【摘要】：目的膿毒癥是機(jī)體對感染產(chǎn)生的一種全身炎癥反應(yīng),至今仍是重癥監(jiān)護(hù)病房引起患者死亡的主要原因之一[1-3]。腸粘膜屏障功能障礙和通透性增加常常伴隨著膿毒癥的發(fā)生和發(fā)展。腸粘膜屏障功能受損后引發(fā)的細(xì)菌移位會成為繼發(fā)膿毒癥的重要原因,而膿毒癥導(dǎo)致的腸組織血液供應(yīng)下降及炎癥因子風(fēng)暴可進(jìn)一步加重腸粘膜的損傷,逐漸形成了惡性循環(huán)。評估腸屏障功能的狀態(tài)已經(jīng)是判斷危重病人預(yù)后的重要環(huán)節(jié),腸道的損傷是啟動MODS的重要因素,因此,如何保護(hù)和維持腸屏障的正常功能已成為當(dāng)前危重癥醫(yī)學(xué)領(lǐng)域重要的研究方向[4,5]。帕瑞昔布鈉(NSAIDs)是一種COX-2特異性抑制劑,屬于非甾體類抗炎藥,可經(jīng)靜脈注射或肌內(nèi)注射,用藥后由肝臟羧酸酯酶水解為伐地昔布而起作用,其主要作用機(jī)制是阻斷花生四烯酸在COX-2作用下合成前列腺素,降低前列腺素(PG)的水平[6]。研究發(fā)現(xiàn),高水平前列腺素可導(dǎo)致腸屏障功能的損傷,而環(huán)氧合酶-2抑制劑通過抑制COX-2的表達(dá)來減少前列腺素合成,可能對膿毒癥導(dǎo)致的腸屏障功能障礙具有改善作用[7-10]。本課題擬研究環(huán)氧合酶-2抑制劑帕瑞昔布鈉對膿毒癥大鼠腸屏障功能的作用,并進(jìn)一步探討帕瑞昔布鈉對腸粘膜屏障功能的保護(hù)作用的相關(guān)機(jī)制。方法第一部分:采用盲腸結(jié)扎穿孔法(CLP)誘導(dǎo)膿毒癥大鼠腸損傷模型。72只Wistar大鼠隨機(jī)分為6組(n=12/組):假手術(shù)組(Sham組)、假手術(shù)組+10mg/kg帕瑞昔布鈉(Sham+10mg/kg Parecoxib組),膿毒癥組(CLP組)、膿毒癥+0.1mg/kg帕瑞昔布鈉(CLP+0.1mg/kg Parecoxib組)、膿毒癥+1.0mg/kg帕瑞昔布鈉(CLP+1.0mg/kg Parecoxib組)、膿毒癥+10mg/kg帕瑞昔布鈉(CLP+10mg/kg Parecoxib組)。帕瑞昔布鈉治療組大鼠于假手術(shù)或CLP后20min腹腔注射帕瑞昔布鈉,建模12h后重復(fù)給藥一次。CLP組和Sham組大鼠僅經(jīng)腹腔注射等量生理鹽水。觀察各組大鼠術(shù)后7天內(nèi)的存活情況,并篩選最佳治療方案。于假手術(shù)或CLP后24h,通過酶聯(lián)免疫吸附法(ELISA)或比色法檢測各組大鼠血漿二胺氧化酶(DAO)和D-乳酸的濃度來觀察各組大鼠腸損傷的情況;通過Western Blot法檢測各組大鼠腸組織緊密連接蛋白ZO-1和Claudin-1的蛋白表達(dá)情況;通過比色法檢測腸組織髓過氧化物酶MPO的活性水平來觀察各組大鼠腸組織的炎癥水平。第二部分:利用盲腸結(jié)扎穿孔法建立膿毒癥大鼠模型。32只Wistar大鼠隨機(jī)分為4組(n=8/組):假手術(shù)組(Sham)、假手術(shù)組+10mg/kg帕瑞昔布鈉(Sham+10mg/kg Parecoxib),膿毒癥組(CLP)、膿毒癥+10mg/kg帕瑞昔布鈉(CLP+10mg/kg Parecoxib)。模型建立后20min,向大鼠腹腔注射帕瑞昔布鈉,每隔12小時重復(fù)注射一次,給予假手術(shù)組和CLP組大鼠腹腔注射等量的生理鹽水。于大鼠頸動脈進(jìn)行插管以連續(xù)監(jiān)測基礎(chǔ)血流動力學(xué)(MAP、HR)且便于行血?dú)夥治霰O(jiān)測,與造模后0h、6h和12h行血?dú)夥治?于造模后6h、12h和24h測定小腸腸系膜的微循環(huán)變化[總血管密度(TVD)、灌注血管密度(PVD)、灌注血管比例(PPV)及微血管血流指數(shù)(MFI)]。第三部分:利用盲腸結(jié)扎穿孔法建立膿毒癥大鼠模型。將48只Wistar雄性大鼠隨機(jī)分為6個實(shí)驗(yàn)組,假手術(shù)組(Sham組)、膿毒癥組(CLP組)、假手術(shù)+帕瑞昔布鈉組(Sham+Parecoxib組)、膿毒癥+帕瑞昔布鈉組(CLP+Parecoxib組)、膿毒癥+NS-398組(CLP+NS-398組)和膿毒癥+帕瑞昔布鈉+NS-398組(CLP+Parecoxib+NS-398組)。CLP+NS-398組和CLP+Parecoxib+NS-398組于術(shù)前2h腹腔注射NS-398 10mg/kg。于假手術(shù)或CLP后6、12和24 h,檢測各組大鼠血清和腸組織中TNF-α,IL-6和IL-10的水平,PGE2、m PGES-1和EP4的蛋白表達(dá)和m RNA水平。給予COX-2抑制劑(NS-398)預(yù)處理后,于術(shù)后24 h檢測各組大鼠腸組織TNF-α,IL-6和IL-10的水平。結(jié)果第一部分:腹腔注射10mg/kg帕瑞昔布鈉能有效提高膿毒癥大鼠7 d內(nèi)的存活率(P0.05)。帕瑞昔布鈉治療明顯降低腸組織病理損傷的評分,減少腸組織MPO的活性(P0.05),降低膿毒癥大鼠腸組織DAO和D-乳酸的水平(P0.05)。帕瑞昔布鈉治療膿毒癥大鼠后腸組織緊密連接蛋白ZO-1和Claudin-1的蛋白表達(dá)上調(diào)(P0.05)。第二部分:環(huán)氧合酶-2抑制劑帕瑞昔布鈉治療可明顯提高膿毒癥大鼠平均動脈壓(MAP)和降低心率(HR)(P0.05),改善血流動力學(xué);環(huán)氧合酶-2抑制劑帕瑞昔布鈉治療可有效改善膿毒癥大鼠的動脈血?dú)庵笜?biāo),降低乳酸水平(P0.05);環(huán)氧合酶-2抑制劑帕瑞昔布鈉治療可增加膿毒癥大鼠腸系膜微循環(huán)總血管的密度、灌注血管密度及微循環(huán)的血流指數(shù)(P0.05)。第三部分:環(huán)氧合酶-2抑制劑帕瑞昔布鈉明顯降低膿毒癥大鼠血清和腸組織中促炎因子TNF-α和IL-6水平(P0.05),提高抗炎因子IL-10水平(P0.05);給予COX-2抑制劑(NS-398)預(yù)處理后抗炎作用更加明顯(P0.01);帕瑞昔布鈉明顯降低膿毒癥大鼠腸組織中PGE2、m PGES-1和EP4的蛋白表達(dá)和m RNA水平(P0.05)。結(jié)論帕瑞昔布鈉治療可減輕腸組織炎性損傷,有效降低腸粘膜的通透性,改善腸系膜的微循環(huán)。其機(jī)制可能與調(diào)控PGE2的表達(dá)有關(guān)。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R459.7

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