒体稀释到5 g·L^-1,取100 μL,加入磷酸盐缓冲液(PBS)0.15 mL,混匀后加入86.4 mmol·L^-1氨基比林0.05 mL,混匀,37 ℃水浴3 min后加入NADPH发生系统0.05 mL,37 ℃孵育60 min后取出,加入30%三氯醋酸0.25 mL终止反应.再加Nash试剂0.25 mL,混匀后60 ℃水浴10 min,室温放置20 min.以不含微粒体的空白体系调零,于412 nm波长处测定吸光度值(A值). ...

... 1.5.4 CYP2E1酶活性的测定采用苯胺羟化酶ANH法,用分光光度法测定对氨基酚的生成量,从而反映CYP2E1的活性^[6].将待测肝微粒体稀释到5 g·L^-1 ,取100 μL加入测定管,再加入苯胺溶液0.05 mL,对照管加入水0.05 mL.37 ℃水浴30 min,加入冰冷20%三氯乙酸溶液1.0 mL,冰浴5 min.取上清液1.0 mL,加入1%酚试剂1.0 mL,混匀,再加入碳酸钠1.0 mL混匀.室温下反应30 min,于630 nm波长处测定吸光度. ...

Pharmacokinetics and metabolic drug interactions

2006

... 细胞色素P₄₅₀是体内重要的Ⅰ相代谢酶之一.主要存在于动物肝脏,中药复方配伍使用时,中药有效成分与其他药物之间产生的代谢性相互作用,会对CYP₄₅₀酶的活性产生影响,从而影响治疗效果或产生药物毒性^[7].参芎葡萄糖注射液是由丹参和盐酸川芎嗪配伍组成的中药注射剂,在临床上广泛应用.本实验是对参芎葡萄糖注射液的主要成分盐酸川芎嗪的研究.实验结果表明,盐酸川芎嗪对小鼠肝脏中的CYP3A11、CYP2D22和CYP2E1的活性及表达无显著影响,对CYP1A2的药物代谢酶的酶活性、mRNA以及蛋白表达具有诱导作用. ...

Effect of aflatoxin B1 on nuclear receptors PXR,CAR,AHR and their target cytochromes P mRNA expression in primary cultures of human hepatocytes

2012

... 近年来,研究人员对细胞色素的调控因子进行了广泛研究,发现大部分CYP1A、2B、2D、2E、3A、4A家族成员的表达受到NR如PXR的调节^[8-9].NR是一类脂溶性配体依赖的转录因子,此类因子在外源性物质对转录水平的基因表达调控中起着重要的作用^[10].AHR调控CYP1A家族的表达,HNF-4α主要调节CYP2D22的水平;PXR主要激活CYP3A异构体;PPARα激活CYP2E亚型等^[11].许多化合物对CYP酶活性的调节作用是通过NR介导的^[12].因此,本文进一步研究了盐酸川芎嗪对CYP3A11、CYP2D22、CYP2E1和CYP1A2及调控因子表达的影响.给药7 d后,盐酸川芎嗪组对CYP3A11 、CYP2D22和CYP2E1的调控因子PXR、HNF-4α、和PPARα的表达无显著变化,但盐酸川芎嗪可以上调AhR表达.这一结果与盐酸川芎嗪对CYP3A11 、CYP2D22、CYP2E1和CYP1A2的作用一致. ...

CAR-mediated up-regulation of CYP3A4 expression in LS174T cells by Chinese herbal compounds

2011

Dose-response of oridonin on hepatic cytochromes P mRNA expression and activities in mice

2014

Effects of tetrahydroxystilbene glucoside on mouse liver cytochrome P enzyme expressions

2015

Human pregnane X receptor agonism by Ginkgo biloba extrat:assessment of the role of individual ginkgolides

2010

Sequence variants at CYP1A1-CYP1A2 and AHR associate with coffee consumption

2011

... CYP1A2在前致癌物的代谢活化中发挥了重要作用,它的活性与许多药物的疗效或毒性以及一些肿瘤的易感性密切相关,是人体参与药物的代谢的重要亚型.诱导剂与AhR相结合,形成复合物进入细胞核与转运因子结合成异二聚体,该异二聚体通过与CYP1A2启动子区上游的特异性结合位点结合,从而调节CYP1A2的转录,改变CYP1A2的表达,起到诱导作用^[13].实验结果表明,盐酸川芎嗪对小鼠肝脏中CYP1A2活性和表达有诱导作用,很可能是由盐酸川芎嗪上调AhR的表达所致. ...

丹红注射液对大鼠肝微粒体5种CYP亚型酶活性的影响

2012

... CYP1A2酶在肝中的含量次于CYP3A和CYP2C^[14],是乙酞氨基酚、非那西丁、华法林、芳香胺、丙咪嗓、乙酞苯胺、雌二醇、茶碱等的药物主要代谢酶^[15].因此当盐酸川芎嗪与这些药物联合使用时,应考虑二者之间可能存在潜在的相互作用.但有文献报道,盐酸川芎嗪对人肝微粒体CYP2E1、CYP1A2等主要的药物代谢酶的活性没有显著影响^[16].对大鼠体外肝细胞CYP1A2无诱导作用^[17].但盐酸川芎嗪对大鼠肝微粒体CYP1A2,CYP2D6,CYP2E1的酶活性有诱导作用^[18].盐酸川芎嗪可以诱导小鼠的CYP1A2的表达,但对其他代谢酶虽有上调趋势,但差异无统计学意义.这表明不同的动物模型、给药方式及给药周期等因素都会影响药物的代谢过程.因此研究中药对CYP酶的影响还需结合临床的试验来验证. ...

稳心颗粒对大鼠细胞色素P酶的影响

2012

人肝微粒体中川芎嗪对CYP酶活性的影响

2008

四物汤效应成分基于CYP酶的相互作用研究

2014

加味佛手散及其配伍对大鼠肝脏P酶活性及肝细胞形态的影响

2015