表没食子儿茶素没食子酸酯在海水淹溺性肺损伤中的作用机制研究

发布时间：2018-05-03 08:15

本文选题：海水淹溺 + ALI　；参考：《第四军医大学》2014年博士论文

【摘要】：研究背景：海水淹溺性急性肺损伤（Seawater aspiration-induced acute lung injury, Seawateraspiration-induced ALI）是海水淹溺后的严重并发症。不可控性的炎症反应是海水淹溺性ALI的主要发病机制，同时海水有着其特殊的理化因素及细胞外液的高渗性。JAK/STAT1是炎症反应中的重要信号转导通路，在诱导上皮细胞凋亡中也起到重要作用，同时JAK，STAT1又能够被细胞外液的高渗环境激活，因此，我们考虑JAK/STAT1通路可能在海水淹溺性ALI炎症反应及上皮细胞凋亡中起到重要的作用。表没食子儿茶素没食子酸酯（Epigallocatechin-3-gallate，EGCG）是绿茶中儿茶素的主要活性成分，是STAT1的抑制剂。近年来大量文献报道EGCG具有抗肿瘤抑制肿瘤生长及转移的作用，除此以外在糖尿病，帕金森，老年痴呆等疾病中都有较好的疗效。研究表明EGCG能够通过抑制STAT1的活性，降低心肌细胞缺血再灌注损伤，而EGCG在肺损伤特别是海水淹溺性ALI中会起到什么样的作用，有待我们进一步研究。研究目的： 1.观察EGCG对海水淹溺性ALI炎症反应及上皮细胞凋亡的作用； 2.探讨JAK/STAT1通路在海水淹溺性ALI发病中的作用； 3.探讨EGCG保护海水淹溺性ALI的作用机制。研究意义：通过离体及活体实验，探讨EGCG保护海水淹溺性ALI的作用机制，明确JAK/STAT1通路在海水淹溺性ALI发病中的作用机制，为海水淹溺性ALI的治疗提供新的思路。实验方法： 1．动物实验（1）雄性SD大鼠（180-220g），暴露气管并气管内滴注配方海水4ml/kg建立海水淹溺性ALI模型。随机分为五组：正常组（n=8），海水损伤后1小时组（n=8），3小时组（n=8），6小时组（n=8），12小时组（n=8）。分别在致伤后1,3,6,12小时检测动脉血氧分压，IL-1，IL-10，TNF-α炎症因子含量，PCR检测JAK，STAT家族各成员的mRNA，Western blot检测STAT1，P-STAT1的蛋白含量。并进行组织病理学检测。（2）雄性SD大鼠（180-220g）。随机分为四组：①正常组：暴露气管并术前半小时腹腔注射1ml生理盐水（n=12）；②EGCG组：暴露气管并术前半小时腹腔注射10mg/kg EGCG溶于1ml生理盐水中（n=12）；③海水组：暴露气管后气管内滴注配方海水4ml/kg并致伤前0.5小时腹腔注射1ml生理盐水（n=12）；④海水+EGCG组：暴露气管后气管内滴注配方海水4ml/kg并致伤前0.5小时腹腔注射10mg/kgEGCG溶于1ml生理盐水中（n=12）。于致伤后0-3小时动态观察动脉血氧分压变化，致伤后6小时检测肺干湿重比，肺泡灌洗液蛋白含量，IL-1，IL-10，TNF-α炎症因子含量，PCR检测STAT1mRNA含量，Western blot检测STAT1，P-STAT1，caspase-3，P21的蛋白含量，荧光TUNEL，免疫组化，电镜观察肺组织超微结构及组织病理学检测。 2．细胞实验（1）培养NR8383细胞，指数生长期的NR8383细胞无血清饥饿16小时后，分为四组：正常组，EGCG组（10цM EGCG干预细胞），海水组（30%海水干预细胞），海水+EGCG组（10цM EGCG干预细胞后0.5小时再给予30%海水干预细胞）。海水干预6小时后取细胞上清液检测IL-1，IL-10，TNF-α炎症因子含量，PCR检测JAK1，JAK2，STAT1的mRNA含量，Western blot检测JAK1，P-JAK1，JAK2，P-JAK2，STAT1，P-STAT1含量。（2）培养A549细胞，指数生长期的A549细胞无血清饥饿16小时后，分为两步实验：第一步用不同浓度海水干预细胞，分为五组：正常组，10%海水组，20%海水组，30%海水组，40%海水组，干预细胞后6小时流式细胞术检测细胞凋亡程度；第二步用EGCG干预细胞，分为四组：正常组，EGCG组（10цM EGCG干预细胞），海水组（30%海水干预细胞），海水+EGCG组（10цM EGCG干预细胞后0.5小时再给予30%海水干预细胞），干预细胞后6小时流式细胞术检测细胞凋亡率。实验结果： 1．海水淹溺性ALI在6小时损伤最重，STAT1在损伤后1小时即升高6小时表达量达最高值。造模成功后检测组织病理学，动脉血氧分压，炎症因子，蛋白表达量指标，结果显示组织病理学检测肺组织明显水肿，炎细胞浸润，随着时间延长肺损伤程度逐渐加重，在6小时点肺损伤程度最重。致伤后出现明显低氧血症。IL-1，TNF-α促炎因子明显升高，IL-10抑炎因子明显下降，差异具有统计学意义（P0.05）。STAT1蛋白表达量在损伤后1小时开始升高6小时达最高，此后开始下降。 2．在体内实验中EGCG预处理能够减轻肺组织炎症并抑制肺组织细胞凋亡。通过体内实验观察组织病理学，动脉血氧分压，肺含水量，，炎症因子，蛋白表达量，凋亡，超微结构等指标明确EGCG对海水淹溺性ALI的保护作用。结果显示EGCG预处理组能够减轻海水淹溺性ALI导致的低氧血症，差异具有统计学意义（P0.05）；减轻肺水肿，降低肺泡灌洗液蛋白含量，差异具有统计学意义（P0.05）；降低IL-1，TNF-α促炎因子，升高IL-10抑炎因子，差异具有统计学意义（P0.05）；Western及免疫组化结果均显示能够降低STAT1，P-STAT1蛋白的表达量；Western结果显示能够降低STAT1下游P21及caspase-3的蛋白表达量；荧光TUNEL结果显示能够减少组织细胞凋亡；电镜观察肺组织超微结构及组织病理学检测均显示能够减轻肺水肿，炎细胞浸润程度。 3.在离体实验中EGCG预处理能够减轻NR8383细胞炎症反应并抑制A549细胞凋亡。通过离体实验观察炎症因子，蛋白表达量，流式检测细胞凋亡率指标明确EGCG在离体细胞中对炎症反应及细胞凋亡的影响。结果显示EGCG预处理能够减轻海水干预NR8383细胞的炎症反应，降低IL-1，TNF-α促炎因子，升高IL-10抑炎因子，差异具有统计学意义（P0.05），降低P-JAK1， P-JAK2，STAT1，P-STAT1的蛋白含量，对JAK1，JAK2总蛋白含量无明显影响。用不同浓度海水干预A549细胞后显示随着海水浓度的升高A549细胞的凋亡率升高（P0.05），EGCG预处理后可以降低海水干预后引起A549细胞的凋亡率（P0.05）。结论： 1.海水淹溺性ALI中STAT1表达明显升高，且STAT1的激活与海水淹溺性ALI的损伤程度相关。 2. EGCG能够减轻海水淹溺性ALI中肺组织炎症反应，减轻肺水肿及血管通透性，这与其负调节JAK/STAT1通路有关。 3. EGCG能够减轻海水淹溺性ALI中肺泡上皮细胞的凋亡，这与其负调节JAK/STAT1通路及凋亡蛋白caspase-3，P21有关。
[Abstract]:Research background:
Seawater drowning acute lung injury (Seawater aspiration-induced acute lung injury, Seawateraspiration-induced ALI) is a serious complication after seawater drowning. The uncontrollable inflammatory reaction is the main pathogenesis of seawater drowning ALI, and the sea water has its special physical and chemical factors and the hypertonic.JAK/STAT1 of the extracellular fluid. The important signal transduction pathway in the inflammatory response also plays an important role in inducing apoptosis of epithelial cells. At the same time, JAK, STAT1 can be activated by the hypertonic environment of extracellular fluid. Therefore, we consider that the JAK/STAT1 pathway may play an important role in the ALI inflammatory response and epithelial cell apoptosis in seawater drowning.
Epigallocatechin gallate (Epigallocatechin-3-gallate, EGCG) is the main active component of catechin in green tea and is an inhibitor of STAT1. In recent years, a large number of literatures have reported that EGCG has the effect of inhibiting tumor growth and metastasis. Besides, it has good curative effect in diabetes, Parkinson, Alzheimer's disease and so on. Studies have shown that EGCG can reduce the myocardial ischemia and reperfusion injury by inhibiting the activity of STAT1, and what role EGCG can play in lung injury, especially in seawater drowning ALI, is to be further studied.
The purpose of the study is:
1. to observe the effect of EGCG on ALI inflammatory response and epithelial cell apoptosis in seawater drowning.
2. to explore the role of JAK/STAT1 pathway in the pathogenesis of seawater drowning ALI.
3. to explore the mechanism of EGCG protecting ALI from seawater drowning.
Research significance:
In vitro and in vivo experiments, the mechanism of EGCG protection for seawater drowning ALI is discussed, and the mechanism of JAK/STAT1 pathway in the pathogenesis of seawater drowning ALI is clearly defined, which provides a new way of thinking for the treatment of seawater drowning ALI.
Experimental methods:
1. animal experiments
(1) the male SD rats (180-220g), exposed to the trachea and intratracheal infusion of seawater 4ml/kg to establish a seawater drowning ALI model, were randomly divided into five groups: normal group (n=8), 1 hour group (n=8) after seawater injury, 3 hour group (n=8), 6 hour group (n=8), 12 hour group (n =8). Levels of inflammatory factors, PCR were detected by JAK, mRNA and Western blot of STAT family members were tested for STAT1 and P-STAT1 protein content, and histopathological examination was performed.
(2) male SD rats (180-220g) were randomly divided into four groups: (1) normal group: exposed trachea and intraperitoneal injection of 1ml saline (n=12) half an hour before operation; (2) EGCG group: exposed trachea and intraperitoneal injection of 10mg/kg EGCG in 1ml physiological saline (n=12) for half an hour before operation; (3) seawater group: intratracheal intratracheal infusion of seawater 4ml/kg and caused by seawater 4ml/kg and cause Intraperitoneal injection of 1ml saline (n=12) 0.5 hours before injury; (4) seawater +EGCG group: intratracheal intratracheal infusion of seawater 4ml/kg and intraperitoneal injection of 10mg/kgEGCG in 1ml physiological saline (n=12) before injury. The changes of arterial blood oxygen pressure were observed at 0-3 hours after injury, and lung dry wet weight ratio and alveolar irrigation were detected at 6 hours after injury. The content of lotion protein, IL-1, IL-10, TNF- alpha inflammatory factor, PCR detection of STAT1mRNA content, Western blot to detect STAT1, P-STAT1, Caspase-3, P21 protein content, fluorescence TUNEL, immunohistochemistry, electron microscopy observation of ultrastructure of lung tissue and histopathological detection.
2. cell experiment
(1) NR8383 cells were cultured, and the NR8383 cells in the exponential growth period were divided into four groups after 16 hours without serum starvation: the normal group, the group EGCG (10 M EGCG intervention cells), the seawater group (30% seawater intervention cells), the seawater +EGCG group (10 0.5 hours after the intervention cells of M EGCG) and the 30% seawater intervention cells after 0.5 hours. The cell supernatant was tested for IL- after the seawater intervention for 6 hours. 1, IL-10, TNF- alpha inflammatory factor content, PCR detection of JAK1, JAK2, STAT1 mRNA content, Western blot detection JAK1, P-JAK1, blot, ",", "," content.
(2) to cultivate A549 cells, the A549 cells in the exponential growth period were divided into two steps after 16 hours without serum starvation: the first step was divided into five groups: normal group, 10% sea water group, 20% seawater group, 30% seawater group, 40% seawater group, and 6 small time flow cytometry to detect cell apoptosis after 6 small time flow cytometry; second steps were performed with EGCG dry. The precells were divided into four groups: normal group, group EGCG (10 M EGCG intervention cells), sea water group (30% seawater intervention cells), and seawater +EGCG group (30% seawater intervened cells after 0.5 hours after 10 M EGCG intervention cells), and 6 hour flow cytometry was used to detect the cell withering rate after 6 hours of flow cytometry.
Experimental results:
1. seawater drowning ALI was the most serious injury at 6 hours, and STAT1 increased 6 hours at 1 hours after injury.
After the success of the model, histopathology, arterial oxygen pressure, inflammatory factors and protein expression were detected. The results showed that the lung tissue was obviously edema, inflammatory cells infiltrated, and the lung injury was gradually increased with time, and the lung injury was the most severe at 6 hours. The obvious hypoxemia.IL-1, TNF- alpha proinflammatory cause appeared after the injury. The IL-10 anti inflammatory factor significantly decreased, and the difference was statistically significant (P0.05), the expression of.STAT1 protein increased at 1 hours after 6 hours, and then began to decline.
2. in vivo experiments, EGCG pretreatment can reduce lung tissue inflammation and inhibit apoptosis in lung tissue.
In vivo observation of histopathology, arterial oxygen pressure, lung water content, inflammatory factors, protein expression, apoptosis, ultrastructure and other indicators of the protective effect of EGCG on seawater drowning ALI. The results showed that the EGCG pretreatment group could reduce the hypoxemia caused by seawater drowning ALI, the difference was statistically significant (P0.05); Pulmonary edema and reducing the protein content of alveolar lavage fluid were statistically significant (P0.05); reducing IL-1, TNF- alpha, and increasing IL-10 anti inflammatory factors were statistically significant (P0.05); Western and immunohistochemical results showed that the expression of STAT1, P-STAT1 protein could be reduced, and Western results showed that the downstream P2 of STAT1 could be reduced. The protein expression of 1 and Caspase-3, and the results of fluorescence TUNEL showed that the apoptosis of the tissue cells could be reduced, and the ultrastructure of the lung tissue and the histopathological examination of the lung tissue showed that the pulmonary edema and the degree of infiltration of inflammatory cells could be reduced by electron microscopy.
3. in vitro experiments, EGCG pretreatment can reduce the inflammatory response of NR8383 cells and inhibit the apoptosis of A549 cells.
The effects of inflammatory factors, protein expression and flow cytometry on the inflammatory response and apoptosis in isolated cells were observed through the experiment in vitro. The results showed that EGCG pretreatment could reduce the inflammatory response of NR8383 cells in seawater intervention, reduce IL-1, TNF- alpha pro-inflammatory factors and increase IL-10 anti inflammatory factors, and the difference was found. Statistical significance (P0.05), reducing the protein content of P-JAK1, P-JAK2, STAT1 and P-STAT1 had no obvious effect on the total protein content of JAK1 and JAK2. The apoptosis rate of A549 cells increased with the increase of seawater concentration (P0.05) with the concentration of seawater in different concentrations (P0.05), and the apoptosis rate of A549 cells could be reduced after the pretreatment of sea water. .05).
Conclusion:
1. the expression of STAT1 in seawater drowning ALI increased significantly, and the activation of STAT1 was related to the degree of ALI damage in seawater.
2. EGCG can reduce the inflammatory reaction of lung tissue and reduce pulmonary edema and vascular permeability in seawater drowning ALI, which is related to the negative regulation of JAK/STAT1 pathway.
3. EGCG can reduce the apoptosis of alveolar epithelial cells in seawater drowning ALI, which is related to the negative regulation of JAK/STAT1 pathway and apoptotic protein caspase-3 and P21.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R649.3

【参考文献】