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医药导报
医药导报, 2017, 36(3): 247-251
doi: 10.3870/j.issn.1004-0781.2017.03.004
齐墩果酸体外肠吸收特征及P-糖蛋白介导跨膜转运机制*
Absorption Characteristics and Mechanism of P-glycoprotein Mediated Transport of Oleanolic Acidin Across the Membrane In Vitro
杨茗钫1,, 程晓华2,, 徐文炜3
1.南昌大学第一附属医院妇产科,南昌 330006
2.南昌大学第一附属医院GCP中心,南昌 330006
3.南昌大学临床药理研究所,南昌 330006
YANG Mingfang1,, CHENG Xiaohua2,, XU Wenwei3
1.Department of Obstetrics and Gynecology
2.Department of GCP Center,the First Affiliated Hospital of Nanchang University,Nanchang 330006,China
3. Institute of Clinical Pharmacology, Nanchang University,Nanchang 330006,China
通信作者 程晓华(1979-),男,江西余干人,主管药师,硕士,主要研究方向:药动学与新药临床试验。电话:0791-88695051,E-mail:ncujg2009@163.com。

作者简介 杨茗钫 (1984-),女,江西瑞金人,主治医师,硕士,主要研究方向:妇科肿瘤及药代动力学。电话:0791-88692250,E-mail:649479518@163.com。
基金资助: 基金项目 国家十二五“重大新药创制”科技重大专项(2011ZX09302-007-03)
中图分类号: R286     文献标识码: A     文章编号: 1004-0781(2017)03-0247-05
摘要:

目的 观察齐墩果酸体外肠吸收特征及P-糖蛋白介导跨膜转运的机制。方法 建立齐墩果酸细胞摄取量以及转运量高效液相色谱-质谱联用仪的定量检测方法,噻唑蓝法确定齐墩果酸对Caco-2细胞的安全浓度范围,考察不同药物浓度、孵育时间、介质pH值、体系温度对Caco-2细胞摄取齐墩果酸的影响;通过构建Caco-2细胞单层模型,评价P-糖蛋白抑制药维拉帕米对齐墩果酸跨膜转运的影响,计算其表观渗透系数(Papp)。结果 在安全浓度范围内,Caco-2细胞对齐墩果酸摄取量呈浓度和时间依赖性,与温度呈负相关,不同pH值对药物摄取影响差异无统计学意义。与对照组比较,加入维拉帕米后,Papp显著改变(P<0.05),表观渗透率由2.90下降至0.95。结论 齐墩果酸吸收转运以被动扩散为主,可能有P-糖蛋白介导外排作用的参与。
关键词: 齐墩果酸 ; Caco-2细胞 ; P-糖蛋白

Abstract:

Objective To explore the absorption characteristics and mechanism of P-glycoprotein (P-gp) mediated transport of oleanolicacid (OA)across membrane in vitro.Methods The intake and transport of OA were evaluated by an HPLC/MS quantitative detection method.Safe concentration range of OA was determined by MTT.The effect of different drug concentration, incubation time, pH and temperature on the intake of OA by Caco-2 cells were investigated.The effect of P-gp inhibitor on the transmembrane transport of OA was investigated by the Caco-2 cell monolayer model, and the apparent permeability coefficient (Papp) was calculated. Results The uptake of OA was concentration-time dependent and correlated negatively to temperaturein Caco-2 cells within the safe concentration range, but was not significantly affected by pH value.Compared with controls, the Pappsignificantly changed when the P-gp inhibitor was added to the model(P<0.05).The apparent permeability ratio decreased from 2.90 to 0.95.Conclusion The absorption of OA was primarily by passive diffusion and might be mediated by the efflux by P-gp.
Key words: Oleanolic acid ; Caco-2 cell ; P-glycoprotein

齐墩果酸(oleanolic acid,OA)具有护肝降酶、抗炎、抗病毒、抗肿瘤等多种药理作用[1-4],临床主要用于急性黄疸型肝炎和慢性病毒性肝炎的治疗。OA疗效稳定,不良反应小,但由于溶解度较差,生物利用度低,限制了其临床应用。针对其生物利用度低的缺点,国内外学者在OA剂型改进及相关剂型药动学研究方面取得不少进展[5],但体外肠吸收机制研究笔者较少见到。

Caco-2 细胞模型重现性好,与药物体内吸收具有良好的相关性,近年来被国内外学者广泛用于研究药物体外肠道吸收过程与机制,也是目前研究体外肠吸收的经典模型[6]。笔者在本研究采用Caco-2细胞模型研究小肠上皮细胞对OA的摄取、跨膜转运及外排作用,评价药物浓度、孵育时间、介质pH值、体系温度对细胞摄取的影响,以及P-糖蛋白(P-gp)抑制药对细胞跨膜转运吸收的影响,以期阐明OA的吸收机制,为合理设计其剂型和深入研究其药动学提供实验依据。

1 材料与方法
1.1 细胞株

Caco-2细胞株购于美国典型菌种保藏中心(American Type Culture Collection,ATCC),实验中使用第30~40代细胞。

1.2 药品与试剂

达尔伯克改良伊格尔培养基(Dulbecco's Modified Eagle Medium,DMEM)(GIBCO公司,批号:31800-022),胎牛血清(Hyclone公司,批号:NXJ0709),0.1%胰蛋白酶(GIBCO公司,批号:27250018),谷氨酰胺(GIBCO公司,批号:25030),非必需氨基酸(GIBCO公司,批号:1232254),维拉帕米(Sigma公司,批号:20120816),罗丹明123 (Sigma公司,批号:R06154),OA(中国食品药品检定研究院,含量:98%,批号:201206),格列喹酮对照品(中国食品药品检定研究院,批号:130824,含量:99.2%),乙腈(TEDIA公司,色谱纯,批号:14025017),胰蛋白酶-EDTA(Sigma公司,批号:S10054),醋酸铵(天津福晨化学试剂厂,批号:20140415),水为超纯水,其他试剂均为分析纯。

1.3 仪器

MCO20-AIC型二氧化碳(CO2)培养箱(日本三洋公司),CKX-41型倒置光学显微镜(日本奥林巴斯公司),EVOM细胞电位仪(美国WPI公司),GS-15R高速冷冻离心机(德国Sigma公司),LCMS-2010EV型高效液相色谱-质谱联用仪(HPLC-MS,日本岛津公司);LCMSsolution色谱工作站(日本岛津公司)。

1.4 细胞培养和细胞单层完整性评估

将Caco-2细胞培养于高糖DMEM培养基中(含10%胎牛血清、非必需氨基酸、1%L-谷氨酰胺、100 U·mL-1青霉素、100 μg·mL-1链霉素),置于37 ℃、含5%CO2、相对湿度90%下培养。隔日换液一次,当细胞融合约达90%后,0.25%胰蛋白酶-1 mmol·L-1乙二胺四乙酸(EDTA)消化传代。取对数生长期细胞,调节密度为6×104个·mL-1,接种于Transwell板,肠腔侧(apical,AP)和基底侧(basolateral,BL)加适量培养液。隔日换液,一周后每天换液,培养21~24 d、待细胞达到基本汇合、跨膜电阻>400 Ω·cm2,以及罗丹明123不能透过细胞间隙时,待确认形成紧密单层后,用于转运实验。

1.5 色谱条件

色谱柱:Shim-pack VP-ODS C18(150 mm×2.1 mm,5 μm);流动相:乙腈-0.1%醋酸铵=68∶32,流速:0.2 mL·min-1,柱温:35 ℃;采用选择性负离子检测及电喷雾离子化(electrospray ionization,ESI);检测对象:OA,m/z:455.3;格列喹酮,m/z:527.1;雾化气流速为1.5 L·min-1;干燥气流量为4.0 L·min-1,检测电压1.60 kV;曲形脱溶剂装置温度为250 ℃;加热块温度:200 ℃。

1.6 MTT细胞毒性实验

取对数生长期Caco-2细胞,调整细胞密度至5×104个·mL-1接种于96孔培养板,24 h后换液,实验组分别加入不同浓度OA培养液,调零空白组加入等体积培养液,每组设4个复孔,继续培养12和24 h后每孔加入噻唑蓝(MTT),每孔加二甲亚砜(DMSO)溶液150 μL,空气恒温振荡器振荡10 min,待结晶物充分溶解,在酶标仪上选择波长570 nm,空白孔调零,测定各孔吸光度值。细胞存活率(%)=(实验组吸光度值/对照组吸光度值)×100%。

1.7 Caco-2细胞摄取实验

取对数生长期Caco-2细胞,调整细胞密度为1×104个·mL-1,接种培养于24孔培养板,实验前2 h缓慢吸弃旧培养液,加入预热Hank'平衡盐溶液(hank's balanced salt solution,HBSS)1.0 mL荡洗细胞3次,最后置于37 ℃培养箱温孵30 min,缓慢吸弃HBSS溶液,洗去细胞单分子层表面杂质,考察药物浓度、孵育时间、体系温度以及介质pH值对药物摄取的影响。不同因素各组均设4个复孔,加入药液1 mL,培养24 h后弃去药液,加入4℃HBSS缓冲液终止摄取。每孔加入超纯水1 mL,细胞刮取器刮下细胞,超声粉碎细胞,取上清液200 μL,加入乙腈,离心,取上清液20 μL用于HPLC-MS分析,另取200 μL进行细胞蛋白质含量测定,OA摄取量以μg·mg-1蛋白质为单位。

1.8 Caco-2细胞单层跨膜转运实验

取符合转运实验要求的Caco-2细胞模型,试验前用预热HBSS清洗生长有Caco-2细胞的Transwell板3次,置于37 ℃培养箱温孵30 min,考察药物浓度、反应时间、转运方向及P-gp抑制药维拉帕米对细胞转运OA的影响。当转运方向为AP→BL时,AP中加入OA供试溶液0.4 mL作为供给液,BL中加入空白HBSS 1.0 mL作为接收液。当转运方向为BL→AP时,AP中加入空白HBSS0.4 mL作为接收液,BP中加入供试药液1.0 mL。将Transwell板置于37 ℃、转速50 r·min-1双层空气恒温振荡器中孵育,分别于10,20,40,60,90,120 min吸取接收液,同时补加空白HBSS 200 μL,每组设4个平行孔。取转运样品200 μL,加入乙腈,离心10 min,取上清液进行HPLC-MS分析,计算其表观渗透系数(Papp)。

1.9 数据处理及统计学方法

细胞的摄取量=C/Cprotein,C是细胞摄取样品中OA的浓度,Cprotein是考马斯亮蓝法测得的蛋白质浓度;Papp=(dC/dt·V)/(A·C0),dC/dt为药物在单位时间转运量,V为接受侧溶液体积,A为Transwell多聚碳酸酯膜表面积(0.33 cm2),C0为OA在供侧初始浓度;表观渗透率(apparent permeability ratio,PDR)的计算:PDR=PappBL→AP/PappAP→BL,表示药物AP→BL表观渗透系数。采用SPSS 17.0版统计软件进行统计分析,计量数据结果均采用均数±标准差( x ̅ ±s)表示,组间比较采用LSD-t检验,P<0.05为差异有统计学意义。

2 结果
2.1 HPLC-MS分析方法的建立

成功建立了测定OA含量专属性强、灵敏度高的HPLC-MS分析方法,OA保留时间约2.3 min,转运液及细胞悬液均无杂质干扰样品的测定。结果见图1。


图1 样品检测质谱图
A.blank HBSS;B.standard solution;C.sample solution;1.OA;2.gliquidone

Fig.1 HPLC-MS chromatograms of the sample

2.2 MTT细胞毒性实验结果

MTT实验结果表明,随着OA浓度增加,细胞存活率逐渐下降,呈剂量依赖性,培养时间对细胞存活率基本无影响。在5~40 μg·mL-1范围内细胞的存活率>80%,OA的浓度>40 μg·mL-1,部分细胞出现细胞毒作用并伴随细胞形态学改变,结果见表1。因此,在Caco-2细胞摄取和转运实验时,选择5~40 μg·mL-1作为实验中OA给药浓度范围。

2.3 培养时间对细胞摄取OA的影响

Caco-2细胞对OA的摄取量随培养时间的延长而变化,0~40 min细胞对其摄取呈增加趋势,40 min后逐渐趋于饱和,细胞摄取量无显著增加(P>0.05),结果见图2。开始时可能由于细胞内外浓度差较大,药液被细胞快速摄取,40 min后细胞内外液OA浓度达到相对平衡,摄取量趋于平缓。因此,在细胞摄取试验中,将摄取时间定为40 min。

表1 OA不同浓度和作用时间对Caco-2细胞存活率的影响
Tab.1 Effects of different concentration and incubation time of OA on the survival rate of Caco-2 cells x¯±s,n=4,%
浓度/(μg·mL-1) 12 h 24 h
5 98.8±25.3 96.5±23.5
10 92.3±18.6 90.6±15.9
20 86.3±12.8 84.4±14.6
40 82.4±15.3 80.3±10.7
80 69.5±12.6 62.8±10.3

表1 OA不同浓度和作用时间对Caco-2细胞存活率的影响

Tab.1 Effects of different concentration and incubation time of OA on the survival rate of Caco-2 cells x¯±s,n=4,%


图1 样品检测质谱图
A.blank HBSS;B.standard solution;C.sample solution;1.OA;2.gliquidone

Fig.1 HPLC-MS chromatograms of the sample


图2 培养时间对Caco-2细胞摄取OA量的影响( x̅±s,n=4)

Fig.2 Effects of incubation time on the uptake OA volumeof Caco-2 cells(x̅±s,n=4)

2.4 药物浓度对细胞摄取OA的影响

将Caco-2细胞分别与5,10,20,40 μg·mL-1的OA供试液培养40 min,考察浓度对细胞摄取OA的影响,结果见图3。摄取量随浓度的增加呈线性上升,细胞对OA的摄取可能主要以被动扩散的方式进行。


图3 药物浓度对Caco-2细胞摄取OA量的影响(x̅±s,n=4)

Fig.3 Effects of different drug concentration on the uptake OA volume of Caco-2 cells (x̅±s,n=4)

2.5 介质pH值对细胞摄取OA影响

将40 μg·mL-1OA供试液在pH值为5,6,7.4,8的条件下培养40 min,测定加药40 min后细胞对OA的吸收量,考察pH值对细胞吸收OA的影响。结果在pH值为5,6,7.4,8条件下,细胞对OA摄取量分别为(1.27±0.25),(1.31±0.35),(1.36±0.33),(1.41±0.28)μg·mL-1,组间比较差异无统计学意义(P>0.05)。本研究中培养液pH值定为7.4。

2.6 培养体系温度对细胞摄取OA的影响

分别考察37 ℃与4 ℃时,作用不同时间Caco-2细胞对40 μg·mL-1OA的摄取量,结果见图4。结果表明,4 ℃时细胞摄取量OA大于37 ℃(P<0.05)。细胞摄取量可能随着温度的升高而降低,呈现一定的温度依赖性。结果预示细胞摄取OA可能受外排蛋白影响,当温度为4 ℃时,外排蛋白活力下降,外排作用减弱,药物的摄取量增加。


图4 不同体系温度对Caco-2细胞摄取OA量的影响(x̅±s,n=4)

Fig.4 Effects of different system temperature on the uptake OA volume of Caco-2 cells(x̅±s,n=4)

2.7 Caco-2细胞转运实验

取OA供试液,分别加入Caco-2细胞单层膜的AP侧和BL侧,不同浓度OA在不同时间点跨膜转运(AP→BL)过程中的转运量变化情况见图5。AP→BL时,OA转运量随浓度和转运时间增加呈线性增加,表现为浓度和时间依赖性,而PappAP→BL随浓度的增加无显著改变(P>0.05);BL→AP时,PappBL→AP随浓度的增加而减小,差异有统计学意义(P<0.05),结果见表2。结果表明,吸收过程(AP→BL)具有明显的浓度依赖性,主要以被动扩散的方式转运,而分泌过程(BL→AP)存在一定的浓度饱和性,PappBL→AP随着浓度升高而下降,PDR随着浓度的增加而下降,可能受载体转运蛋白外排作用的影响。

为证实是否存在P-gp外排作用对OA转运的影响,转运液中加入P-gp抑制药维拉帕米 (Ver,100 μmol·L-1),孵育30 min后加入40 μg·mL-1OA进行转运实验。结果见图6。结果表明,吸收方向(AP→BL)OA转运量显著上升,差异有统计学意义(P<0.05),OA分泌过程中(BL→AP)转运量则有所下降,PDR值由2.90下降为0.95。结果见表3。该结果进一步说明OA在吸收过程中受到P-gp外排作用影响。


图5 OA浓度与作用时间对AP→BL转运量的影响(x̅±s,n=4)

Fig.5 Effects of time and concentration of OA on AP→BL transport volume (x̅±s,n=4)

表2 OA浓度对Papp和PDR的影响
Tab.2 Effects of concentration of OA on Papp and PDR x¯±s,n=4
浓度/
(μg·mL-1)		 Papp/(×10-6cm·s-1) PDR
AP→BL BL→AP
10 0.87±0.22 3.88±0.89 4.46
20 0.95±0.26 3.31±0.55 3.48
40 0.81±0.18 2.35±0.46*1 2.90

Compared with 10 μg·mL-1 group,*1P<0.05

与10 μg·mL-1组比较,*1P<0.05

表2 OA浓度对Papp和PDR的影响

Tab.2 Effects of concentration of OA on Papp and PDR x¯±s,n=4


图6 Ver对AP→BL转运量的影响( x ̅ ±s,n=4)

Fig.6 Effects of Ver on AP→BL transportvolumn of OA(x̅±s,n=4)

表3 Ver对Papp和PDR的影响
Tab.3 Effects of P-gp on Papp and PDRx¯±s,n=4
转运液 Papp/(×10-6cm·s-1) PDR
AP→BL BL→AP
OA 0.81±0.18 2.35±0.46 2.90
OA+Ver 1.71±0.37*1 1.62±0.39 0.95

与OA组比较,*1P<0.05

Compared with OA group,*1P<0.05

表3 Ver对Papp和PDR的影响

Tab.3 Effects of P-gp on Papp and PDRx¯±s,n=4

3 讨论

Caco-2细胞来源于人结肠癌细胞,其结构和生化特点类似于人类小肠上皮细胞,体外培养一定时间后可以分化成具有多种药物载体和酶的小肠微绒毛结构,能够在细胞水平提供关于药物分子通过小肠黏膜的吸收、代谢信息,因此被广泛用于研究药物吸收机制[7]。

本研究结果显示,在0~24 h、OA浓度为5~40 μg·mL-1范围内,Caco-2细胞存活率均>80%,因此选取40 μg·mL-1为最大药物浓度应用于本实验,以避免浓度过高导致细胞死亡,影响对药物吸收的判断。通过建立灵敏度高、特异性强的细胞摄取液和转运液中OA浓度的HPLC-MS测定方法,评价不同药物浓度、反应时间、介质pH值等因素对OA摄取以及P-gp抑制药对OA跨膜转运的影响。研究结果表明,OA摄取存在时间和浓度依赖性,随着OA浓度上升细胞摄取量呈线性增加,表明OA主要以被动扩散方式被摄取。快速摄取在开始时出现,40 min后细胞内外液浓度达到相对平衡,摄取量趋于平缓并达到相对饱和,不同介质pH值对OA的摄取量无显著影响;细胞摄取量随着温度的升高而降低,呈现一定的温度依赖性,4 ℃时OA细胞摄取量明显大于37 ℃,预示OA细胞摄取可能受外排蛋白质影响,当温度4 ℃时,外排蛋白质活力下降,外排作用减弱,故药物摄取量增加。

Papp的数量级是反映药物透过Caco-2细胞单层能力及药物吸收难易程度的一般标准[8]。本研究结果表明,OA的吸收方向Papp在10-6~10-7数量级,PappAP→BL在(0.81~0.95)×10-6 cm·s-1之间,表明其肠黏膜的渗透性低,吸收不佳,这与其口服生物利用度较低的结果基本一致。吸收转运实验结果显示:细胞跨膜转运具有明确方向性,吸收方向(AP→BL)累积转运量随着OA浓度(10~40 μg·mL-1)及孵育时间增加而增加,Papp差异无统计学意义,提示药物吸收主要以被动扩散的方式转运; 而分泌(BL→AP)方向,Papp随浓度增加而减小,呈现一定的浓度饱和性,且Papp AP→BL和PappBL→AP两组比较差异有统计学意义,不同浓度PDR值均>1.5,提示可能存在载体介导的外排转运过程[9]。P-gp是能量依赖性外排转运蛋白,能将许多结构不同的化合物逆向转运出细胞,从而培养形成完整细胞单层的Caco-2细胞能表达此类外排蛋白[10]。有研究证实,部分同类五环三萜类化合物是P-gp底物,其吸收过程受到P-gp外排作用影响[11]。为考察是否存在P-gp对OA吸收的外排作用,在转运液中加入P-gp专属性抑制药Ver,与对照组比较,Ver能显著增加OA吸收方向(AP→BL)的转运量,提高吸收方向Papp,降低分泌方向(BL→AP)Papp,且PDR由2.90下降为0.95,该研究结果进一步证实OA肠吸收过程可能受P-gp外排作用的影响,降低了药物的吸收,OA可能是P-gp底物。综上结论,OA口服生物利用度低可能受现有剂型溶解度及溶出速率的影响,肠黏膜低渗透性和吸收转运中存在外排蛋白(P-gp)作用,导致吸收较差也是其中原因之一。

The authors have declared that no competing interests exist.
参考文献
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篇首： 评估药物口服吸收效果的常用方法是整体动物实验.但由于动物有个体差异,测定建立分析生物样本中药物的方法难度大,整体动物吸收代谢的耗药量大,周期较长.
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We applied a parallel pore permeation model based on the Renkin molecular sieving function by using two different-sized pathways to analyze the permeation-enhancing effects of poly-L-arginine (PLA) or a mixed system of spermine (SPM) and sodium taurocholate (STC). Four paracellular markers were simultaneously applied to Caco-2 cell monolayers, and a set of apparent permeability coefficient (P) values was used to obtain membrane parameters. For PLA treatment, the pore occupancy/length ratio (epsilon/L) of the large pathways increased while the pore radius (R) did not, suggesting that the number of large pathways for the relatively large hydrophilic molecules in the monolayers could be increased by the addition of PLA. In contrast, application of the mixed system comprising SPM and STC significantly increased not only the R of the large pathways but also epsilon/L of the small pathways. Such changes in membrane parameters could be related to the enhancing mechanism of these compounds. The simulation curves for molecular weight (MW)-P calculated from the membrane parameters could be used to predict the P of drugs with different MWs.
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熊果酸（Ursolicacid，UA），又名乌索酸，乌苏酸，属α-香树脂醇（α- amyri）型五环三萜类化合物。在自然界分布非常广泛，据目前所知，至少存在于26个科70多种天然植物中。大量的研究表明，UA具有广泛的药理作用和 重要的生物活性，尤其在抗炎、护肝、抗肿瘤以及机体免疫调节等方面已经显现出令人关注的药理特性”。
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关键词（key words）
齐墩果酸
Caco-2细胞
P-糖蛋白
Oleanolic acid
Caco-2 cell
P-glycoprotein
作者
杨茗钫
程晓华
徐文炜
YANG Mingfang
CHENG Xiaohua
XU Wenwei