辣椒素的肠道转运特性与TRPV1通道及多药耐药蛋白间的关系
摘要
背景和目的
辣椒素(capsaicin, CAP,(反式)8-甲基-N-香草基-6-壬烯酰胺)是辣椒中产生辛辣的主要成分,被广泛用作调味料。分子量：305.42。辣椒素不仅广泛用于食品添加,也是临床治疗神经性疼痛的有效药物。美国食品药品监督管理局(FDA)已经通过了含8%辣椒素的贴剂(Qutenza)用于治疗带状孢疹后神经痛。不仅如此,也有诸多研究表明辣椒素在低于细胞毒性的浓度时就能够抑制P-糖蛋白(P-gp)活性,并且这种抑制水平与已公认的P-gp抑制剂维拉帕米相当。辣椒素能够逆转阿霉素在人类多重耐药癌细胞KB-C2中的耐药性,而且在很低的浓度时既可显著增加P-gp底物如地高辛等的透过。同时,也有研究表明辣椒素可以通过影响肠粘膜的通透性而使某些抗生素药物如头孢唑林,头孢氨苄的血药浓度增加。基于辣椒素同一些药物的相互作用以及辣椒素是许多人日常食用品中的主要成分之一,故有必要首先了解辣椒素的经肠转运机制。然而目前对于辣椒素的研究及应用主要集中在体外经皮吸收方面,而对于其在体内的吸收转运机制几乎未见报道。
因此,本文通过研究辣椒素经不同肠段的透过性来考察辣椒素与肠粘膜上一些转运体或通道之间的关系。这些转运体或通道的确定是基于以下考虑：
辣椒素的镇痛应用主要得益于它对瞬态电压感受器阳离子通道(TRPV1)具有高度的选择性。TRPV1是一种温度和疼觉感受器,一些小的金属离子的透过依赖于TRPV1通道的活性,但TRPV1通道可能会被一些化学调节剂激活并打开,从而促使一些大的阳离子化合物的快速透过。这说明TRPV1通道能够作为透过途径而被激活从而促进药物的转运。此外,有报道指出辣椒素与TRPV1至少存在两个结合位点,胞内和胞外,并且它与其中一个位点的结合会加速其对另一个位点的结合。所以我们猜测辣椒素对TRPVl通道的激活是否会促进其自身的转运呢?因此,我们假设TRPV1通道参与了辣椒素经肠粘膜的透过,并用TRPV1非竞争性抑制剂钌红(RR)阻塞TRPV1通道来研究TRPV1通道对辣椒素在不同肠粘膜透过及吸收的影响。
此外,Yi Han等研究者用Caco-2细胞研究发现,辣椒素是一种P-gp抑制剂。Loo, T.W等研究者提出由于辣椒素的抑制作用是能够刺激ATP苷酶的激活产生的,因此辣椒素也可能是P-gp的一种底物。由于Caco-2细胞本身过表达P-gp以及研究部位的局限性,本文我们设想通过体外chamber的方法研究了辣椒素在不同肠段透过时受P-gp的影响。另一方面,同属于多药耐药蛋白的MRP2和BCRP与P-gp之间的底物具有很高的重叠性,而对于MRP2或BCRP与辣椒素之间的相互关系还未见报道。我们进一步分别用丙磺舒和新生霉素作为MRP2和BCRP抑制剂,研究这两种转运体对辣椒素经肠粘膜转运时的影响。
由于TRPV1以及ABC转运体在肠粘膜的表达上具有区段差异性,我们考虑这种差异是否也会造成辣椒素在肠段的转运存在一种区段差异。因此,在本研究中,我们比较了辣椒素在不同肠段透过的差异性。并以荧光素钠作为旁细胞通路指示剂揭示了辣椒素经不同肠段肠黏膜透过时上皮细胞紧密连接的打开情况,以了解肠粘膜的状态。用钌红或抗辣椒碱作为阻塞剂,研究了TRPV1通道在辣椒素透过中所起的作用。并用维拉帕米,新生霉素和丙磺舒分别作为外排转运体抑制剂评价了这些外排转运体对辣椒素经空回结肠转运的影响。
为了评价辣椒素体外经不同肠段透过与体内实验的相关性,本研究进行了In situ肠封闭实验,In situ体内实验中,应用LC-MS/MS建立了大鼠血浆中检测辣椒素的方法学,期望通过在体环境下对辣椒素经不同肠段的吸收情况进行检测来验证体外实验与体内试验的相关性。
内容和结果：
首先我们建立了辣椒素的高效液相检测方法,用于测定体外经肠粘膜透过的样品中辣椒素的含量。体外透过样品不需要特殊处理,取样后直接15000r/m高速离心即可进样,测定条件确定为：检测器为紫外检测器(SPD-20A),色谱柱为Ecosil C18(150mm×4.6mm,5μm),进样量为20μL,柱温箱30℃。流动相：甲醇-水(70：30),流速：1.2mL·min-1,分析时间：7min,检测波长：280nnm。
应用荧光分光光度计,建立R123及CF检测方法学。R123的激发波长为485nm,发射波长为535nm。R123在15.625-500μg·L-1,荧光强度对浓度进行回归,回归方程为Y=1.0435X-1.0989(R2=0.9982),线性关系良好,回收率和日内精密度分别为：98.9%和1.9%。CF的激发波长为480nm,发射波长为520nm,可在此波长下测定CF的荧光强度,空白无干扰。CF在7.8125-500μg·L-1,荧光强度对浓度进行回归,回归方程为Y=0.639X+0.068(R2=0.9997),线性关系良好,回收率和日内精密度分别为：97.8%和2.7%。
应用高效液相质谱联用建立血浆中辣椒素的检测方法。血浆样本100μL加入终浓度为50ng/ml的内标,用混合溶剂乙酸乙酯：丙酮(85：15)萃取后用安捷伦6460三重四级杆质谱系统进行分析。采用多离子反应监测(MRM)扫描方式,在ESI源正离子模式下,选择m/z306→137作为MRM监测的离子对,m/z455→165为内标物维拉帕米的MRM监测离子对。结果：样品分析时间仅需2.5min,方法线性范围在1.85-370ng/ml (R2=0.9997),最低检测限为1.85ng/ml。辣椒素在三个质控浓度(3.7,37,370ng/ml)检测下的提取回收率为77.34%,70.64%及78.02%。
使用体外Ussing chamber实验评价辣椒素经不同肠段透过的差异,计量资料统计结果用均数±标准差(X±S)表示。各组间不同方向,不同区段的Papp比较用析因设计资料的方差分析,不同组间多重比较采用SNK法检测。显著性标准为P<0.05。
对不同区段M-S方向的Papp进行One way ANOVA方差分析,结果显示,辣椒素经不同肠段M-S方向透过的Papp具有显著性差异(F=12.290,P=0.001)。进一步多重比较结果显示,辣椒素经三个肠段粘膜M-S方向透过的Papp之间均存在显著性差异,表现为结肠>回肠>空肠。对不同区段S-M方向的Papp进行One way ANOVA方差分析,结果显示,辣椒素经不同肠段S-M方向透过的Papp具有显著性差异(F=5.749,P=0.027)。进一步多重比较结果显示,辣椒素经结肠粘膜S-M方向透过的Papp与空肠相比,具有显著性差异,空肠与回肠、结肠与回肠相比,没有显著性差异(P=0.324、P=0.261)。上述统计结果表明辣椒素体外在结肠段具有特异性透过的特性。当辣椒素的浓度增高时,辣椒素经各肠段吸收方向的透过趋势线拟合方程良好,随着浓度的增高,其Papp并没有大幅增加。
对R123、CF经不同区段肠粘膜的经时吸收方向和分泌方向的影响,用累计透过率和表观渗透系数(Papp)表示。计量资料统计结果用均数±标准差(X±S)表示。药物不同区段的Papp比较用析因设计资料的方差分析,不同组间多重比较采用SNK法检验；各组累计透过率的比较用重复测量数据的方差分析,各组间不同方向,不同区段的Papp的比较用析因设计资料的方差分析。显著性标准为P<0.05。
结果显示,经空肠粘膜透过时,辣椒素对R123经空肠粘膜M-S方向和S-M方向透过的影响具有显著性,辣椒素提高了M-S方向的R123透过,降低了其S-M方向的透过。经回肠粘膜透过时,辣椒素对R123经回肠粘膜M-S方向和S-M方向透过的影响不具有显著性。经结肠黏膜透过时,辣椒素对R123经结肠粘膜M-S方向和S-M方向透过的影响不具有显著性。由此说明,辣椒素通过抑制P-gp,从而介导R123透过性质改变的这种影响仅存在于空肠,在回肠和结肠没有影响。
辣椒素对CF经空肠、回肠和结肠粘膜M-S方向和S-M方向的影响结果显示。经空肠粘膜透过时,辣椒素对CF经空肠粘膜M-S方向和S-M方向透过的影响具有显著性(F=24.239,P=0.000),辣椒素提高了CF的双向透过性。经回肠粘膜透过时,辣椒素对CF经回肠粘膜M-S方向和S-M方向透过的影响不具有显著性(F=2.260,P=0.152)。经结肠黏膜透过时,辣椒素对CF经结肠粘膜M-S方向和S-M方向透过的影响不具有显著性(F=0.882,P=0.362)。由此说明,辣椒素可能打开了空肠粘膜上的紧密连接,从而使得经旁细胞通道透过的CF的肠粘膜双向透过增加,而对回肠和结肠粘膜上的紧密连接没有影响。
TRPV1通道竞争性抑制剂钌红(RR)对辣椒素体外透过的影响中,所有样品均用平均值±标准差来表示。LDH含量测定的均数比较采用独立样本t检验对数据的显著性进行分析。当数据为P<0.05时则认为具有显著性。钌红对辣椒素经各肠段透过的影响结果用one way ANO VA进行分析,方差不齐时做方差分析的稳健估计welch检验。方差分析具有显著性差异时,方差齐时采用SNK法,不齐时用DunnetT3法进行不同区段间的多重比较检测检验。
在完成2h体外透过实验后,分别用LDH试剂盒检测了辣椒素存在或不存在情况下的LDH水平。从空肠,回肠和结肠释放的LDH分别于与对照组相比均没有显著性差异(t=-0.918,P=0.394；t=0.402,P=0.702；t=-2.002,P=0.092)。说明辣椒素没有引起各段肠粘膜的损伤。
通过将TRPV1通道竞争性抑制剂钌红(RR)与辣椒素同时进行体外透过,考察TRPV1在辣椒素经肠粘膜透过中的作用。结果显示,20μM RR组显著降低了辣椒素经结肠粘膜的双向透过,10μM RR组显著降低了辣椒素经结肠粘膜吸收方向的透过。在对M-S方向的不同肠段透过分别用One way ANOVA进行统计学分析发现：在对照组中：不同肠段间的Papp具有显著性差异(F=15.096,P=0.000),多重比较结果显示,结肠和空肠,结肠和回肠间具有显著性差异；空肠和回肠间不具有显著性差异(P=0.075)。在10μM组中：不同肠段间的Papp没有显著性差异(F=3.401,P=0.063)。在20μM组中：不同肠段间的Papp没有显著性差异(F=2.359,P=0.137)。说明RR可能通过抑制TRPV1通道从而对辣椒素经结肠粘膜的透过产生了影响。
肠粘膜上多药耐药蛋白对辣椒素经肠粘膜转运的影响中,所有样品均用平均值±标准差来表示。当数据为P<0.05时则认为具有显著性。维拉帕米/丙磺舒/新生霉素对辣椒素经各肠段透过的影响结果用独立样本t检验进行分析,维拉帕米/丙磺舒/新生霉素组的辣椒素经三个肠段吸收方向的透过用one way ANOVA分析。
P-gp对辣椒素经肠粘膜透过的影响结果显示,在经空肠、回肠和结肠粘膜的双向透过中,辣椒素均未受到维拉帕米的影响,并且辣椒素经三个肠段吸收方向结肠特异性透过特性没有被改变。
MRP2对辣椒素经肠粘膜透过的影响结果显示,在经空肠、回肠和结肠粘膜的双向透过中,辣椒素均未受到丙磺舒的影响,并且辣椒素经三个肠段吸收方向结肠特异性透过特性没有被改变。
BCRP对辣椒素经肠粘膜透过的影响结果显示,新生霉素显著降低了辣椒素经回肠和结肠粘膜吸收方向的透过,但对辣椒素经空肠和回肠粘膜的双向透过没有影响。
在体In situ实验测定辣椒素经不同肠段吸收后的血药浓度,研究辣椒素在体吸收情况,计算各种药动学参数。实验资料结果用均数±标准差表示,采用SPSS13.0统计软件包软件进行统计分析。辣椒素各组浓度与峰面积的效应关系用线性回归分析(Linear Regression)。辣椒素在肠道内吸收后Cmax、Tmax、 AUC0-240min、F的均数比较各自均采用单向方差分析(One-Way ANOVA),如果有显著性差异,则组间两两比较采用SNK法(方差齐性时)或Dunnett T3法(方差不齐时)；
实验发现,辣椒素经不同肠段吸收后,结肠的曲线下面积AUC0-240min和最高血浆浓度Cmax的平均值大于回肠和空肠,但经One way ANOVA统计学分析,三个肠段定位吸收后血浆中辣椒素的Cmax、Tmax、AUC0-240无统计学差异。体内研究结果并没有出现体外研究中所呈现的具有显著性的结肠特异性吸收特性。
结论和讨论：
辣椒素体外透过结果显示,辣椒素的经肠粘膜透过与其他许多药物不同,在肠道内的转运具有区段性差异,在结肠部位的透过显著性大于空肠和回肠。然而体内试验虽显示辣椒素的结肠曲线下面积AUC0-240min和最高血浆浓度Cmax的平均值大于回肠和空肠,但却没有统计学差异,这可能与体内研究的干扰因素要比体外复杂有关,而这些因素都可能间接影响了辣椒素的吸收。提示我们,对于体外研究和体内研究的完全相关性仍然是值得我们进行深层次,多方面的考察。
在研究肠黏膜上转运体与辣椒素转运间的相互影响中发现,辣椒素能够通过抑制P-gp影响R123的透过,并能够打开肠黏膜上紧密连接增加CF的双向透过。但辣椒素抑制P-gp功能从而影响P-gp底物R123经肠粘膜透过的作用具有肠段差异性,对R123的影响仅仅存在于空肠,而在回肠和结肠却影响很小。此外辣椒素影响经旁细胞通路透过的药物CF的经肠粘膜透过时也仅在空肠有显著性影响,在回肠和结肠均无影响,这些可能都与P-gp及紧密连接在肠粘膜上的分布是从空肠到结肠逐渐增高的分布有关。辣椒素对经旁细胞通路透过的药物CF的经回肠和结肠粘膜透过没有影响,这也暗示了辣椒素的结肠特异性透过性与肠黏膜上紧密连接的打开可能没有关系,而存在着其他转运蛋白的影响。
其次,TRPV1通道抑制剂钌红显著的降低了辣椒素经结肠段吸收方向的透过,但是对辣椒素经空肠或回肠的透过没有影响。这说明TRPV1参与了辣椒素经结肠粘膜在吸收方向的透过。更有趣的是,我们发现在钌红存在时,辣椒素经不同肠粘膜的透过性没有差异。而没有钌红时,辣椒素经不同肠段的透过却呈现出区段差异性。说明钌红通过抑制TRPV1改变了辣椒素经肠粘膜透过的特性。
第三,新生霉素能够通过抑制BCRP这种外排蛋白而增加其底物的透过。但结果却相反,辣椒素在所有肠段吸收方向的透过均有降低的趋势,尤其是在回肠和结肠吸收方向的透过显著降低。这似乎说明新生霉素抑制了某种介导辣椒素转运的转运体或通道,这也许是辣椒素经肠粘膜转运的一种尚未发现的新途径,值得我们进一步的研究。
Background&Objective
Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a primary pungent and irritating principle present in chilies and red peppers. Mol Wt:305.42. It has been widely used not only as food additive, but also as a potent drug to treat neuropathic pain in clinical. The Food and Drug Administration (FDA) has approved the approval of Qutenza (capsaicin)8%patch to treat postherpetic neuralgia. In addition, there is some researches showed that capsaicin at sub-cytotoxic concentrations was capable of inhibiting the P-gp efflux activity to a comparable level as the established P-gp inhibitor, verapamil. It has demonstrated that capsaicin could reverse doxorubicin resistance in human multidrug resistant carcinoma KB-C2cells by inhibiting the P-gp efflux pump, and increased AB digoxin transport and inhibited BA digoxin transport across the Caco-2cells at low concentration. Also, capsaicin can alters both transcellular and paracellular pathways of intestinal cephalexin and cefazolin absorption. As hot chilli is consumed on a daily basis in many populations, it is necessary to understand the transport mechanism of capsaicin through intestine. However, the research and application of capsaicin mainly concentrated in percutaneous absorption, it is still unclear about the mechanism of capsaicin on intestinal absorption. Therefore, we studied the permeation of capsaicin across different intestinal segments to understand the relationship between capsaicin and transporters or channel on the membrane. This article selects transporters or channel is mainly considered based on below:The application of capsaicin as an analgesic to treat neuropathic pain is benefiting from its highly selective activating lig and for transient receptor potential vanilloid1(TRPV1). TRPV1is a detector for noxious heat (>43℃) and inflammatory pain. The permeation of some small metal ions acrossTRPVlis dependent on the activation of TRPV1. It has been found that TRPV1could be opened to increase the permeation of large cations by some chemical regulators such as capsaicin. Moreover, it has been proved that the TRPV1channel has at least two binding sites for CAP, extracellular and intracellular sides of the membrane. The binding of CAP to one site of the channel may interact the binding of CAP to another site. Consequently, we conjecture whether the action of TRPV1by capsaicin could also mediate the permeation of itself. The hypothesis can be judged by blocking the TRPV1with ruthenium red (a TRPV channel blocker) in the transport of capsaicin across different intestinal segments, including jejunum, ileum and colon. Besides, it has been reported that capsaicin is an inhibitor of P-gp. So far, it has been suggested that a P-gp inhibitor usually be also a P-gp substrate such as mibefradil. Additionally, most P-gp substrates are of relatively hydrophobic nature because they are able to access the hydrophobic binding site. Therefore, we suspected that capsaicin might be a P-gp substrate because of its hydrophobicity. We investigated the effect of P-gp on the transport of capsaicin across different intestinal regions in the study. Furthermore, the substrates of multidrug resistance proteins such as multidrug resistance-associated protein2(MRP2) and breast cancer resistance protein (BCRP) are often overlapped with those of p-gp, and no report has been published to evaluate the effect of MRP2or BCRP on the permeation of capsaicin. Therefore, we designed to use probenecid (MRP2inhibitor) and novobiocin (BCRP inhibitor) to study the probable impact of these transporters on the permeability of capsaicin across different intestinal segments.In the study, capsaicin with absence of the inhibitors was set as the control group. As the expression levels of TRPV1and multidrug resistance proteins along the intestine track appear to be difference, all of the experiments were performed across jejunal, ileac and colonic membranes. Additionally, Na-fluorescein (Na-Flu), an indicator of the paracellular pathway, was used to reveal the condition of the intestinal epithelial TJ barrier on different intestine. In all, in this study we evaluated the permeation differences of capsaicin across three intestinal segments. The contribution of TRPV1in the intestinal permeation of capsaicin was evaluated by co-using RR as a TRPV1blocker. Moreover, the roles of multidrug resistance proteins in the transporting of capsaicin were investigated by co-using verapamil, probenecid, and novobiocin, respectively. And in order to validate the correlation between in vitro and in vivo absorption of capsaicin, in situ closed loop method has been used to measure the plasma levels.
Content&Results:
First, we establish the HPLC method for the determination of capsaicin in the transdermal solution obtained from the intestinal transport study. There is no need to have Special Handling for the transdermal solution, it can be injected into HPLC after high speed centrifugation on15000r/m. Capsaicin was analyzed using a Ecosil C18(150mmx4.6mm,5μm) with a flow rate of1.2ml/min at30℃. The mobile phase consisted of methanol-water (70:30, v/v), detection wavelength were280nm in7min.
A fluorospectro photometer method for determining the concenteation of R123and CF was established. The EX was set at485nm and Em was set at535nm for R123. Calibration Curve of R123was the follow:Y=1.0435X-1.0989(R2=0.9982), and the linearity was good in the range of15.625～500μg·L-1, with the approach recovery and within-day RSD of R123were98.9%and1.9%. The EX was set at490nm and Em was set at520nm for CF. Calibration Curve of CF was the follow: Y=0.639X+0.068(R2=0.9997), and the linearity was good in the range of7.8125～500μg·L-1, with the approach recovery and within-day RSD of CF were97.8%and2.7%.
A HPLC-MS/MS detector method for determining the concenteation of capsaicin was established in rat plasma. Capsaicin was extracted from aliquots of plasma with acetone and ethyl acetate (15:85) as the solvent and chromatographic separation was performed using a ZorbaxEclipse Plus C18column. To quantify capsaicin and IS respectively, multiple reaction monitoring (MRM) transition of m/z306→137and m/z455→165was performed. The analysis time was2.5min in positive mode; the calibration curve was linear in the concentration range of1.85-370ng/ml (R2=0.9997) ng/ml. The lowest limit of quantification (LLOQ) reachedl.85ng/ml. The recovery for the QC (3.7,37,370ng/ml) were77.34%,70.64%and78.02%, respectively.
The permeability of capsaicin via different intestinal membranes was evaluated by an in vitro diffusion chamber system at different concentrations. Data in all experiments were presented as the mean±SD. Statistical differences were tested by Factorial design analysis of variance. Differences were considered to be significant when P<0.05.
There were regional differences in the in vitro permeability of capsaicin(F=17.808, P=0.000). The Papp of capsaicin in colon was also significantly higher than that in jejunum or ileum in the M-S direction.
The permeability of R123or CF via different intestinal membranes was evaluated by an in vitro diffusion chamber system, with or without the co-administration of capsaicin. Data in all experiments were presented as the mean±SD. Statistical differences were tested by Factorial design analysis of variance. Differences were considered to be significant when P<0.05.
The mucosal-to-serosal (M-S) of R123was increased and its serosal-to-mucosal transport (S-M) transport was decreased with co-administration of capsaicin across jejunum transport. However, no action of capsaicin at test concentration was found on the transport of R123across ileum or colon.
The mucosal-to-serosal (M-S) and serosal-to-mucosal transport (S-M) transport of CF were both increased with co-administration of capsaicin across jejunum transport. However, no action of capsaicin at test concentration was found on the transport of CF across ileum or colon.
The contribution of TRPV1to the transport of capsaicin was performed by co-administrating RR as a blocker of the TRPV1. Data in all experiments were presented as the mean±SD. Statistical differences were tested by one-way ANOVA and independent-sample test. Differences were considered to be significant when P<0.05.
The activity of LDH at2h after completing the permeation of capsaicin (200μM) through the intestine was detected by LDH kit. There is no significant effect on the release of LDH from jejunal, ileac or colonic membrane (t=-0.918, P=0.394; t=0.402, P=0.702; t=-2.002, P=0.092). It suggests that capsaicin may not cause intestinal membrane damage in the study.
RR significantly reduced the M-S direction transport of capsaicin across colon region, while no effect on the transport of capsaicin across jejunum or ileum. However, there were no statistical differences in the M-S permeation of capsaicin among different intestinal regions in presence of ruthenium red. Additionally, there was no further decrease of the permeation of capsaicin across colon when the concentration of RR was increased to a higher concentration.
The influence of transporter proteins in the intestinal bidirectional transportation of capsaicin, jejunum, ileum and colon were performed respectively. Data in all experiments were presented as the mean±SD. Statistical differences were tested by one-way ANOVA and independent-sample test. Differences were considered to be significant when P<0.05.
Verapamil was applied as P-gp inhibitor, which showed it had no significant effect on M-S or S-M direction of three intestinal segments for the transport of capsaicin.
There is no effect on the absorption of capsaicin across the different intestinal regions by co-using MRP2inhibitor probenecid. In addition, the efflux of capsaicin was not significantly reduced by probenecid.
However, the BCRP inhibitor novobiocin significantly reduced the M-S transport of capsaicin across ileum and colon, while no effect on the M-S transport of capsaicin across jejunum. It did not affect the S-M transport of capsaicin in each intestinal region.
Rat plasma concentrations and pharmacokinetic parameters of capsaicin were examined by in situ closed loop method. Data in all experiments were presented as the meaniSD. Statistical differences were tested by one-way ANOVA. Differences were considered to be significant when P<0.05.
It was found that the largest plasma concentration (Cmax), area under curve (AUC) of capsaicin absorbed in colon were higher than it in jejunum or ileum. However, there is no statistics difference when analyzed by one-way ANOVA.
Conclusion&Discussion
In this primary study, we excitedly realized that it was the first time to find the permeability of capsaicin across the colonic mucosa in M-S direction is remarkably higher than that across jejunal or ileac mucosa. However, there is no statistics difference for the absorption of capsaicin by in situ across different intestinal regions. As the influencing factors are much complex in in situ, some indirect interaction may affect the absorption of capsaicin, which have not been studied clarity. The result suggests that there is still a further study for the correlation between in vitro and in situ method.
It shows that capsaicin could affect the permeation of R123by inhibiting P-gp, and increased the permeation of CF by opening the paracellular pathway. However, these effects were only appeared through the jejunal membrane. This may because the regional distribution difference of P-gp and tight junction (TJ) along the intestine were both increased from jejunum to colon.
Secondly, RR significantly reduced the M-S direction transport of capsaicin across colon region, while no effect on the transport of capsaicin across jejunum or ileum. However, there were no statistical differences in the M-S permeation of capsaicin among different intestinal regions in presence of ruthenium red. Additionally, there was no further decrease of the permeation of capsaicin across colon when the concentration of RR was increased to a higher concentration (20μM). It seems that RR alters the characteristic of capsaicin transport across the intestinal membranes.
BCRP is also an efflux transporter belonging to multidrug resistance proteins, and the inhibition of BCRP by novobiocin could increase the permeability of its substrates. However, our data showed that the permeability of capsaicin was decreased dramatically in all the segments in presence of novobiocin, especially in ileal colonic mucosa. It seemed that the transport of capsaicin was not the substrate of BCRP. There should be another transporter, which could pump capsaicin from mucosal to serosal side, and the transporter could be inhibited by novobiocin.
辣椒素(capsaicin, CAP,(反式)8-甲基-N-香草基-6-壬烯酰胺)是辣椒中产生辛辣的主要成分,被广泛用作调味料。分子量：305.42。辣椒素不仅广泛用于食品添加,也是临床治疗神经性疼痛的有效药物。美国食品药品监督管理局(FDA)已经通过了含8%辣椒素的贴剂(Qutenza)用于治疗带状孢疹后神经痛。不仅如此,也有诸多研究表明辣椒素在低于细胞毒性的浓度时就能够抑制P-糖蛋白(P-gp)活性,并且这种抑制水平与已公认的P-gp抑制剂维拉帕米相当。辣椒素能够逆转阿霉素在人类多重耐药癌细胞KB-C2中的耐药性,而且在很低的浓度时既可显著增加P-gp底物如地高辛等的透过。同时,也有研究表明辣椒素可以通过影响肠粘膜的通透性而使某些抗生素药物如头孢唑林,头孢氨苄的血药浓度增加。基于辣椒素同一些药物的相互作用以及辣椒素是许多人日常食用品中的主要成分之一,故有必要首先了解辣椒素的经肠转运机制。然而目前对于辣椒素的研究及应用主要集中在体外经皮吸收方面,而对于其在体内的吸收转运机制几乎未见报道。
因此,本文通过研究辣椒素经不同肠段的透过性来考察辣椒素与肠粘膜上一些转运体或通道之间的关系。这些转运体或通道的确定是基于以下考虑：
辣椒素的镇痛应用主要得益于它对瞬态电压感受器阳离子通道(TRPV1)具有高度的选择性。TRPV1是一种温度和疼觉感受器,一些小的金属离子的透过依赖于TRPV1通道的活性,但TRPV1通道可能会被一些化学调节剂激活并打开,从而促使一些大的阳离子化合物的快速透过。这说明TRPV1通道能够作为透过途径而被激活从而促进药物的转运。此外,有报道指出辣椒素与TRPV1至少存在两个结合位点,胞内和胞外,并且它与其中一个位点的结合会加速其对另一个位点的结合。所以我们猜测辣椒素对TRPVl通道的激活是否会促进其自身的转运呢?因此,我们假设TRPV1通道参与了辣椒素经肠粘膜的透过,并用TRPV1非竞争性抑制剂钌红(RR)阻塞TRPV1通道来研究TRPV1通道对辣椒素在不同肠粘膜透过及吸收的影响。
此外,Yi Han等研究者用Caco-2细胞研究发现,辣椒素是一种P-gp抑制剂。Loo, T.W等研究者提出由于辣椒素的抑制作用是能够刺激ATP苷酶的激活产生的,因此辣椒素也可能是P-gp的一种底物。由于Caco-2细胞本身过表达P-gp以及研究部位的局限性,本文我们设想通过体外chamber的方法研究了辣椒素在不同肠段透过时受P-gp的影响。另一方面,同属于多药耐药蛋白的MRP2和BCRP与P-gp之间的底物具有很高的重叠性,而对于MRP2或BCRP与辣椒素之间的相互关系还未见报道。我们进一步分别用丙磺舒和新生霉素作为MRP2和BCRP抑制剂,研究这两种转运体对辣椒素经肠粘膜转运时的影响。
由于TRPV1以及ABC转运体在肠粘膜的表达上具有区段差异性,我们考虑这种差异是否也会造成辣椒素在肠段的转运存在一种区段差异。因此,在本研究中,我们比较了辣椒素在不同肠段透过的差异性。并以荧光素钠作为旁细胞通路指示剂揭示了辣椒素经不同肠段肠黏膜透过时上皮细胞紧密连接的打开情况,以了解肠粘膜的状态。用钌红或抗辣椒碱作为阻塞剂,研究了TRPV1通道在辣椒素透过中所起的作用。并用维拉帕米,新生霉素和丙磺舒分别作为外排转运体抑制剂评价了这些外排转运体对辣椒素经空回结肠转运的影响。
为了评价辣椒素体外经不同肠段透过与体内实验的相关性,本研究进行了In situ肠封闭实验,In situ体内实验中,应用LC-MS/MS建立了大鼠血浆中检测辣椒素的方法学,期望通过在体环境下对辣椒素经不同肠段的吸收情况进行检测来验证体外实验与体内试验的相关性。
内容和结果：
首先我们建立了辣椒素的高效液相检测方法,用于测定体外经肠粘膜透过的样品中辣椒素的含量。体外透过样品不需要特殊处理,取样后直接15000r/m高速离心即可进样,测定条件确定为：检测器为紫外检测器(SPD-20A),色谱柱为Ecosil C18(150mm×4.6mm,5μm),进样量为20μL,柱温箱30℃。流动相：甲醇-水(70：30),流速：1.2mL·min-1,分析时间：7min,检测波长：280nnm。
应用荧光分光光度计,建立R123及CF检测方法学。R123的激发波长为485nm,发射波长为535nm。R123在15.625-500μg·L-1,荧光强度对浓度进行回归,回归方程为Y=1.0435X-1.0989(R2=0.9982),线性关系良好,回收率和日内精密度分别为：98.9%和1.9%。CF的激发波长为480nm,发射波长为520nm,可在此波长下测定CF的荧光强度,空白无干扰。CF在7.8125-500μg·L-1,荧光强度对浓度进行回归,回归方程为Y=0.639X+0.068(R2=0.9997),线性关系良好,回收率和日内精密度分别为：97.8%和2.7%。
应用高效液相质谱联用建立血浆中辣椒素的检测方法。血浆样本100μL加入终浓度为50ng/ml的内标,用混合溶剂乙酸乙酯：丙酮(85：15)萃取后用安捷伦6460三重四级杆质谱系统进行分析。采用多离子反应监测(MRM)扫描方式,在ESI源正离子模式下,选择m/z306→137作为MRM监测的离子对,m/z455→165为内标物维拉帕米的MRM监测离子对。结果：样品分析时间仅需2.5min,方法线性范围在1.85-370ng/ml (R2=0.9997),最低检测限为1.85ng/ml。辣椒素在三个质控浓度(3.7,37,370ng/ml)检测下的提取回收率为77.34%,70.64%及78.02%。
使用体外Ussing chamber实验评价辣椒素经不同肠段透过的差异,计量资料统计结果用均数±标准差(X±S)表示。各组间不同方向,不同区段的Papp比较用析因设计资料的方差分析,不同组间多重比较采用SNK法检测。显著性标准为P<0.05。
对不同区段M-S方向的Papp进行One way ANOVA方差分析,结果显示,辣椒素经不同肠段M-S方向透过的Papp具有显著性差异(F=12.290,P=0.001)。进一步多重比较结果显示,辣椒素经三个肠段粘膜M-S方向透过的Papp之间均存在显著性差异,表现为结肠>回肠>空肠。对不同区段S-M方向的Papp进行One way ANOVA方差分析,结果显示,辣椒素经不同肠段S-M方向透过的Papp具有显著性差异(F=5.749,P=0.027)。进一步多重比较结果显示,辣椒素经结肠粘膜S-M方向透过的Papp与空肠相比,具有显著性差异,空肠与回肠、结肠与回肠相比,没有显著性差异(P=0.324、P=0.261)。上述统计结果表明辣椒素体外在结肠段具有特异性透过的特性。当辣椒素的浓度增高时,辣椒素经各肠段吸收方向的透过趋势线拟合方程良好,随着浓度的增高,其Papp并没有大幅增加。
对R123、CF经不同区段肠粘膜的经时吸收方向和分泌方向的影响,用累计透过率和表观渗透系数(Papp)表示。计量资料统计结果用均数±标准差(X±S)表示。药物不同区段的Papp比较用析因设计资料的方差分析,不同组间多重比较采用SNK法检验；各组累计透过率的比较用重复测量数据的方差分析,各组间不同方向,不同区段的Papp的比较用析因设计资料的方差分析。显著性标准为P<0.05。
结果显示,经空肠粘膜透过时,辣椒素对R123经空肠粘膜M-S方向和S-M方向透过的影响具有显著性,辣椒素提高了M-S方向的R123透过,降低了其S-M方向的透过。经回肠粘膜透过时,辣椒素对R123经回肠粘膜M-S方向和S-M方向透过的影响不具有显著性。经结肠黏膜透过时,辣椒素对R123经结肠粘膜M-S方向和S-M方向透过的影响不具有显著性。由此说明,辣椒素通过抑制P-gp,从而介导R123透过性质改变的这种影响仅存在于空肠,在回肠和结肠没有影响。
辣椒素对CF经空肠、回肠和结肠粘膜M-S方向和S-M方向的影响结果显示。经空肠粘膜透过时,辣椒素对CF经空肠粘膜M-S方向和S-M方向透过的影响具有显著性(F=24.239,P=0.000),辣椒素提高了CF的双向透过性。经回肠粘膜透过时,辣椒素对CF经回肠粘膜M-S方向和S-M方向透过的影响不具有显著性(F=2.260,P=0.152)。经结肠黏膜透过时,辣椒素对CF经结肠粘膜M-S方向和S-M方向透过的影响不具有显著性(F=0.882,P=0.362)。由此说明,辣椒素可能打开了空肠粘膜上的紧密连接,从而使得经旁细胞通道透过的CF的肠粘膜双向透过增加,而对回肠和结肠粘膜上的紧密连接没有影响。
TRPV1通道竞争性抑制剂钌红(RR)对辣椒素体外透过的影响中,所有样品均用平均值±标准差来表示。LDH含量测定的均数比较采用独立样本t检验对数据的显著性进行分析。当数据为P<0.05时则认为具有显著性。钌红对辣椒素经各肠段透过的影响结果用one way ANO VA进行分析,方差不齐时做方差分析的稳健估计welch检验。方差分析具有显著性差异时,方差齐时采用SNK法,不齐时用DunnetT3法进行不同区段间的多重比较检测检验。
在完成2h体外透过实验后,分别用LDH试剂盒检测了辣椒素存在或不存在情况下的LDH水平。从空肠,回肠和结肠释放的LDH分别于与对照组相比均没有显著性差异(t=-0.918,P=0.394；t=0.402,P=0.702；t=-2.002,P=0.092)。说明辣椒素没有引起各段肠粘膜的损伤。
通过将TRPV1通道竞争性抑制剂钌红(RR)与辣椒素同时进行体外透过,考察TRPV1在辣椒素经肠粘膜透过中的作用。结果显示,20μM RR组显著降低了辣椒素经结肠粘膜的双向透过,10μM RR组显著降低了辣椒素经结肠粘膜吸收方向的透过。在对M-S方向的不同肠段透过分别用One way ANOVA进行统计学分析发现：在对照组中：不同肠段间的Papp具有显著性差异(F=15.096,P=0.000),多重比较结果显示,结肠和空肠,结肠和回肠间具有显著性差异；空肠和回肠间不具有显著性差异(P=0.075)。在10μM组中：不同肠段间的Papp没有显著性差异(F=3.401,P=0.063)。在20μM组中：不同肠段间的Papp没有显著性差异(F=2.359,P=0.137)。说明RR可能通过抑制TRPV1通道从而对辣椒素经结肠粘膜的透过产生了影响。
肠粘膜上多药耐药蛋白对辣椒素经肠粘膜转运的影响中,所有样品均用平均值±标准差来表示。当数据为P<0.05时则认为具有显著性。维拉帕米/丙磺舒/新生霉素对辣椒素经各肠段透过的影响结果用独立样本t检验进行分析,维拉帕米/丙磺舒/新生霉素组的辣椒素经三个肠段吸收方向的透过用one way ANOVA分析。
P-gp对辣椒素经肠粘膜透过的影响结果显示,在经空肠、回肠和结肠粘膜的双向透过中,辣椒素均未受到维拉帕米的影响,并且辣椒素经三个肠段吸收方向结肠特异性透过特性没有被改变。
MRP2对辣椒素经肠粘膜透过的影响结果显示,在经空肠、回肠和结肠粘膜的双向透过中,辣椒素均未受到丙磺舒的影响,并且辣椒素经三个肠段吸收方向结肠特异性透过特性没有被改变。
BCRP对辣椒素经肠粘膜透过的影响结果显示,新生霉素显著降低了辣椒素经回肠和结肠粘膜吸收方向的透过,但对辣椒素经空肠和回肠粘膜的双向透过没有影响。
在体In situ实验测定辣椒素经不同肠段吸收后的血药浓度,研究辣椒素在体吸收情况,计算各种药动学参数。实验资料结果用均数±标准差表示,采用SPSS13.0统计软件包软件进行统计分析。辣椒素各组浓度与峰面积的效应关系用线性回归分析(Linear Regression)。辣椒素在肠道内吸收后Cmax、Tmax、 AUC0-240min、F的均数比较各自均采用单向方差分析(One-Way ANOVA),如果有显著性差异,则组间两两比较采用SNK法(方差齐性时)或Dunnett T3法(方差不齐时)；
实验发现,辣椒素经不同肠段吸收后,结肠的曲线下面积AUC0-240min和最高血浆浓度Cmax的平均值大于回肠和空肠,但经One way ANOVA统计学分析,三个肠段定位吸收后血浆中辣椒素的Cmax、Tmax、AUC0-240无统计学差异。体内研究结果并没有出现体外研究中所呈现的具有显著性的结肠特异性吸收特性。
结论和讨论：
辣椒素体外透过结果显示,辣椒素的经肠粘膜透过与其他许多药物不同,在肠道内的转运具有区段性差异,在结肠部位的透过显著性大于空肠和回肠。然而体内试验虽显示辣椒素的结肠曲线下面积AUC0-240min和最高血浆浓度Cmax的平均值大于回肠和空肠,但却没有统计学差异,这可能与体内研究的干扰因素要比体外复杂有关,而这些因素都可能间接影响了辣椒素的吸收。提示我们,对于体外研究和体内研究的完全相关性仍然是值得我们进行深层次,多方面的考察。
在研究肠黏膜上转运体与辣椒素转运间的相互影响中发现,辣椒素能够通过抑制P-gp影响R123的透过,并能够打开肠黏膜上紧密连接增加CF的双向透过。但辣椒素抑制P-gp功能从而影响P-gp底物R123经肠粘膜透过的作用具有肠段差异性,对R123的影响仅仅存在于空肠,而在回肠和结肠却影响很小。此外辣椒素影响经旁细胞通路透过的药物CF的经肠粘膜透过时也仅在空肠有显著性影响,在回肠和结肠均无影响,这些可能都与P-gp及紧密连接在肠粘膜上的分布是从空肠到结肠逐渐增高的分布有关。辣椒素对经旁细胞通路透过的药物CF的经回肠和结肠粘膜透过没有影响,这也暗示了辣椒素的结肠特异性透过性与肠黏膜上紧密连接的打开可能没有关系,而存在着其他转运蛋白的影响。
其次,TRPV1通道抑制剂钌红显著的降低了辣椒素经结肠段吸收方向的透过,但是对辣椒素经空肠或回肠的透过没有影响。这说明TRPV1参与了辣椒素经结肠粘膜在吸收方向的透过。更有趣的是,我们发现在钌红存在时,辣椒素经不同肠粘膜的透过性没有差异。而没有钌红时,辣椒素经不同肠段的透过却呈现出区段差异性。说明钌红通过抑制TRPV1改变了辣椒素经肠粘膜透过的特性。
第三,新生霉素能够通过抑制BCRP这种外排蛋白而增加其底物的透过。但结果却相反,辣椒素在所有肠段吸收方向的透过均有降低的趋势,尤其是在回肠和结肠吸收方向的透过显著降低。这似乎说明新生霉素抑制了某种介导辣椒素转运的转运体或通道,这也许是辣椒素经肠粘膜转运的一种尚未发现的新途径,值得我们进一步的研究。
Background&Objective
Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a primary pungent and irritating principle present in chilies and red peppers. Mol Wt:305.42. It has been widely used not only as food additive, but also as a potent drug to treat neuropathic pain in clinical. The Food and Drug Administration (FDA) has approved the approval of Qutenza (capsaicin)8%patch to treat postherpetic neuralgia. In addition, there is some researches showed that capsaicin at sub-cytotoxic concentrations was capable of inhibiting the P-gp efflux activity to a comparable level as the established P-gp inhibitor, verapamil. It has demonstrated that capsaicin could reverse doxorubicin resistance in human multidrug resistant carcinoma KB-C2cells by inhibiting the P-gp efflux pump, and increased AB digoxin transport and inhibited BA digoxin transport across the Caco-2cells at low concentration. Also, capsaicin can alters both transcellular and paracellular pathways of intestinal cephalexin and cefazolin absorption. As hot chilli is consumed on a daily basis in many populations, it is necessary to understand the transport mechanism of capsaicin through intestine. However, the research and application of capsaicin mainly concentrated in percutaneous absorption, it is still unclear about the mechanism of capsaicin on intestinal absorption. Therefore, we studied the permeation of capsaicin across different intestinal segments to understand the relationship between capsaicin and transporters or channel on the membrane. This article selects transporters or channel is mainly considered based on below:The application of capsaicin as an analgesic to treat neuropathic pain is benefiting from its highly selective activating lig and for transient receptor potential vanilloid1(TRPV1). TRPV1is a detector for noxious heat (>43℃) and inflammatory pain. The permeation of some small metal ions acrossTRPVlis dependent on the activation of TRPV1. It has been found that TRPV1could be opened to increase the permeation of large cations by some chemical regulators such as capsaicin. Moreover, it has been proved that the TRPV1channel has at least two binding sites for CAP, extracellular and intracellular sides of the membrane. The binding of CAP to one site of the channel may interact the binding of CAP to another site. Consequently, we conjecture whether the action of TRPV1by capsaicin could also mediate the permeation of itself. The hypothesis can be judged by blocking the TRPV1with ruthenium red (a TRPV channel blocker) in the transport of capsaicin across different intestinal segments, including jejunum, ileum and colon. Besides, it has been reported that capsaicin is an inhibitor of P-gp. So far, it has been suggested that a P-gp inhibitor usually be also a P-gp substrate such as mibefradil. Additionally, most P-gp substrates are of relatively hydrophobic nature because they are able to access the hydrophobic binding site. Therefore, we suspected that capsaicin might be a P-gp substrate because of its hydrophobicity. We investigated the effect of P-gp on the transport of capsaicin across different intestinal regions in the study. Furthermore, the substrates of multidrug resistance proteins such as multidrug resistance-associated protein2(MRP2) and breast cancer resistance protein (BCRP) are often overlapped with those of p-gp, and no report has been published to evaluate the effect of MRP2or BCRP on the permeation of capsaicin. Therefore, we designed to use probenecid (MRP2inhibitor) and novobiocin (BCRP inhibitor) to study the probable impact of these transporters on the permeability of capsaicin across different intestinal segments.In the study, capsaicin with absence of the inhibitors was set as the control group. As the expression levels of TRPV1and multidrug resistance proteins along the intestine track appear to be difference, all of the experiments were performed across jejunal, ileac and colonic membranes. Additionally, Na-fluorescein (Na-Flu), an indicator of the paracellular pathway, was used to reveal the condition of the intestinal epithelial TJ barrier on different intestine. In all, in this study we evaluated the permeation differences of capsaicin across three intestinal segments. The contribution of TRPV1in the intestinal permeation of capsaicin was evaluated by co-using RR as a TRPV1blocker. Moreover, the roles of multidrug resistance proteins in the transporting of capsaicin were investigated by co-using verapamil, probenecid, and novobiocin, respectively. And in order to validate the correlation between in vitro and in vivo absorption of capsaicin, in situ closed loop method has been used to measure the plasma levels.
Content&Results:
First, we establish the HPLC method for the determination of capsaicin in the transdermal solution obtained from the intestinal transport study. There is no need to have Special Handling for the transdermal solution, it can be injected into HPLC after high speed centrifugation on15000r/m. Capsaicin was analyzed using a Ecosil C18(150mmx4.6mm,5μm) with a flow rate of1.2ml/min at30℃. The mobile phase consisted of methanol-water (70:30, v/v), detection wavelength were280nm in7min.
A fluorospectro photometer method for determining the concenteation of R123and CF was established. The EX was set at485nm and Em was set at535nm for R123. Calibration Curve of R123was the follow:Y=1.0435X-1.0989(R2=0.9982), and the linearity was good in the range of15.625～500μg·L-1, with the approach recovery and within-day RSD of R123were98.9%and1.9%. The EX was set at490nm and Em was set at520nm for CF. Calibration Curve of CF was the follow: Y=0.639X+0.068(R2=0.9997), and the linearity was good in the range of7.8125～500μg·L-1, with the approach recovery and within-day RSD of CF were97.8%and2.7%.
A HPLC-MS/MS detector method for determining the concenteation of capsaicin was established in rat plasma. Capsaicin was extracted from aliquots of plasma with acetone and ethyl acetate (15:85) as the solvent and chromatographic separation was performed using a ZorbaxEclipse Plus C18column. To quantify capsaicin and IS respectively, multiple reaction monitoring (MRM) transition of m/z306→137and m/z455→165was performed. The analysis time was2.5min in positive mode; the calibration curve was linear in the concentration range of1.85-370ng/ml (R2=0.9997) ng/ml. The lowest limit of quantification (LLOQ) reachedl.85ng/ml. The recovery for the QC (3.7,37,370ng/ml) were77.34%,70.64%and78.02%, respectively.
The permeability of capsaicin via different intestinal membranes was evaluated by an in vitro diffusion chamber system at different concentrations. Data in all experiments were presented as the mean±SD. Statistical differences were tested by Factorial design analysis of variance. Differences were considered to be significant when P<0.05.
There were regional differences in the in vitro permeability of capsaicin(F=17.808, P=0.000). The Papp of capsaicin in colon was also significantly higher than that in jejunum or ileum in the M-S direction.
The permeability of R123or CF via different intestinal membranes was evaluated by an in vitro diffusion chamber system, with or without the co-administration of capsaicin. Data in all experiments were presented as the mean±SD. Statistical differences were tested by Factorial design analysis of variance. Differences were considered to be significant when P<0.05.
The mucosal-to-serosal (M-S) of R123was increased and its serosal-to-mucosal transport (S-M) transport was decreased with co-administration of capsaicin across jejunum transport. However, no action of capsaicin at test concentration was found on the transport of R123across ileum or colon.
The mucosal-to-serosal (M-S) and serosal-to-mucosal transport (S-M) transport of CF were both increased with co-administration of capsaicin across jejunum transport. However, no action of capsaicin at test concentration was found on the transport of CF across ileum or colon.
The contribution of TRPV1to the transport of capsaicin was performed by co-administrating RR as a blocker of the TRPV1. Data in all experiments were presented as the mean±SD. Statistical differences were tested by one-way ANOVA and independent-sample test. Differences were considered to be significant when P<0.05.
The activity of LDH at2h after completing the permeation of capsaicin (200μM) through the intestine was detected by LDH kit. There is no significant effect on the release of LDH from jejunal, ileac or colonic membrane (t=-0.918, P=0.394; t=0.402, P=0.702; t=-2.002, P=0.092). It suggests that capsaicin may not cause intestinal membrane damage in the study.
RR significantly reduced the M-S direction transport of capsaicin across colon region, while no effect on the transport of capsaicin across jejunum or ileum. However, there were no statistical differences in the M-S permeation of capsaicin among different intestinal regions in presence of ruthenium red. Additionally, there was no further decrease of the permeation of capsaicin across colon when the concentration of RR was increased to a higher concentration.
The influence of transporter proteins in the intestinal bidirectional transportation of capsaicin, jejunum, ileum and colon were performed respectively. Data in all experiments were presented as the mean±SD. Statistical differences were tested by one-way ANOVA and independent-sample test. Differences were considered to be significant when P<0.05.
Verapamil was applied as P-gp inhibitor, which showed it had no significant effect on M-S or S-M direction of three intestinal segments for the transport of capsaicin.
There is no effect on the absorption of capsaicin across the different intestinal regions by co-using MRP2inhibitor probenecid. In addition, the efflux of capsaicin was not significantly reduced by probenecid.
However, the BCRP inhibitor novobiocin significantly reduced the M-S transport of capsaicin across ileum and colon, while no effect on the M-S transport of capsaicin across jejunum. It did not affect the S-M transport of capsaicin in each intestinal region.
Rat plasma concentrations and pharmacokinetic parameters of capsaicin were examined by in situ closed loop method. Data in all experiments were presented as the meaniSD. Statistical differences were tested by one-way ANOVA. Differences were considered to be significant when P<0.05.
It was found that the largest plasma concentration (Cmax), area under curve (AUC) of capsaicin absorbed in colon were higher than it in jejunum or ileum. However, there is no statistics difference when analyzed by one-way ANOVA.
Conclusion&Discussion
In this primary study, we excitedly realized that it was the first time to find the permeability of capsaicin across the colonic mucosa in M-S direction is remarkably higher than that across jejunal or ileac mucosa. However, there is no statistics difference for the absorption of capsaicin by in situ across different intestinal regions. As the influencing factors are much complex in in situ, some indirect interaction may affect the absorption of capsaicin, which have not been studied clarity. The result suggests that there is still a further study for the correlation between in vitro and in situ method.
It shows that capsaicin could affect the permeation of R123by inhibiting P-gp, and increased the permeation of CF by opening the paracellular pathway. However, these effects were only appeared through the jejunal membrane. This may because the regional distribution difference of P-gp and tight junction (TJ) along the intestine were both increased from jejunum to colon.
Secondly, RR significantly reduced the M-S direction transport of capsaicin across colon region, while no effect on the transport of capsaicin across jejunum or ileum. However, there were no statistical differences in the M-S permeation of capsaicin among different intestinal regions in presence of ruthenium red. Additionally, there was no further decrease of the permeation of capsaicin across colon when the concentration of RR was increased to a higher concentration (20μM). It seems that RR alters the characteristic of capsaicin transport across the intestinal membranes.
BCRP is also an efflux transporter belonging to multidrug resistance proteins, and the inhibition of BCRP by novobiocin could increase the permeability of its substrates. However, our data showed that the permeability of capsaicin was decreased dramatically in all the segments in presence of novobiocin, especially in ileal colonic mucosa. It seemed that the transport of capsaicin was not the substrate of BCRP. There should be another transporter, which could pump capsaicin from mucosal to serosal side, and the transporter could be inhibited by novobiocin.
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