罗氟司特对脓毒血症小鼠的保护作用及机制研究

发布时间：2018-07-07 08:58

本文选题：罗氟司特 + 脓毒血症　；参考：《南方医科大学》2017年硕士论文

【摘要】：目的:脓毒血症是指由感染所引起的全身炎症反应综合征,常伴随多器官功能障碍,具有高发病率和高死亡率的特点。罗氟司特作为一种选择性磷酸二酯酶抑制剂,具有强大的抗炎作用。于是,本课题采用盲肠结扎穿刺(cecal ligation and puncture,CLP)的方法建立脓毒血症小鼠模型,考察罗氟司特对脓毒血症小鼠的作用及可能涉及的作用机制。方法:1.采用CLP方法建立脓毒血症小鼠模型。动物分组包括:假手术对照组、CLP模型组、CLP+罗氟司特低、中、高剂量组(0.3mg/kg、1.0mg/kg、3.0 mg/kg)。在造模前7天开始灌胃给药处理,每天一次。造模后,每天观察小鼠一次,连续观察7天,记录小鼠死亡时间,观察存活率变化;按照评分标准对小鼠状态进行评分,观察临床得分的变化。采用统计学方法检验存活率的组间差异、临床观察得分的组间差异是否具有统计学意义,考察罗氟司特对脓毒血症小鼠是否具有保护作用。2.造模24 h后,收集血液、腹腔灌洗液、肝、肺、脾,将组织进行匀浆处理,采用平板表面涂布方法对样品进行细菌计数;将血液离心后收集血浆,组织进行匀浆处理,采用ELISA方法检测五种样品中TNF-α、IL-6的含量;检测血浆中ALT、AST、LDH的含量,同时对肝、肺、脾组织切片进行HE染色,在光学显微镜下观察组织病理变化。观察这些指标在造模条件下的变化,罗氟司特对这些指标的影响,考察罗氟司特对脓毒血症小鼠的保护作用可能涉及的途径。3.造模24 h后,收集肝脏,将肝脏进行匀浆处理,采用ELISA方法检测肝脏中cAMP的含量,采用Western blot方法检测cAMP下游信号分子(p-CREB、CREB)、NF-κB通路相关蛋白(IκB-α、全蛋白NF-κB p65、胞浆蛋白NF-κB p65、胞核蛋白 NF-κB p65)、p38MAPK 通路相关蛋白(p-p38MAPK、p38MAPK)、STAT3 通路相关蛋白(p-JAK1、JAK1、p-JAK2、JAK2、p-STAT3、全蛋白 STAT3、胞浆蛋白STAT3、胞核蛋白STAT3)的蛋白表达。观察这些蛋白在造模条件下的表达情况,以及罗氟司特对这些蛋白表达的影响,考察罗氟司特对脓毒血症小鼠的保护作用可能涉及的信号通路。结果:1.罗氟司特对脓毒血症小鼠具有保护作用。存活率实验结果显示,假手术组小鼠没有出现死亡,造模后小鼠的存活率明显下降,罗氟司特处理能够显著提高小鼠存活率。临床观察得分结果显示,假手术组的分数是最低的,模型组小鼠的评分在造模后显著升高,罗氟司特处理可以显著地降低评分,将评分维持在较低水平。2.罗氟司特通过作用于脓毒血症的多个环节介导对脓毒血症小鼠的保护作用。细菌计数结果显示,与假手术组相比,模型组各种样本的细菌量均显著增高。罗氟司特处理可以显著降低腹腔灌洗液的细菌量,即局部细菌量,也可以明显降低血液中的细菌量,即全身扩散的细菌量。此外,罗氟司特处理还可以有效降低肝、肺、脾中的细菌量。炎症因子结果显示,与假手术组相比,模型组小鼠各种样本中IL-6、TNF-α的含量均显著升高。罗氟司特处理能够显著降低血液、腹腔灌洗液、肝脏中的IL-6、TNF-α水平。这提示罗氟司特能够抑制脓毒血症小鼠全身炎症反应、肝脏炎症反应。此外,罗氟司特对肺和脾的炎症因子升高也有一定抑制作用。酶活力结果显示,与假手术组相比,模型组小鼠AST、ALT水平显著上升,而罗氟司特处理显著降低ALT、AST水平,这提示罗氟司特能够缓解脓毒血症小鼠肝组织损伤,HE切片结果进一步证实了罗氟司特的保护作用。此外,罗氟司特还可以一定程度缓解脓毒血症肺损伤、脾损伤。3.罗氟司特对脓毒血症小鼠的保护作用涉及多个信号通路。首先,结果显示,与假手术组相比,造模后的cAMP含量和CREB磷酸化显著下降,而罗氟司特能够显著抑制蛋白的下降,表明罗氟司特保护作用可能与cAMP/CREB的激活有关。此外,造模后IκB-α的降解、NF-κB p65核位移、p38 MAPK磷酸化均显著增强,而罗氟司特处理可以显著抑制这些蛋白的变化,表明而且罗氟司特的保护作用可能与NF-κB、p38MAPK的抑制有关。同时,造模后JAK1、JAK2、STAT3的磷酸化、STAT3核位移均显著增强,而罗氟司特处理可以显著抑制这些蛋白的变化,表明而且罗氟司特的保护作用可能与STAT3的抑制有关。结论:1.罗氟司特能够提高脓毒血症模型小鼠的存活率、缓解脓毒血症症状,具有保护作用。2.罗氟司对特脓毒血症模型小鼠的保护作用可能通过降低小鼠体内的细菌量;降低炎症因子IL-6、TNF-α水平,抑制炎症:降低ALT、AST、LDH水平,减轻组织损伤。3.罗氟司特对脓毒血症模型小鼠的保护作用可能通过激活cAMP/CREB信号通路,抑制NF-κκB、p38MAPK、STAT3信号通路。
[Abstract]:Objective: sepsis is a systemic inflammatory response syndrome caused by infection, often accompanied by multiple organ dysfunction, with high morbidity and high mortality. Roflurox is a selective phosphodiesterase inhibitor and has a strong anti-inflammatory effect. Therefore, cecal ligation and punct is used in this study. Ure, CLP) to establish a mouse model of sepsis to investigate the effect and possible mechanism of rolflurox on sepsis mice. Methods: 1. the mice model of sepsis was established by CLP method. The group of animals included the sham operation control group, the CLP model group, the low, middle, and high dose group (0.3mg/kg, 1.0mg/kg, and 3 mg/kg) of CLP+. After 7 days before the model, the gavage was given to the stomach. After the model was made, the mice were observed once a day for 7 days. The time of death and the change of survival rate were recorded. The state of the mice was evaluated according to the scoring standard and the changes of clinical scores were observed. The differences in the survival rate were examined by the statistical method and the clinical observation score was observed. Whether the difference between groups was statistically significant, to investigate whether rofluusate had protective effect on sepsis in mice with.2. 24 h, collect blood, peritoneal lavage fluid, liver, lung, spleen, homogenate the tissue, use the plate surface coating method to count the bacteria, collect blood after centrifugation, and homogenate the tissue. Treatment, ELISA method was used to detect the content of TNF- alpha and IL-6 in five samples; the content of ALT, AST, LDH in plasma was detected, and the liver, lung, and spleen tissue sections were stained with HE, and the pathological changes were observed under the optical microscope. The changes of these indexes under the model conditions and the effect of roflulesite on these indexes were observed and roflulein was examined. The protective effect of sepsis in mice may be involved in the pathway.3. 24 h, the liver is collected, the liver is homogenized, and the content of cAMP in the liver is detected by ELISA method. The Western blot method is used to detect the cAMP downstream signal molecules (p-CREB, CREB) and NF- kappa B pathway Guan Danbai. Nuclear protein NF- kappa B p65), p38MAPK pathway related proteins (p-p38MAPK, p38MAPK), STAT3 pathway related proteins (p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3, whole protein, Cytoplasmic Protein, cytoplasmic nucleoprotein). The protective effect of rolflurox on sepsis mice might be involved. Results: 1. rolllice had protective effect on sepsis in mice. The results of the survival rate test showed that the mice in the sham operation group had no death, the survival rate of the mice was significantly decreased, and the rolllethe treatment could significantly improve the survival rate of mice. The score of the sham operation group was the lowest, the score of the model group was significantly higher after the model, and the rolllethe treatment could significantly reduce the score, and the score was maintained at the lower level of.2. roflurox in the protective effect of multiple rings on sepsis in sepsis mice. The count results showed that the amount of bacteria in all the samples in the model group was significantly higher than that in the sham group. Roflurox treatment could significantly reduce the amount of bacteria in the peritoneal lavage fluid, that is, the amount of bacteria in the local bacteria and the amount of bacteria in the blood, that is, the amount of bacteria in the whole body. In addition, roflulein treatment can also effectively reduce the liver and lung, The amount of bacteria in the spleen. The results of inflammatory factors showed that the levels of IL-6 and TNF- a in all the samples of the model group were significantly higher than those in the sham group. The rolluset treatment could significantly reduce the blood, peritoneal lavage fluid, IL-6 and TNF- alpha levels in the liver. This suggests that rollluset can inhibit the systemic inflammatory response of mice with sepsis, liver, and liver. In addition, roflurox also inhibited the increase of inflammatory factors in the lungs and spleen. The results of enzyme activity showed that the AST, ALT level of the model mice increased significantly compared with the sham operation group, while rolllleat treatment significantly reduced ALT and AST levels, which suggested that rolllrex could relieve the liver tissue damage in mice with sepsis and HE section. The results further confirmed the protective effect of roflurolash. In addition, rolllrex could also relieve the pulmonary injury of sepsis to a certain extent. The protective effect of.3. rollrex on sepsis mice involved multiple signal pathways. First, the results showed that the content of cAMP and the phosphorylation of CREB after the model were significantly lower than those in the sham operation group. Roflurox could significantly inhibit the decrease of protein, indicating that the protection of rofluleat may be related to the activation of cAMP/CREB. In addition, the degradation of I kappa B- alpha, the nuclear shift of NF- kappa B p65, and the phosphorylation of p38 MAPK significantly increased, while roflurox treatment could significantly inhibit the changes of these proteins, indicating and the protective effect of rofluleat. It may be related to the inhibition of NF- kappa B and p38MAPK. At the same time, after the phosphorylation of JAK1, JAK2, STAT3, the nuclear displacement of STAT3 is significantly enhanced, and roflulethe treatment can significantly inhibit the changes of these proteins, indicating that the protective effect of rofluleat may be related to the inhibition of STAT3. Conclusion: 1. roflurox can improve the sepsis model small. The survival rate of rats, relieving the symptoms of sepsis, the protective effect of.2. rofluus on the mice with sepsis may be reduced by reducing the amount of bacteria in the mice; reducing the level of inflammatory factors IL-6, TNF- alpha, and inhibiting inflammation: lowering the level of ALT, AST, LDH, and reducing the tissue injury of the.3. rofluleus on the sepsis model mice. Protective effect may inhibit NF- kappa B, p38MAPK and STAT3 signaling pathway by activating cAMP/CREB signaling pathway.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965

【参考文献】