脓毒症大鼠肠道肠三叶因子mRNA表达的变化

目的：探讨脓毒症大鼠肠道肠三叶因子（trefoil factor family,TFF3）mRNA表达的变化。方法：32只SD大鼠随机分为对照组（n=8）和脓毒症组（n=24）。采用盲肠结扎穿孔术（cecal ligation and puncture,CLP）制作大鼠脓毒症模型。于模型建立后3h,6h及12h（n=8）取回肠黏膜,以RT-PCR法检测TFF3 mRNA的表达。结果：脓毒症模型建立3h始TFF3 mRNA表达即显著下降（P〈0.01）,随着时间延长表达进一步下降。结论：脓毒症大鼠肠道TFF3 mRNA表达明显下降,可能是脓毒症肠屏障功能障碍的机制之一并延缓肠道黏膜修复。     

乌司他丁对脓毒症大鼠肠黏膜上皮防御屏障的影响

目的 探讨乌司他丁(UTI)对脓毒症大鼠肠黏膜上皮防御屏障的影响.方法 50只SD大鼠被随机分为对照组、模型组、UTI预处理组及UTI治疗组,后3组再按盲肠结扎穿孔术(CLP)后3、6和12 h分为3个亚组,每个亚组5只.采用改良的CLP制备大鼠脓毒症模型,预处理组在CLP前2 h经尾静脉注射UTI 25 kU/kg,治疗组在CLP后2 h经尾静脉注射UTI 50 kU/kg.分别于CLP后3、6和12 h活杀大鼠,取回肠黏膜光镜下观察组织病理学变化,用逆转录-聚合酶链反应(RT-PCR)检测肠黏膜防御素-5(RD-5)和肠三叶因子家族3(TFF3)的mRNA表达水平.结果 模型组小肠绒毛顶端上皮脱落,固有层崩解,毛细血管出血和溃疡形成,UTI治疗组和预处理组能显著减轻上述改变.模型组RD-5和TFF3的mRNA表达水平较对照组显著降低,UTI治疗组和预处理组能显著升高其表达水平(P<0.05或P<0.01);预处理组CLP后12 h较治疗组升高最为显著,差异有统计学意义(P均<0.05).结论 脓毒症时肠黏膜上皮屏障功能受损,UTI对其有保护作用,从而遏制脓毒症的发生发展.     

连续胸椎硬膜外麻醉可以改善脓毒症鼠的肠黏膜微循环

微循环功能障碍可明显加重脓毒症时组织缺氧和多器官功能衰竭。肠道缺血、缺氧引起的黏膜屏障功能受损可导致细菌／毒素移位，促进脓毒症发展。由于在生理条件下交感神经阻滞能增加肠内灌注，德国学者假设胸椎硬膜外麻醉（TEA）可减轻脓毒症对微循环的影响。他们研究了连续TEA对脓毒症鼠盲肠结扎穿孔术（CLP）模型肠微循环的作用。     

丙酮酸乙酯对脓毒症大鼠肠黏膜屏障功能保护作用的研究

目的 观察丙酮酸乙酯(ethyl pyruvate ,EP)干预治疗对脓毒症大鼠生存率和肠黏膜屏障的影响.方法 ①EP对脓毒症大鼠生存率的影响:无特定病原雄性SD大鼠100只随机分为假手术组(A组)、脓毒症组(B组)、EP早期治疗组(C组)及EP延迟治疗组(D组),每组25只,利用盲肠结扎穿孔法(cecal ligation and puncture,CLP)制作大鼠脓毒症模型,各组均于术后6、12、18、24、36、48、60、72 h腹腔内注射给药3 mL,C、D组分别于术后6、12 h开始予EP(40 mg/kg),A、B两组同法予等量林格乳酸钠溶液(ringer lactate solution ,RLS),每隔12 h记录死亡情况,分析比较5 d生存率;②EP对脓毒症大鼠肠黏膜屏障的影响:80只无特定病原雄性SD大鼠随机分为四组,每组20只,分组及给药方法与方法一相同,术后24、48 h各处死10只.测定各时间点血浆D-乳酸、DAO的变化,同时用透射电镜观察术后48 h肠黏膜上皮细胞超微结构的变化.采用Kaplan- Meier生存分析法进行生存分析,多组均数间比较采用单因素方差分析的方法, 多组均数间两两比较采用SNK-q检验,P＜0.05为差异有统计学意义.结果 A、B、C、D四组大鼠5 d生存率分别为100%、24%、68%、56%,与B组相比,C、D组大鼠5 d生存率明显提高(P<0.05),C、D组间差异无统计学意义(P＞0.05);与B组相比,C、D组术后24 h和48 h血浆D-乳酸含量明显下降(P<0.01);与B组相比,C组、D术后24 h和48 h血浆DAO活性明显下降(P<0.01),C、D组术后24、48 h血浆D-乳酸含量、DAO活性差异无统计学意义(P＞0.05);电镜下C、D组肠黏膜上皮细胞损伤较B组明显减轻,细胞间紧密连接较清楚.结论 脓毒症时肠黏膜损伤严重,EP早期与延迟干预治疗能有效保护肠黏膜屏障,提高5 d生存率,具有抗脓毒症作用 .     

影响脓毒症受损肠黏膜修复的机制

目的 探讨影响脓毒症肠黏膜损害后修复的因素。方法 采用肓肠结扎穿孔(CLP)所致脓毒症模型,分别以CLP后6,24,48 h不同时间段观测肠黏膜损伤程度和修复过程,前者包括形态学观察及细胞凋亡的测定,后者包括肠黏膜修复的杯状细胞变化、黏膜肠三叶因子3(TFF3)、转化生长因子β1(TGF-β1)以及TNF-α、IL-1含量。结果 形态学观察显示肠黏膜呈持续损害状态,6h的损害积分明显小于24h,48 h组(P＜0.05),后两组之间差异无统计学意义(P＞0.05);磷酸化caspase-3蛋白在3组均高于sham组4倍以上;黏膜IL-1,TNF-α含量明显高于sham组3～4倍,其中24h及48 h组明显高于6h组。肠黏膜的修复过程不明显,损伤黏膜未见到明显的杯状细胞积聚;TFF3在6h组轻度增高,24h及48 h组表达下降;杯状细胞数量在CLP的3个组明显减少;TGF-β1在6h组增高,其他两组均接近于sham组。结论 严重脓毒症肠黏膜持续的高炎症状态、杯状细胞功能以及黏膜重建能力下降,影响了受损肠屏障的修复。     

乌司他丁对脓毒症大鼠肠道防御素5 mRNA表达的影响

目的 探讨乌司他丁(ulinastatin,LTI)对脓毒症大鼠肠道潘氏细胞防御素5(rat defemin-5,RD-5)mRNA表达的影响.方法 实验在中山大学医学院药理实验室完成.60只SD大鼠随机分为对照组、脓毒症组、预处理组及治疗组(n=15).后三组采用盲肠结扎穿孔术(cecal ligation andpuncture,CLP)制作大鼠脓毒症模型.预处理组在CLP前2h经尾静脉注射UTI 25 000 U/kg,治疗组在CLP后2 h经尾静脉注射UTI 50 000 U/kg.于模犁建立后12 h取回肠黏膜,观察其病理改变并以RT-PCR法检测RD-5 mRNA的表达.数据以SPSS 13.0统计软件处理,采用方差分析及LSD-t检验进行数据分析.结果 RD-5 mRNA在脓毒症组显著下降(P<0.05),预处理组及治疗组较脓毒症组有明显升高(P<0.05),预处理组较治疗组明显升高(P<0.05).结论 脓毒症大鼠RD-5mRNA表达明显下降,乌司他丁可显著上调其表达,保护肠黏膜,预防用药较治疗给药可能更有意义.     

肠黏膜屏障研究进展

肠道黏膜是机体自身与外环境接触的最大界面，具有选择性渗透吸收营养物质和防御肠道内微生物及致炎因子入侵等屏障功能。肠黏膜通透性受多种因子高度精密调节，它们共同构成黏膜屏障而发挥生理作用。     

阻滞交感神经改善脓毒症大鼠肠道微循环

因胃肠道局部缺血、缺氧、黏膜屏障作用受损导致的肠道内细菌／毒素移位与脓毒症发生发展密切相关，在生理条件下阻滞交感神经可提高肠道灌注压。瑞士学者研究了硬膜外麻醉阻滞交感神经对脓毒症大鼠肠黏膜微循环障碍的影响。研究者采用大鼠盲肠结扎穿孔术制备脓毒症模型，然后经胸椎硬膜外导管连续24h给予质量分数为0.125％的布比卡因（n=10）和生理盐水（n=9），     

中暑时肠黏膜机械屏障功能的变化

中暑是由于外在的高温环境或活动引起内在代谢产热作用于机体,导致机体不能去除这些因素以保持核心体温恒定,继而核心体温升高引起的潜在致命性机体紊乱[1],其特点是核心体温＞40℃,伴有中枢神经系统异常,如谵妄、抽搐或昏迷.按照致病因素,中暑分为经典型中暑(高热环境导致)和劳力性中暑(高强度的体力活动导致),其高热和(或)体力活动都可影响肠道,造成屏障功能障碍[2].     

血必净对脓毒症大鼠肠黏膜屏障及巨噬细胞抗体表达的作用

目的 观察血必净对脓毒症大鼠肠黏膜屏障和巨噬细胞抗体表达的变化的影响作用,探讨其在脓毒症发病中的作用机制及意义.方法 Wistar大鼠150只,分成脓毒症组、血必净干预组和假手术组.采用肓肠结扎穿孔术建立脓毒症大鼠模型,模型标准是发热、呼吸频率增加、心率加快和白细胞总数改变.血必净组大鼠术前12 h、术后腹腔内注射4 mL/kg血必净,1次/12 h,共3 d;其余两组大鼠同时注射相同体积的生理盐水.利用图像分析系统检测三组大鼠肠黏膜病理变化和巨噬细胞的表达水平,并运用等级资料秩和检验和方差分析进行统计学分析.结果 假手术组大鼠小肠黏膜多为基本正常黏膜,12 h,24 h,48 h,72 h脓毒症组、血必净组大鼠肠道黏膜出现病变;脓毒症组病变较血必净组更为严重,差异具有统计学意义(H=19.732,P<0.01).在0 h三组小肠黏膜巨噬细胞抗体的表达数基本一致,12 h,24 h,48 h,72 h脓毒症组和血必净组间差异具有统计学意义,且均较C组变化差异具有统计学意义(F=560.13,P<0.05).结论 脓毒症大鼠肠道黏膜机械屏障和免疫屏障均有不同程度的损害,血必净注射液可部分保护肠道黏膜机械屏障和免疫屏障.     

Changes of the immunological barrier of intestinal mucosa in rats with sepsis

BACKGROUND:

Sepsis has become the greatest threat to in-patients, with a mortality of over 25%. The dysfunction of gut barrier, especially the immunological barrier, plays an important role in the development of sepsis. This dysfunction occurs after surgery, but the magnitude of change does not differentiate patients with sepsis from those without sepsis. Increased intestinal permeability before surgery is of no value in predicating sepsis. The present study aimed to observe the changes of intestinal mucosal immunologic barrier in rat models of sepsis induced by cecal ligation and puncture.

METHODS:

Sixty Sprague-Dawley rats were randomly divided into a sepsis group (n=45) and a control group (n=15). The rats in the sepsis group were subjected to cecal ligation and puncture (CLP), whereas the rats in the control group underwent a sham operation. The ileac mucosa and segments were harvested 3, 6 and 12 hours after CLP, and blood samples were collected. Pathological changes, protein levels of defensin-5 (RD-5) and trefoil factor-3 (TFF₃) mRNA, and lymphocytes apoptosis in the intestinal mucosa were determined. In an additional experiment, the gut-origin bacterial DNA in blood was detected.

RESULTS:

The intestinal mucosa showed marked injury with loss of ileal villi, desquamation of epithelium, detachment of lamina propria, hemorrhage and ulceration in the sepsis group. The expression of TFF₃ mRNA and level of RD-5 protein were decreased and the apoptosis of mucosal lymphocyte increased (P<0.05) in the sepsis group compared with the control group. Significant differences were observed in RD-5 and TFF₃ mRNA 3 hours after CLP and they were progressively increased 6 and 12 hours after CLP in the sepsis group compared with the control group (P<0.05, RD-5 F=11.76, TFF₃ F=16.86 and apoptosis F=122.52). In addition, the gut-origin bacterial DNA detected in plasma was positive in the sepsis group.

CONCLUSION:

The immunological function of the intestinal mucosa was impaired in septic rats and further deteriorated in the course of sepsis.KEY WORDS: Sepsis, Mucosal immunology, Defensin-5, Trefoil factor family 3, Cecal ligation and puncture     

δ阿片受体激动剂D-丙2,D-亮5脑啡肽对脓毒症大鼠死亡率的影响

目的观察δ阿片受体激动剂D-丙2,D-亮5脑啡肽（[D-Ala2,D-Leu5]enkephalin,DADLE）对脓毒症大鼠存活时间和死亡率的影响。方法采用盲肠结扎穿孔（CLP）方法制备大鼠脓毒症模型。120只SD大鼠（雌雄不拘）,按随机数字表法分为假手术组（n=20）、模型对照组（n=20）,及4个DADLE治疗组。各DADLE组按给药剂量及方式分为制模前给药3mg/kg组（n=20）、制模前给药5mg/kg组（n=20）、制模后给药3mg/kg组（n=20）、制模后给药5mg/kg组（n=20）。观察各组大鼠CLP术后的存活时间,计算7d累积死亡率。结果与CLP模型未治疗组比较,各DADLE治疗组大鼠CLP术后的存活时间显著延长（P〈0.05）,各组7d死亡率分别为85%、60%、70%及75%,均较CLP组明显降低 但各DADLE治疗组之间无统计学差异（P〉0.05）。结论δ阿片受体激动剂DADLE能够明显延长脓毒症大鼠的存活时间,降低脓毒症大鼠的死亡率。     

Imipramine reverses the depressive symptoms in sepsis survivor rats

Objective To evaluate the antidepressant effect of imipramine on depressive symptoms observed in sepsis survivors rats. Design and setting Prospective, controlled experiment in an animal basic science laboratory. Subjects Male Wistar rats weighing 300–350 g. Interventions The rats underwent cecal ligation and perforation (CLP; sepsis group) with “basic support” (saline at 50 ml/kg immediately and 12 h after CLP plus ceftriaxone at 30 mg/kg and clindamycin at 25 mg/kg 6, 12, and 18 h after CLP) or sham-operated (control group). After 10 days of recovery rats received intraperitoneal injections of imipramine 10 mg/kg or saline and were subjected to the forced swimming test. Measurements and results The observed increase in the immobility time in the forced swimming test in animals subjected to CLP, as a parameter of depressive behavior, was reversed by imipramine. Conclusions The depressive symptoms evaluated by forced swimming test had been reversed after imipramine administration. Our data provide evidence that CLP-induced depressive symptoms are sensitive to antidepressants. This research was supported in part by UNESC (Brazil), FAPESC (Brazil), and CNPq (Brazil).     

Esmolol reduces apoptosis and inflammation in early sepsis rats with abdominal infection

Background

Esmolol is a highly selective beta 1 receptor blocker with various effects such as slowing heart rate, lowering blood pressure and reducing myocardial oxygen consumption. However, few studies have reported the use of beta blockers in sepsis with multiple organ dysfunctions. This study aimed to investigate the effects of esmolol on reducing apoptosis and inflammation in early sepsis rats with abdominal infection.

脓毒症早期大鼠下丘脑-垂体-肾上腺轴超微结构变化与功能的关系

目的 观察脓毒症早期下丘脑-垂体-肾上腺(HPA)轴结构及功能的变化,并探讨其相互关系.方法 雄性SD大鼠30只,按随机数字表法均分为正常对照组、假手术组和脓毒症组.采用盲肠结扎穿孔术(CLP)制作脓毒症大鼠模型,术后6 h取血并处死动物,检测血浆促肾上腺皮质激素(ACTH)、皮质酮(CoRT)以及下丘脑促肾上腺皮质激素释放激素(CRH)水平;透射电镜下观察HPA轴的超微结构改变.结果 脓毒症组血浆ACTH、CORT和下丘脑CRH水平均明显高于正常对照组和假手术组[ACTH(pmol/L):5.78±0.36比1.94±0.31、2.51±0.10;CORT(nmol/L):88.48±4.47比22.02±1.62、34.20±2.51,CRH(μg/L):101.92±6.61比61.65±6.05、66.65±4.03,P＜0.05或P＜0.01].透射电镜下观察:脓毒症组大鼠下丘脑粗面内质网囊状扩张、脱颗粒,高尔基体肿胀,垂体ACTH细胞分泌颗粒增多,肾上腺组织脂滴减少.结论 脓毒症早期大鼠HPA轴处于过度激活状态,血浆ACTH、CORT和下丘脑CRH水平明显升高;HPA轴的超微结构明显改变,且与功能变化有密切联系.

Abstract：

Objective To observe the changes in ultrastructure and function of hypothalamicpituitary-adrenal axis (HPAA), and to approach the relationship between them in early stage of sepsis in rats. Methods Thirty male Sprague-Dawley (SD) rats were randomly divided into normal control group,sham group, sepsis group. The sepsis model was reproduced by cecal ligation and puncture (CLP). The rats were sacrificed after collection of blood at 6 hours after CLP, and the levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) in the plasma, and the corticotropin release hormone (CRH) in the tissue of hypothalamus were detected. The histopathological changes in HPAA were observed with transmission electron microscopy. Results The levels of ACTH and CORT in plasma, and the CRH in hypothalamus tissue of sepsis group were increased in the early stage of sepsis compared with the normal control group or sham group [ACTH (pmol/L): 5. 78 ± 0. 36 vs. 1.94 ±0.31, 2. 51 ± 0.10; CORT (nmol/L), 88.48±4.47 vs. 22.02±1.62, 34.20±2.51; CRH (μg/L): 101. 92±6. 61 vs. 61.65±6.05,66. 65±4. 03, P＜0. 05 or P＜0. 01]. The changes in ultrastructure of the hypothalamus, pituitary and adrenal were also found. In sepsis group, the ultrastructure of hypothalamus was as follows. Rough endoplasmic reticulum expansion and degranulation of rough endoplasmic reticulum, and swelling of Golgi complex were found. A large number of endocrine granules could be seen in ATCH cells in the pituitary with depletion of adrenal lipid droplets. Conclusion In septic rats, the HPAA was excessively activated, and ACTH and CORT in plasma, and CRH in hypothalamus were significantly increased in early stage of sepsis.The changes in ultrastructure of HPAA were obvious, and the change in function was closely related to the ultrastructural changes.     

Repair of damaged intestinal mucosa in a mouse model of sepsis

BACKGROUND:

The intestine is not only the main target attacked by sepsis but also the vital organ which mediated sepsis. The recovery of the damaged intestinal barrier structure and function is related to the occurrence and outcome of multiple organ dysfunction syndrome (MODS). How to protect and reduce the damage of the intestinal mucosa and how to promote the reconstruction of the intestinal mucosa have been the important topics in sepsis for many years. This study aimed to investigate the influential factors of intestinal mucosal reconstruction after intestinal epithelial injury in vivo in a mouse model of sepsis.

METHODS:

Mice were subjected to cecal ligation and puncture (CLP) for induction of sepsis to assess intestinal mucosal damage, epithelial cell apoptosis, and transformed number of goblet cells, and to detect the concentration of TNF-α, IL-1 and TGF-β1 and TFF3 (trefoil factor 3) expression in the small intestinal mucosa. All above were performed by HE staining, western blot, ELISA and immunohistochemistry respectively. The experimental animals were divided into a sepsis group and a sham-operation group. The animals with sepsis were separately killed at 6 (7 animals), 24 (7 animals) and 48 hours (7 animals) after CLP.

RESULTS:

Injured intestinal mucosa was observed in the 3 groups under a light microscope, in which damage scores in the 24-hour and 48-hour groups were higher than in the 6-hour group and no difference was found between the two groups. Moreover, less of goblet cells or other epithelial cells adjacent to the injured surface migrated into the wound to cover the denuded area. The number of goblet cells was substantially decreased in the three CLP groups compared with the sham-operation group. Protein levels of IL-1 and TNF-α were significantly increased by 3–4 fold at all time points when compared with the sham-operation group, and cleaved caspase-3 by 4 fold. Although TFF3 expression was modestly increased for 6 hours after the onset of CLP, it appeared to decline at 24 hours and 48 hours as shown by Western blot. A similar tendency was observed upon TGF-β1, i.e. the protein level was not elevated at 24 hours and 48 hours, but increased modestly at 6 hours.

CONCLUSIONS:

Sepsis from CLP shows less restitution on the surface of injured intestinal mucosa. There is evidence that both constant inflammatory reaction and epithelial cell apoptosis may affect mucosal reestablishment of the intestine at the onset of sepsis. Mucosa after severe sepsis showed the state of high inflammation, and declined goblet cell function and mucosal reconstruction, which affected the repair of damaged intestinal barrier. Constant inflammatory reaction, and declined goblet cell function and mucosal reconstruction ability may affect the reestablishment of intestinal mucosa at the onset of sepsis.KEY WORDS: Sepsis, Cecal ligation and puncture, Intestinal mucosa, Restitution, Goblet cells, Intestinal trefoil factor 3, Transforming growth factor β1, Cysteine-containing aspartate-specific proteases     

免气腹单孔腹腔镜辅助盲肠结扎穿孔术建立脓毒症大鼠模型

目的 基于免气腹单孔腹腔镜辅助盲肠结扎穿孔(CLP)术建立脓毒症大鼠模型.方法 将64只大鼠分为A组(传统CLP术对照组)、B组(免气腹单孔腹腔镜辅助下CLP术对照组)、C组(传统CLP术穿孔组)及D组(免气腹单孔腹腔镜辅助下CLP术穿孔组),每组各16只.记录建模后24h各组大鼠的死亡情况,并检测各组大鼠血清白细胞介...     

重组人促红细胞生成素对感染致急性肝脏损伤大鼠的保护作用

目的 探讨重组人促红细胞生成素(recombinant human erythropoietin,rHuEPO)对盲肠结扎穿孔术(CLP)所致大鼠急性肝损伤的保护作用.方法 96只雄性SD大鼠随机(随机数字法)分为:假CLP组(对照组),CLP组(脓毒症组),rHuEPO组.采用盲肠结扎穿孔术复制急性肝损伤的动物模型,rHuEPO组造模后即刻经尾静脉推注rHuEPO 5000U/kg.手术后连续观察24h,选择术后2h、6h、12h、24h为观察点.分别在各时点随机处死大鼠,采用酶联免疫法(ELISA法)检测血清TNF-α、iNOS水平变化;同时检测肝功能(ALT、AST);采用光镜和透射电镜观察肝组织形态学变化.结果 ①各时间点大鼠血清中ALT、AST、TNF-α、iNOS水平的变化为脓毒症组及rHuEPO组上述指标明显高于对照组;rHuEPO组与脓毒症组相比,上述指标明显降低,差异具有统计学意义(P＜0.01).②光镜及电镜观察发现,脓毒症时肝脏病理学改变为局灶性肝细胞坏死,炎性细胞浸润,小叶间静脉充血扩张,肝细胞核固缩,线粒体、内质网明显减少.rHuEPO组肝脏病理学改变较CLP组明显减轻.结论 rHuEPO能够降低血清中ALT、AST、TNF-α、iNOS水平,发挥调节炎症反应进而改善肝功能的作用,对感染所致急性肝损伤具有一定的保护作用.