帕瑞昔布钠对脓毒症肠屏障功能的保护效应及机制研究

发布时间：2018-07-23 12:13

【摘要】：目的脓毒症是机体对感染产生的一种全身炎症反应,至今仍是重症监护病房引起患者死亡的主要原因之一[1-3]。肠粘膜屏障功能障碍和通透性增加常常伴随着脓毒症的发生和发展。肠粘膜屏障功能受损后引发的细菌移位会成为继发脓毒症的重要原因,而脓毒症导致的肠组织血液供应下降及炎症因子风暴可进一步加重肠粘膜的损伤,逐渐形成了恶性循环。评估肠屏障功能的状态已经是判断危重病人预后的重要环节,肠道的损伤是启动MODS的重要因素,因此,如何保护和维持肠屏障的正常功能已成为当前危重症医学领域重要的研究方向[4,5]。帕瑞昔布钠(NSAIDs)是一种COX-2特异性抑制剂,属于非甾体类抗炎药,可经静脉注射或肌内注射,用药后由肝脏羧酸酯酶水解为伐地昔布而起作用,其主要作用机制是阻断花生四烯酸在COX-2作用下合成前列腺素,降低前列腺素(PG)的水平[6]。研究发现,高水平前列腺素可导致肠屏障功能的损伤,而环氧合酶-2抑制剂通过抑制COX-2的表达来减少前列腺素合成,可能对脓毒症导致的肠屏障功能障碍具有改善作用[7-10]。本课题拟研究环氧合酶-2抑制剂帕瑞昔布钠对脓毒症大鼠肠屏障功能的作用,并进一步探讨帕瑞昔布钠对肠粘膜屏障功能的保护作用的相关机制。方法第一部分:采用盲肠结扎穿孔法(CLP)诱导脓毒症大鼠肠损伤模型。72只Wistar大鼠随机分为6组(n=12/组):假手术组(Sham组)、假手术组+10mg/kg帕瑞昔布钠(Sham+10mg/kg Parecoxib组),脓毒症组(CLP组)、脓毒症+0.1mg/kg帕瑞昔布钠(CLP+0.1mg/kg Parecoxib组)、脓毒症+1.0mg/kg帕瑞昔布钠(CLP+1.0mg/kg Parecoxib组)、脓毒症+10mg/kg帕瑞昔布钠(CLP+10mg/kg Parecoxib组)。帕瑞昔布钠治疗组大鼠于假手术或CLP后20min腹腔注射帕瑞昔布钠,建模12h后重复给药一次。CLP组和Sham组大鼠仅经腹腔注射等量生理盐水。观察各组大鼠术后7天内的存活情况,并筛选最佳治疗方案。于假手术或CLP后24h,通过酶联免疫吸附法(ELISA)或比色法检测各组大鼠血浆二胺氧化酶(DAO)和D-乳酸的浓度来观察各组大鼠肠损伤的情况;通过Western Blot法检测各组大鼠肠组织紧密连接蛋白ZO-1和Claudin-1的蛋白表达情况;通过比色法检测肠组织髓过氧化物酶MPO的活性水平来观察各组大鼠肠组织的炎症水平。第二部分:利用盲肠结扎穿孔法建立脓毒症大鼠模型。32只Wistar大鼠随机分为4组(n=8/组):假手术组(Sham)、假手术组+10mg/kg帕瑞昔布钠(Sham+10mg/kg Parecoxib),脓毒症组(CLP)、脓毒症+10mg/kg帕瑞昔布钠(CLP+10mg/kg Parecoxib)。模型建立后20min,向大鼠腹腔注射帕瑞昔布钠,每隔12小时重复注射一次,给予假手术组和CLP组大鼠腹腔注射等量的生理盐水。于大鼠颈动脉进行插管以连续监测基础血流动力学(MAP、HR)且便于行血气分析监测,与造模后0h、6h和12h行血气分析,于造模后6h、12h和24h测定小肠肠系膜的微循环变化[总血管密度(TVD)、灌注血管密度(PVD)、灌注血管比例(PPV)及微血管血流指数(MFI)]。第三部分:利用盲肠结扎穿孔法建立脓毒症大鼠模型。将48只Wistar雄性大鼠随机分为6个实验组,假手术组(Sham组)、脓毒症组(CLP组)、假手术+帕瑞昔布钠组(Sham+Parecoxib组)、脓毒症+帕瑞昔布钠组(CLP+Parecoxib组)、脓毒症+NS-398组(CLP+NS-398组)和脓毒症+帕瑞昔布钠+NS-398组(CLP+Parecoxib+NS-398组)。CLP+NS-398组和CLP+Parecoxib+NS-398组于术前2h腹腔注射NS-398 10mg/kg。于假手术或CLP后6、12和24 h,检测各组大鼠血清和肠组织中TNF-α,IL-6和IL-10的水平,PGE2、m PGES-1和EP4的蛋白表达和m RNA水平。给予COX-2抑制剂(NS-398)预处理后,于术后24 h检测各组大鼠肠组织TNF-α,IL-6和IL-10的水平。结果第一部分:腹腔注射10mg/kg帕瑞昔布钠能有效提高脓毒症大鼠7 d内的存活率(P0.05)。帕瑞昔布钠治疗明显降低肠组织病理损伤的评分,减少肠组织MPO的活性(P0.05),降低脓毒症大鼠肠组织DAO和D-乳酸的水平(P0.05)。帕瑞昔布钠治疗脓毒症大鼠后肠组织紧密连接蛋白ZO-1和Claudin-1的蛋白表达上调(P0.05)。第二部分:环氧合酶-2抑制剂帕瑞昔布钠治疗可明显提高脓毒症大鼠平均动脉压(MAP)和降低心率(HR)(P0.05),改善血流动力学;环氧合酶-2抑制剂帕瑞昔布钠治疗可有效改善脓毒症大鼠的动脉血气指标,降低乳酸水平(P0.05);环氧合酶-2抑制剂帕瑞昔布钠治疗可增加脓毒症大鼠肠系膜微循环总血管的密度、灌注血管密度及微循环的血流指数(P0.05)。第三部分:环氧合酶-2抑制剂帕瑞昔布钠明显降低脓毒症大鼠血清和肠组织中促炎因子TNF-α和IL-6水平(P0.05),提高抗炎因子IL-10水平(P0.05);给予COX-2抑制剂(NS-398)预处理后抗炎作用更加明显(P0.01);帕瑞昔布钠明显降低脓毒症大鼠肠组织中PGE2、m PGES-1和EP4的蛋白表达和m RNA水平(P0.05)。结论帕瑞昔布钠治疗可减轻肠组织炎性损伤,有效降低肠粘膜的通透性,改善肠系膜的微循环。其机制可能与调控PGE2的表达有关。
[Abstract]:Objective sepsis is a systemic inflammatory response to infection, which is still one of the main causes of death in the intensive care unit (ICU). [1-3]. intestinal mucosal barrier dysfunction and increased permeability often accompany the occurrence and development of sepsis. Bacterial translocation caused by impaired intestinal mucosal barrier function becomes secondary purulent. The important cause of toxic disease, and the decrease of blood supply in intestinal tissue caused by sepsis and inflammatory factor storm can further aggravate the injury of intestinal mucosa, and gradually form a vicious cycle. Evaluation of the function of intestinal barrier is an important link in judging the prognosis of critically ill patients. Intestinal damage is an important factor to start MODS. Therefore, how to protect the intestinal tract is how to protect it The normal function of the intestinal barrier has become an important research direction in the current critical medical field [4,5]. parinoxib sodium (NSAIDs) is a COX-2 specific inhibitor, which is a non steroidal anti-inflammatory drug, which can be injected via intravenous or intramuscular injection. After the use of carboxylesterase from liver, it plays a role in the main mechanism of action. The inhibition of prostaglandins (PG) by blocking the prostaglandin (COX-2) and reducing the prostaglandin (PG) level by COX-2 shows that high level prostaglandins can cause damage to the intestinal barrier function, and the cyclooxygenase -2 inhibitor reduces the prostaglandin synthesis by inhibiting the expression of COX-2, and may be associated with the intestinal barrier dysfunction caused by sepsis. The role of parexoxib sodium (parexoxib), an inhibitor of cyclooxygenase -2, on the intestinal barrier function of rats with sepsis and the mechanism of protective effect of parexib sodium on intestinal mucosal barrier function in [7-10]. is studied. Method first part: using the cecum ligation and perforation (CLP) to induce the intestinal injury model of septic rats.72 only Wistar rats were randomly divided into 6 groups (group n=12/): sham operation group (group Sham), sham operation group +10mg/kg parinoxib sodium (group Sham+10mg/kg Parecoxib), sepsis group (group CLP), sepsis +0.1mg/kg parinoxib sodium (CLP+0.1mg/kg Parecoxib group), +1.0mg/kg palioxib sodium in sepsis, sepsis palioxib sodium. (CLP+10mg/kg Parecoxib group). Parinoxib sodium treatment group rats were intraperitoneally injected with pareoxib sodium after sham operation or CLP after CLP. After modeling 12h, the rats were repeated to a.CLP group and Sham group only by intraperitoneal injection of equal amount of saline. The survival of the rats in 7 days after operation was observed and the best treatment was screened. In sham operation or after CLP 24 H, the concentrations of plasma two amine oxidase (DAO) and D- lactate were detected by enzyme linked immunosorbent assay (ELISA) or colorimetric assay to observe the intestinal damage in rats of each group. The expression of the protein ZO-1 and Claudin-1 in intestinal tissue of the rats was detected by Western Blot method, and the peroxidation of intestinal tissue was detected by colorimetric assay. The activity level of enzyme MPO was observed in the intestinal tissue of rats in each group. The second part: the rat model of sepsis was established by cecum ligation and perforation method.32 rats were randomly divided into 4 groups (group n=8/): sham operation group (Sham), sham operation group +10mg/kg palioxib sodium (Sham+10mg/kg Parecoxib), sepsis group (CLP), +10mg/kg PA of sepsis. CLP+10mg/kg Parecoxib. After the model was established, 20min was injected into the abdominal cavity of rats by intraperitoneal injection of parecoxib sodium and injected once every 12 hours to give the sham operation group and the CLP group the same amount of normal saline. The carotid artery was intubated in rats to monitor the basic hemodynamics (MAP, HR) and facilitate the blood gas analysis monitoring. Blood gas analysis was performed with 0h, 6h and 12h after modeling, and the microcirculation changes in the mesentery of the small intestine were measured by 6h, 12h and 24h after modeling. The third part: the rat model of sepsis was established by cecal ligature and perforation. 48 Wistar male rats were established with the cecal ligation perforation method. The machine was divided into 6 experimental groups, sham operation group (group Sham), sepsis group (group CLP), sham operation + palioxib sodium group (group Sham+Parecoxib), sepsis + pareximab sodium group (group CLP+Parecoxib), +NS-398 group of sepsis (group CLP+NS-398), sepsis + pararexime sodium + NS-398 group (CLP+Parecoxib+NS-398 group).CLP+NS-398 group and CLP+Parecoxib+NS-398 group. The levels of TNF- a, IL-6 and IL-10 in the serum and intestinal tissues of rats were detected by intraperitoneal injection of NS-398 10mg/kg. before 2h, and the levels of IL-6 and IL-10 in serum and intestinal tissues, PGE2, m PGES-1 and protein expression and the level of CLP were measured. After the pretreatment, the levels of intestinal tissue were detected in the 24 groups of rats after the operation. Results first part: intraperitoneal injection of 10mg/kg parecoxib sodium can effectively improve the survival rate of 7 d in septic rats (P0.05). Palioxib sodium treatment significantly reduces the score of intestinal tissue pathological injury, reduces the activity of MPO in intestinal tissue (P0.05), and reduces the level of DAO and D- lactate in intestinal tissue of sepsis rats (P0.05). Parecoxib sodium is used in the treatment of sepsis. The protein expression of close connexin ZO-1 and Claudin-1 in the rat hindgut tissue is up regulated (P0.05). Second: the treatment of cyclooxygenase -2 inhibitor parinoxib can obviously improve the mean arterial pressure (MAP) and the decrease of heart rate (HR) (P0.05) of septic rats and improve the hemodynamics. The treatment of the cyclooxygenase -2 inhibitor pareoxib sodium can effectively improve the purulent. The arterial blood gas index of the poisoned rats decreased the lactate level (P0.05), and the treatment of the cyclooxygenase -2 inhibitor parexib sodium could increase the density of the microcirculation in the mesenteric microcirculation, the density of perfusion vessels and the blood flow index of microcirculation (P0.05). The third part: parinoxib sodium of cyclooxygenase -2 inhibitor significantly reduced the blood of sepsis rats The level of proinflammatory factor TNF- alpha and IL-6 (P0.05) and the level of anti-inflammatory factor IL-10 (P0.05) in the clear and intestinal tissues (P0.05); the anti-inflammatory effect of COX-2 inhibitor (NS-398) preconditioning was more obvious (P0.01); parinoxib sodium significantly reduced the expression of PGE2, m PGES-1, and EP4 protein in the intestinal tissue of sepsis rats. Treatment can reduce inflammatory injury of intestinal tissue, reduce intestinal permeability and improve mesenteric microcirculation. The mechanism may be related to the regulation of PGE2 expression.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R459.7

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