NMDA受体的激活引起肺损伤及机制研究

发布时间：2018-05-06 18:45

本文选题：N-甲基-D-天门冬氨酸 + 急性肺损伤　；参考：《中南大学》2006年博士论文

【摘要】： 第一章NMDA受体激活引起小鼠急性肺损伤研究背景 N-甲基-D-天门冬氨酸(NMDA)受体是谷氨酸受体的重要亚型。NMDA受体过度激活引起的兴奋性神经毒性是引起神经细胞损伤的最后共同通路，在多种急慢性脑损伤的发生中起有重要作用。近年来发现NMDA受体在肺脏也有较高水平的表达，但生物学意义尚不清楚。急性呼吸窘迫综合征(ARDS)是临床常见危重疾病，发病机制尚未完全阐明。肺内中性粒细胞的滞留和肺内中性粒细胞、巨噬细胞的激活在ARDS的发生发展中起关键性作用。中性粒细胞和巨噬细胞可释放谷氨酸。脓毒症时肺静脉中谷氨酸浓度明显高于肺动脉，提示在ARDS等疾病状态下，肺组织中的NMDA受体有可能被过度激活。Said等人发现NMDA可引起离体灌流肺发生高通透性肺水肿。本室前期工作观察到NMDA受体阻断剂MK-801对腹腔注射谷氨酸引起的小鼠急性肺损伤具有明显的保护作用，提示在器官水平和整体水平，NMDA受体激活可引起肺损伤。在整体水平研究NMDA受体激活后对肺组织的影响及可能的作用机制，将为深入研究肺脏等外周组织中NMDA受体的病理生理意义，拓展肺部疾病的防治研究提供新的思路。方法 1．测量肺湿重和干重比值(W／D)并观察组织病理变化，以反映肺组织的损伤程度。 2．生化试剂盒测定肺组织髓过氧化物酶(MPO)活性的改变以反映肺内中性粒细胞的浸润程度。 3．尾静脉取血，记数中性粒细胞百分比，反映外周血中性粒细胞的数目。 4．Western blot方法测定肺组织proSP-C和CCTα蛋白水平，反映肺泡Ⅱ型上皮细胞的功能。结果 1．NMDA(50 mg／kg，ip)可引起肺组织W／D增加，病理切片炎症渗出明显，，NMDA受体的阻断剂MK-801(0.1 mg／kg，ip)预处理30 min可明显减轻由于NMDA引起的炎症渗出。 2．NMDA(50 mg／kg，ip)能引起肺组织MPO活性增加，MK-801可抑制NMDA引起的MPO活性变化。 3．长春新碱(5 mg／kg，iv)预处理4d可引起昆明小鼠外周血白细胞总数、中性粒细胞总数和中性粒细胞百分比的明显减少，对NMDA引起的肺组织W／D的升高有明显的抑制作用。 3．NMDA(50 mg／kg，ip)引起肺组织proSP-C和CCTα蛋白水平下降，NMDA受体阻断剂MK-801可缓解NMDA对proSP-C和CCTα的影响。结论在整体条件下，NMDA可引起急性肺损伤，其机制与中性粒细胞的募集和肺泡Ⅱ型上皮细胞的功能障碍有关。第二章NMDA受体在肺泡Ⅱ型上皮细胞的表达研究背景第一部分研究首次在整体水平证实NMDA受体的激活具有肺毒性效应，但其肺毒性机制未完全阐明。肺泡Ⅱ型上皮细胞(ATⅡ)具有合成分泌表面活性物质(PS)的特性，是维持肺泡正常功能和结构的重要细胞。表面活性物质结合蛋白-C的前体蛋白proSP-C仅存在于ATⅡ中，是ATⅡ的特征性蛋白。CTP：磷酸胆碱二胞苷酰基转移酶α(CCTα)是合成PS主要脂质成分磷脂酰胆碱(PC)的关键酶。腹腔注射NMDA可以使肺组织proSP-C和CCTα表达减少，表明肺组织合成PS脂质成分和蛋白成分的能力下降，提示ATⅡ受损。但NMDA是直接对ATⅡ发挥损伤作用，还是通过作用于其他细胞启动损伤效应、释放损伤性介质从而间接导致ATⅡ的损害，尚有待于进一步的探索。 NR1是构成NMDA受体的必需组份。研究表明在肺组织的外周、中段、主干均有NR1的mRNA及蛋白的表达。放射配基结合实验也证实在肺泡壁上有NMDA受体表达，但在ATⅡ细胞，NMDA受体有无表达尚无直接的证据。方法 1．免疫组织化学(SABC法和双标记法)技术检测在人肺组织ATⅡ细胞上NR1的表达。 2．原代分离的大鼠ATⅡ细胞经免疫黏附法纯化2次后，用碱性磷酸酶(AKP)染色鉴定纯度。 3．RT-PCR法和Western blot分别检测ATⅡ细胞上NR1的mRNA和蛋白分子。免疫细胞化学观察ATⅡ细胞上NR1的表达。结果正常人肺组织石蜡切片免疫组化结果显示ATⅡ细胞上有NR1的表达。原代分离纯化的ATⅡ细胞经AKP染色鉴定纯度为90％以上，可检测到NR1的mRNA和蛋白分子，免疫细胞化学方法显示ATⅡ细胞呈NR1阳性。结论 ATⅡ细胞有NMDA受体的NR1亚单位表达。第三章NMDA对肺表面活性物质合成的影响研究背景肺泡Ⅱ型上皮细胞(ATⅡ)是肺泡上皮的主要组成细胞，可以合成分泌肺表面活性物质(PS)。ATⅡ细胞或PS的受损在ARDS、肺纤维化多种肺部疾病的发生发展中起着重要作用。磷脂酰胆碱(PC)是PS中脂质的主要成分。CTP：磷酸胆碱二胞苷酰基转移酶(CCTα)是PC合成的限速酶，也是PC生物合成的重要调控部位。第三部分的研究发现ATⅡ有NMDA受体表达。前期研究已经在整体水平证实NMDA受体的激活可导致肺组织CCTα蛋白水平下降；在组织培养水平证实NMDA受体的激活可降低肺组织CCTαmRNA的水平，抑制肺组织PC生物合成；但是在细胞水平NMDA可否直接影响PS的合成仍有待于进一步的研究。方法液闪记数测定[~3H]氯化胆碱的掺入量，反映PC合成的变化。 RT-PCR和Western blot分别测定CCTα的mRNA水平和微粒体中的CCTα蛋白水平，揭示PC合成变化的生物化学机制。 Real time PCR测定CCTα和SP-A、SP-B、SP-C、SP-D的mRNA水平，反映CCTα和SPs转录水平的变化。结果浓度为30μmol／L～100μmol／L的NMDA处理12 h，可以剂量依赖方式抑制A549细胞[~3H]氯化胆碱的掺入量。 10μmol／L～1000μmol／L的NMDA处理12 h可以剂量依赖方式降低A549细胞CCTα的mRNA水平和微粒体中的CCTα蛋白水平。100μmol／L的NMDA可以引起ATⅡ细胞CCTα的mRNA水平降低。 100μmol／L的NMDA处理12 h可以引起ATⅡ细胞SP-B、SP-C、SP-D的mRNA水平降低，而对SP-A mRNA的影响不明显。结论 NMDA可通过减少CCTα的转录，降低高活性的CCTα蛋白水平，导致PC合成减少。NMDA对SP-B、SP-C、SP-D的转录也有抑制作用。第四章NMDA抑制CTP：磷酸胆碱二胞苷酰基转移酶表达的信号转导通路的研究研究背景肺泡Ⅱ型上皮细胞(ATⅡ)合成分泌的肺表面活性物质(PS)是维持正常呼吸功能不可缺少的生物活性物质。CTP：磷酸胆碱二胞苷酰基转移酶α(CCTα)是PS主要脂质成分磷脂酰胆碱(PC)合成的限速酶。在CCTα基因近端启动子存在核因子κB(NFκB)的结合位点，但NFκB对CCTα转录有何影响未见报道。神经细胞在受到兴奋性毒性损伤时核内NFκB活性增强，在此过程中一氧化氮合酶(NOS)活化是NFκB激活的上游事件。在整体实验和离体灌流肺模型的研究表明，NOS的激活是导致NMDA肺组织损伤的重要环节。本室在细胞水平的研究中发现NMDA可以抑制ATⅡ细胞CCTα的转录，推测在该效应中，与兴奋性神经毒性相似，NMDA激活NOS并由此导致NFκB活化，从而调节CCTα的转录。方法 Western blot测定细胞核中P65蛋白水平的变化，反映NFκB的激活；测定CCTα蛋白水平，反映CCTα的表达变化。生化试剂盒测定细胞中一氧化氮合酶(NOS)活性和释放到培养液中的一氧化氮(NO)量；Western blot检测NOS阻断剂L-NNA预处理对细胞核中P65蛋白水平的影响，反映NOS在NFκB活化过程中的作用。结果 NMDA(100μmol／L)处理2 h，8 h均检测到A549细胞核中P65蛋白增加。 NFκB的核转位抑制剂SN-50(360μmol／L)预处理2 h可明显抑制NMDA(100μmol／L)所致的A549细胞核中P65的增加，并对CCTα蛋白的减少具有明显的保护作用。 NMDA(100μmol／L)处理2 h，A549细胞中NOS的活性和释放到培养液中的NO无明显变化；而处理7 h则可使A549细胞中NOS的活性升高，NO释放量增多。 L-NNA(2.5 m mol／L)预处理3 h可明显抑制NMDA(100μmol／L)处理后8 h A549细胞核中P65的增加。结论 NMDA通过激活NFκB抑制CCTα的表达，NOS活化是激活NFκB的上游途径。
[Abstract]:Chapter 1 activation of NMDA receptor causes acute lung injury in mice
Research background
N- methyl -D- aspartic acid (NMDA) receptor, an important subtype of glutamate receptor, is an important subtype of.NMDA receptor excitability induced neurotoxicity, the final common pathway that causes nerve cell damage, and plays an important role in the occurrence of a variety of acute and chronic brain damage. In recent years, it has been found that NMDA receptors have high levels of expression in the lungs, but they have been produced in the lung. The significance of physical science is not yet clear.
Acute respiratory distress syndrome (ARDS) is a common critical disease in the clinic. The pathogenesis is not fully elucidated. The retention of neutrophils in the lung and the activation of neutrophils and macrophages in the lung play a key role in the development of ARDS. Neutrophils and macrophages can release glutamate. The degree of the pulmonary artery was significantly higher than that of the pulmonary artery. It is suggested that the NMDA receptor in the lung tissue may be excessively activated by.Said and others in the condition of ARDS and other diseases. It is found that NMDA can cause high permeability pulmonary edema in the isolated perfusion lung. The early work of this room has observed that the NMDA receptor blocker MK-801 is evident in the acute lung injury induced by the intraperitoneal injection of glutaric acid in mice. The protective effect of NMDA receptor activates lung injury at organ level and overall level. The study of the effect and possible mechanism of NMDA receptor activation on lung tissue at the whole level will provide a new idea for the in-depth study of the pathophysiological significance of NMDA receptors in the peripheral tissue of the lung and the prevention and treatment of pulmonary diseases.
Method
1. to measure lung wet weight and dry weight ratio (W / D) and observe histopathological changes to reflect the degree of lung tissue injury.
2. biochemical assay kit was used to measure the activity of myeloperoxidase (MPO) in lung tissue to reflect the infiltration of neutrophils in the lung.
3. tail venous blood was taken, counting the percentage of neutrophils, reflecting the number of neutrophils in peripheral blood.
4.Western blot method was used to detect proSP-C and CCT alpha protein levels in lung tissue, reflecting the function of alveolar type II epithelial cells.
Result
1.NMDA (50 mg / kg, IP) can cause the increase of W / D in lung tissue, and the inflammatory exudation of pathological section is obvious. The pretreatment of NMDA receptor blocker MK-801 (0.1 mg / kg, IP) can obviously reduce the inflammatory exudation caused by the 30 min.
2.NMDA (50 mg / kg, IP) could increase the activity of MPO in lung tissue, and MK-801 could inhibit the change of MPO activity induced by NMDA.
3. vincristine (5 mg / kg, IV) pretreated 4D could cause the total number of white blood cells in the peripheral blood of Kunming mice, the total number of neutrophils and the percentage of neutrophils decreased significantly, which had obvious inhibitory effect on the increase of W / D in the lung tissue caused by NMDA.
3.NMDA (50 mg / kg, IP) reduced the level of proSP-C and CCT alpha protein in lung tissue, and NMDA receptor blocker MK-801 could alleviate the effect of NMDA on proSP-C and CCT alpha.
conclusion
Under the overall conditions, NMDA can cause acute lung injury, which is related to the recruitment of neutrophils and dysfunction of alveolar type II epithelial cells.
The second chapter is the expression of NMDA receptor in alveolar type II epithelial cells.
Research background
The first part of the study showed that the activation of NMDA receptor had lung toxicity for the first time, but the mechanism of lung toxicity was not fully elucidated. The pulmonary alveolar type II epithelial cells (AT II) had the characteristics of synthetic and secretory surfactant (PS), an important cell to maintain normal function and structure of the alveoli. The precursor of surfactant binding protein -C Protein proSP-C only exists in AT II and is the characteristic protein.CTP of AT II: phosphachylcholine two cytidine acyltransferase alpha (CCT alpha) is a key enzyme in the synthesis of PS major lipid component phosphatidylcholine (PC). Intraperitoneal injection of NMDA can reduce the expression of proSP-C and CCT alpha in lung tissue, indicating the ability of lung tissue to synthesize PS lipid and protein components. Decrease, suggesting that AT II is damaged. But NMDA is a direct damage to AT II, or by acting on the damage effect of other cells, releasing damaging media and indirectly causing the damage of AT II. It still remains to be further explored.
NR1 is an essential component of the NMDA receptor. The study shows that there are NR1 mRNA and protein expressions in the peripheral, middle, and main arteries of the lung tissue. The radioligand binding assay also confirmed the expression of NMDA receptor on the alveolar wall, but there is no direct evidence for the expression of NMDA receptor in AT II cells.
Method
1. immunohistochemical method (SABC and double labelling) was used to detect the expression of NR1 in human lung AT II cells.
2. the purified rat AT II cells were purified by immunological adhesion for 2 times, and their purity was identified by alkaline phosphatase (AKP) staining.
3.RT-PCR and Western blot were used to detect the mRNA and protein molecules of NR1 on AT II cells. The expression of NR1 on AT II cells was observed by immunocytochemistry.
Result
Immunohistochemical staining of paraffin sections of normal lung tissue showed NR1 expression on AT II cells.
The purity of the original purified AT II cells was more than 90% by AKP staining, and the mRNA and protein molecules of NR1 were detected. The immunocytochemical method showed that AT II cells were NR1 positive.
conclusion
AT II cells have the expression of NR1 subunit of the NMDA receptor.
The third chapter is about the effect of NMDA on the synthesis of pulmonary surfactant.
Research background
Alveolar type II epithelial cells (AT II) are the main constituent cells of alveolar epithelium. The damage of PS.AT II cells or PS can be synthesized in ARDS, and it plays an important role in the development and development of pulmonary fibrosis of various pulmonary diseases.
Phosphatidylcholine (PC) is the main component of lipid in PS,.CTP: phosphocholine two cytidine acyltransferase (CCT alpha) is a speed limiting enzyme in PC synthesis, and is also an important regulatory part of PC biosynthesis.
The third part of the study found that AT II has NMDA receptor expression. Earlier studies have confirmed that the activation of NMDA receptor can lead to a decrease in the level of CCT alpha protein in lung tissue at the overall level; at the level of tissue culture, the activation of NMDA receptor can reduce the level of CCT alpha mRNA in lung tissue and inhibit PC biosynthesis in lung tissue, but at the cell level NMDA can be found. No direct impact on the synthesis of PS remains to be further studied.
Method
The amount of choline chloride in [~3H] was determined by liquid scintillation counting, which reflected the change of PC synthesis.
RT-PCR and Western blot were used to measure the mRNA level of CCT alpha and the CCT alpha protein level in microsomes, revealing the biochemical mechanism of PC synthesis.
Real time PCR measured the mRNA level of CCT alpha and SP-A, SP-B, SP-C, SP-D, reflecting the change of CCT alpha and SPs transcriptional level.
Result
The NMDA concentration of 30 mol / L to 100 mol / L treated with 12 h could inhibit the incorporation of [~3H] choline chloride in A549 cells in a dose-dependent manner.
The NMDA treatment of 10 mol / L ~ 1000 mu mol / L can reduce the mRNA level of CCT alpha in A549 cells in a dose-dependent manner and CCT alpha protein level in microsomes.
The treatment of 12 h with 100 mol / L NMDA could cause the mRNA level of SP-B, SP-C and SP-D in AT II cells to decrease, but the effect on SP-A mRNA was not obvious.
conclusion
NMDA can reduce the transcription of CCT alpha and reduce the level of high activity CCT alpha protein, resulting in decreased PC synthesis..NMDA also inhibits SP-B, SP-C and SP-D transcription.
Fourth chapter NMDA inhibits CTP: signal transduction pathway of phosphorylcholine two cytidine acyltransferase expression.
Research background
Pulmonary surfactant (PS), which is synthesized and secreted from alveolar type II epithelial cells (AT II), is an essential bioactive substance to maintain normal respiratory function,.CTP: phosphocholine two cytidine acyltransferase alpha (CCT alpha) is a limiting enzyme in the synthesis of phosphatidylcholine (PC), the main lipid component of PS.
There is a binding site of nuclear factor kappa B (NF kappa B) in the proximal promoter of the CCT alpha gene, but there is no report on the effect of NF kappa B on the transcription of CCT a. The activation of NF kappa B in the nucleus is enhanced during excitotoxicity damage. During this process, the activation of nitric oxide synthase (NOS) is the upstream event of NF kappa B excitability. In the whole experiment and in vitro perfusion lung model The study showed that activation of NOS was an important link in the injury of NMDA lung tissue. In the cell level study, it was found that NMDA could inhibit the transcription of CCT alpha in AT II cells. In this effect, it is similar to excitatory neurotoxicity. NMDA activates NOS and thus leads to the activation of NF kappa B, thus regulating the transcription of CCT alpha.
Method
Western blot was used to measure the level of P65 protein in the nucleus, reflecting the activation of NF kappa B, and measuring the level of CCT alpha protein, reflecting the change of CCT alpha expression.
The activity of nitric oxide synthase (NOS) in cells and the amount of nitric oxide (NO) released into the culture medium were measured by biochemical kits, and the effect of NOS blocker L-NNA preconditioning on the level of P65 protein in the nucleus was detected by Western blot, reflecting the role of NOS in the activation of NF kappa B.
Result
NMDA (100 mol / L) treated 2 h, and 8 h detected A549 protein increased in the nucleus of A549.
The pretreatment of NF kappa B nuclear transposition inhibitor SN-50 (360 Mu mol / L) pretreated 2 h could obviously inhibit the P65 increase in A549 nuclei induced by NMDA (100 u mol / L), and had a significant protective effect on the reduction of CCT alpha protein.
NMDA (100 mu mol / L) treated 2 h. The activity of NOS in A549 cells and the NO released into the culture medium did not change obviously, while the treatment of 7 h could increase the activity of NOS in A549 cells and increase the NO release.
Pretreatment of 3 h with L-NNA (2.5 m mol / L) significantly inhibited the increase of P65 in 8 h A549 nuclei after NMDA (100 mol mol / L) treatment.
conclusion
NMDA inhibits CCT alpha expression by activating NF kappa B, and NOS activation is the upstream pathway to activate NF kappa B.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R363

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