【作者】 赵文新；

【导师】 陈道中； 陈良万；

【作者基本信息】 福建医科大学 ， 外科学， 2008， 博士

【摘要】 体外循环（cardiopulmonary bypass,CPB）对机体是一种全身性的强刺激,能引起机体缺血缺氧再灌注损害并产生强烈的应激反应。应激引起过度的炎症反应造成组织损害、全身性炎症反应综合征（systemic inflammation responsesyndrome,SIRS）甚至多器官功能障碍（multiple organs dysfunction syndrome,MODS）,是心脏外科术后死亡的主要原因之一。CPB过程中器官保护是心血管外科基础临床研究热点之一。一直以来,人们对CPB所致的心、脑、肺、肾等重要器官的研究较多,也比较深入,而近来逐步认识到肠道是应激反应的始动器官和重要靶器官之一,其关键在于肠粘膜屏障功能损害导致细菌/内毒素移位。CPB后肠粘膜屏障功能损害属于学科交叉范畴,尚未引起广泛重视,目前对其发生及其机制研究较少。临床CPB后消化系统严重并发症的发生率低0.41～3.7%,但死亡率高13.9～52%,因此研究CPB过程中肠屏障损伤的发生机制及探讨相应的保护措施具有重要的临床意义。正常肠屏障功能的维持依赖于完整的肠粘膜上皮、肠道内正常菌群、肠道内分泌物、蠕动、肠道免疫功能等,其中最关键的是肠粘膜上皮屏障。在肠粘膜高度旺盛的新陈代谢背景下,严重创伤缺血缺氧—再灌注后,过度的粘膜上皮细胞凋亡和坏死,必然导致粘膜屏障的损伤。既往认为应激状态后肠粘膜坏死是肠屏障功能损害的病理基础,有研究表明肠粘膜上皮细胞凋亡可能是肠屏障功能损害的主要原因。小动物特制微型膜肺和转流泵装置的生产和采用闭胸穿刺法手术操作技术,使得建立经济实用、微创、操作简便可靠、更接近临床过程的全流量、常温大鼠CPB模型成为可能,为CPB后早期全身炎症反应多器官功能障碍及其防治策略的研究提供理想载体。乌司他丁是具有多种生物学功能的广谱酶抑制剂,抑制多种水解酶的活性,抑制炎症介质的过度释放及改善微循环和组织灌注的药理作用,在临床危重患者的抢救中取得良好的疗效,对重要器官有保护作用。本研究目的在于探讨CPB后大鼠肠粘膜屏障形态和功能的损害,以及炎症应激反应的发生与肠粘膜上皮的过度凋亡在肠粘膜损害中的机制,并通过乌司他丁预处理研究其预防作用和机制。本研究分三部分展开论述,研究方法、结果和结论如下。1.插管法建立大鼠常温体外循环动物模型1.1目的插管法建立大鼠经济实用、微创、操作简便可靠、更接近临床过程的全流量、常温大鼠体外循环实验模型。1.2方法雄性成年SD大鼠14只350～500g,麻醉后给予气管穿刺插管,呼吸机辅助通气,股动脉置管接监护仪,实时监测动脉血压并按时采集动脉血气标本。血液经蠕动泵输送至特制微型膜肺氧合后经由右颈动脉实施灌注。模型采用林格氏液、中分子羟乙基淀粉（130/0.4）、少量大鼠供血进行预充,总量为22ml,晶胶比为1:1,灌注流量120～160ml·kg^-1·min^-1,平均转流时间60min。1.3结果14只大鼠中2只死于CPB建立过程中的出血,2只因颈静脉插管遇阻力,导致深度不够引流不通畅失败。模型成功率71%,CPB平均转流时间60min,转流量达到120～160ml·kg^-1·min^-1,接近大鼠的心排出量,符合完全转流的要求。实验中动物血流动力学稳定,氧合器氧合性能完全符合满意CPB标准。CPB期间大鼠血气显示常常有酸中毒,其余血气和电解质基本正常。在CPB停止过程中,大鼠血压、心率开始下降,虽经应用血管活性药物,但仍明显低于转流前,加之血液严重稀释,大鼠不能长期存活,一般在7小时内因循环呼吸衰竭死亡。1.4结论采用插管法,通过右颈静脉腔房引流、右颈动脉灌注的方法可建立大鼠闭合胸腔的全流量CPB实验动物模型,手术操作简便,易于管理,经济可行。微型化的环路设计能够为建立稳定的大鼠CPB提供保证,微型膜肺和蠕动泵能够满足大鼠CPB的氧合和血流动力学要求。这一模型术后短期存活率高,充分发挥相关病理、生理、生化及分子生物学指标的检测能力,可使与CPB相关的基础研究系列化、规模化,有较大应用前景。2.大鼠体外循环后早期肠粘膜屏障功能的损害及其机制2.1目的本研究第二部分通过观察CPB后大鼠回肠粘膜形态和屏障功能的损伤以及血浆炎症因子的浓度变化,探讨CPB后肠粘膜屏障的损害的临床意义。通过蛋白和基因水平的检测,探讨是否存在大量炎症因子的产生和过度凋亡,在CPB大鼠肠粘膜屏障损害的可能机制。2.2方法健康成年雄性清洁级Spranue-Dawley（SD）大鼠30只随机分为正常对照组（N组,n=10）,假手术组（sham-operated group,SH组,n=10）体外循环组（cardiopulmonary bypass group,CPB组,n=10）;SH组大鼠在相应部位插管,同时经股动脉10min内缓慢推注预充液22ml,但不进行CPB转流,2h后活杀动物采取标本;CPB组转流结束后2h活杀动物采取标本。光镜、检查末段回肠粘膜绒毛高度和Chiu损害评分:电镜检查肠粘膜超微结构改变:化学比色法检测血浆DAO活性、D-乳酸和NO浓度改变;ElISA法检测血浆TNFα、IL-6浓度变化。血浆检测数值予以校正,以排除预充液稀释的干扰。TUNEL法检测肠粘膜凋亡指数,免疫组化图像分析检测肠粘膜TNFα、iNOS、Bcl-2、Bax、Caspase-3蛋白表达变化,PT-PCR法检测肠粘膜TNFα、iNOS、Bcl-2、Bax、Caspase-3mRNA表达变化。2.3结果本研究中CPB组小肠部分绒毛水肿、绒毛两侧大量分离伴部分绒毛顶端破损,片状坏死脱落,固有层血管充血、淋巴细胞浸润伴固有层毛细血管暴露。N组和SH组形态学明显好于CPB组,小肠绒毛形态较正常。Chiu评分显示CPB后肠粘膜损害较N组和SH组明显（P＜0.01）。电镜观察N组和SH组小肠粘膜上皮细胞微绒毛排列整齐,柱状上皮细胞结构完整、细胞器结构未见明显异常;CPB组主要表现为小肠上皮细胞死亡的两种不同形式:凋亡和肿胀性死亡,微绒毛稀疏、排列不整、呈倒伏状、部分缺如、上皮细胞间连接增宽、部分紧密连接开放、线粒体肿胀,内质网扩张,部分细胞凋亡,表现为细胞核边集于核膜下并可见凋亡小体脱落于肠腔内,固有层水肿较明显并可见到增多的淋巴细胞,中性粒细胞浸涧。部分凋亡细胞继发坏死,表现为胞浆内细胞器崩解,丢失,胞膜不完整。还可见肿胀性死亡的上皮细胞,其核膜及胞膜破坏,胞浆内线粒体等细胞器崩解。大鼠经历CPB后出现肠屏障功能损害,CPB组血浆DAO活性、D-乳酸浓度较N组和SH组明显升高（P＜0.01）,SH组较N组略有升高但差异无显著性。大鼠CPB后处于全身炎症应激反应状态,血浆TNFα、IL-6、NO浓度较N组和SH组明显升高（P＜0.01）;SH组血浆TNFα、NO浓度较N组也明显升高（P＜0.05）。线性回归及相关分析表明:肠粘膜绒毛高度与血浆DAO活性、D-乳酸浓度高度负相关;血浆DAO活性、D-乳酸浓度与血浆TNFα、IL-6、NO浓度之间高度正相关。CPB组大鼠肠粘膜上皮细胞AI较N组和SH组明显升高（P＜0.01）,相关分析显示AI与血浆DAO活性、D-乳酸浓度、血浆TNFα、IL-6、NO浓度之间显著正相关。CPB组大鼠肠粘膜上皮细胞TNFα、iNOS、Bax、Caspase-3蛋白和mRNA表达均较N组和SH组明显升高（P＜0.01）:N组和SH组之间差异无显著性。Bcl-2蛋白和mRNA表达有轻度升高但无统计学意义,而Bcl-2/Bax比值下降与N组和SH组之比较有显著性。2.4结论大鼠CPB后肠粘膜出现形态学损害和屏障功能损害并处于全身炎症反应状态,线性回归及相关分析表明肠粘膜形态和屏障功能损害在CPB后全身炎症反应中起重要作用。CPB后肠粘膜上皮细胞凋亡上调局部缺血缺氧-再灌注、炎症因子TNFα,自由基NO生成过度以及促凋亡因子Bax上调有关,肠粘膜上皮细胞凋亡上调是CPB后肠粘膜屏障功能损害的主要机制。3.乌司他丁预防大鼠体外循环后早期肠粘膜屏障功能损害机制研究3.1目的采用转流前大、小不同剂量两组乌司他丁预处理,通过观察大鼠CPB后小肠粘膜损害的病理、生理指标和炎症因子、细胞凋亡的改变,并探讨其应用于CPB后肠粘膜屏障保护的可行性和机制。3.2方法健康成年雄性清洁级SD大鼠40只随机分为假手术组（SH组,n=10）体外循环组（CPB组,n=10）:乌司他丁小剂量组（U1组,n=10）,乌司他丁大剂量组（U2组,n=10）,CPB开始转流前U1组经股动脉给予乌司他丁4万U/kg,U2组经股动脉给予乌司他丁10万u/kg,乌司他丁两组转流和采取标本同CPB组,SH组与CPB组同前。持续监测大鼠CPB后平均动脉压与心率2h;光镜检查末段回肠粘膜绒毛高度和Chiu损害评分:电镜检查肠粘膜超微结构改变;化学比色法检测血浆DAO活性、D-乳酸、NO浓度改变:ELISA法检测血浆TNFα、IL-6浓度变化;TUNEL法检测大鼠小肠粘膜凋亡指数,免疫组化图像分析检测肠粘膜TNFα、iNOS、Bcl-2、Bax、Caspase-3蛋白表达变化,PT-PCR法检测肠粘膜TNFα、iNOS、Bcl-2、Bax、Caspase-3mRNA表达变化。3.3结果乌司他丁干预组大鼠CPB后平均动脉压与心率下降较CPB组改善,以U2组为著（P＜0.05）。U1、U2组大鼠肠粘膜充血,间质小血管扩张,仍可见中性粒细胞浸润,间质水肿,绒毛变矮、局灶性绒毛上皮脱落,但形态明显好于CPB组,绒毛高度和Chiu评分显示U1、U2组肠粘膜损害较CPB组改善明显（P＜0.05）,但U1、U2组间无明显差异。电镜观察见:U1、U2组较CPB组小肠粘膜上皮细胞微绒毛排列较整齐,柱状上皮细胞结构大部分完整、炎症细胞浸润明显,部分紧密连接开放、线粒体肿胀减轻、内质网轻度扩张,仍可见少量凋亡细胞,较CPB组明显改善。大鼠经乌司他丁干预CPB后出现肠屏障功能损害,U1、U2组血浆DAO活性、D-乳酸浓度较SH组仍明显升高（P＜0.05）,但较CPB组明显下降差异显著（P＜0.05）,U2组较U1组下降更明显差异（P＜0.05）。U1、U2组大鼠CPB后全身炎症反应减轻,U1、U2组血浆TNFα、IL-6、NO浓度较CPB组明显下降（P＜0.01）:U2组较U1下降更明显差异（P＜0.05）,U2组血浆TNFα、NO浓度较SH组升高不明显（P＞0.05）;U1、U2组大鼠肠粘膜上皮细胞AI较CPB组明显下降（P＜0.01）,但仍明显高于SH组,U1、U2组间无明显差异。U1、U2组较CPB组大鼠肠粘膜上皮细胞TNFα、iNOS、Bax、Caspase-3蛋白表达和mRNA表达均有明显下降（P＜0.05）;而Bcl-2仅略有下降与CPB组比较没有显著性,U2组TNFα、iNOS、Bax、Caspase-3蛋白和mRNA表达均较U1组有明显下降（P＜0.05）。3.4结论乌司他丁能有效改善大鼠CPB后循环稳定,减轻肠粘膜形态改变和屏障功能的损害,减轻过度全身炎症反应,乌司他丁通过改善CPB后肠粘膜上皮对局部缺血缺氧再灌注的耐受、抑制炎症因子TNFα、自由基NO过度生成以及促凋亡因子Bax、Caspase-3下调来减少肠粘膜上皮细胞凋亡,保护CPB后肠粘膜屏障功能。同时显示乌司他丁的预防作用存在量效关系。更多还原

【Abstract】 Cardiopulmonary bypass （CPB） is a powerful systemic stimulation to the body, generating ischemia, anoxemia and reperfusion injury and intense stress reaction. Excessive inflammatory reaction induced by stress is one of the main causes of death in cardiac surgery, leading to tissue damage, systemic inflammation response syndrome （SIRS）, and even multiple organs dysfunction syndrome （MODS）. Organ protection during CPB is one of the hot areas in both basic and clinical research of cardiovascular surgery. Many in-depth studies have long focused on the function of important organs like heart, brain, lung and kidney in the CPB. It was not until recently that researchers have realized that intestine is not only one of the organs that initiate stress but also one of the paramount target organs of stress. The key point is that the dysfunction of intestinal mucosal barrier results in the bacteria/endotoxin translocation. The impairment of intestinal mucosal barrier after CPB is a marginal area of study with little attention, and the studies on its occurrence and mechanism are fairly few. The incidence of serious complications of digestive system after clinical CPB is low （0.41～3.7%）, while the mortality rate is rather high（13.9～52%）. Therefore, it is clinically important to study the mechanism of impairment of intestine barrier in CPB, and to discuss the corresponding protective measures.The maintenance of normal intestine barrier depends on the intact intestinal mucosal epithelia, normal flora and secretion in intestine, peristalsis and immune function of intestine, with intestinal mucosal barrier, the most critical part. Vigorous metabolic ability of intestinal mucosa along with severe traumatic ischemia, anoxemia and reperfusion thereafter contributes to apoptosis of large amount of excessive mucosal epithelial cells leading to the damage of mucosal barrier. Necrosis of intestinal mucosa was believed to be the pathological basis of intestinal barrier dysfunction, yet increasing studies have suggested that the main cause is apoptosis of mucosal epithelial cells. Recently, the production of specially made micro membrane lung of small animals and the closed-chest puncture technique have enabled the establishment of full-flow CPB rat model in common temperature, which is economic, practical, minimally invasive, easy to operate, reliable, and closer to clinical process, providing ideal model for the research of early stage systemic inflammation reaction, multiple organs dysfunction syndrome and tactics of prevention and care.Ulinastatin is a kind of broad spectrum enzyme inhibitor with multidunious biological functions. It inhibits the activity of various proteins, glucose and lipidic hydrolase, hampers excessive secretion of inflammatory mediators, improves microcirculation and pharmacological action of tissue perfusion. It is effective in the rescue of critical patients, protecting important organs.The research is to discuss the form of intestinal mucosal barrier of rats that went through CPB and its dysfunction, as well as the role of inflammatory reaction and apoptosis of intestinal mucosal epithelial cells in the impairment of intestinal mucosa, and to study preventive function and mechanism of ulinastatin by treating the rats with it. The research is discussed in three parts with methods, results and conclusion as follows.1. Establishment of CPB animal model in common temperature on rats with intubation.1.1 ObjectiveTo set up the full-flow CPB experiment model on rats in common temperature with intubation, which is economic, practical, minimally invasive, easy to operate, reliable, and closer to clinical process.1.2 Methods14 male adult SD rats （350～500g） were anesthetized and given tracheal intubation and assisted ventilation by breathing machine. The tube placed in the femoral artery was connected to the monitor for real-time blood pressure monitoring, and arterial blood gas was collected on time. Blood was transmitted through peristaltic pump to specially made micro membrane lung for oxidation before its infusion into right cervical artery. The model was pre-inflated with Ringer’s solution, middle molecular hydroxyethly starch and small amount of rat blood. The total amount was 22ml, the ratio of crystal and colloid 1:1, infusion flow 120～160ml·kg^-1·min^-1, and mean bypass time 60 minutes.1.3 ResultsTwo out of fourteen rats died of hemorrhage during the establishment of CPB, another two failed to establish the model because of the obstructed drainage as a result of resistance in jugular vein intubation and inadequate length of the catheter in the vein. Success rate of model establishment was 71% with mean bypass time 60 minutes and bypass flow approaching 120～160ml·kg^-1·min^-1, approximate to rats’cardiac output and in accordance with the requirements of complete bypass. During the experiment, hemodynamics was stable, and oxygenation of oxygenator fully met CPB standard. Blood gas test during CPB indicated frequent acidosis, while other indices of blood gas and electrolytes were normal. During the discontinuance of CPB, blood pressure and heart rate began to drop, the level was remarkably lower than before the bypass even with the help of vasoactive agents, and serious dilution of blood worsened the condition, rats did not live long and generally died of circulation and respiratory failure in 7 hours.1.4 ConclusionFull-flow CPB experimental animal model could be established by intubation, drainage through right jugular vein cavity and right jugular artery infusion. The surgery was simple, easy to manage, economic and practical. Miniaturized circuit design provided guarantee for the establishment of stable CPB, and micro membrane lung and peristaltic pump met the requirements of oxygenation and hemodynamics of CPB rats. The model resulted in high short-term survival rate, exhibiting relevant detection capability for pathological, biological, biochemical and molecular biological index, and enabled the serialization and formularization of CPB related basic research, promising comprehensive application perspective. 2. Research on the early stage dysfunction of intestinal mucosal barrier, inflammatory reaction and mechanism after CPB.2.1 ObjectiveTo discuss clinical significance of the intestinal mucosal barrier impairment after CPB by observing the ileal mucosa morphous, barrier impairment and the variance of serum inflammatory factor concentration. And by detecting the level of protein and gene, to approach the existence of excess apoptosis and generation of inflammatory factors as well as the prospective mechanism of intestinal mucosal barrier impairment.2.2 Methods30 clean healthy adult SD rats were divided randomly into normal control group （group N,n=10）, sham-operated group（SH,n=10）, CPB group（n=10）. Rats in SH group received intubation in corresponding area while being injected 22ml of pre-inflated fluid slowly through femoral artery in 10 minutes. CPB was not performed on these rats which were killed 2 hours later for sample collection. Rats in CPB group were executed 2 hours after bypass for sample collection. The height of end-piece ileal mucosa villi was examined by light microscope and graded according to Chiu’s score, ultramicrostructural change of intestinal mucosa was observed by electron microscope, and serum DAO activity, the variance of D-lactic acid and NO. The change of serum TNFα, IL-6 concentration was detected by ELISA. The serum values were corrected to eliminate the interference caused by the dilution of pre-inflation. Then, apoptotic index of small intestinal mucosa of rats was tested by TUNEL, the changes of TNFα, iNOS, Bcl-2, Bax, Caspas-3 protein expression of intestinal mucosa went through image analysis by immunohistochemical. The expression variance of TNFα, iNOS, Bcl-2, Bax, Caspase-3mRNA was tested by RT-PCR.2.3 ResultsAccording to the study, obvious mucous hyperemia and dilation of small vessels in interstitium were observed in rats in CPB group, as well as massive infiltration of neutrophils, interstitial edema, expansion of intestinal canal, shortening and deformity of villi, and frequent focal exfoliation of chorioepithelia. Besides, necrosis and exfoliation of mucosal epithelia exacerbated, and some parts of proper layer exposed, with notable decrease in the number of glands in proper layer. The condition of rats in N group and SH group was much better than that of CPB group with fairly normal villi of small intestine. Chiu’s score demonstrated that the intestinal mucosal impairment was obvious in CPB group than that of N group and SH group（P<0.05）. Electron microscope showed that in N group and SH group, microvilli of intestinal mucosal epithelial cells were lined up in order with the complete structure of cellula columnoepithelialis, and that no abnormality was detected in cell organs, and that tight junction, intermediate junction desmosome and gap junction were clear without being widened. in N group and SH group, microvilli of intestinal mucosal epithelial cells were lined up in order with the complete structure of cellula columnoepithelialis, and that no abnormality was detected in cell organs, and that tight junction, intermediate junction, desmosome and gap junction were clear without being widened. The structure of glands and various cells in proper layer was normal. In CPB group, two different ways of apoptosis and swelling death of small intestine epithelial cells were presented. Rarefaction of microvilli was observed whose arrangement was not in order but downward, and some of which was absent. Junctions between epithelial cells widened with opening of some tight junctions, mitochondria swollen, and endoplasmic reticulum expanded. Several apoptotic cells were seen with the cell nucleus gathering under the nuclear envelop and apoptotic bodies exfoliated into the enteric cavity. Edema of proper layer was obvious with increased lymphocytes and neutrophils. Some apoptotic cells resulted in further necrosis with disintegration, loss and incomplete membranes of intracytoplasm cell organs. Epithelial cells died of swelling were also observed with the destruction of nuclear membrane and cell membrane, and disintegration of intracytoplasm cell organs like mitochodria. Rats that went through CPB presented the impairment of intestinal barrier with serum DAO activity, D-lactic acid concentration higher than those of N group and SH group （P<0.05）. DAO activity, D-lactic acid concentration in SH group was higher than those of N group, yet no significance of difference was detected. Rats that went through CPB were in systemic inflammatory state with serum TNFα, IL-6 and NO concentration remarkably higher than those of N group and SH group （P<0.05）. Serum TNFαand NO concentration in SH group were higher than those of N group （P<0.05）. Linear regression and relevant analysis indicated that height of intestinal mucosal villi and Chiu’s score were positively correlated with serum DAO activity and D-lactic acid concentration, and that serum DAO activity and D-lactic acid concentration were positively correlated with serum TNFα, IL-6 and NO concentration. Apoptotic index of intestinal mucosal epithelial cells was significantly higher than that of N group and SH group （P<0.01）. Apoptotic index was positively correlated with Chiu’s score, serum DAO activity, D-lactic acid, serum TNFα, IL-6 and NO concentration. TNFα, iNOS, Caspase-3 protein and mRNA expression of intestinal mucosal epithelial cells in CPB group stepped up notably compared with those of N group and SH group. Yet no significance of difference was detected between N group and SH group. Decrease of Bcl-2 protein and mRNA expression, increase of Bax protein expression and mRNA expression were of no statistical significance, and yet the drop of Bcl-2/Bax was of significance compared with that of N group and SH group.2.4 ConclusionRats that went through CPB presented the morphous and barrier dysfunction of intestinal mucosa with systemic inflammatory state. Linear regression and relevant analysis indicated the important role of the morphous and barrier dysfunction of intestinal mucosa in inflammatory reaction after CPB. Apoptosis of intestinal mucosal epithelial cells is one of the main ways of death for epithelial cells after CPB. Ischemia, anoxemia and reperfusion after CPB and excessive secretion of inflammatory factors and increase of NO synthesis after stress promoted apoptosis, and the decrease of Bcl-2/Bax also contributed to the apoptosis of intestinal mucosa.3. Research on the mechanism of ulinastatin prevention of the early stage dysfunction of intestinal mucosal barrier3.1 ObjectiveTo discuss feasibility and mechanism of intestinal mucosal barrier after CPB by observing the pathological, physiological index and inflammatory factors and control of apoptosis pretreated with large and small dose groups of ulinastatin before the establishment of bypass.3.2 Methods40 clean healthy male adult Spranue-Dawley （SD） rats were divided randomly into sham-operated group （SH,n=10）, CPB group（n=10）, ulinastatin small dose group（U1 group,n=10）, ulinastatin large dose group（U2 group,n=10）. Before the bypass, U1 group was given 40,000U/kg of ulinastatin through femoral artery, while U2 group was given 100,000U/kg of ulinastatin. The bypass and sample collection methods were identical to those taken in CPB group. SH group was the same as CPB group. Mean blood pressure and heart rate of rats that went through CPB were monitored continuously. The height of end-piece ileal mucosa villi was examined by light microscope and graded according to Chiu’s score, ultramicrostructural change of intestinal mucosa was observed by electron microscope, and serum DAO activity, the variance of D-lactic acid and NO concentration. The change of serum TNFα, IL-6 concentration was detected by ELISA. Apoptotic index of small intestinal mucosa of rats was tested by TUNEL, the changes of TNFα, iNOS, bcl-2, Bax, Caspas-3 protein expression of intestinal mucosa went through image analysis by immunohistochemical . The expression variance of TNFα, iNOS, Bcl-2, Bax, Caspase-3mRNA was tested by RT-PCR.3.3 ResultsThe drop of mean blood pressure and heart rate of rats that went through CPB in ulinastatin interference group improved compared with that of CPB, U2 group in particular （P<0.05）. In U1, U2 groups, intestinal mucosal hyperemia and dilation of small vessels in interstitium were observed, as well as infiltration of neutrophils, interstitial edema, expansion of intestinal canal, shortening and deformity of villi, and focal exfoliation of chorioepithelia, all of which were better than those of CPB group, in accordance with the indication by villi height and Chiu’s score that the improvement of intestinal mucosal damage in U1, U2 groups was more obvious than that of CPB group （P<0.05）, yet there was no difference between U1 and U2 groups. Electron microscope showed that in U1 and U2 groups, microvilli of intestinal mucosal epithelial cells were lined up in order with most of cellula columnoepithelialis remained complete structure, and with opening of some tight junction. The swelling of mitochondria mitigated, and endoplasmic reticulum slightly expanded with few apoptotic cells. Rats that went through CPB and received ulinastatin treatment showed intestinal barrier impairment. Serum DAO activity, D-lactic acid concentration were remarkably higher than those of SH group （P<0.05）, yet the drop of the values was significant compared with that of CPB group （P<0.05）, and the drop in U2 group was greater than that of U1 group （P<0.05）. Systemic inflammatory reaction of rats that went through CPB and received ulinastatin treatment alleviated. Serum TNFα,IL-6 and NO concentration in U1 and U2 groups extremely descended than those of CPB group（P<0.01）; The drop in U2 group was greater than that of U1 group（P<0.05）.The increase of serum TNFαand NO concentration in U2 group was not obvious than that of SH group（P>0.05）. Apoptotic index of intestinal mucosal epithelial cells dropped notably compared with that of CPB group （P<0.01）, higher than that of SH group, yet no difference between U1 and U2 groups was detected. TNFα, iNOS, Caspase-3 protein and mRNA expression of intestinal mucosal epithelial cells in U1 and U2 groups decreased remarkably compared with those of CPB group, while the drop of Bcl-2/Bax was significant in comparison with that of CPB group. TNFα, iNOS, Caspase-3 protein expression and mRNA expression of U2 group dropped remarkably compared with those of U1 group.3.4 ConclusionUlinastatin effectively enhanced the condition of rats that went through CPB, not only alleviating the morphological change and impairment of intestinal mucosa barrier, but also mitigating systemic inflammatory reaction. Besides, it downregulated the apoptosis of intestinal mucosal epithelial cells by inhibiting the secretion of inflammatory factors and lessening stress reaction which to some extent prevents the early stage dysfunction of intestinal mucosal barrier after CPB, indicating dose-effective relationship of ulinastatin prevention.更多还原