Study On The Mechanism Of Promoting Jejunal Paracellular Absorption Of Octreotide By Sinomenine In Cirrhosis With Portal Hypertension

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 absorptionIt 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 IStudy on Sinomenine-mediated reversible opening of intestinalepithelial tight junction and its molecular mechanismObject: 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 ofClaudin-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 IIThe effects of portal hypertension on tight junction in jejunalepitheliumObject: 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 IIIThe research on pormoting intestinal absorption ofOctreotide by Sinomenine under cirrhosis with portalhypertensionObject: 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.