本文選題：海水 + 急性肺損傷��； 參考：《第四軍醫(yī)大學》2013年碩士論文

【摘要】：研究背景： 淹溺是一個重大但時常被忽視的公共衛(wèi)生問題，據(jù)不完全統(tǒng)計，每年約有50萬人死于淹溺。肺臟是吸入海水后首先受到侵害的重要臟器，研究表明吸入海水所致肺損傷明顯比吸入淡水嚴重。海水吸入型肺損傷（Seawater Inhalation Induced AcuteLung Injury, SWI-ALI）主要表現(xiàn)為肺部炎癥反應和肺水腫，具體發(fā)生機制較為復雜，可以肯定的是損傷的嚴重程度取決于液體的吸入量和清潔程度。海水吸入型肺損傷病情發(fā)展迅速，如未及時治療或處置不當易發(fā)展為海水吸入型呼吸窘迫綜合征（Seawater Inhalation Induced Acute Respiratory Distress Syndrome, SWI-ARDS）。近年來，，對海水吸入型肺損傷發(fā)病機制和救治的研究日益增多，但是對其發(fā)病機制尚未形成統(tǒng)一認識，未能提出切實有效、有針對性的治療措施。 核轉錄因子κB（NF-κB）是一種幾乎存在于所有哺乳動物細胞內(nèi)的轉錄因子，參與細胞增殖、分化、凋亡和癌變等多種病理生理過程。通常細胞中NF-κB與IκB結合存在于細胞漿中；當受到LPS、TNF-α和IL-1等刺激時，IκB被迅速降解，游離狀態(tài)的NF-κB轉移入細胞核，與相應目的基因結合并促使其轉錄，發(fā)揮相應的生物學功能。NF-κB調(diào)節(jié)多種效應分子的表達，介導組織損傷與修復；其中缺氧誘導因子1α（HIF-1α）和誘生型一氧化氮合酶（iNOS）是其重要的下游調(diào)節(jié)因子，三者共同參與多種疾病的發(fā)生于發(fā)展。在此理論基礎上，NF-κB相關信號通路在海水吸入型肺損傷中的作用成為本課題的研究重點。 白藜蘆醇（Resveratrol）是廣泛存在于多種植物體內(nèi)的多酚化合物，是一種植物抗毒素，具有多種生物活性，如抗炎、抗氧化、抗腫瘤細胞增殖和抗細胞老化等；因此，近年來對白藜蘆醇的研究較為多見。然而，由于白藜蘆醇生物半衰期短和血藥濃度低等原因，其臨床應用始終面臨種種困難。乙�；邹继J醇是白藜蘆醇三個羥基被乙�；〈蟮漠a(chǎn)物，在體內(nèi)轉化為白藜蘆醇發(fā)揮藥理活性，并且能夠增加白藜蘆醇在肺部蓄積濃度同時延長生物半衰期，前期研究表明乙�；邹继J醇能夠通過抑制活性氧類（reactive oxygen species，ROS）的生成降低γ-射線照射時小鼠的死亡率。在本研究中，我們通過體內(nèi)試驗觀察大鼠吸入海水后肺部損傷性變化及乙�；邹继J醇的保護作用；在細胞實驗中，我們采用白藜蘆醇，乙�；邹继J醇的代謝產(chǎn)物，研究其保護作用的具體機制。 實驗目的： （1）明確乙酰化白藜蘆醇能否抑制炎癥反應和氧化損傷減輕海水吸入型肺損傷； （2）進一步探討乙�；邹继J醇抗炎、抗氧化的具體信號轉導機制，尤其是NF-κB、iNOS和HIF-1α等信號分子在其中的作用； （3）細胞試驗驗證相關信號通路在海水吸入型肺損傷中的作用。 實驗方法： 實驗一： 健康雄性SD大鼠按照完全隨機的方法分為6組：A組：空白對照組；B組：海水吸入型肺損傷模型組；C組：乙�；邹继J醇低劑量（50mg/kg）組；D組：中劑量組（150mg/kg）；E組：高劑量組（450mg/kg）預處理組；和F組：PDTC預處理組（100mg/kg）。各預處理組從造模前7天開始每天灌胃相應藥物，最后一次灌胃90分鐘后造模，向?qū)嶒灤笫髿夤軆?nèi)滴注配方海水4ml/kg。造模4小時后放血處死取肺組織標本。采用HE染色的方法觀察各組大鼠肺組織結構損傷變化；通過ELISA的方法檢測肺組織中TNF-α和IL-1β的含量，分別采用硫代巴比妥酸（ThibabituricAcid TBA）法和黃嘌呤氧化酶法測定肺組織中MDA含量和T-SOD活性，用WesternBlot的方法檢測NF-κB、HIF-1α和iNOS的表達量。以評價乙酰化白藜蘆醇對海水吸入后所致炎癥和氧化損傷的改善作用。 實驗二： 將處于指數(shù)生長期的A549細胞分為4組，A：正常培養(yǎng)組；B：海水刺激組；C：白藜蘆醇干預組；D：PDTC預處理組。C組和D組在分別加入200μmol/L白藜蘆醇和PDTC30分鐘后進行海水刺激。同時向B、C和D組加入含有25%配方海水的培養(yǎng)基，正常培養(yǎng)4小時后棄去培養(yǎng)基進行免疫熒光染色操作，檢測細胞中NF-κB、HIF-1α和iNOS的表達量。 實驗結果： 實驗一： 與空白對照組相比，大鼠吸入海水后肺組織結構受到破壞，肺泡壁增厚并伴有局部塌陷；肺泡腔和肺間質(zhì)內(nèi)可見大量水腫液滲出和炎細胞浸潤；肺組織內(nèi)代表損傷效應的TNF-α、IL-1β、MDA和NO等表達量增加（P0.05），而代表抗損傷效應的T-SOD活性降低（P0.05）；同時，海水吸入能夠?qū)е翹F-κB、HIF-1α和iNOS的表達量增高（P0.05）。然而，乙�；邹继J醇預處理組大鼠肺組織中TNF-α、IL-1β、MDA和NO表達量相對降低（P0.05），T-SOD表達量增高（P0.05），且高劑量組效果最為明顯（P0.01）；同時，乙�；邹继J醇降低肺組織中NF-κB、HIF-1α和iNOS的表達量（P0.05），且干預效果與PDTC無明顯差異（P0.05）。 實驗二： 與正常培養(yǎng)的細胞相比，海水刺激后A549細胞中NF-κB、HIF-1α和iNOS的表達量明顯增高，而乙�；邹继J醇體內(nèi)代謝產(chǎn)物白藜蘆醇預處理后NF-κB、HIF-1α和iNOS的表達量顯著降低，與PDTC效果無明顯差異。 結論： NF-κB及其下游的信號分子HIF-1α和iNOS構成的信號通路參與海水吸入型肺損傷的發(fā)生與發(fā)展；乙�；邹继J醇能夠通過干預此信號通路的表達，抑制下游炎癥因子等的表達減輕海水吸入所致的肺部炎癥和肺水腫。
[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.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R563

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本文編號：1754150