内毒素导致的肺肾损伤和西洛他唑减轻肺肾损伤的实验研究

发布时间：2018-05-31 01:03

本文选题：LPS + 脓毒症　；参考：《吉林大学》2015年博士论文

【摘要】：脓毒症，是临床上死亡率较高的疾病之一，在临床各科室尤其是呼吸科、ICU科中发病率很高。临床上导致败血症的细菌有很多种，以大肠埃希菌更为常见，且死亡率很高。脓毒症导致患者死亡的最主要原因是导致急性多器官功能损伤，最终导致多器官功能衰竭死亡。对于脓毒症的发病机制及治疗的探讨目前仍然是国内外研究的热门问题，希望通过对机制的研究，为临床治疗脓毒症提供有效的辅助治疗药物。参与急性炎症的主要细胞成分有中性粒细胞、单核细胞、巨噬细胞等。常见的炎症因子有白介素（IL）、肿瘤坏死因子（TNF）、血小板活化因子（PAF）、前列腺素等。血管反应被认为是炎症过程的中心环节。在脓毒症中，中性粒细胞等炎症细胞激活，可导致多种促炎症因子及粘附分子增多，血管通透性增高，白细胞渗出并导致组织水肿和炎症。内毒素导致的炎症反应的主要信号通路有NF-kB pathway，MAPK/ERKpathway等等。NF-kB可与多种基因启动子或增强子部位的kB位点特异性结合，促进基因转录，是炎症反应的主要介质和转录因子。MAPK的持续激活可以导致炎症因子不断产生，诱导“瀑布”效应，引起炎症持续及损伤不断加重。其中较为重要的是p38MAPK信号，它与NF-kB pathway关系密切，是激活NF-kB pathway的主要因素。活化的NF-kB pathway和p38MAPK pathway可以导致炎症因子瀑布式级联反应、氧自由基增多，导致细胞损伤甚至死亡。西洛他唑是常见的一种抗血小板凝集的药物，在近期国内外的很多实验中均证明西洛他唑对于减轻炎症损伤具有重要作用。为了进一步研究LPS诱导的脓毒症进展过程中的炎症改变情况，进一步研究西洛他唑减轻LPS诱导的肺肾损伤的机制，我们以C57小鼠为研究对象，，通过腹腔内注射LPS的方法制作脓毒症模型，并通过西洛他唑腹腔内注射的方法进行研究，应用病理及免疫荧光染色、ELISA、Western Blot等方法进行检测。实验第一部分：对于LPS腹腔内注射导致肺损伤和肾损伤的研究。我们通过腹腔内注射内毒素（LPS）的方法，成功制作了脓毒症的模型。基于不同的研究目的，我们制作了两种模型：为观察组织病理改变，我们制作了第一模型组（低剂量组）：腹腔内注射LPS5mg/kg造模，24小时后处死小鼠并留取组织标本，通过肾组织和肺组织病理染色和冰冻切片的免疫荧光染色，观察内毒素损伤时对于肺组织和肾组织的结构改变的影响；为了研究促炎症因子的变化情况，我们制作了第二模型组（高剂量组），腹腔内注射LPS7.5mg/kg造模，分别于注射LPS6小时和24小时后处死小鼠，收集血液及肺组织及肾组织样本，测定肺组织MPO和血清BUN水平，评价脏器损伤程度变化，通过ELISA方法，测定促炎症因子IL-6和TNF-α的水平变化，通过Western Blot测定炎症相关因子ICAM-1和VCAM-1、炎症通路ERK激活情况以及Mst-1的变化。通过实验我们发现，在LPS诱导的脓毒症模型早期，病理上主要以炎症细胞浸润为主，炎症细胞透过血管壁向组织内浸润，局部血管上皮及组织细胞损伤，但对于组织整体结构的破坏并不明显。肺组织及肾脏功能损伤，肺组织MPO6小时和24小时均明显增高，但两者无统计学差异，肾功能BUN水平随时间推移，明显增高。促炎症细胞因子IL-6和TNF-α均明显增高，IL-66小时和24小时增加量无明显统计差异，而TNF-α6小时含量高于24小时；Mst-1随时间推移，明显减少。实验第二部分：西洛他唑减轻肺及肾脏损伤的研究。为了明确西洛他唑减轻炎症损伤的机制，我们应用西洛他唑治疗脓毒症。腹腔内注射LPS（5mg/Kg）造模，对照组腹腔内注射PBS，在注射LPS1小时后注射西洛他唑0.3mg/只,为避免西洛他唑溶剂DMSO的实验干扰，PBS组和LPS组1小时后注射同等体积的DMSO。造模24小时后收集血液及组织，通过ELISA方法，测定促炎症因子TNF-α的水平变化。对于肾组织，我们通过Western Blot测定西洛他唑对炎症相关因子VCAM-1、p-ERK、p-p38的影响，了解西洛他唑对炎症通路ERK、p-p38MAPK途径激活情况的影响；为了明确西洛他唑对氧化应激的作用，测定了HO-1、MnSOD及TBARS。同时，我们还应用Western Blot及ELISA方法对肺组织进行了研究。通过实验我们发现，治疗组小鼠（LPS/CSZ组）与模型组（LPS组）小鼠血清中的TNF-α水平相比明显下降。LPS/CSZ组与LPS组相比，肺组织中MPO检测无明显差异，ICAM-1、VCAM-1表达无明显差异；肾组织中，LPS/CSZ组HO-1含量降低，TBARS实验中与硫代巴比妥酸物质反应减少，MnSOD的水平较LPS组有所升高，三者证明西洛他唑具有抗氧化应激作用；ERK，p38MAPK通路激活减少，VCAM-1减少，肾脏损伤减轻。实验证明，西洛他唑可以减轻LPS导致的肾脏损伤，但对于肺损伤的减轻作用不明显。
[Abstract]:Sepsis is one of the diseases with high mortality in clinic. The incidence of ICU is very high in all clinical departments, especially in the Department of respiration. There are many bacteria in the clinic which cause sepsis. Escherichia coli is more common and the death rate is high. The most important reason for the death of patients with sepsis is the cause of acute multiple organ damage. Finally, the cause of acute multiple organ damage is caused by sepsis. The pathogenesis and treatment of sepsis are still a hot issue at home and abroad. We hope to provide effective adjuvant therapy for clinical treatment of sepsis through the study of the mechanism.
The main components involved in acute inflammation are neutrophils, monocytes, macrophages, etc.. Common inflammatory factors include IL, tumor necrosis factor (TNF), platelet activating factor (PAF), prostaglandin and so on. Vascular response is considered to be the central link in the inflammatory process. In sepsis, inflammatory cells like neutrophils are activated in sepsis. It can lead to a variety of proinflammatory and adhesion molecules, vascular permeability, leukocyte exudation and tissue edema and inflammation.
The main signal pathways of endotoxin induced inflammatory response are NF-kB pathway, MAPK/ERKpathway and so on, which can specifically bind to the kB loci of various gene promoters or enhancers, promote gene transcription, which is the main medium of the inflammatory response and the sustained activation of the transcription factor.MAPK, which can lead to the continuous production of inflammatory factors. The effect of waterfall causes inflammation and damage continuously. The more important of which is the p38MAPK signal, which is closely related to NF-kB pathway, which is the main factor to activate NF-kB pathway. The activated NF-kB pathway and p38MAPK pathway can lead to the cascade cascade of inflammatory factors, the increase of oxygen free radicals, and cell damage and even death.
Cilostazol is a common antiplatelet aggregation drug. It has been proved to play an important role in reducing inflammatory damage in a number of experiments both at home and abroad. In order to further study the inflammatory changes in the progress of LPS induced sepsis, cilostazol was further studied to reduce LPS induced lung and renal injury. The mechanism, we take C57 mice as the research object, by intraperitoneal injection of LPS method to make sepsis model, and through the method of intraperitoneal injection of cilostazol to study, using pathological and immunofluorescence staining, ELISA, Western Blot and other methods to detect.
Part 1: the study of LPS intraperitoneal injection of lung injury and renal injury. We successfully made a model of sepsis by intraperitoneal injection of endotoxin (LPS). Based on different research purposes, we made two models: To observe histopathological changes, we made the first model group (low dose group). Intraperitoneal injection of LPS5mg/kg model, 24 hours after the death of mice and left tissue specimens, through the renal tissue and lung tissue pathological staining and frozen section immunofluorescence staining, observe the effects of endotoxin injury on the structural changes in the lung tissue and renal tissue, in order to study the changes in the proinflammatory factors, we made second The model group (high dose group), intraperitoneally injected with LPS7.5mg/kg model, killed mice after LPS6 hours and 24 hours respectively, collected blood and lung tissue and renal tissue samples, measured the level of MPO and serum BUN in lung tissue, evaluated the changes in the degree of organ damage, and measured the level of the level of pro-inflammatory factor IL-6 and TNF- alpha by ELISA method, through Weste. RN Blot was used to measure inflammation related factors ICAM-1 and VCAM-1, ERK activation in inflammatory pathway and Mst-1 changes.
In the early stage of LPS induced sepsis, we found that in the early stage of the sepsis model, the main pathological changes were infiltration of inflammatory cells, inflammatory cells infiltrated into the tissue through the vascular wall, local vascular epithelium and tissue cells were damaged, but the damage to the whole structure of the tissues was not obvious. Lung tissue and kidney function damage, lung tissue MPO6 hours and 24 small There was no significant difference between the two groups, but the BUN level of renal function increased significantly as time went on. The proinflammatory cytokines IL-6 and TNF- alpha increased significantly, and there was no significant statistical difference between IL-66 hours and 24 hours, while the content of TNF- alpha 6 hours was higher than 24 hours, and Mst-1 decreased with time.
The second part of the experiment: cilostazol alleviates the injury of lung and kidney. To clarify the mechanism of cilostazol alleviating inflammatory damage, we use cilostazol in the treatment of sepsis. Intraperitoneal injection of LPS (5mg/Kg) model, intraperitoneal injection of PBS in the control group, injection of cilostazol 0.3mg/ only after LPS1 hours, in order to avoid cilostazol solvent DM SO experimental interference, group PBS and LPS group 1 hours after the injection of the same volume of DMSO. model 24 hours after the collection of blood and tissue, the ELISA method, the determination of the level of the level of pro-inflammatory factor TNF- alpha. For renal tissue, we used Western Blot to determine the effect of cilostazol on the inflammatory related factors, VCAM-1, p-ERK, p-p38, to understand cilostazol. The effect of ERK, p-p38MAPK pathway activation in the inflammatory pathway; to determine the effect of cilostazol on oxidative stress, and to determine HO-1, MnSOD and TBARS., we also used Western Blot and ELISA methods to study lung tissue.
Through the experiment, we found that the mice in the treatment group (group LPS/CSZ) and the model group (group LPS) had a significant decrease in the level of TNF- alpha in the serum of the mice. There was no significant difference in the MPO detection in the lung tissue between the group.LPS/CSZ and the LPS group, and there was no significant difference in the expression of ICAM-1 and VCAM-1 in the lung tissue; the HO-1 content in the LPS /CSZ group was lower in the renal tissue and in the TBARS experiment with the thiobarbituric acid. The level of MnSOD was higher than that in group LPS. The three showed that cilostazol had antioxidant stress, ERK, p38MAPK pathway activation decreased, VCAM-1 decreased, and renal injury alleviated. The experiment proved that cilostazol could reduce the renal damage caused by LPS, but the reduction of lung injury was not obvious.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R459.7

【共引文献】