乙酰化白藜芦醇对大鼠海水吸入型肺损伤的保护作用研究

发布时间：2018-04-15 12:39

本文选题：海水 + 急性肺损伤　；参考：《第四军医大学》2013年硕士论文

【摘要】：研究背景：淹溺是一个重大但时常被忽视的公共卫生问题，据不完全统计，每年约有50万人死于淹溺。肺脏是吸入海水后首先受到侵害的重要脏器，研究表明吸入海水所致肺损伤明显比吸入淡水严重。海水吸入型肺损伤（Seawater Inhalation Induced AcuteLung Injury, SWI-ALI）主要表现为肺部炎症反应和肺水肿，具体发生机制较为复杂，可以肯定的是损伤的严重程度取决于液体的吸入量和清洁程度。海水吸入型肺损伤病情发展迅速，如未及时治疗或处置不当易发展为海水吸入型呼吸窘迫综合征（Seawater Inhalation Induced Acute Respiratory Distress Syndrome, SWI-ARDS）。近年来，，对海水吸入型肺损伤发病机制和救治的研究日益增多，但是对其发病机制尚未形成统一认识，未能提出切实有效、有针对性的治疗措施。核转录因子κB（NF-κB）是一种几乎存在于所有哺乳动物细胞内的转录因子，参与细胞增殖、分化、凋亡和癌变等多种病理生理过程。通常细胞中NF-κB与IκB结合存在于细胞浆中；当受到LPS、TNF-α和IL-1等刺激时，IκB被迅速降解，游离状态的NF-κB转移入细胞核，与相应目的基因结合并促使其转录，发挥相应的生物学功能。NF-κB调节多种效应分子的表达，介导组织损伤与修复；其中缺氧诱导因子1α（HIF-1α）和诱生型一氧化氮合酶（iNOS）是其重要的下游调节因子，三者共同参与多种疾病的发生于发展。在此理论基础上，NF-κB相关信号通路在海水吸入型肺损伤中的作用成为本课题的研究重点。白藜芦醇（Resveratrol）是广泛存在于多种植物体内的多酚化合物，是一种植物抗毒素，具有多种生物活性，如抗炎、抗氧化、抗肿瘤细胞增殖和抗细胞老化等；因此，近年来对白藜芦醇的研究较为多见。然而，由于白藜芦醇生物半衰期短和血药浓度低等原因，其临床应用始终面临种种困难。乙酰化白藜芦醇是白藜芦醇三个羟基被乙酰基取代后的产物，在体内转化为白藜芦醇发挥药理活性，并且能够增加白藜芦醇在肺部蓄积浓度同时延长生物半衰期，前期研究表明乙酰化白藜芦醇能够通过抑制活性氧类（reactive oxygen species，ROS）的生成降低γ-射线照射时小鼠的死亡率。在本研究中，我们通过体内试验观察大鼠吸入海水后肺部损伤性变化及乙酰化白藜芦醇的保护作用；在细胞实验中，我们采用白藜芦醇，乙酰化白藜芦醇的代谢产物，研究其保护作用的具体机制。实验目的：（1）明确乙酰化白藜芦醇能否抑制炎症反应和氧化损伤减轻海水吸入型肺损伤；（2）进一步探讨乙酰化白藜芦醇抗炎、抗氧化的具体信号转导机制，尤其是NF-κB、iNOS和HIF-1α等信号分子在其中的作用；（3）细胞试验验证相关信号通路在海水吸入型肺损伤中的作用。实验方法：实验一：健康雄性SD大鼠按照完全随机的方法分为6组：A组：空白对照组；B组：海水吸入型肺损伤模型组；C组：乙酰化白藜芦醇低剂量（50mg/kg）组；D组：中剂量组（150mg/kg）；E组：高剂量组（450mg/kg）预处理组；和F组：PDTC预处理组（100mg/kg）。各预处理组从造模前7天开始每天灌胃相应药物，最后一次灌胃90分钟后造模，向实验大鼠气管内滴注配方海水4ml/kg。造模4小时后放血处死取肺组织标本。采用HE染色的方法观察各组大鼠肺组织结构损伤变化；通过ELISA的方法检测肺组织中TNF-α和IL-1β的含量，分别采用硫代巴比妥酸（ThibabituricAcid TBA）法和黄嘌呤氧化酶法测定肺组织中MDA含量和T-SOD活性，用WesternBlot的方法检测NF-κB、HIF-1α和iNOS的表达量。以评价乙酰化白藜芦醇对海水吸入后所致炎症和氧化损伤的改善作用。实验二：将处于指数生长期的A549细胞分为4组，A：正常培养组；B：海水刺激组；C：白藜芦醇干预组；D：PDTC预处理组。C组和D组在分别加入200μmol/L白藜芦醇和PDTC30分钟后进行海水刺激。同时向B、C和D组加入含有25%配方海水的培养基，正常培养4小时后弃去培养基进行免疫荧光染色操作，检测细胞中NF-κB、HIF-1α和iNOS的表达量。实验结果：实验一：与空白对照组相比，大鼠吸入海水后肺组织结构受到破坏，肺泡壁增厚并伴有局部塌陷；肺泡腔和肺间质内可见大量水肿液渗出和炎细胞浸润；肺组织内代表损伤效应的TNF-α、IL-1β、MDA和NO等表达量增加（P0.05），而代表抗损伤效应的T-SOD活性降低（P0.05）；同时，海水吸入能够导致NF-κB、HIF-1α和iNOS的表达量增高（P0.05）。然而，乙酰化白藜芦醇预处理组大鼠肺组织中TNF-α、IL-1β、MDA和NO表达量相对降低（P0.05），T-SOD表达量增高（P0.05），且高剂量组效果最为明显（P0.01）；同时，乙酰化白藜芦醇降低肺组织中NF-κB、HIF-1α和iNOS的表达量（P0.05），且干预效果与PDTC无明显差异（P0.05）。实验二：与正常培养的细胞相比，海水刺激后A549细胞中NF-κB、HIF-1α和iNOS的表达量明显增高，而乙酰化白藜芦醇体内代谢产物白藜芦醇预处理后NF-κB、HIF-1α和iNOS的表达量显著降低，与PDTC效果无明显差异。结论： NF-κB及其下游的信号分子HIF-1α和iNOS构成的信号通路参与海水吸入型肺损伤的发生与发展；乙酰化白藜芦醇能够通过干预此信号通路的表达，抑制下游炎症因子等的表达减轻海水吸入所致的肺部炎症和肺水肿。
[Abstract]:Research background:
Drowning is a major but often neglected public health problems, according to incomplete statistics, there are about 500 thousand people died of drowning each year. The lung is an important organ in water after the first violation, research shows that inhalation lung injury induced by seawater than freshwater seawater inhalation inhalation. Severe lung injury type (Seawater Inhalation Induced AcuteLung Injury, SWI-ALI) mainly manifested as pulmonary inflammatory reaction and pulmonary edema and its mechanism is complex, to be sure the severity of injury depends on the liquid intake and clean degree of seawater aspiration induced lung injury. The disease develops rapidly, if not timely treatment or improper disposal of inhaled seawater susceptible to respiratory distress syndrome (Seawater Inhalation Induced Acute Respiratory with Distress Syndrome, SWI-ARDS). In recent years, the study on seawater inhalation lung injury pathogenesis and treatment is Increased, but its pathogenesis has not yet formed a unified understanding, to put forward effective and targeted treatment measures.
Nuclear factor kappa B (NF- K B) is a transcription factor that exists in almost all mammalian cells, involved in cell proliferation, differentiation, apoptosis and carcinogenesis in many pathophysiological process. Usually cells NF- kappa B and I kappa B binding in the cytoplasm; when exposed to LPS, TNF- and IL-1 when stimulated, I kappa B was rapidly degraded and free NF- kappa B transfer into the nucleus, combined with the corresponding gene and the transcription, expression play the biological functions of.NF- kappa B regulation of the corresponding variety of effector molecules, mediated tissue injury and repair; hypoxia inducible factor 1 alpha (HIF-1 alpha) and inducible nitric oxide synthase (iNOS) is an important downstream factor, the three jointly participate in a variety of diseases in development. On the basis of this theory, NF- kappa B signaling pathway in the seawater inhalation lung injury has become the key point of this research.
Resveratrol is a polyphenol compound (Resveratrol) exists widely in many plants, is a phytoalexin, has many biological activities, such as anti-inflammatory, antioxidant, anti proliferative and anti aging cells; therefore, in recent years the study of resveratrol is rare. However, because of resveratrol the short half-life and low blood concentration, its clinical application has always been faced with difficulties. The acetylation of resveratrol is resveratrol three hydroxy acetyl substituted by the product after the in vivo into resveratrol play a pharmacological activity, and can increase the accumulation concentration of resveratrol in lung and prolong the half-life of. The preliminary study suggests that acetylation of resveratrol can inhibit reactive oxygen species (reactive oxygen, species, ROS) to reduce the mortality of mice generated when gamma ray irradiation. In this study, we through the body In the test rats to observe the protective effect of inhaling water after lung injury changes and acetyl resveratrol; in vitro experiments, we used the resveratrol metabolite acetyl resveratrol, study the specific mechanism of the protective effect.
The purpose of the experiment:
(1) clear acetyl resveratrol can inhibit inflammatory reaction and reduce oxidative damage of seawater inhaled lung injury;
(2) to further explore the acetyl resveratrol anti-inflammatory, specific signal transduction mechanism of antioxidant, especially NF- kappa B, iNOS and HIF-1 alpha molecules in which the role of;
(3) test cell signaling pathway in the seawater inhaled lung injury.
Experiment method:
Experiment 1:
Healthy male SD rats were randomly divided into 6 groups: group A: control group; group B: model group type seawater inhalation lung injury; group C: acetyl resveratrol low dose group (50mg/kg); group D: middle dose group (150mg/kg); E group: high dose group (450mg/kg) preconditioning group; and group F: PDTC preconditioning group (100mg/kg). The pretreatment group from 7 days before modeling to intragastric administration with corresponding drugs every day, the last gavage 90 minutes after modeling, the rats to tracheal instillation of seawater 4ml/kg. formula model 4 hours after bloodletting rats were sacrificed and lung tissues. To observe the changes of structure damage lung tissue of rats in each group by HE staining; the content detected by ELISA in lung tissue of TNF- alpha and IL-1 beta, respectively by thiobarbituric acid (ThibabituricAcid TBA) determination of MDA content and T-SOD activity in lung tissue method and xanthine oxidase method, West ErnBlot method for detection of NF- kappa B, expression of HIF-1 alpha and iNOS. To evaluate the acetylation of resveratrol on the effect caused by inflammation and oxidative stress after seawater inhalation.
Experiment two:
In the exponential growth phase of A549 cells were divided into 4 groups, A: normal culture group; B: seawater group; C: Resveratrol intervention group; D:PDTC group.C group and D group were added with 200 mol/L resveratrol and PDTC30 minutes after seawater stimulation. At the same time to B, C and the D group containing 25% medium water, normal after 4 hours of incubation, discard the culture medium by immunofluorescence staining, cells were detected in NF- kappa B, expression of HIF-1 alpha and iNOS.
The experimental results:
Experiment 1:
Compared with the control group, the lung tissue damage after seawater inhalation in rats, alveolar wall thickening and partial collapse; alveolar and interstitial lung edema showed a large amount of exudation and inflammatory cell infiltration in lung tissue injury; effect on behalf of TNF- alpha, IL-1 beta, MDA and NO was increased (table P0.05), and anti injury effect decreased the activity of T-SOD (P0.05); at the same time, seawater inhalation can lead to NF- expression of alpha kappa B, HIF-1 and iNOS increased (P0.05). However, TNF- alpha, acetyl resveratrol preconditioning group in the lung tissues of rats with IL-1 beta, the expression of MDA and NO the relative reduction (P0.05), T-SOD expression (P0.05), and the effect of high dose group was most significant (P0.01); at the same time, acetyl resveratrol decreased in the lung tissues of NF- kappa B, expression of HIF-1 alpha and iNOS (P0.05), and the intervention effect of PDTC with no significant difference (P0.05).
Experiment two:
Compared with the normal cultured cells, A549 cells in seawater after stimulation NF- K B expression of HIF-1 alpha and iNOS were significantly increased, while the acetylation of resveratrol metabolites of resveratrol after pretreatment of NF- kappa B, expression of HIF-1 alpha and iNOS decreased significantly, no significant difference with the PDTC effect.
Conclusion:
The signal pathway of NF- kappa B and its downstream signal molecules of HIF-1 alpha and iNOS constitute participate in the occurrence and development of type seawater inhalation lung injury; acetyl resveratrol intervention through the expression of this signaling pathway, inhibiting the expression of downstream inflammatory factors reduce seawater inhalation pulmonary inflammation and pulmonary edema.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563

【参考文献】