活性氧在休克肠淋巴液介导急性肺损伤中的作用与机制

发布时间：2018-04-17 13:15

本文选题：失血性休克 + 急性肺损伤　；参考：《河北北方学院》2017年硕士论文

【摘要】：急性肺损伤(acute lung injury,ALI)是由多种致病因素造成的肺实质细胞出现严重炎症反应,导致肺通透性增高、发生急性肺水肿的结果。由于失血性休克引起的微循环障碍以及肺的血流动力学特点,使得失血性休克患者最易发生ALI,可迅速发展为急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS),严重危及患者生命。我室前期研究发现,减少肠淋巴液回流,可减轻肺损伤,在降低肺组织氧化应激的同时,降低了肺组织Toll样受体2(toll-like receptor 2,TLR2)、TLR4的表达以及高迁移率族蛋白B1(high-mobility group box1,HMGB1)的表达。研究表明,失血性休克后,活性氧(reactive oxygen species,ROS)大量产生与释放,通过激发组织机体的炎性反应、介导细胞凋亡及增加血管通透性等方面导致ALI。但失血性休克后肠淋巴液(post-hemorrhagic shock mesenteric lymph,PHSML)介导ALI过程中是否有ROS的参与,尚不清楚。减少PHSML回流是否可降低肺组织ROS的产生与释放,PHSML介导肺组织ROS的产生与释放是否受TLR2、TRL4、HMGB1的影响(和/或影响TLR2、TLR4、HMGB1的表达)?有待研究。因此,本文在前期研究基础上,采用失血性休克小鼠模型,观察PHSML引流对失血性休克小鼠肺组织结构以及ROS含量变化的影响,观察静脉回输PHSML是否引起小鼠(野生型、TLR2-/-、TLR4-/-)肺组织结构损伤、ROS产生与释放及TLR2、TLR4分子表达,并进一步从细胞层面观察ROS抑制剂(NAC)、TLR2及TLR4封闭性抗体处理小鼠肺微血管内皮细胞(pulmonary microvascular endothelial cells,PMVECs)后,PHSML对PMVECs产生ROS的影响,从而验证PHSML是肺组织ROS产生增多的作用因素之一,该作用是通过TLR2、TLR4、HMGB1介导的。首先,将野生型(WT)C57BL/6J小鼠18只随机均分为假手术组(Sham)、失血性休克组(Shock)、休克+引流组(Shock+drainage)组;常规方法建立失血性休克模型,观察PHSML对失血性休克小鼠肺组织形态与ROS含量的作用。结果显示,Sham组小鼠肺组织结构基本正常;Shock组小鼠肺组织严重破坏,间隔增宽,多见炎性渗出及出血,肺泡腔大量融合;Shock+drainage组小鼠肺组织损伤较轻。同时可见,失血性休克引起了小鼠肺组织ROS显著增高,PHSML引流显著降低了Shock组小鼠肺组织ROS含量。随后,应用TLR2-/-、TLR4-/-小鼠各12只,均分为Sham、Shock组,常规方法建立失血性休克模型,观察TLR2、TLR4基因敲除对失血性休克小鼠肺组织形态与ROS含量的作用。结果显示,TLR2-/-、TLR4-/-的Shock组小鼠肺组织肺间隔炎性物质渗出,间隔增宽,肺泡部分融合,但程度较WT小鼠低,且肺组织ROS含量均显著低于WTShock组小鼠。然后,应用50只WT小鼠,引流正常淋巴液(normal mesenteric lymph,NML)和PHSML(与前述相同的失血性休克模型),分别静脉输入至WT、TLR2-/-、TLR4-/-小鼠,每组6只,观察静脉输入PHSML对各型小鼠肺组织形态、ROS含量以及TLR2、TLR4 mRNA表达的影响。结果显示,静脉输入NML对WT、TLR2-/-、TLR4-/-小鼠肺组织结构无明显影响,静脉输入PHSML引起了WT正常小鼠出现了比较严重的肺组织损伤,而静脉输入PHSML对TLR2-/-、TLR4-/-小鼠肺组织损伤的程度较轻。同时可见,静脉输入PHSML引起了WT正常小鼠肺组织ROS含量以及TLR2、TLR4的mRNA表达显著高于输入NML组;静脉输入PHSML至TLR2-/-、TLR4-/-小鼠的肺组织ROS含量显著低于WT小鼠,高于输入NML的同型小鼠;静脉输入PHSML至TLR4-/-小鼠肺组织的TLR2 mRNA表达显著高于输入NML的TLR4-/-小鼠以及输入PHSML的WT小鼠;同样,静脉输入PHSML至TLR2-/-小鼠肺组织的TLR4 mRNA表达显著高于输入NML的TLR2-/-小鼠以及输入PHSML的WT小鼠。为了进一步验证ROS在PHSML介导ALI中的作用及其与TLR2、TLR4的关系,本文以小鼠肺微血管内皮细胞(PMVECs)作为研究对象,观察了ROS特异性抑制剂NAC、TLR2和TLR4封闭性抗体对PHSML损伤PMVECs以及促进PMVECs产生ROS的作用。结果显示,PHSML引起了PMVECs损伤、ROS产生增多,NAC、TLR2和TLR4封闭性抗体均减轻了PHSML导致的PMVECs结构损伤,抑制了PHSML增加PMVECs产生ROS的作用。此外,针对HMGB1在PHSML介导ALI中的作用,本文进一步观察了正常小鼠腹腔注射HMGB1抑制剂甘草酸(Glycyrrhizic acid,GL)处理后,静脉输入PHSML后肺组织形态与ROS含量的变化。结果显示,GL预先处理显著降低了静脉输入PHSML损伤正常小鼠肺组织、增加ROS产生的作用。说明HMGB1参与了PHSML介导ALI的ROS机制。上述研究表明,PHSML引起肺组织ROS的过度产生是失血性休克ALI的重要机制之一;PHSML介导肺组织ROS过度产生与释放的机制与TLR2、TLR4、HMGB1有关。以PHSML、ROS为干预靶点,对于防治重症休克导致的ALI具有积极的作用。
[Abstract]:Acute lung injury (acute lung, injury, ALI) is caused by various pathogenic factors of lung parenchyma cells appeared severe inflammation, leading to pulmonary permeability, acute pulmonary edema results. The microcirculation in hemorrhagic shock caused by pulmonary and hemodynamic characteristics of the patients with hemorrhagic shock were most prone to ALI, can be quickly for the development of acute respiratory distress syndrome (acute respiratory distress syndrome, ARDS), seriously endanger the lives of patients. Our previous study found that reducing the intestinal lymph circulation, can reduce lung injury, reduce oxidative stress in lung tissue and reduce the Toll like receptor in lung tissue of 2 (Toll-like receptor 2, TLR2, TLR4) the expression of high mobility group protein B1 (high-mobility group box1, HMGB1) expression. The results show that after hemorrhagic shock, reactive oxygen species (reactive oxygen, species, ROS) a large number of production and release, The inflammatory reaction to stimulate body tissue, mediated cell apoptosis and increased vascular permeability in hemorrhagic shock rats but ALI. (post-hemorrhagic shock mesenteric lymph, lymph PHSML) mediated whether ALI participates in the process of ROS, is not clear. Whether PHSML can reduce reflux reduced production and release of ROS in lung tissue. The production and release of PHSML mediated by ROS in lung tissue by TLR2, TRL4, HMGB1 (the effect of expression and / or effects of TLR2, TLR4, HMGB1)? To study. Therefore, this paper based on the previous studies, the hemorrhagic shock model in mice, observe the PHSML drainage on the effect of hemorrhagic shock in the lung tissue of mice the structure and the change of the content of ROS, observe the intravenous infusion of PHSML can cause mice (wild type, TLR2-/-, TLR4-/-) of lung tissue damage, ROS release and TLR2, TLR4 expression, and further from the cellular level observation ROS inhibitor (NAC), TLR2 and TLR4 blocking antibody treatment of mouse pulmonary microvascular endothelial cells (pulmonary microvascular endothelial cells, PMVECs), PHSML ROS effect on PMVECs, which validates that PHSML is one of ROS in lung tissue increased the role of factors, the effect is through TLR2, TLR4, HMGB1 mediated. First of all, the wild type (WT) 18 C57BL/6J mice were randomly divided into sham operation group (Sham), hemorrhagic shock group (Shock), the shock + drainage group (group Shock+drainage); conventional method to establish the model of hemorrhagic shock, to observe the effects of PHSML on hemorrhagic shock in mice lung tissue morphology and ROS content. Show, Sham group of mice lung tissue structure was normal; lung tissue of Shock group were severely damaged, septum, rare inflammatory exudation and hemorrhage, alveolar fusion; Shock+drainage group of lung tissue injury in mice. At the same time less visible, hemorrhagic Hugh G caused the lung tissue of mice ROS significantly increased, PHSML drainage decreased the content of ROS in group Shock, the lung tissue of mice. Then, the application of TLR2-/- in TLR4-/- mice, 12 rats each, divided into Sham, Shock group, the conventional method to establish the model of hemorrhagic shock, observation of TLR2, TLR4 gene knockout on hemorrhagic shock in mice lung tissue with the content of ROS. The results showed that TLR2-/-, TLR4-/- in Shock group lung interval inflammatory substance exudation, alveolar septum, partial fusion, but more WT mice is low, and the content of ROS in lung tissue were significantly lower than those in WTShock group. Then, the application of 50 WT mice (normal lymph drainage. Normal mesenteric lymph, NML) and PHSML (hemorrhagic shock model with the same), respectively, intravenous to WT, TLR2-/-, TLR4-/- mice, 6 rats in each group. The observation of intravenous infusion of PHSML of various types of mouse lung tissue morphology, content of ROS, TLR2, TLR4 Effect of mRNA expression. The results showed that intravenous infusion of NML of WT, TLR2-/-, TLR4-/- had no effect on lung tissue structure in mice, intravenous infusion of PHSML caused by WT in normal mice appeared more serious lung injury, and intravenous infusion of PHSML of TLR2-/- and TLR4-/- in mouse lung tissue injury to a lesser degree. At the same time, visible, vein enter the PHSML WT in the lung tissue of normal mice caused by the content of ROS, TLR2, TLR4 mRNA expression was significantly higher than that of the input NML group; intravenous infusion of PHSML to TLR2-/- and ROS in lung tissue of TLR4-/- mice was significantly lower than that in WT mice, the same type of mice is higher than the input NML; intravenous infusion of PHSML into TLR4-/- mice lung tissue TLR2 mRNA expression was significantly higher than that of the input of the NML input PHSML TLR4-/- mice and WT mice; similarly, intravenous infusion of PHSML and TLR2-/- in lung tissue of mice TLR4 mRNA expression was significantly higher than that of the input NML TLR2-/- mice and PHSML input WT mice. In order to verify the ROS in PHSML mediated ALI and its interaction with TLR2, TLR4, the mouse pulmonary microvascular endothelial cells (PMVECs) as the research object, the effect of ROS specific inhibitor of NAC, TLR2 and TLR4 blocking antibody effects on PHSML and PMVECs to promote the production of PMVECs ROS. The results showed that PHSML caused PMVECs damage, increased ROS production, NAC, TLR2 and TLR4 blocking antibody can reduce the damage caused by PMVECs PHSML, PHSML ROS inhibited the increase of PMVECs effect. In addition, the HMGB1 in PHSML mediated ALI effect, we further observed in normal mice by intraperitoneal injection the inhibitor of HMGB1 (Glycyrrhizic acid GL, glycyrrhizic acid) after treatment, the morphological changes of lung tissue and ROS content after intravenous infusion of PHSML. The results showed that GL pretreatment significantly decreased the intravenous infusion of PHSML injury of the normal lung tissues of mice, The increase of ROS production. It indicated that HMGB1 is involved in ROS mechanism mediated by PHSML ALI. The results of the study showed that PHSML induced overproduction of ROS in lung tissue is one of the important mechanisms of hemorrhagic shock ALI; PHSML mediated lung ROS overproduction and release mechanism and TLR2, TLR4, HMGB1. PHSML have to. ROS is the target for intervention, plays a positive role in the prevention and treatment of severe shock caused ALI.

【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.8

【参考文献】