活化蛋白-1在大鼠内毒素休克急性肺损伤时血红素氧合酶-1表达中的作用
发布时间：2018-08-08 20:24
【摘要】：内毒素诱发的急性肺损伤／急性呼吸窘迫综合征(ALI/ARDS)是临床上常见的急危重症,是感染性休克导致死亡的主要原因之一[1],因此探索其有效的防治措施具有一定的临床意义。 血红素氧合酶-1(HO-1)为诱导型,是一种应激蛋白,又被称为热休克蛋白-32(heat shock protein-32, HSP-32),其广泛存在于各种细胞和组织。HO-1是催化血红素降解为CO、胆红素和铁的限速酶,HO-1及其催化产物是机体重要的内源性防御保护系统,在维持细胞稳定和调节炎症反应中发挥重要作用[2-3]。除血红素外,内毒素、休克、应激、高氧等也可诱导HO-1表达。有研究发现,LPS诱导的ALI大鼠中,α硫辛酸可通过上调HO-1的表达发挥肺组织保护作用[4]。本课题组前期研究发现,内毒素休克大鼠肺组织中HO-1表达上调并对肺脏发挥保护作用[5-6]。 活化蛋白-1(AP-1)是信号转导通路中重要的转录因子复合物,能与许多基因上的AP-1位点结合,在细胞中发挥转录作用[7]。脂多糖(LPS)刺激大鼠RAW264.7巨噬细胞时,活化的转录因子AP-1与HO-1基因启动子区的AP-1结合位点结合,可导致HO-1表达上调并发挥细胞保护作用[8-1ol。然而在LPS诱导的大鼠内毒素休克急性肺损伤模型中,AP-1是否也参与了HO-1表达上调尚未定论。因此本研究拟在建立大鼠内毒素休克急性肺损伤模型的基础上,评价转录因子AP-1在HO-1表达中的作用,为探讨内毒素休克急性肺损伤时机体内源性保护机制提供参考。 目的探讨转录因子AP-1在大鼠内毒素休克ALI时HO-1表达中的作用。 方法健康清洁级雄性Sprague Dawley (SD)大鼠48只,体重200-220g,2.5-3.0月龄,采用随机数字表法,将其随机分为4组(n=12)：正常对照组(C组)、内毒素休克急性肺损伤组(ES组)、姜黄素+内毒素休克急性肺损伤组(Cur+ES组)、姜黄素组(Cur组)。正常对照组(C组)腹腔注射0.1%二甲基亚砜(姜黄素溶媒)0.5ml,30min时股静脉注射生理盐水(LPS溶媒)0.5ml；内毒素休克肺损伤组(ES组)腹腔注射0.1%二甲基亚砜0.5ml,30min时股静脉注射10mg/kg LPS0.5ml;姜黄素+内毒素休克肺损伤组(Cur+ES组)腹腔注射20mg/kg姜黄素0.5ml,30min时股静脉注射10mg/kg LPS0.5ml;姜黄素组(Cur组)腹腔注射姜黄素20mg/kg,30min时股静脉注射生理盐水0.5ml。观察动物一般情况,持续监测MAP和心率变化,静脉注射LPS2h内平均动脉压(MAP)较基础值降低25%以下并维持该水平为休克模型制备成功标志。本实验中若给予LPS后未满足上述条件或6h内死亡者均排除本观察组。静脉注射LPS6h时采集动脉血0.5ml行血气分析计算氧合指数,随后处死大鼠留取肺组织,观察肺组织病理学结果,行肺损伤程度评分、计算肺组织含水率、测定肺组织MDA含量和SOD活性；采用Western blot免疫印迹法分别测定大鼠肺组织HO-1和AP-1蛋白的表达；采用反转录酶-聚合酶链锁反应(reverse transcription-polymerase chain reaction,RT-PCR)法测定大鼠肺组织HO-1mRNA表达。 结果与C组比较,ES组和Cur+ES组肺损伤程度评分、肺组织含水率、氧合指数和MDA含量升高,SOD活性降低,AP-1、HO-1和HO-1mRNA表达上调(P0.05),Cur组上述各指标差异无统计学意义(P0.05)；与ES组比较,Cur+ES组肺损伤程度评分、肺组织含水率和MDA含量升高,SOD活性降低,AP-1、HO-1和HO-1mRNA表达下调(P0.05)；与Cur+ES组比较,Cur组肺损伤程度评分、肺组织含水率和MDA含量降低,氧合指数和SOD活性升高,AP-1、HO-1和HO-1mRNA表达下调(P0.05)。 结论LPS诱导大鼠内毒素休克ALI时,转录因子AP-1参与调节HO-1表达上调。
[Abstract]:Acute lung injury / acute respiratory distress syndrome (ALI/ARDS) induced by endotoxin is a common acute critical disease in clinic. It is one of the main causes of death in septic shock, [1]. Therefore, it is of certain clinical significance to explore its effective prevention and treatment measures.
Heme oxygenase -1 (HO-1) is an inducible type, which is a stress protein, also known as the heat shock protein -32 (heat shock protein-32, HSP-32). It widely exists in all kinds of cells and tissue.HO-1 to catalyze heme degradation to CO, bilirubin and iron, and HO-1 and its catalytic products are important endogenous defensive protection systems in the body. HO-1 expression can also be induced by endotoxin, shock, stress, hyperoxia and so on, which plays an important role in cell stability and regulation of inflammatory response. It is found that in LPS induced ALI rats, alpha lipoic acid can play a protective role in lung tissue by up regulation of HO-1 expression [in the early study of the group of 4]. subjects, endotoxic shock rats The expression of HO-1 in lung tissue was up-regulated and the lung was protected. [5-6].
Activated protein -1 (AP-1) is an important transcription factor complex in signal transduction pathway. It can be combined with AP-1 loci on many genes. When [7]. lipopolysaccharide (LPS) stimulates rat RAW264.7 macrophages in cells, the activated transcription factor AP-1 is combined with the AP-1 binding site in the promoter region of HO-1 gene, which can lead to the expression of HO-1. [8-1ol., however, plays an important role in LPS induced acute lung injury induced by endotoxin shock in rats, and whether AP-1 has also been involved in the up regulation of HO-1 expression. Therefore, the purpose of this study is to evaluate the role of the transcription factor AP-1 in the expression of HO-1 on the basis of the model of acute lung injury in rats with endotoxic shock. The endogenous protective mechanisms of toxin shock in acute lung injury provide reference.
Objective to investigate the role of transcription factor AP-1 in the expression of HO-1 during endotoxin shock ALI in rats.
Methods 48 healthy and clean male Sprague Dawley (SD) rats, weight 200-220g, 2.5-3.0 month old, were randomly divided into 4 groups (n=12): normal control group (C group), acute lung injury group (ES group), curcumin + endotoxin shock group (Cur+ES group), curcumin group (Cur group). Normal control group (C). Group) intraperitoneal injection of 0.1% two methyl sulfoxide (curcumin solvent) 0.5ml, 30min femoral vein injection of physiological saline (LPS solvent) 0.5ml; endotoxin shock lung injury group (ES group) intraperitoneal injection of 0.1% two methyl sulfoxide (0.5ml), 30min in the femoral vein injection 10mg/kg LPS0.5ml; curcumin + endotoxic shock lung injury group (Cur+ES group) intraperitoneal injection 20mg/kg curcumin 0.5ml, 30min, 10mg/kg LPS0.5ml were injected into the femoral vein, the curcumin group (group Cur) was injected with curcumin 20mg/kg, and the normal saline 0.5ml. was injected into the femoral vein in 30min to observe the animal general condition, and the changes of MAP and heart rate were monitored continuously. The mean arterial pressure (MAP) within the intravenous injection LPS2h was lower than the base value by 25% and maintained the level as a shock model. In this experiment, if LPS was not satisfied with the above conditions or the deaths in 6h were all excluded from the observation group, the blood gas analysis of arterial blood was collected to calculate the oxygenation index, and then the rats were sacrificed to leave the lung tissue, to observe the pathological results of the lung tissue, to score the degree of lung injury, to calculate the water content of the lung tissue and to determine the lung tissue. MDA content and SOD activity were organized and the expression of HO-1 and AP-1 protein in lung tissue of rats was measured by Western blot immunoblotting, and the lung tissue HO-1mRNA expression was measured by reverse transcriptase polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR).
Results compared with group C, the level of lung injury in group ES and group Cur+ES, lung tissue water content, oxygenation index and MDA content increased, SOD activity decreased, AP-1, HO-1 and HO-1mRNA expression up up (P0.05), and there was no statistical significance (P0.05) in the above-mentioned indexes of Cur group (P0.05). High SOD activity decreased, AP-1, HO-1 and HO-1mRNA were down regulated (P0.05). Compared with group Cur+ES, the degree of lung injury in Cur group, the decrease of lung tissue water content and MDA content, the increase of oxygenation index and SOD activity, AP-1, HO-1, and HO-1mRNA expression downregulation.
Conclusion transcription factor AP-1 participates in the regulation of HO-1 expression in LPS induced endotoxin shock ALI in rats.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R459.7
本文编号：2172950
[Abstract]:Acute lung injury / acute respiratory distress syndrome (ALI/ARDS) induced by endotoxin is a common acute critical disease in clinic. It is one of the main causes of death in septic shock, [1]. Therefore, it is of certain clinical significance to explore its effective prevention and treatment measures.
Heme oxygenase -1 (HO-1) is an inducible type, which is a stress protein, also known as the heat shock protein -32 (heat shock protein-32, HSP-32). It widely exists in all kinds of cells and tissue.HO-1 to catalyze heme degradation to CO, bilirubin and iron, and HO-1 and its catalytic products are important endogenous defensive protection systems in the body. HO-1 expression can also be induced by endotoxin, shock, stress, hyperoxia and so on, which plays an important role in cell stability and regulation of inflammatory response. It is found that in LPS induced ALI rats, alpha lipoic acid can play a protective role in lung tissue by up regulation of HO-1 expression [in the early study of the group of 4]. subjects, endotoxic shock rats The expression of HO-1 in lung tissue was up-regulated and the lung was protected. [5-6].
Activated protein -1 (AP-1) is an important transcription factor complex in signal transduction pathway. It can be combined with AP-1 loci on many genes. When [7]. lipopolysaccharide (LPS) stimulates rat RAW264.7 macrophages in cells, the activated transcription factor AP-1 is combined with the AP-1 binding site in the promoter region of HO-1 gene, which can lead to the expression of HO-1. [8-1ol., however, plays an important role in LPS induced acute lung injury induced by endotoxin shock in rats, and whether AP-1 has also been involved in the up regulation of HO-1 expression. Therefore, the purpose of this study is to evaluate the role of the transcription factor AP-1 in the expression of HO-1 on the basis of the model of acute lung injury in rats with endotoxic shock. The endogenous protective mechanisms of toxin shock in acute lung injury provide reference.
Objective to investigate the role of transcription factor AP-1 in the expression of HO-1 during endotoxin shock ALI in rats.
Methods 48 healthy and clean male Sprague Dawley (SD) rats, weight 200-220g, 2.5-3.0 month old, were randomly divided into 4 groups (n=12): normal control group (C group), acute lung injury group (ES group), curcumin + endotoxin shock group (Cur+ES group), curcumin group (Cur group). Normal control group (C). Group) intraperitoneal injection of 0.1% two methyl sulfoxide (curcumin solvent) 0.5ml, 30min femoral vein injection of physiological saline (LPS solvent) 0.5ml; endotoxin shock lung injury group (ES group) intraperitoneal injection of 0.1% two methyl sulfoxide (0.5ml), 30min in the femoral vein injection 10mg/kg LPS0.5ml; curcumin + endotoxic shock lung injury group (Cur+ES group) intraperitoneal injection 20mg/kg curcumin 0.5ml, 30min, 10mg/kg LPS0.5ml were injected into the femoral vein, the curcumin group (group Cur) was injected with curcumin 20mg/kg, and the normal saline 0.5ml. was injected into the femoral vein in 30min to observe the animal general condition, and the changes of MAP and heart rate were monitored continuously. The mean arterial pressure (MAP) within the intravenous injection LPS2h was lower than the base value by 25% and maintained the level as a shock model. In this experiment, if LPS was not satisfied with the above conditions or the deaths in 6h were all excluded from the observation group, the blood gas analysis of arterial blood was collected to calculate the oxygenation index, and then the rats were sacrificed to leave the lung tissue, to observe the pathological results of the lung tissue, to score the degree of lung injury, to calculate the water content of the lung tissue and to determine the lung tissue. MDA content and SOD activity were organized and the expression of HO-1 and AP-1 protein in lung tissue of rats was measured by Western blot immunoblotting, and the lung tissue HO-1mRNA expression was measured by reverse transcriptase polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR).
Results compared with group C, the level of lung injury in group ES and group Cur+ES, lung tissue water content, oxygenation index and MDA content increased, SOD activity decreased, AP-1, HO-1 and HO-1mRNA expression up up (P0.05), and there was no statistical significance (P0.05) in the above-mentioned indexes of Cur group (P0.05). High SOD activity decreased, AP-1, HO-1 and HO-1mRNA were down regulated (P0.05). Compared with group Cur+ES, the degree of lung injury in Cur group, the decrease of lung tissue water content and MDA content, the increase of oxygenation index and SOD activity, AP-1, HO-1, and HO-1mRNA expression downregulation.
Conclusion transcription factor AP-1 participates in the regulation of HO-1 expression in LPS induced endotoxin shock ALI in rats.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R459.7
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2 黄新莉;周晓红;周君琳;羡晓辉;丁春华;;CCK-8上调LPS诱导的大鼠肺组织HO-1表达的信号转导机制[J];中国病理生理杂志;2009年12期
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