【作者】 孙晓宇；

【导师】 段志军；

【作者基本信息】 大连医科大学 ， 内科学， 2013， 博士

【摘要】 乙型肝炎是肝硬化主要病因，我国乙肝患者及乙肝病毒感染者众多，每年新发肝硬化者超过百万。门脉高压（portal hypertension, PH）是肝硬化失代偿期患者典型的临床表现，是肝硬化多种并发症发生的根源及主要致死原因。对PH进行积极有效防治是改善患者预后及降低病死率的关键。血红素氧合酶(heme oxygense, HO)/一氧化碳(carbon monoxide, CO)系统在肝硬化中晚期可加重PH程度，促多组织器官的进行性病变，加速肝硬化进程，我们曾通过肝硬化PH大鼠研究模型得到了证实。但目前对HO/CO系统在肝硬化PH时期作用临床研究较少，我们曾发现肝硬化PH患者血中可反映HO/CO系统水平的碳氧血红蛋白指标升高，本研究拟通过临床前瞻性研究进一步揭示该系统对PH患者伴多种并发症及对各脏器作用影响。奥曲肽（Octreotide,OCT）是公认的理想降门脉压药物，但其降压机制尚不清楚。因OCT可收缩内脏血管，降门脉血流达降压作用，而内源性CO维持PH的机理之一为扩张内脏血管，故推测OCT可能通过影响HO/CO系统而起到降压作用。本实验对OCT通过HO/CO系统降门脉压的可能机制进行探讨，并进一步证实HO/CO系统的关键作用。由上述实验并结合既往研究基础证明了HO/CO系统在肝硬化PH发展过程中的重要意义，OCT可通过抑制该系统表达而起到降低门脉压目的。结合OCT众多药物优点，若能实现其口服降压治疗，有利于解决PH防治的难题。但蛋白多肽类物质OCT，虽可抵抗胃肠道中酸或酶降解，但分子量大，脂溶性差，受肠肝首过效应及肠道吸收屏障影响，口服生物利用度极低。目前通过改变OCT的化学结构或加入促吸收剂等方法，实验结果均并不理想，且未研究PH病理情况下OCT吸收机制及促吸收研究。我们考虑直接研究影响OCT吸收的首过效应的因素，可能更有利于发现OCT促吸收策略。因在PH状态下门体静脉分流形成，侧支循环的建立及肝功异常等诸多因素，肝首过效应对OCT口服吸收影响较小，而肠首过效应影响可能更为显著。我们通过对主要的肠上皮转运蛋白P-糖蛋白、多耐药相关蛋白2和代谢酶细胞色素P4503A4进行研究，揭示OCT肠首过效应机制，及探及在肝硬化PH情况下，上述转运蛋白及代谢酶的变化对OCT肠道吸收影响，拟寻求实现OCT口服降压策略。此外对PH状态下寡肽类转运蛋白PepT1对OCT的作用做了初步研究，寻求该病理状态下的OCT直接转运蛋白。本研究旨在通过基础及临床多角度揭示HO/CO系统在肝硬化PH时期重要作用，了解在该系统介导下的OCT降门脉压机制，并明确OCT肠道吸收障碍机理，为提高OCT口服生物利用度提供有效方法及途径，有利于实现其口服降压治疗，同时为OCT临床合理用药提供理论依据。本研究共分三部分第一部分血红素氧合酶/一氧化碳系统介导的奥曲肽降门脉压机制研究第一节血红素氧合酶/一氧化碳系统对门脉高压并发症的影响第二节血红素氧合酶/一氧化碳系统介导的奥曲肽降门脉压机制探讨第二部分肠道转运蛋白及代谢酶对奥曲肽肠吸收的影响第三部分肝硬化门脉高压状态下奥曲肽空肠促吸收及降门脉压研究第一部分血红素氧合酶/一氧化碳系统介导的奥曲肽降门脉压机制研究第一节血红素氧合酶/一氧化碳系统对门脉高压并发症的影响目的：分析乙肝后肝硬化（hepatitis B virus-related cirrhosis, HBC）伴肝性脑病（hepatic encephalopathy, HE）患者碳氧血红蛋白（carboxyhemoglobin, COHb）水平及其在肝硬化常见门脉高压（portal hypertension, PH）并发症时的水平差异，并了解其与HE分期等的关系，揭示血红素氧合酶(heme oxygenase, HO)/一氧化碳（carbon monoxide, CO)系统对PH患者伴多种并发症及对各脏器的作用影响。方法：根据排除及诊断标准，68例入院治疗的HBC伴HE的大连医科大学附属第一医院患者纳入本研究入选标准（H组）；除外了重要器官异常的急诊室患者（血气分析等所有检查数据均在正常范围以内）作为对照组（N组），共22例。收集患者临床资料，进行相关检查，收集COHb、氧分压（partial pressure ofoxygen, PaO2）、氧饱和度（oxygen saturation, SaO2）等检测数据，对患者进行HE分期、食管胃底静脉曲张、肝肾综合征(hepatic renal syndrom, HRS)、自发性腹膜炎(spontaneous bacterial peritonitis, SBP)、低氧血症等诊断分析。比较HE患者COHb水平变化，伴不同PH并发症时COHb水平差异及分析COHb水平与HE分期、PaO2及SaO2关系。结果1. H组患者COHb水平较N组明显升高（（2.102±1.021）%vs.（0.983±0.231）%，p=0.000），COHb水平与HE分期呈正相关（p=0.003，rS=0.358）。2. HE伴HRS患者COHb水平较未发生HRS者明显降低（（1.981±1.020）%vs.（2.502±1.073）%，p=0.029）；COHb水平在患者有无食管胃底静脉曲张或SBP中无差异（p>0.05）。3. HE患者COHb水平与PaO2呈负相关（P=0.006, r=-0.336），与SaO2无关(P=0.576, r=-0.072)。HE患者达到低氧血症标准时，COHb水平升高（（2.72±0.362）%vs.（1.93±0.242）%，p=0.012）。结论血红素氧合酶/一氧化碳系统在肝硬化门脉高压时期病理生理过程中起到重要作用，其表达具有组织特异性，对该时期患者具有重要临床意义。第二节血红素氧合酶/一氧化碳系统介导的奥曲肽降门脉压机制探讨目的：探讨血红素氧合酶(heme oxygense, HO)/一氧化碳(carbon monoxide, CO)系统介导下的奥曲肽（Octreotide, OCT）降门脉压机制，并进一步证实在肝硬化门脉高压（portal hypertension, PH）阶段HO/CO系统的关键作用。方法：将34只健康成年雄性Sprague-Dawley大鼠随机分为3组，即假手术组（Sham组）10只，肝硬化门脉高压组（PH组）12只及奥曲肽组（OCT组）12只。大鼠肝硬化PH模型采用胆总管结扎方法。术后4周，OCT及PH组制模完成，前者予OCT腹腔注射（100μg/kg/d，日2次）3天，PH组及Sham组给予同等剂量的生理盐水。给药结束后，禁食水24h，对三组大鼠进行门静脉压力测定，后处死大鼠，取血、取肝，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染色对肝脏进行病理形态学观察；通过免疫组织化学、western blot及RT-PCR方法观察大鼠肝脏HO-1蛋白及基因表达情况。结果1.对比ALT和AST值，PH组较Sham组明显升高(P﹤0.01)；OCT组较PH组明显下降(p﹤0.01)，但较Sham组升高(p﹤0.05)。2.大鼠肝脏HE染色结果显示PH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。OCT组纤维组织增生程度较PH组明显减轻，炎细胞浸润减少(p﹤0.05)，但较Sham组程度重（p<0.05）。3.大鼠门脉压力比较，PH组较Sham组明显升高（（18.52±1.83）mmHg vs.（9.08±0.52）mmHg, p<0.01）；OCT组（13.17±1.12mmHg）较PH组明显下降（p<0.05），但较SH组升高（p<0.05）。4.免疫组织化学方法及western blot方法结果显示HO-1蛋白表达, PH组较Sham组增高（p﹤0.01）；OCT组表达较PH组显著减少，但较Sham组高（p﹤0.05）。应用RT-PCR方法对上述HO-1蛋白表达结果在基因水平进行了验证。结论1.奥曲肽可抑制肝硬化门脉高压时期血红素氧合酶-1在肝脏的表达，其对血红素氧合酶/一氧化碳系统进行调控可能是其降门脉压机制之一。2.血红素氧合酶/一氧化碳系统在门脉高压形成中具有重要作用。第二部分肠道转运蛋白及代谢酶对奥曲肽肠吸收的影响目的：研究肠上皮转运蛋白P-糖蛋白(P-glycoprotein, P-gp)、多药耐药相关蛋白2（multidrug resistance associated protein2, MRP2）及代谢酶细胞色素CYP4503A4（cytochrome P4503A4, CYP3A4）与奥曲肽（Octreotide, OCT）肠吸收关系，并了解在肝硬化门脉高压（portal hypertension, PH）状态下，P-gp、MRP2及CYP3A4变化对OCT肠吸收作用影响。方法1.运用Caco-2细胞转运模型，分别给予OCT（10μM）及加入P-gp抑制剂罗丹明123（1mM）或MRP2抑制剂丙磺舒（1mM）后（n=4），考察OCT在Caco-2细胞肠腔侧（apical side, AP）和基底侧（basolateral side, BL）双向转运的表观渗透系数（apparent permeability coefficient, Papp）及渗透方向率（permeability directionrate, PDR）；考察OCT药物浓度(1μM或50μM)对转运的影响。2.建立胆管结扎肝硬化PH大鼠模型，运用离体翻转肠实验，考察P-gp抑制剂维拉帕米（1mM）及MRP2抑制剂丙磺舒（1mM）对浆膜侧OCT浓度影响。正常大鼠分组（n=4）：单独予OCT（A组）；予维拉帕米+OCT（B组）；予丙磺舒+OCT（C组）；予维拉帕米及丙磺舒+OCT（D组），OCT浓度10μM。PH大鼠分组同上，对应设为A1组，B1组，C1组及D1组（n=4）。3.运用大鼠在体空肠灌流实验，考察抑制剂对血中OCT浓度影响情况。正常大鼠分组（n=4）：予OCT（A组）；予P-gp抑制剂维拉帕米+OCT（B组）；予丙磺舒+OCT（C组）；同时予维拉帕米及丙磺舒（D组）。PH大鼠分组同上，对应分组为A1组，B1组，C1组及D1组。给药浓度同翻转肠实验。4.制备正常及PH大鼠肠微粒体，考察CYP3A抑制剂酮康唑对OCT代谢影响。正常大鼠微粒体实验分组（n=6）：予OCT（N+OCT组），予OCT+酮康唑（N+OCT+K组）；PH大鼠微粒体实验分组同正常大鼠，对应地设为PH+OCT组，PH+OCT+K组；对照组：未加微粒体，仅予OCT孵育（OCT组）。OCT浓度100μM，酮康唑浓度10μM。5.人CYP3A4重组酶实验，在2.5mg/mL重组微粒体酶中加入OCL（100mM），考察不同孵育时间对OCT代谢影响，选择最佳孵育时间。孵育10min，考察不同CYP3A4蛋白浓度（1.25,2.5,5,10,20mg/mL）对OCT（100mM）代谢影响，选择最佳重组酶的蛋白浓度。根据最适重组酶的蛋白浓度和最适反应时间，将不同浓度OCT（20,50,100,200,400mM）加入孵育体系中，检测剩余OCT浓度，考察孵育体系中是否有代谢物生成。结果1. OCT在Caco-2细胞转运无浓度依赖性，10μM是考察P-gp、MRP2主动转运最适宜浓度，分别单独予P-gp和MRP2抑制剂，PDR均>1.5。正常组Papp（B-A）较Papp（A-B）明显升高（p<0.05），予P-gp及MRP2抑制剂后，抑制剂组Papp（A-B）均高于Papp（B-A）（p<0.05）；各抑制剂组Papp（A-B）较正常组明显升高，在两种抑制剂联用情况下升高更为显著（p<0.01）；合用两种抑制剂，Papp（A-B）高于单用一种抑制剂组（p<0.05），且Papp（B-A）低于正常组（p<0.01）。2.离体翻转肠实验，对比OCT浓度，D组在三组中最高(p<0.05)，B组较C组升高(p<0.05)，PH大鼠离体翻转肠实验提示上述相同结果，且PH大鼠各组OCT浓度较相应正常大鼠各组下降(p<0.05)。正常大鼠B，C和D组AUC值分别是A组的176.22%，151.39%和220.63%。PH大鼠B1，C1和D1组AUC值分别是A1组的151.82%，128.23%，和165.33%。A1组的AUC值是A组的21.54%。3.在体空肠灌流实验，对比OCT浓度，D组较其他三组升高(p<0.05)，B组较C组升高(p<0.05)，PH大鼠实验结果与之相同，且PH大鼠各组OCT浓度较相应正常大鼠各组下降(p<0.05)。B，C和D组AUC值分别是组A的521.08%，418.95%和618.40%。B1，C1和D1组AUC值分别是组A1的384.61%，236.28%和461.78%。A1组的AUC值是A组的96%。4.肠微粒体实验，PH+OCT组OCT浓度低于N+OCT组(p<0.05)；予抑制剂酮康唑后，OCT浓度显著升高(N+OCT组<N+OCT+K组, PH+OCT组<PH+OCT+K组,p<0.01)。OCT组OCT浓度较N+OCT组及PH+OCT组升高（p<0.01)，但较N+OCT+K组及PH+OCT+K组均无明显差异(p>0.05)。5.人重组CYP3A4酶实验，最佳孵育时间为10min，最佳重组酶CYP3A4蛋白浓度为2.5mg/mL。随着体系中加入OCT浓度降低，残余OCT量减少，孵育体系中可检测到小分子OCT代谢产物。结论奥曲肽是肠上皮转运蛋白P-糖蛋白、多药耐药相关蛋白2及代谢酶细胞色素CYP4503A4的底物，三者具有明显抑制奥曲肽肠吸收效应。在肝硬化门脉高压病理状态下，上述转运蛋白及代谢酶表达或活性升高，对OCT肠吸收的抑制作用更为显著。第三部分肝硬化门脉高压状态下奥曲肽空肠促吸收及降门脉压研究目的：考察应用肠上皮转运蛋白P-糖蛋白(P-glycoprotein, P-gp)、多药耐药相关蛋白2（multidrug resistance associated protein2, MRP2）及代谢酶细胞色素CYP4503A（4cytochrome P4503A4, CYP3A4）抑制剂后对门脉高压（portal hypertension, PH）大鼠奥曲肽（Octreotide, OCT）肠吸收影响及降门脉压效果测定；了解PH状态下，寡肽转运蛋白PepT1变化对OCT肠吸收影响，期待寻求实现OCT口服用药的有效方法。方法1.建立胆管结扎肝硬化PH大鼠模型，进行在体吸收实验，观察加入P-gp，MRP2及CYP3A抑制剂后，大鼠肠吸收效果变化。正常大鼠分2组（n=4）：OCT静脉给药及OCT十二指肠给药组；PH大鼠分组：OCT静脉给药组，OCT十二指肠给药组及OCT联合抑制剂（维拉帕米4.9mg/kg,丙磺舒300mg/kg，酮康唑5.3mg/kg）十二指肠给药组（n=4）。OCT十二指肠给药剂量1mg/kg，静脉给药剂量0.1mg/kg。给药后不同时间点于颈静脉取血，计算药代动力学参数。2.正常大鼠分为2组，未加药物组（N组），予OCT组（N+OCT组）；PH大鼠分3组，无药物组（PH组），予OCT组（PH+OCT组），及OCT+混合抑制剂组（抑制剂剂量同上），OCT给药剂量1mg/kg，日1次，连续给药3天，处死大鼠，取空肠，用western blot、免疫组化及逆转录聚合酶链式反应（reverse transcriptionpolymerase chain reaction, RT-PCR）方法观察各组大鼠空肠上皮P-gp，MRP2及CYP3A4蛋白和基因的表达。3.测压实验：正常大鼠不给药（N组），PH大鼠分为无药物组（PH组），OCT组（PH+OCT组）及OCT+混合抑制剂（抑制剂剂量同上）组(PH+OCT+I组)（n=4），OCT给药剂量为1mg/kg，日1次，连续3天灌胃给药后，测定门静脉压力。4.对PH状态下，PepT1对OCT转运效果考察。正常大鼠分不给药组（N组），及OCT给药组（N+OCT组），PH大鼠分组相同，设为PH及PH+OCT组（n=4），OCT给药剂量为1mg/kg，日1次，连续灌胃给药3天，处死大鼠，取空肠，通过westernblot、免疫组化及RT-PCR方法观察各组大鼠PepT1蛋白及基因表达情况。运用Caco-2细胞转运模型，考察PepT1抑制剂甘氨酰肌氨酸（glycylsarcosine，Gly-Sar）(1mM)对OCT（10μM）在肠腔侧（apical side, AP）和基底侧（basolateral side, BL）双向转运的表观渗透系数（apparent permeability coefficient, Papp）及渗透率（permeability direction rate, PDR）。建立离体翻转肠模型（n=4），正常大鼠分为给予OCT（10μM）组（N组）及OCT+Gly-Sar(1mM)组（N+G组），PH大鼠给药及分组相同，设为PH及PH+G组。建立大鼠空肠灌流模型，分组及给药同翻转肠实验。结果1.大鼠在体吸收实验，静脉注射后OCT浓度从血浆中快速清除，t1/2较短，正常大鼠Vd、AUC较PH大鼠升高，表明药物清除缓慢。空肠输注OCT后，PH大鼠较正常大鼠Tmax延长，Cmax和AUC(0-12h)下降。OCT联合P-gp/MRP2/CYP3A混合抑制剂后Tmax减少，同时Cmax, AUC(0-12h)较正常大鼠及未应用抑制剂的PH大鼠明显升高，F及ER约是未应用抑制剂的PH大鼠的4倍。2. western blot、免疫组化结果均发现PH组P-gp，MRP2和CYP3A4蛋白均较N组升高(p<0.05)，予OCT后显著升高(N+OCT组> N组, PH+OCT组> PH组, p<0.05)，予OCT联合混合抑制剂后蛋白表达最少(p<0.01)，N+OCT组和PH组表达无差异(p>0.05)。RT-PCR方法见基因表达与蛋白表达结果一致。3.考察混合抑制剂对大鼠门脉压力影响发现，PH组大鼠平均门脉压力较正常组（(15.56±2.36mmHg) vs.(9.24±0.76mmHg)明显升高（p<0.01)，且高于PH+OCT组(12.51±1.50mmHg，p<0.05)。PH+OCT+I组门脉压力(6.95±1.12mmHg)较PH组及PH+OCT组明显下降(p<0.01)，在正常范围内。4.考察PepT1与OCT肠吸收关系，免疫组化及western blot检测发现对比PepT1蛋，PH组较N组表达升高（p<0.05），应用OCT组较未应用组无差异(N+OCT组vs. N组, PH+OCT组vs. PH组, p>0.05)， PH+OCT组高于N+OCT组（p<0.01)。RT-PCR基因检测结果与蛋白表达结果一致。Caco-2细胞研究发现，Gly-Sar抑制PepT1后，Papp（A-B）较Papp（B-A）仍明显降低，无升高趋势，且PDR<1.5，故表明PepT1不能介导OCT在Caco-2细胞的主动转运。翻转肠实验见较N组，N+OCT组浆膜腔中OCT浓度无明显差异，在PH大鼠中，亦无明显改变（p>0.05）。PH大鼠各组OCT浓度较相应正常大鼠各组下降(p<0.05)，PH组是N组的52.4%。在体空肠灌流模型，OCT浓度在N组和N+G组中无差异，在PH组和PH+G组中亦无差异（p>0.05）。PH大鼠各组OCT浓度较相应正常大鼠各组下降(p<0.05)，PH组是N组的62.7%。结论1. P-糖蛋白，多药耐药相关蛋白2及细胞色素CYP4503A4在奥曲肽肠吸收中具有重要作用，对其进行抑制可以明显促肝硬化门脉高压大鼠肠吸收，降压效果理想。2.寡肽转运蛋白PepT1在肝硬化门脉高压时期表达增强，但对奥曲肽无转运作用。更多还原

【Abstract】 Objective: Hepatitis B is an important reason to cause liver cirrhosis. There arehundreds of patients with hepatitis B or with hepatitis B virus infection in China, andmillions of them can develop to cirrhosis very year. Portal hypertension (PH) is thetypical manifestation of patients at the decompensated stage of cirrhosis, and it is themain reason to result in various complications and death. So it is important to preventand treat of patients with PH, thus improving the prognosis and decreasing the mortalityof patients.It is revealed that heme oxygenase/carbon monoxide (HO/CO) system canaggravate PH during the middle and end stage of cirrhosis, and induce progressivedamages of various organs and tissues, thus accelerating cirrhosis process. The resultswere also confirmed by our studies on the basis of cirrhotic rats with PH. But up to now,the researches focused on HO/CO system mostly are basic studies, and lack of clinicalstudies. We had found that carboxyhemoglobin, which could reflect the level of HO/COsystem was obviously increased in patients with PH. The current research was the firsttime to perform perspective study to reveal the importance of HO/CO system onpatients with PH complicated by various complications and the effects of it on organs.As a kind of Somatostatin, Octrotide (OCT) is an ideal drug for decrease portal pressure,but the detail mechanism is unclear. Due to the effects of vascular contraction, OCT canreduce portal blood volumn and decrease portal pressure. Due to the mechanism ofendogenous OCT to maintain PH is dilation of splanchnic vessels, we thought OCTmight play effects in decreasing portal pressure by influence on HO/CO system. Thisstudy was to deeply research the mechanism of OCT effects on PH by HO/CO system,and further confirm the key role of HO/CO system on cirrhosis.On the basis of the two parts of experiments and results of previous studies, we found that HO/CO system played great role during PH development and OCT coulddecrease portal pressure by inhibition of the system. Consideration of variouspharmacological advantages of OCT, it is useful to realize oral administration of OCTin order to resolve the difficulties of prevention and treatment of PH. 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 prohibited by intestinal absorption barrier, soits oral bioavailability is low. Up to now, many researchers are focused on increasingOCT absorption by change its chemical structures or increase paracellular absorptionsthrough adding absorption enhancers, and enhancing permeability of biologicalmembrane, but the absorption effects are not ideal. In addition, the mechanisms of OCTabsorption and ways to increase absorption have not been studied underwent PH state.We thought studies on factors which directly influence on first-pass effects of OCTabsorption might be useful for finding out the absorbed strategies. Because ofportosystemic shunt formation, collateral circulation establishment and abnormal liverfunction, hepatic first-pass effects plays little influence on OCT absorption, while thefirst-pass effects of intestine may take obvious roles. The current study was to studyp-glycoprotein (P-gp) and multidrug resistance associated protein2(MRP2) which arethe transport proteins located on intestines, and intestinal metabolic enzyme cytochromeP4503A4(CYP3A4), in order to reveal the absorption mechanisms of OCT inintestines. In addition, whether OCT absorption would be affected by changes of thesetransporters and enzyme underwent cirrhosis with PH was also been considered.Moreover, the effects of PepT1changes underwent PH on OCT absorption were alsobeen studied, for finding a transporter which could transport OCT directly.The aim of this study was to reveal the pivotal effects of HO/CO system duringcirrhosis with PH by basic and clinical researches from various angles, in order to findout the mechanism of decreasing portal pressure by OCT mediated by this system,definite the reason hindered the intestinal absorption of OCT and provide the solutionsfor reducing intestinal-pass effects. It was also useful to find out the effective ways forincreasing oral bioavailability of OCT, realize portal pressure reduction by oraladministration of it, and provide theory evidence for rational use of OCT in clinic at thesame time. Part IStudy on the mechanism of decreasing portal pressure by Octreotidemediated by heme oxygenase/carbon monoxide systemQuarter IThe effects of heme oxygenase/carbon monoxide system on portal hypertensionObject: The aim of this study is to detect changes of carboxyhemoglobin (COHb)levels and the relationship between them and portal hypertension (PH) complicated byadvanced complications, and to know the correlation of COHb levels and hepaticencephalopathy (HE) degrees on patients of hepatitis B related cirrhosis with HE,hoping for revealing the effects of heme oxygenase/carbon monoxide (HO/CO) systemon patients with PH complicated by complications and on various organs.Methods: According to the diagnostic and excluding criterions,68inpatients withhepatitis B virus-related cirrhosis (HBC) complicated by HE who were admitted to theGastrointestinal Department of the First Affiliated Hospital of Dalian MedicalUniversity without treatment were enrolled (group H).22patients who were inemergency department and excluded vital organs disorders (all related clinical testswere normal) were selected as control group (group N). The information of clinical datawas collected. The patients were performed related tests, and the data ofcarboxyhemoglobin (COHb) levels, partial pressure of oxygen (PaO2), oxygensaturation (SaO2) and so on were gathered. The patients were received diagnosticanalyses of HE stages, esophagogastric varices, hepatic renal syndrome (HRS),spontaneous bacterial peritonitis (SBP) and hypoxemia. The differentiations of COHb inHE patients with PH complications and none were compared, and the relationship ofCOHb level among grades of HE, PaO2and SaO2were analyzed.Results: The level of COHb in Group H was higher than in Group N((2.102±1.021)%vs.(0.983±0.231)%, P=0.000）. The COHb standard has positivecorrelation with Grade of HE (p=0.003, r=0.358). The content of COHb was increasedin patients without HRS comparison to patients with HRS ((1.981±1.020)%vs.(2.502±1.073)%，p=0.029), with no differences between patients with SBP or not(p=0.75), and with esophagogastric varices or none (p>0.05). COHb level has anegative correlation with PaO2(p=0.006, r=-0.336) and no relative to SaO2(p=0.576,r=-0.072). It was higher when the two parameters met diagnostic criteria of hypoxemia ((2.72±0.362)%vs.(1.93±0.242)%，p=0.012).Conclusions：Heme oxygenase/carbon monoxide system played an important rolein pathophysiological process of hepatic cirrhosis with portal hypertension, and its levelhas tissue-specific. This system has great clinical meanings in cirrhotic patients with PHcomplicated by various complications. Quarter IIThe research on the mechanism of decreasing portal hypertension by Octreotidemediated by heme oxygense/carbon monoxide systemObject: The aim was to understand the mechanism of reducing portal pressure byOCT, to observe whether the process was mediated by heme oxygenase/carbonmonoxide (HO/CO) system and to confirm the pivotal effects of HO/CO on cirrhosiswith portal hypertension (PH).Methods:34Male Sprague-Dawley rats were divided randomly into a Sham group(n=10), PH group (n=12) and OCT group (n=12). The cirrhotic rats with PH wereestablished by common bile duct ligation. The group which intraperitoneallyadministrated Otreotide (100μg/kg/d) was regarded as group OCT. The rats in group PHand group Sham was treated with the same doses of saline. After drugs administrationfor3days, all rats were measured portal venous pressure and then were killed forcollection of blood and liver tissues. HE staining was performed to observe thepathomorphology of liver tissues. The expressions of HO-1protein and mRNA weretested by the ways of immunohistochemistry, western blot and reverse transcriptionpolymerase chain reaction (RT-PCR), respectively. The levels of alanineaminotransferase (ALT) and aspartate aminotransferase (AST) were assayed byautomatic blood biochemistry analyzer.Results: In group PH, the levels of ALT and AST were much higher, and wascharacterized by fibrosis proliferation, amount of inflammatory cells infiltration,pseudolobule formation, and proliferation of interlobular bile duct, moreover, the portalpressure ((18.52±1.83) mmHg) was greatly increased, all the results were exited statistical difference between group Sham (p<0.01). In group OCT, the contents of ALTand AST, the lesions degree of liver tissues by HE staining and portal pressure((13.17±1.12mmHg) were greatly lightened (p<0.05), but worse than in group Sham(p<0.05). The methods of immunohistochemistry and western blot showed that theprotein expressions of HO-1in group OCT was evidently lower than in group PH(P<0.01), but higher than in group Sham (p<0.05), moreover, the way of RT-PCR wasconfirmed the results form mRNA levels.Conclusions: The expressions of heme-1in liver can be inhibited by OCT at thestage of cirrhosis with portal hypertension. The regulation of OCT to heme oxygense/carbon monoxide system may be regarded as a mechanism of it effects on portalpressure. The heme oxygense/carbon monoxide system plays an important role on theformation of portal hypertension. Part IIThe effects of transports and metabolic enzymes onOctreotide absorption in intestinesObject: The transports, such as P-glycoprotein (P-gp) and multidrug resistanceassociated protein2(MRP2), and metabolic enzyme cytochrome P4503A4(CYP3A4)located on intestines are regared as key factors for affecting on drugs transport andmetabolism. The aim of this study was to know the relationship of them and Octreotide(OCT), and to understand whether their changes underwent cirrhosis with portalhypertension (PH) can influence on OCT absorption.Methods: The effects of P-gp or Mrp2inhibitors rhodamine123(1mM) andprobenecid (1mM) on the uptake and transepithelial transport of OCT (10μM) wereobserved by permeability direction rate (PDR) and from apparent permeabilitycoefficient (Papp) received by OCT bidirectional transportation from apical side (AP) orbasolateral side (BL). The effects of OCT concentrations (1μM and50μM) ontransportation were also considered. The cirrhotic rats with PH were established bycommon bile duct ligation. The effects of P-gp or MRP2inhibitors verapamil (1mM)and probenecid (1mM) on transportation of OCT (10μM) were observed by the model of everted intestinal sacs. The normal rats were divided into group A, B, C and D (n=4).The PH rats were did the same experiments and in accordance divided into group A1,B1, C1and D1(n=4). In situ jejunal perfusions of rats were also performed, and thedoses and grouping were the same as in the experiments of everted intestinal sacs (n=4).The effect of CYP3A inhibitor ketoconazole on intestinal metabolism of OCT wasdetermined by intestinal microsomes obtained from both kinds of rats. Each group wasadded OCT (100μM). According to whether ketoconazole (10μM) was added, thenormal rats were separated to group N+OCT and group N+OCT+K. The same drugs’doses and grouping were performed to PH rats, and in accordance divided into groupPH+OCT and group PH+OCT+K (n=6). The group without microsomes was regardedas control (group OCT).The experiment of human recombinant CYP3A4was applied for determinewhether OCT was one of substrates of CYP3A4. Firstly, the best incubation time wasdetermined by changes of reaction time by limitation of protein content of recombinantCYP3A4(2.5mg/mL) and the OCT concentration (100mM). Secondly, the bestincubated protein concentration of recombinant CYP3A4was observed by limitation ofother two factors. According to the optimized protein contents of CYP3A4and reactiontime, different OCT concentrations (20,50,100,200,400mM) were put into thereaction mixture and found out the metabolic productsResults1. It was no concentration dependence of OCT transportation across Caco-2cells.The permeability direction rate PDR>1.5when P-gp or/and MRP2were inhibited at theconcentrtation of OCT10μM. The Pappof transportation for10μM OCT from side B toA mediated by P-gp and MRP2was significantly increased than the Pappoftransportation from side A to B, and Papp (A-B)s were much increased when inhibitedP-gp or MRP2, especially for inhibition P-gp and MRP2at the same time, on thecontrary, Papp (B-A)s were decreased obviously.2. In the model of everted intestinal sacs, the concentration of OCT in group D wasincreased comparison to other three groups (p<0.05). Moreover, compared with group C,the OCT content in group B was much higher (p<0.05). The same phenomenon couldbe seen in PH rats. Moreover, the OCT contents in PH rats were decreased obviouslycompared with those of the corresponding normal group (p<0.05). The AUC values ingroup B, C and D were176.22%,151.39%, and220.63%of that of the group A,respectively. The AUC values of the group B1, C1and D1were151.82%,128.23%, and165.33%of that in the group A1. While the AUC of the group A1was21.54%ofthat in the group A.3. In the model of in situ jejunal perfusions of rats, the levels of OCT in Group Dwere increased significantly (p<0.05). The OCT content in group C was much higherthan in group C (p<0.05). The same phenomenon could be seen in PH rats. In addition,the OCT contents in PH rats were decreased compared with those of the correspondingnormal group (p<0.05). The AUCs of the group B, C and D were521.08%,418.95%,618.40%of that of the respective control group (group A). The AUCs of the Group B1,C1and D1were384.61%,236.28%,461.78%of that in the group A1. While the AUCof the group A1was96%of that of the group A.4. In the experiments of intestinal microsomes, the levels of OCT were eminentlylowered in group PH+OCT than in group N+OCT (p<0.05), were obviously increasedwhen usage of ketoconazole (group N+OCT<group N+OCT+K, group PH+OCT<group PH+OCT+K, p<0.01). The contents of OCT in group OCT were much higherthan in Group N+OCT and in group PH+OCT (p<0.01). There was no difference ofOCT contents between group N+OCT+K or PH+OCT+K with group OCT (p>0.05).5. In the experiments of human recombinant CYP3A4, the best incubation timewas10min and the best protein was2.5mg/mL. Under the optimized incubation timeand protein content of CYP3A4, the residual OCT contents were decreased along withreduced initial concentration of OCT (p<0.05), and also many micromolecularmetabolites were found by test of Liquid chromatography-tandem mass spectrometry.Conclusions: P-glycoprotein, multidrug resistance associated protein2, andmetabolic enzyme cytochrome P4503A4were played inhibitory effects on Octreotideintestinal absorption, and Octreotide is the substrate of them. Up-regulated expressionsor activities of them were found under cirrhosis with portal hypertension, and enhancedblocking effects of OCT in intestines. Part IIIThe research on pormoting intestinal absorption ofOctreotide and decreasing portal pressuer under cirrhosiswith portal hypertensionObject: The aim was to know the influence of inhibitors of P-glycoprotein (P-gp),multidrug resistance associated protein2(MRP2) and cytochrome P4503A4(CYP3A4)on intestinal absorption of Octreotide (OCT) and the effects of them on decreasingportal pressuer. In addition, the impact of oligopeptide transporter PepT1on OCTabsorption was also studied, in order to find an effective way for realizaion of OCT byoral administration.Methods:1. The in vivo absorption experiment in rats was performed. The Normal rats weredivided randomly into two groups:1) intra-jejunal (IJ) injection of OCT (1mg/kg),2) i.v.infusion of OCT (0.1mg/kg)(n=4). PH rats were divided randomly into three groups:1)IJ injection of OCT (1mg/kg),2) i.v. infusion of OCT (0.1mg/kg),3) IJ injection ofOCT (1mg/kg) and mixed inhibitors (verapamil hydrochloride4.9mg/kg, probenecid300mg/kg and ketoconazole5.3mg/kg)(n=4, separately). The pharmacokineticparameters were caculated.2. The normal rats were divided into three groups according to if administratedOCT or not (group N and group N+OCT, respectively). PH rats were divided inaccordance to group PH and group PH+OCT, and the rats with OCT and mixedinhibitors (verapamil hydrochloride4.9mg/kg, probenecid300mg/kg and ketoconazole5.3mg/kg, once a day) were regared as group PH+OCT+I (n=4, separately). The dose ofOCT was1mg/kg, once a day. After3days, all rats were killed and the ways of westernblot, immunohistochemistry and reverse transcription polymerase chain reaction(RT-PCR) were performed to test the protein and mRAN expressions of P-gp, MRP2and CYP3A4on jejumns of each group.3. The PH rats were divided into three groups according to whether oraladministration of OCT (1mg/kg, once a day), and if orally administrated mixedinhibitors or not (group PH, group OCT, and group PH+OCT+I). The normal rats weregiven routine food and water without any drug. The doses of inhibitors were the same asabove (n=4, separately). After3days, the portal pressure of each rat was measured.4. The normal rats were divided into two groups according to whether oral administration of OCT (1mg/kg, once a day) or not (group N and group N+OCT). ThePH rats were did the same experiments and were grouping in accordance to group PHand group PH+OCT. After3days, the rats were killed and the ways of western blot,immunohistochemistry and RT-PCR were performed to test the protein and mRANexpressions of PepT1on jejumns of each group. The effects of PepT1inhibitorglycylsarcosine (Gly-Sar)(1mM) on the uptake and transepithelial transport of OCT(10μM) in Caco-2cells were observed by permeability direction rate (PDR) and fromapparent permeability coefficient (Papp) received by OCT bidirectional transportationfrom apical side (AP) or basolateral side (BL)(n=4, respectively). The models ofeverted intestinal sacs were established, and according to whether Gly-Sar wasadministrated, normal rats were divided into group N and group N+G. The PH rats weredid the same experiments, and in accordance to divided as group PH and group PH+G(n=4). In situ jejunal perfusions of rats were also performed, the doses and groupingwere the same as in the experiments of everted intestinal sacs (n=4).Results1. In vivo experiments, OCT was rapidly dispelled from plasma after i.v. infusion,and it underwent slower elimination or easier absorption in normal rats than in PH rats.Increased expression or activities of transporters and metabolic enzymes in liver mayoccur, leading to quicker elimination in PH state. By IJ administration of OCT, longerTmax, lower Cmaxand AUC0-12hwere found in PH rats than in normal rats, indicating thatincreased expressions or activities of transporters and metabolic enzymes might exist inintestines of PH rats to inhibit OCT absorption. Shorter Tmax, higher Cmaxand AUC0-12hwere observed in the group of OCT with P-gp/MRP2/CYP3A mixed inhibitorscomparing to normal rats and PH rats without inhibitors. In addition, F and ER wereobviously increased about4times for PH rats when usage of mixed inhibitors.2. The results from western blot and immunohistochemistry found that proteinexpressions of P-gp/MRP2/CYP3A4were significantly increased in Group PH than inGroup N (p<0.05), were obviously increased when usage of OCT (GroupN+OCT>Group N, Group PH+OCT> Group PH, p<0.05), and were the least in GroupPH+OCT+I among all the groups because of inhibitors usage (p<0.01), whereas noeminent difference between Group N+OCT and Group PH (p>0.05). The results ofRT-PCR were the same as western blot showed from mRNA levels.3. The average PVP level of Group PH (15.56±2.36mmHg) was increased thannormal rats (9.24±0.76mmHg, p<0.01) and was higher than that of Group PH+OCT (12.51±1.50mmHg, p<0.05). The PVP level of Group PH+OCT+I (6.95±1.12mmHg)was much lower than that of Group PH and Group PH+OCT (p<0.01). It was indicatedthat OCT with mixed inhibitors by oral via could decrease PVP effectively.4. RT-PCR showed that the expressive levels of PepT1mRNA were significantlyhigher in PH group than in group N (p<0.05). Compared the groups without OCTtreatment, the mRNA expression levels of PepT1in groups with OCT were no obviousdifference (group N+OCT vs. group N, group PH+OCT vs. group PH, p>0.05), whilein Group PH+OCT was higher than in group N+OCT (p>0.05). The results of westernblot and immunohistochemistry were the same as RT-PCR showed from protein levels.Caco-2cell model was used to examine the effects of PepT1inhibitor on OCT uptake.The Papp of transportation for OCT from side A to B mediated by influx pumptransporter PepT1was significantly decreased than the Papp of transportation from sideB to A. The PDR<1.5revealed that PepT1could not mediate OCT transport in Caco-2cells. On the basis of everted intestinal sacs, compared with Group N, the serosal sideconcentration of OCT in Group N+G was no obvious difference (p>0.05). The samephenomenon could be seen in PH rats. These results suggested PepT1could not mediateOCT transport, not only in normal rats, but also for rats with PH. While the AUC of thegroup PH was52.4%of that in the group N. In the model of in situ perfusions of rats,there was no obvious difference of OCT concentration between Group N and GroupN+G (p>0.05). The same phenomenon could be seen in PH rats. The results revealedthe similar conclusions of experiments of everted small intestinal sacs that PepT1couldnot transport the OCT uptake in situ, either for normal rats or PH rats. While the AUCof the group PH was67.2%of that in the group N.Conclusions: P-glycoprotein, multidrug resistance associated protein2andcytochrome P4503A4play great important role in intestinal absorption of Octreotide.Inhibition of them can induce OCT absorption of rats with portal hypertension, andrealize ideal effects of decreasing portal pressure. The expressions of oligopeptidetransporter PepT1were enhanced on the stage of cirrhosis with portal hypertension, butit has no effect on transportation of OCT in intestines of rats.更多还原