肝硬化门脉高压时青藤碱促奥曲肽空肠胞旁吸收机制研究
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摘要
中国肝硬化人口十分庞大，而门脉高压是肝硬化失代偿期患者典型的临床表现，又是肝硬化多种并发症发生的根源及致死的主因。对门脉高压进行积极有效防治是改善患者预后及降低病死率的关键，但目前尚无理想的口服药物来预防门脉高压。
奥曲肽（octreotide, OCT）是目前公认的治疗肝硬化门脉高压的有效药物，虽可抵抗胃肠道中酸、酶的降解，但因分子量大，脂溶性差，受肠粘膜屏障和肝肠首过效应的影响，口服生物利用度极低，临床上仅限于注射给药，不利于作为预防性药物长期应用。OCT口服可以避免长期反复注射给药带来的痛苦和不便，临床意义重大。我们期待能像治疗高血压一样，实现OCT口服，达到早期、持续服药来防治门脉高压。国内外学者对此做了大量工作，但仍无任何一项研究成果可应用于临床。
本课题组前期实验建立了部分门脉结扎的门脉高压大鼠模型，证实广泛侧支循环的建立可有效地降低OCT肝首过效应，故肝首过效应并非门脉高压下OCT口服吸收的主要障碍。随后，又进一步研究了肝硬化门脉高压状态时空肠首过效应中起着关键作用的转运蛋白P-糖蛋白（p-glycoprotein, P-gp）、多耐药相关蛋白2（multidrug resistance-associated protein2, MRP2）和代谢酶细胞色素P4503A4（cytochrome P4503A4, CYP3A4），探讨了抑制肠上皮首过效应来增加OCT肠吸收的机制，故肠上皮首过效应问题也可得到解决。而对于OCT肠吸收至关重要的胞旁吸收途径则是本次研究的重点。本研究通过对肝硬化门脉高压大鼠空肠粘膜的形态学及分子水平的研究表明，门脉高压状态时空肠上皮紧密连接（tightjunction, TJ）蛋白Claudin-1表达减少，TJ松弛，肠粘膜屏障功能破坏，可使肠壁通透性增加，利于OCT的肠吸收。而通过Caco-2细胞模型证实促吸收剂青藤碱（sinomenine, SN）能够通过激活蛋白激酶C（protein kinase C, PKC）信号转导通路来调控TJ蛋白Claudin-1的表达，实现短暂可逆的调节胞旁TJ，有效提高OCT在Caco-2细胞单层中的渗透率。预实验初步证实SN能够增加正常大鼠OCT的肠吸收率，本研究则进一步在肝硬化门脉高压大鼠体内及体外吸收模型上证实SN有效的提高了OCT口服生物利用度，且在肝硬化门脉高压状态时其促吸收效果更为显著。
本研究旨在通过细胞及动物实验，揭示SN可逆性调控胞旁TJ促进OCT肠吸收的机制，为提高OCT口服生物利用度提供有效可行的方法及途径，为OCT临床合理用药提供理论依据，为实现口服OCT预防门脉高压开创新纪元。
本研究共分三部分
第一部分青藤碱可逆调控肠上皮细胞紧密连接促奥曲肽吸收的机制研究
第二部分肝硬化门脉高压对大鼠空肠上皮细胞紧密连接影响的研究
第三部分肝硬化门脉高压时青藤碱促奥曲肽空肠吸收研究
第一部分
青藤碱可逆调控肠上皮细胞紧密连接促奥曲肽吸收的机制研究
目的：在Caco-2细胞单层模型上评估SN对OCT肠吸收的影响，明确SN可逆调控肠上皮细胞TJ的分子机制。
方法：1.建立Caco-2细胞单层模型，并通过形态学、跨膜电阻（transepithelialelectrical resistance, TEER）测量及胞旁漏出标记物异硫氰酸荧光素标记的右旋糖酐-4000（FD-4）的渗透量来考察Caco-2细胞单层的完整性。
2.通过乳酸脱氢酶（lactate dehydrogenase, LDH）试验考察不同浓度组（0.5%、1%、2%w/v) SN对Caco-2细胞单层的损伤情况。
3.通过TEER的测量及FD-4渗透量的计算明确SN对TJ的可逆性调节作用。
4. SN对OCT肠吸收的影响。分组：Control组（10μM OCT）；SN组（0.5%SN+10μM OCT）。考察各组OCT的表观渗透系数（apparent permeability coefficient,Papp）。
5. SN对TJ蛋白Claudin-1表达的影响。分组为：Control组；SN组（0.5%SN）、SN去除组（SN removal）及PKC抑制剂组(0.5%SN+10μM Ro318220)。运用蛋白免疫印迹（western blot）、实时荧光定量聚合酶链式反应（Real-Time-PolymeraseChain Reaction, Real-Time-PCR）、免疫荧光染色（Immunofluorescent Staining, IF）方法分别考察各组中Claudin-1的表达。
6. SN对PKC信号转导通路的影响。分组：Control组，SN组（0.5%SN），运用western blot的方法分别考察PKC-α在对照组及SN组中细胞膜及胞浆中的表达。
结果：1.细胞培养21天后，倒置显微镜可见细胞之间的接触；经过测量和计
算，Caco-2细胞单层的TEER值均达到700Ω·cm2以上；1mg/ml的FD-4在120min
内的渗透量（AP-BL侧）无明显变化，一直处于较低水平（<0.5pmol/cm2），说明细胞间已形成完整的TJ。
2. LDH试验显示120min后，中浓度组（1%）及高浓度组（2%）的SN对细胞均有损伤，与对照组相比P<0.05；而低浓度组（0.5%）SN对细胞无明显损伤，与对照组相比P>0.05。
3.0.5%SN作用120min时，TEER%降至最低点，为起始TEER值的43%，FD-4的流出量达到最高，与对照组相比，P<0.05；去除SN的作用后，TEER%又迅速回升，在SN去除120min（总时间的240min）时恢复到起始水平，与对照组相比，P>0.05。
4.共同给予0.5%SN和10μM OCT120min时，OCT的Papp为5.89±0.038×106cm/s，是对照组（单给10μM OCT）的2.2倍（P<0.05）。
5. IF显示Control组中Claudin-1蛋白均匀表达于细胞膜，显示较强的绿色荧光；0.5%SN作用120min后，绿色荧光强度明显减弱，细胞间的连接出现断断续续的改变；去除SN120min时，细胞膜上的绿色荧光再次增强，细胞间的TJ完整性又恢复；而在SN+Ro318220共同作用120min时，细胞膜上的绿色荧光强度及细胞间TJ形态较给药前无明显变化。Western blot及Real-Time-PCR结果显示，0.5%SN组较Control组Claudin-1蛋白表达明显降低（P<0.05），SN removal组及0.5%SN+10μM Ro318220组Claudin-1蛋白表达均较Control组无明显差异（P>0.05）。
6.对照组中，PKC-α蛋白主要在细胞质内表达，细胞膜上表达量很少；而0.5%SN组，PKC-α则从细胞质中转移出来，主要表达于细胞膜上，表达量与Control组相比具有统计学意义(P<0.05)，这种PKC-α的转膜现象意味着PKC通路的激活。
结论：1.在Caco-2单层细胞模型上，SN能够促进OCT的吸收。
2. TJ蛋白Claudin-1在SN可逆性调控TJ中发挥着重要作用，且很可能与PKC信号转导通路的激活有关。
第二部分
肝硬化门脉高压对大鼠空肠上皮细胞紧密连接影响的研究
目的：在肝硬化门脉高压大鼠模型上，从形态学及分子水平对OCT胞旁吸收途径的主要结构—TJ进行研究，探讨肝硬化门脉高压对肠粘膜屏障的影响及机制。
方法：大鼠肝硬化门脉高压模型采用胆总管结扎（bile duct ligation, BDL）方法来制备。将健康成年雄性Sprague-Dawley（SD）大鼠随机分为2组，即Control和BDL组。BDL术后4周，对2组大鼠进行门静脉压力测定，之后处死大鼠，取血、取肝、取空肠，采用全自动生化分析仪检测血清丙氨酸氨基转移酶(alanineaminotransferase, ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase, AST)水平；HE染色观察肝脏病理形态学；透射电镜（transmission electron microscope,TEM）观察空肠上皮细胞间TJ形态；免疫组织化学染色（ImmunohistochemistryStaining, IHC）、western blot及Real-Time-PCR方法观察大鼠空肠Claudin-1蛋白及基因表达情况。
结果：1.大鼠门脉压力比较，BDL组较Control组明显升高（P<0.01）；对比ALT和AST值，BDL组较Control组明显升高(P<0.01)。
2.大鼠肝脏HE染色结果显示BDL组肝小叶结构紊乱，大量纤维组织增生，炎性细胞侵润，见多个假小叶，小叶间胆管增生明显。
3．TEM观察显示BDL大鼠空肠上皮细胞间TJ松弛，细胞间隙增宽。
4. IHC及western blot结果显示BDL组Claudin-1蛋白表达较Control组降低（P<0.01）；Real-Time-PCR方法显示BDL组Claudin-1基因表达较Control组降低（P<0.01）。
结论：肝硬化门脉高压大鼠空肠粘膜超微结构改变，TJ蛋白Claudin-1表达减少，TJ松弛，为增加OCT的胞旁渗透提供了可能。
第三部分肝硬化门脉高压时青藤碱促奥曲肽空肠吸收研究
目的：通过肝硬化门脉高压大鼠离体翻转肠、肠灌流及灌胃给药等多种药物吸收模型，明确SN在门脉高压状态时对OCT肠吸收的促进作用。
方法：1.建立BDL大鼠模型，运用离体翻转肠实验，考察SN对浆膜侧OCT浓度影响。实验大鼠随机分组及给药浓度如下（n=4）：Control+OCT组：单给10μMOCT；Control+OCT+SN组：给予10μM OCT+0.5%SN；BDL+OCT组：单给10μMOCT；BDL+OCT+SN组：给予10μM OCT+0.5%SN。
2.运用大鼠在体空肠灌流实验，考察SN对血中OCT浓度影响。分组及给药浓度同翻转肠实验。
3.通过大鼠灌胃实验，考察SN对OCT生物利用度的影响。实验大鼠随机分组及给药浓度如下（n=4）：Control+OCT静脉给药组：每只大鼠单给OCT20μg；Control+OCT口服给药组：每只大鼠单给OCT200μg；Control+OCT+SN口服给药组：每只大鼠给予OCT200μg+SN30mg；BDL+OCT静脉给药组：每只大鼠单给OCT20μg；BDL+OCT口服给药组：每只大鼠单给OCT200μg；BDL+OCT+SN口服给药组：每只大鼠给予OCT200μg+SN30mg。
结果：1.离体翻转肠实验，对比OCT浓度，BDL+OCT+SN组较BDL+OCT组明显升高(P<0.05)；Control+OCT+SN组较Control+OCT组明显升高(P<0.05)；BDL+OCT+SN组较Control+OCT+SN组也有所升高(P<0.05)； BDL+OCT组与Control+OCT组无明显差异(P>0.05)。
2.体空肠灌流实验，对比OCT浓度，结果同离体翻转肠组。
3．口服吸收组均于给药后30min出现峰值，对比Cmax，Control+OCT+SN组是Control+OCT组的3.6倍（P<0.05）；BDL+OCT+SN组是BDL+OCT组的7.0倍（P<0.05），是Control+OCT+SN组的1.6倍（P<0.05），而Control+OCT组和BDL+OCT组无明显差异（P>0.05）。
对比AUC, Control+OCT+SN是Control+OCT组的4.8倍（P<0.05）；BDL+OCT+SN组是BDL+OCT组的6.8倍（P<0.05），是Control+OCT+SN组的1.4倍（P<0.05），而Control+OCT组和BDL+OCT组的无明显差异（P>0.05）。
对比绝对生物利用度（absolute bioavailability, F），Control+OCT+SN组是Control+OCT组的3.79倍（P<0.05）；BDL+OCT+SN组是BDL+OCT组的6.76倍（P<0.05），是Control+OCT+SN组的1.6倍（P<0.05），而Control+OCT组和BDL+OCT组的无明显差异（P>0.05）。
静脉对照组大鼠均在给药后10min出现峰值，Control+OCT组与BDL+OCT组的Cmax和AUC均无明显差异（P>0.05）。
结论：1.在大鼠离体与在体实验中，BDL大鼠与正常大鼠OCT肠吸收率及生物利用度无明显差异。
2.在大鼠离体与在体实验中，无论是BDL大鼠还是正常大鼠，SN均能提高OCT的肠吸收率及口服生物利用度，且在BDL大鼠中SN的促吸收作用更为显著。
The population of patients with liver cirrhosis is large, especially in China. Portalhypertension is not only the typical manifestation of patients at the decompensated stageof cirrhosis, but also the main reason to result in various complications and death. So itis important to prevent and treat of patients with portal hypertension, thus improving theprognosis and decreasing the mortality of patients. However, there are no ideal oraldrugs for prevention of portal hypertension.
Octrotide (OCT) is an acknowledged drug for decrease portal pressure. Due to itsstabilized structure against enzymatic degradation, OCT can partially overcome theproblems of therapeutically active peptides often limited by their short biologicalhalf-lives. But OCT has high molecular weight, less fat-soluble quality, influenced byfirst-pass effect of intestines and liver, and it is prohibited by intestinal absorptionbarrier, so its oral bioavailability is low. The oral delivery of OCT would have theimportant clinical significance of avoiding multiple daily injections for patients withportal hypertension. Up to now, many researchers are focused on increasing the OCTintestinal absorption, but to the best of our knowledge, no versions have becomecommercially available.
Our preliminary experiments established partial portal vein ligation of portalhypertension rats’ model, which were proved that portosystemic shunt formation andcollateral circulation establishment can effectively reduce the OCT liver first effect, sothe hepatic first-pass effect plays little influence on OCT absorption. Then we studiedp-glycoprotein (P-gp) and multidrug resistance associated protein2(MRP2) which arethe transport proteins located on intestines, and intestinal metabolic enzyme cytochromeP4503A4(CYP3A4), thus revealing the absorption mechanisms of OCT by inhibition of intestinal first-pass effect. So the focus of our current study was to study the crucialparacellular route of intestinal OCT absorption.
By means of the morphological study with transmission electron microscope (TEM)and molecular studies with western blot and Real-Time-Polymerase Chain Reaction(Real-Time-PCR), our study demonstrated that the expression of Claudin-1proteindecreased and Tight Junction (TJ) were loosen in the enterocytes of portalhypertension rats, thus enhancing the paracellular permeability. This is a favorablefactor for OCT intestinal absorption via paracellular route. The study on Caco-2cellmodel indicated that Sinomenine (SN) was capable of enhancing the intestinalpermeability of OCT, which is at least in part related to the mechanism of TJ proteinClaudin-1and the protein kinase C (PKC) signaling pathway for the paracellular route.Preliminary experiment preliminarily confirmed SN could increase the intestinal absorption rate ofOCT in normal rat. Our further in vivo studies which examined the effects of SN on theintestinal transport and the absorptive kinetics of OCT using the everted rat gut sacsystem, rat intestinal perfusion system and oral gavaged system indicated that theintestinal absorption rate and oral bioavailability of OCT were markedly improved bySN under cirrhosis with portal hypertension. And in the condition of portal hypertension,the promoting absorption effect of SN is more significant.
The aim of this study is to reveal the SN-mediated reversible TJ opening and itsmolecular mechanism by cellular and creatural researches, in order to find out themechanisms of improving intestinal OCT absorption
It is also useful to find out the effective ways for increasing oral bioavailability ofOCT, realize portal pressure reduction by oral administration of it, and provide theoryevidence for rational use of OCT in clinic at the same time.
Part I
Study on Sinomenine-mediated reversible opening of intestinalepithelial tight junction and its molecular mechanism
Object: The aim of this study is to assess the effects of SN on intestinal OCTabsorption in Caco-2cell monolayer, investigate the molecular mechanisms of TJdisruption and recovery by SN-mediated changes in the Claudin-1and the PKCsignaling pathway.
Methods:1. Caco-2cell monolayer model was established, and the integrity of it was evaluated by the morphology, transepithelial electrical resistance (TEER)measurement and fluorescein isothiocyanate dextran40,00(FD-4) permeability.
2. The cytotoxic effects of different concentrations of SN (0.5%,1%,2%w/v) onCaco-2cell monolayers were examined using Lactate dehydrogenase (LDH) assay.
3. The SN-mediated reversible TJ opening was evaluated by measuring the TEER,and the assessment of the potential OCT penetration through TJ was evaluated bymeasuring FD-4permeability.
4. The effect of SN on the transport of OCT across the Caco-2cell monolayer bycalculated apparent permeability coefficient (Papp). The cells were divided into Controlgroup (10μM OCT) and SN group (0.5%SN+10μM OCT).
5. The effect of SN on the gene and protein expression of Claudin-1by westernblot、Real-Time-PCR、Immunofluorescent Staining (IF). The cells were divided intoControl,0.5%SN, SN removal and0.5%SN+10μM Ro318220group.
6. The effect of SN on the PKC protein on the activation of the PKC signalingpathway was revealed by westerm blot. The cells were divided into Control and0.5%SN group.
Results:1. The TEER values were746.11±46.57·cm2, and the FD-4flux（apical-to-basolateral direction）was below0.5pmol/cm2, indicating good monolayerintegrity when subjected to experimentation.
2. After120min,2%and1%SN group showed markedly increased LDH activity(P<0.05), only0.5%of the SN group did not differ from the Control (P>0.05). Thisresult indicated that a0.5%concentration of SN was safe for Caco-2cell monolayer.
3. The0.5%SN treatment group resulted in a strong reduction of TEER (P<0.05)at120min; after SN removal, a gradual recovery in TEER was observed at240min,and there was no difference compared with the control (P>0.05).
4. The calculated Pappof OCT in the0.5%w/v SN treatment group is higher than inthe OCT alone group (P<0.05), which indicated that0.5%w/v SN significantlyincreased the transport of OCT in the apical-to-basolateral direction across the Caco-2cell monolayer.
5. Control cells displayed the expected TJ protein localization pattern; Claudin-1was concentrated at the sites of cell-cell contact and formed a belt-like structure,showing continuous and a strong intensity of staining. Treatment with0.5%SN for2hinduced the apparent loss of Claudin-1from some distinct membrane regions, indicatingthe loss of functional TJ from such areas. The removal of SN from the culture medium resulted in a significant recovery of Claudin-1at TJ at2h. However, the incubation ofthe cells with0.5%SN and10μM Ro318220together resulted in a complete retentionof the intensity and staining pattern, as observed in the Control group.
After exposure to0.5%SN for2h, a distinct loss of Claudin-1gene and proteinexpression were observed in Caco-2cell monolayer when compared with Control (P<0.05), but after SN removal for2h, the expression of Claudin-1returned to its normallevel (P>0.05). Strangely, there was no significant difference in the expression of
Claudin-1, when Caco-2cell monolayers were treated with0.5%SN and10μMRo318220together for2h (P>0.05).
6. PKC-αprotein on cytomembrane was higher in0.5%SN group than in Controlgroup(P>0.05).
Conclusions: We conclude that SN has the ability to enhance intestinal OCTabsorption and that these mechanisms are related at least in part to the important role ofClaudin-1in SN-mediated, reversible TJ opening via PKC activation.
Part II
The effects of portal hypertension on tight junction in jejunalepithelium
Object: The aim of this study is to investigate the alteration of the intestinalepithelium TJ via morphology and the molecular level by common bile duct ligation(BDL) rats, in order to probe the effect of cirrhotic portal hypertension on the intestinalmucosal barrier.
Methods: The healthy male Sprague-Dawley (SD) rats, weighing250-300g, weredivided into Control and BDL group. The cirrhotic rats with portal hypertension wereestablished by BDL. After surgery for4weeks, all rats were measured portal venouspressure and then were killed for collection of blood, liver and jejunum tissues. HEstaining was performed to observe the pathomorphology of liver tissues. The TJbetween the jejunum epithelial cells was observed by TEM. The expressions ofClaudin-1protein and mRNA were tested by the ways of ImmunohistochemistryStaining (IHC), western blot and Real-Time-PCR, respectively. The levels of alanineaminotransferase (ALT) and aspartate aminotransferase (AST) were assayed byautomatic blood biochemistry analyzer.
Results:1. In group BDL, the levels of ALT and AST were much higher, theportal pressure ((18.52±1.83) mmHg) was greatly increased, and all the results wereexited statistical difference between Control group (P<0.05).
2. HE staining for liver in BDL group was characterized by fibrosis proliferation,amount of inflammatory cells infiltration, pseudolobule formation, and proliferation ofinterlobular bile duct.
3. The obtained TEM images showed that TJ was located at the apical side ofenterocytes, and the membranes of enterocytes were in close proximity, appearing tofuse at the apical side in control rat, and distinct ultra-structural changes in TJmorphology, such as dilation of the intercellular space, were observed in BDL rat.
4.The methods of IHC and western blot showed that the protein expressions ofClaudin-1in group BDL was evidently lower than in group Control (P<0.05), moreover,the way of Real-Time-PCR was confirmed the results form mRNA levels.
Conclusions: The jejunum mucosal ultra-structure is changed in liver cirrhosiswith portal hypertension rats, the expression of Claudin-1in the intestinal mucosa ofBDL rats is descended, and the TJs are loosen in the enterocytes of BDL rats. This is
Part III
The research on pormoting intestinal absorption ofOctreotide by Sinomenine under cirrhosis with portalhypertension
Object: The aim is to know the influence of SN on intestinal absorption of OCTunder cirrhosis with portal hypertension via multiple absorption models (such as theeverted rat gut sac model, the perfused rat intestinal model and the oral gavaged ratmodel), in order to find an effective way for realization of OCT by oral administration.
Methods:1. The cirrhotic rats with portal hypertension were established by BDL.The effects of SN on intestinal absorption of OCT were observed by the model ofeverted intestinal sacs. The rats were divided into Control+OCT group (10μM OCT)；Control+OCT+SN group (10μM OCT+0.5%SN)；BDL+OCTgroup (10μM OCT)；BDL+OCT+SN group (10μM OCT+0.5%SN).
2. In situ jejunal perfusions of rats were also performed, and the doses and grouping were the same as in the experiments of everted intestinal sacs (n=4).
3. The in vivo absorption experiment in rats was performed. The Normal rats weredivided randomly into Control+OCT i.v. group (OCT20μg per rat); Control+OCT p.o.group (OCT200μg per rat); Control+OCT+SN p.o. group (OCT200μg+SN30mg perrat); BDL+OCT i.v. group (OCT20μg per rat); BDL+OCT p.o. group (OCT200μg perrat); BDL+OCT+SN p.o. group (OCT200μg+SN30mg per rat). The absolutebioavailability (F) of OCT was determined by measuring the area under the uptakecurve (AUC) for each group.
Results：1. In the model of everted intestinal sacs, the absorption of OCT wasincreased approximately2.2-fold (P<0.05) in BDL+OCT+SN group when comparedwith BDL+OCT group, and the absorption of OCT was increased only to1.6-fold(P<0.05) in Control+OCT+SN group when compared with Conrtol+OCT group. Andthe absorption of OCT in BDL+OCT+SN group was significantly increased whencompared with Conrtol+OCT+SN group (P<0.05). However, no significant differencewas found between the Control+OCT and BDL+OCT group (P>0.05).
2. In the model of in situ jejunal perfusions of rats, the absorption of OCT wasincreased approximately3.3-fold (P<0.05) in BDL+OCT+SN group when comparedwith BDL+OCT group, and the absorption of OCT was increased only to1.9-fold(P<0.05) in Control+OCT+SN group when compared with Conrtol+OCT group. Andthe absorption of OCT in BDL+OCT+SN group was significantly increased whencompared with Conrtol+OCT+SN group (P<0.05). However, no significant differencewas found between the Control+OCT and BDL+OCT group (P>0.05).
3. In vivo experiments, uptake profiles following i.v. administration of OCTshowed a single peak immediately at10min (Tmax) with a Cmax of307±6ng/mL inConrtol+OCT group and310ng/ml in the BDL+OCT group. Uptake profilesfollowing p.o. administration of OCT showed a single peak later at30min (Tmax) with aCmaxof44.3±6.66ng/mL in Conrtol+OCT group,35±3.61ng/mL in BDL+OCTgroup,158±10.33ng/mL in Conrtol+OCT+SN group and246±13.53ng/mL inBDL+OCT+SN group.
The pharmacokinetic parameters of the i.v. and p.o. administered OCT for600minobservation showed that SN provoked approximately a6-fold increase in Cmax, the AUCand F in BDL+OCT+SN group when compared with BDL+OCT group(P<0.05), onlyprovoked approximately a3-fold increase in Cmax, AUC and F in Control+OCT+SNgroup when compared with Control+OCT group(P<0.05), and the Cmax, AUC and F of OCT in BDL+OCT+SN group was significantly increased when compared withConrtol+OCT+SN group (P<0.05). However, there was no statistics difference betweenConrtol+OCT group and BDL+OCT group (P>0.05).
Conclusions: SN can improve intestinal OCT absorption of rats with portalhypertension. The oral bioavailability of OCT in rats with portal hypertension isimproved by SN.
奥曲肽（octreotide, OCT）是目前公认的治疗肝硬化门脉高压的有效药物，虽可抵抗胃肠道中酸、酶的降解，但因分子量大，脂溶性差，受肠粘膜屏障和肝肠首过效应的影响，口服生物利用度极低，临床上仅限于注射给药，不利于作为预防性药物长期应用。OCT口服可以避免长期反复注射给药带来的痛苦和不便，临床意义重大。我们期待能像治疗高血压一样，实现OCT口服，达到早期、持续服药来防治门脉高压。国内外学者对此做了大量工作，但仍无任何一项研究成果可应用于临床。
本课题组前期实验建立了部分门脉结扎的门脉高压大鼠模型，证实广泛侧支循环的建立可有效地降低OCT肝首过效应，故肝首过效应并非门脉高压下OCT口服吸收的主要障碍。随后，又进一步研究了肝硬化门脉高压状态时空肠首过效应中起着关键作用的转运蛋白P-糖蛋白（p-glycoprotein, P-gp）、多耐药相关蛋白2（multidrug resistance-associated protein2, MRP2）和代谢酶细胞色素P4503A4（cytochrome P4503A4, CYP3A4），探讨了抑制肠上皮首过效应来增加OCT肠吸收的机制，故肠上皮首过效应问题也可得到解决。而对于OCT肠吸收至关重要的胞旁吸收途径则是本次研究的重点。本研究通过对肝硬化门脉高压大鼠空肠粘膜的形态学及分子水平的研究表明，门脉高压状态时空肠上皮紧密连接（tightjunction, TJ）蛋白Claudin-1表达减少，TJ松弛，肠粘膜屏障功能破坏，可使肠壁通透性增加，利于OCT的肠吸收。而通过Caco-2细胞模型证实促吸收剂青藤碱（sinomenine, SN）能够通过激活蛋白激酶C（protein kinase C, PKC）信号转导通路来调控TJ蛋白Claudin-1的表达，实现短暂可逆的调节胞旁TJ，有效提高OCT在Caco-2细胞单层中的渗透率。预实验初步证实SN能够增加正常大鼠OCT的肠吸收率，本研究则进一步在肝硬化门脉高压大鼠体内及体外吸收模型上证实SN有效的提高了OCT口服生物利用度，且在肝硬化门脉高压状态时其促吸收效果更为显著。
本研究旨在通过细胞及动物实验，揭示SN可逆性调控胞旁TJ促进OCT肠吸收的机制，为提高OCT口服生物利用度提供有效可行的方法及途径，为OCT临床合理用药提供理论依据，为实现口服OCT预防门脉高压开创新纪元。
本研究共分三部分
第一部分青藤碱可逆调控肠上皮细胞紧密连接促奥曲肽吸收的机制研究
第二部分肝硬化门脉高压对大鼠空肠上皮细胞紧密连接影响的研究
第三部分肝硬化门脉高压时青藤碱促奥曲肽空肠吸收研究
第一部分
青藤碱可逆调控肠上皮细胞紧密连接促奥曲肽吸收的机制研究
目的：在Caco-2细胞单层模型上评估SN对OCT肠吸收的影响，明确SN可逆调控肠上皮细胞TJ的分子机制。
方法：1.建立Caco-2细胞单层模型，并通过形态学、跨膜电阻（transepithelialelectrical resistance, TEER）测量及胞旁漏出标记物异硫氰酸荧光素标记的右旋糖酐-4000（FD-4）的渗透量来考察Caco-2细胞单层的完整性。
2.通过乳酸脱氢酶（lactate dehydrogenase, LDH）试验考察不同浓度组（0.5%、1%、2%w/v) SN对Caco-2细胞单层的损伤情况。
3.通过TEER的测量及FD-4渗透量的计算明确SN对TJ的可逆性调节作用。
4. SN对OCT肠吸收的影响。分组：Control组（10μM OCT）；SN组（0.5%SN+10μM OCT）。考察各组OCT的表观渗透系数（apparent permeability coefficient,Papp）。
5. SN对TJ蛋白Claudin-1表达的影响。分组为：Control组；SN组（0.5%SN）、SN去除组（SN removal）及PKC抑制剂组(0.5%SN+10μM Ro318220)。运用蛋白免疫印迹（western blot）、实时荧光定量聚合酶链式反应（Real-Time-PolymeraseChain Reaction, Real-Time-PCR）、免疫荧光染色（Immunofluorescent Staining, IF）方法分别考察各组中Claudin-1的表达。
6. SN对PKC信号转导通路的影响。分组：Control组，SN组（0.5%SN），运用western blot的方法分别考察PKC-α在对照组及SN组中细胞膜及胞浆中的表达。
结果：1.细胞培养21天后，倒置显微镜可见细胞之间的接触；经过测量和计
算，Caco-2细胞单层的TEER值均达到700Ω·cm2以上；1mg/ml的FD-4在120min
内的渗透量（AP-BL侧）无明显变化，一直处于较低水平（<0.5pmol/cm2），说明细胞间已形成完整的TJ。
2. LDH试验显示120min后，中浓度组（1%）及高浓度组（2%）的SN对细胞均有损伤，与对照组相比P<0.05；而低浓度组（0.5%）SN对细胞无明显损伤，与对照组相比P>0.05。
3.0.5%SN作用120min时，TEER%降至最低点，为起始TEER值的43%，FD-4的流出量达到最高，与对照组相比，P<0.05；去除SN的作用后，TEER%又迅速回升，在SN去除120min（总时间的240min）时恢复到起始水平，与对照组相比，P>0.05。
4.共同给予0.5%SN和10μM OCT120min时，OCT的Papp为5.89±0.038×106cm/s，是对照组（单给10μM OCT）的2.2倍（P<0.05）。
5. IF显示Control组中Claudin-1蛋白均匀表达于细胞膜，显示较强的绿色荧光；0.5%SN作用120min后，绿色荧光强度明显减弱，细胞间的连接出现断断续续的改变；去除SN120min时，细胞膜上的绿色荧光再次增强，细胞间的TJ完整性又恢复；而在SN+Ro318220共同作用120min时，细胞膜上的绿色荧光强度及细胞间TJ形态较给药前无明显变化。Western blot及Real-Time-PCR结果显示，0.5%SN组较Control组Claudin-1蛋白表达明显降低（P<0.05），SN removal组及0.5%SN+10μM Ro318220组Claudin-1蛋白表达均较Control组无明显差异（P>0.05）。
6.对照组中，PKC-α蛋白主要在细胞质内表达，细胞膜上表达量很少；而0.5%SN组，PKC-α则从细胞质中转移出来，主要表达于细胞膜上，表达量与Control组相比具有统计学意义(P<0.05)，这种PKC-α的转膜现象意味着PKC通路的激活。
结论：1.在Caco-2单层细胞模型上，SN能够促进OCT的吸收。
2. TJ蛋白Claudin-1在SN可逆性调控TJ中发挥着重要作用，且很可能与PKC信号转导通路的激活有关。
第二部分
肝硬化门脉高压对大鼠空肠上皮细胞紧密连接影响的研究
目的：在肝硬化门脉高压大鼠模型上，从形态学及分子水平对OCT胞旁吸收途径的主要结构—TJ进行研究，探讨肝硬化门脉高压对肠粘膜屏障的影响及机制。
方法：大鼠肝硬化门脉高压模型采用胆总管结扎（bile duct ligation, BDL）方法来制备。将健康成年雄性Sprague-Dawley（SD）大鼠随机分为2组，即Control和BDL组。BDL术后4周，对2组大鼠进行门静脉压力测定，之后处死大鼠，取血、取肝、取空肠，采用全自动生化分析仪检测血清丙氨酸氨基转移酶(alanineaminotransferase, ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase, AST)水平；HE染色观察肝脏病理形态学；透射电镜（transmission electron microscope,TEM）观察空肠上皮细胞间TJ形态；免疫组织化学染色（ImmunohistochemistryStaining, IHC）、western blot及Real-Time-PCR方法观察大鼠空肠Claudin-1蛋白及基因表达情况。
结果：1.大鼠门脉压力比较，BDL组较Control组明显升高（P<0.01）；对比ALT和AST值，BDL组较Control组明显升高(P<0.01)。
2.大鼠肝脏HE染色结果显示BDL组肝小叶结构紊乱，大量纤维组织增生，炎性细胞侵润，见多个假小叶，小叶间胆管增生明显。
3．TEM观察显示BDL大鼠空肠上皮细胞间TJ松弛，细胞间隙增宽。
4. IHC及western blot结果显示BDL组Claudin-1蛋白表达较Control组降低（P<0.01）；Real-Time-PCR方法显示BDL组Claudin-1基因表达较Control组降低（P<0.01）。
结论：肝硬化门脉高压大鼠空肠粘膜超微结构改变，TJ蛋白Claudin-1表达减少，TJ松弛，为增加OCT的胞旁渗透提供了可能。
第三部分肝硬化门脉高压时青藤碱促奥曲肽空肠吸收研究
目的：通过肝硬化门脉高压大鼠离体翻转肠、肠灌流及灌胃给药等多种药物吸收模型，明确SN在门脉高压状态时对OCT肠吸收的促进作用。
方法：1.建立BDL大鼠模型，运用离体翻转肠实验，考察SN对浆膜侧OCT浓度影响。实验大鼠随机分组及给药浓度如下（n=4）：Control+OCT组：单给10μMOCT；Control+OCT+SN组：给予10μM OCT+0.5%SN；BDL+OCT组：单给10μMOCT；BDL+OCT+SN组：给予10μM OCT+0.5%SN。
2.运用大鼠在体空肠灌流实验，考察SN对血中OCT浓度影响。分组及给药浓度同翻转肠实验。
3.通过大鼠灌胃实验，考察SN对OCT生物利用度的影响。实验大鼠随机分组及给药浓度如下（n=4）：Control+OCT静脉给药组：每只大鼠单给OCT20μg；Control+OCT口服给药组：每只大鼠单给OCT200μg；Control+OCT+SN口服给药组：每只大鼠给予OCT200μg+SN30mg；BDL+OCT静脉给药组：每只大鼠单给OCT20μg；BDL+OCT口服给药组：每只大鼠单给OCT200μg；BDL+OCT+SN口服给药组：每只大鼠给予OCT200μg+SN30mg。
结果：1.离体翻转肠实验，对比OCT浓度，BDL+OCT+SN组较BDL+OCT组明显升高(P<0.05)；Control+OCT+SN组较Control+OCT组明显升高(P<0.05)；BDL+OCT+SN组较Control+OCT+SN组也有所升高(P<0.05)； BDL+OCT组与Control+OCT组无明显差异(P>0.05)。
2.体空肠灌流实验，对比OCT浓度，结果同离体翻转肠组。
3．口服吸收组均于给药后30min出现峰值，对比Cmax，Control+OCT+SN组是Control+OCT组的3.6倍（P<0.05）；BDL+OCT+SN组是BDL+OCT组的7.0倍（P<0.05），是Control+OCT+SN组的1.6倍（P<0.05），而Control+OCT组和BDL+OCT组无明显差异（P>0.05）。
对比AUC, Control+OCT+SN是Control+OCT组的4.8倍（P<0.05）；BDL+OCT+SN组是BDL+OCT组的6.8倍（P<0.05），是Control+OCT+SN组的1.4倍（P<0.05），而Control+OCT组和BDL+OCT组的无明显差异（P>0.05）。
对比绝对生物利用度（absolute bioavailability, F），Control+OCT+SN组是Control+OCT组的3.79倍（P<0.05）；BDL+OCT+SN组是BDL+OCT组的6.76倍（P<0.05），是Control+OCT+SN组的1.6倍（P<0.05），而Control+OCT组和BDL+OCT组的无明显差异（P>0.05）。
静脉对照组大鼠均在给药后10min出现峰值，Control+OCT组与BDL+OCT组的Cmax和AUC均无明显差异（P>0.05）。
结论：1.在大鼠离体与在体实验中，BDL大鼠与正常大鼠OCT肠吸收率及生物利用度无明显差异。
2.在大鼠离体与在体实验中，无论是BDL大鼠还是正常大鼠，SN均能提高OCT的肠吸收率及口服生物利用度，且在BDL大鼠中SN的促吸收作用更为显著。
The population of patients with liver cirrhosis is large, especially in China. Portalhypertension is not only the typical manifestation of patients at the decompensated stageof cirrhosis, but also the main reason to result in various complications and death. So itis important to prevent and treat of patients with portal hypertension, thus improving theprognosis and decreasing the mortality of patients. However, there are no ideal oraldrugs for prevention of portal hypertension.
Octrotide (OCT) is an acknowledged drug for decrease portal pressure. Due to itsstabilized structure against enzymatic degradation, OCT can partially overcome theproblems of therapeutically active peptides often limited by their short biologicalhalf-lives. But OCT has high molecular weight, less fat-soluble quality, influenced byfirst-pass effect of intestines and liver, and it is prohibited by intestinal absorptionbarrier, so its oral bioavailability is low. The oral delivery of OCT would have theimportant clinical significance of avoiding multiple daily injections for patients withportal hypertension. Up to now, many researchers are focused on increasing the OCTintestinal absorption, but to the best of our knowledge, no versions have becomecommercially available.
Our preliminary experiments established partial portal vein ligation of portalhypertension rats’ model, which were proved that portosystemic shunt formation andcollateral circulation establishment can effectively reduce the OCT liver first effect, sothe hepatic first-pass effect plays little influence on OCT absorption. Then we studiedp-glycoprotein (P-gp) and multidrug resistance associated protein2(MRP2) which arethe transport proteins located on intestines, and intestinal metabolic enzyme cytochromeP4503A4(CYP3A4), thus revealing the absorption mechanisms of OCT by inhibition of intestinal first-pass effect. So the focus of our current study was to study the crucialparacellular route of intestinal OCT absorption.
By means of the morphological study with transmission electron microscope (TEM)and molecular studies with western blot and Real-Time-Polymerase Chain Reaction(Real-Time-PCR), our study demonstrated that the expression of Claudin-1proteindecreased and Tight Junction (TJ) were loosen in the enterocytes of portalhypertension rats, thus enhancing the paracellular permeability. This is a favorablefactor for OCT intestinal absorption via paracellular route. The study on Caco-2cellmodel indicated that Sinomenine (SN) was capable of enhancing the intestinalpermeability of OCT, which is at least in part related to the mechanism of TJ proteinClaudin-1and the protein kinase C (PKC) signaling pathway for the paracellular route.Preliminary experiment preliminarily confirmed SN could increase the intestinal absorption rate ofOCT in normal rat. Our further in vivo studies which examined the effects of SN on theintestinal transport and the absorptive kinetics of OCT using the everted rat gut sacsystem, rat intestinal perfusion system and oral gavaged system indicated that theintestinal absorption rate and oral bioavailability of OCT were markedly improved bySN under cirrhosis with portal hypertension. And in the condition of portal hypertension,the promoting absorption effect of SN is more significant.
The aim of this study is to reveal the SN-mediated reversible TJ opening and itsmolecular mechanism by cellular and creatural researches, in order to find out themechanisms of improving intestinal OCT absorption
It is also useful to find out the effective ways for increasing oral bioavailability ofOCT, realize portal pressure reduction by oral administration of it, and provide theoryevidence for rational use of OCT in clinic at the same time.
Part I
Study on Sinomenine-mediated reversible opening of intestinalepithelial tight junction and its molecular mechanism
Object: The aim of this study is to assess the effects of SN on intestinal OCTabsorption in Caco-2cell monolayer, investigate the molecular mechanisms of TJdisruption and recovery by SN-mediated changes in the Claudin-1and the PKCsignaling pathway.
Methods:1. Caco-2cell monolayer model was established, and the integrity of it was evaluated by the morphology, transepithelial electrical resistance (TEER)measurement and fluorescein isothiocyanate dextran40,00(FD-4) permeability.
2. The cytotoxic effects of different concentrations of SN (0.5%,1%,2%w/v) onCaco-2cell monolayers were examined using Lactate dehydrogenase (LDH) assay.
3. The SN-mediated reversible TJ opening was evaluated by measuring the TEER,and the assessment of the potential OCT penetration through TJ was evaluated bymeasuring FD-4permeability.
4. The effect of SN on the transport of OCT across the Caco-2cell monolayer bycalculated apparent permeability coefficient (Papp). The cells were divided into Controlgroup (10μM OCT) and SN group (0.5%SN+10μM OCT).
5. The effect of SN on the gene and protein expression of Claudin-1by westernblot、Real-Time-PCR、Immunofluorescent Staining (IF). The cells were divided intoControl,0.5%SN, SN removal and0.5%SN+10μM Ro318220group.
6. The effect of SN on the PKC protein on the activation of the PKC signalingpathway was revealed by westerm blot. The cells were divided into Control and0.5%SN group.
Results:1. The TEER values were746.11±46.57·cm2, and the FD-4flux（apical-to-basolateral direction）was below0.5pmol/cm2, indicating good monolayerintegrity when subjected to experimentation.
2. After120min,2%and1%SN group showed markedly increased LDH activity(P<0.05), only0.5%of the SN group did not differ from the Control (P>0.05). Thisresult indicated that a0.5%concentration of SN was safe for Caco-2cell monolayer.
3. The0.5%SN treatment group resulted in a strong reduction of TEER (P<0.05)at120min; after SN removal, a gradual recovery in TEER was observed at240min,and there was no difference compared with the control (P>0.05).
4. The calculated Pappof OCT in the0.5%w/v SN treatment group is higher than inthe OCT alone group (P<0.05), which indicated that0.5%w/v SN significantlyincreased the transport of OCT in the apical-to-basolateral direction across the Caco-2cell monolayer.
5. Control cells displayed the expected TJ protein localization pattern; Claudin-1was concentrated at the sites of cell-cell contact and formed a belt-like structure,showing continuous and a strong intensity of staining. Treatment with0.5%SN for2hinduced the apparent loss of Claudin-1from some distinct membrane regions, indicatingthe loss of functional TJ from such areas. The removal of SN from the culture medium resulted in a significant recovery of Claudin-1at TJ at2h. However, the incubation ofthe cells with0.5%SN and10μM Ro318220together resulted in a complete retentionof the intensity and staining pattern, as observed in the Control group.
After exposure to0.5%SN for2h, a distinct loss of Claudin-1gene and proteinexpression were observed in Caco-2cell monolayer when compared with Control (P<0.05), but after SN removal for2h, the expression of Claudin-1returned to its normallevel (P>0.05). Strangely, there was no significant difference in the expression of
Claudin-1, when Caco-2cell monolayers were treated with0.5%SN and10μMRo318220together for2h (P>0.05).
6. PKC-αprotein on cytomembrane was higher in0.5%SN group than in Controlgroup(P>0.05).
Conclusions: We conclude that SN has the ability to enhance intestinal OCTabsorption and that these mechanisms are related at least in part to the important role ofClaudin-1in SN-mediated, reversible TJ opening via PKC activation.
Part II
The effects of portal hypertension on tight junction in jejunalepithelium
Object: The aim of this study is to investigate the alteration of the intestinalepithelium TJ via morphology and the molecular level by common bile duct ligation(BDL) rats, in order to probe the effect of cirrhotic portal hypertension on the intestinalmucosal barrier.
Methods: The healthy male Sprague-Dawley (SD) rats, weighing250-300g, weredivided into Control and BDL group. The cirrhotic rats with portal hypertension wereestablished by BDL. After surgery for4weeks, all rats were measured portal venouspressure and then were killed for collection of blood, liver and jejunum tissues. HEstaining was performed to observe the pathomorphology of liver tissues. The TJbetween the jejunum epithelial cells was observed by TEM. The expressions ofClaudin-1protein and mRNA were tested by the ways of ImmunohistochemistryStaining (IHC), western blot and Real-Time-PCR, respectively. The levels of alanineaminotransferase (ALT) and aspartate aminotransferase (AST) were assayed byautomatic blood biochemistry analyzer.
Results:1. In group BDL, the levels of ALT and AST were much higher, theportal pressure ((18.52±1.83) mmHg) was greatly increased, and all the results wereexited statistical difference between Control group (P<0.05).
2. HE staining for liver in BDL group was characterized by fibrosis proliferation,amount of inflammatory cells infiltration, pseudolobule formation, and proliferation ofinterlobular bile duct.
3. The obtained TEM images showed that TJ was located at the apical side ofenterocytes, and the membranes of enterocytes were in close proximity, appearing tofuse at the apical side in control rat, and distinct ultra-structural changes in TJmorphology, such as dilation of the intercellular space, were observed in BDL rat.
4.The methods of IHC and western blot showed that the protein expressions ofClaudin-1in group BDL was evidently lower than in group Control (P<0.05), moreover,the way of Real-Time-PCR was confirmed the results form mRNA levels.
Conclusions: The jejunum mucosal ultra-structure is changed in liver cirrhosiswith portal hypertension rats, the expression of Claudin-1in the intestinal mucosa ofBDL rats is descended, and the TJs are loosen in the enterocytes of BDL rats. This is
Part III
The research on pormoting intestinal absorption ofOctreotide by Sinomenine under cirrhosis with portalhypertension
Object: The aim is to know the influence of SN on intestinal absorption of OCTunder cirrhosis with portal hypertension via multiple absorption models (such as theeverted rat gut sac model, the perfused rat intestinal model and the oral gavaged ratmodel), in order to find an effective way for realization of OCT by oral administration.
Methods:1. The cirrhotic rats with portal hypertension were established by BDL.The effects of SN on intestinal absorption of OCT were observed by the model ofeverted intestinal sacs. The rats were divided into Control+OCT group (10μM OCT)；Control+OCT+SN group (10μM OCT+0.5%SN)；BDL+OCTgroup (10μM OCT)；BDL+OCT+SN group (10μM OCT+0.5%SN).
2. In situ jejunal perfusions of rats were also performed, and the doses and grouping were the same as in the experiments of everted intestinal sacs (n=4).
3. The in vivo absorption experiment in rats was performed. The Normal rats weredivided randomly into Control+OCT i.v. group (OCT20μg per rat); Control+OCT p.o.group (OCT200μg per rat); Control+OCT+SN p.o. group (OCT200μg+SN30mg perrat); BDL+OCT i.v. group (OCT20μg per rat); BDL+OCT p.o. group (OCT200μg perrat); BDL+OCT+SN p.o. group (OCT200μg+SN30mg per rat). The absolutebioavailability (F) of OCT was determined by measuring the area under the uptakecurve (AUC) for each group.
Results：1. In the model of everted intestinal sacs, the absorption of OCT wasincreased approximately2.2-fold (P<0.05) in BDL+OCT+SN group when comparedwith BDL+OCT group, and the absorption of OCT was increased only to1.6-fold(P<0.05) in Control+OCT+SN group when compared with Conrtol+OCT group. Andthe absorption of OCT in BDL+OCT+SN group was significantly increased whencompared with Conrtol+OCT+SN group (P<0.05). However, no significant differencewas found between the Control+OCT and BDL+OCT group (P>0.05).
2. In the model of in situ jejunal perfusions of rats, the absorption of OCT wasincreased approximately3.3-fold (P<0.05) in BDL+OCT+SN group when comparedwith BDL+OCT group, and the absorption of OCT was increased only to1.9-fold(P<0.05) in Control+OCT+SN group when compared with Conrtol+OCT group. Andthe absorption of OCT in BDL+OCT+SN group was significantly increased whencompared with Conrtol+OCT+SN group (P<0.05). However, no significant differencewas found between the Control+OCT and BDL+OCT group (P>0.05).
3. In vivo experiments, uptake profiles following i.v. administration of OCTshowed a single peak immediately at10min (Tmax) with a Cmax of307±6ng/mL inConrtol+OCT group and310ng/ml in the BDL+OCT group. Uptake profilesfollowing p.o. administration of OCT showed a single peak later at30min (Tmax) with aCmaxof44.3±6.66ng/mL in Conrtol+OCT group,35±3.61ng/mL in BDL+OCTgroup,158±10.33ng/mL in Conrtol+OCT+SN group and246±13.53ng/mL inBDL+OCT+SN group.
The pharmacokinetic parameters of the i.v. and p.o. administered OCT for600minobservation showed that SN provoked approximately a6-fold increase in Cmax, the AUCand F in BDL+OCT+SN group when compared with BDL+OCT group(P<0.05), onlyprovoked approximately a3-fold increase in Cmax, AUC and F in Control+OCT+SNgroup when compared with Control+OCT group(P<0.05), and the Cmax, AUC and F of OCT in BDL+OCT+SN group was significantly increased when compared withConrtol+OCT+SN group (P<0.05). However, there was no statistics difference betweenConrtol+OCT group and BDL+OCT group (P>0.05).
Conclusions: SN can improve intestinal OCT absorption of rats with portalhypertension. The oral bioavailability of OCT in rats with portal hypertension isimproved by SN.
引文
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