【作者】 段玉忠；

【导师】 陈正堂；

【作者基本信息】 第三军医大学 ， 内科学， 2003， 博士

【摘要】 急性肺损伤(acute lung injury，ALI)是由于致病因子激活中性粒细胞、肺巨噬细胞等炎症效应细胞，引发多种细胞因子和炎症介质的失控性释放，从而引发肺脏的过度炎症反应，常可并发多器官功能障碍（multiple organ dysfunction syndrome，MODS），病死率高。目前对其发病机理仍未完全阐明。近年来研究发现，核转录因子-κB(nuclear factor-kappa B，NF-κB)居于许多细胞信号转导通路的交汇处，其诱导性活化可影响许多前炎症介质如TNFα、IL-1 β、IL-6及IL-8等释放，以及诱生性一氧化氮合酶（inducible nitric oxide synthases，iNOs）、环氧合酶-2（cyclooxygenase-2，COX-2）等合成和分泌。 IκBα磷酸化、泛素化降解是NF-κB活化的重要步骤，而蛋白磷酸化-去磷酸化是细胞信号转导调节的基本机制。本课题在既往研究的基础上，通过调节猪肺泡巨噬细胞（swine alveolar macrophage，s-AM）内蛋白磷酸化-去磷酸化动态平衡，干预NF-κB活化，试图抑制炎症介质的释放，阻断炎症介质“瀑布效应”形成，为ALI的治疗提供新的理论依据。本研究课题应用荧光检测术、Western Blot、EMSA、RT-PCR和ELISA等实验技术，观察内毒素脂多糖（lipopolysaccharide，LPS）刺激后，猪肺泡巨噬细胞内蛋白激酶C（protein kinase C，PKC）、蛋白酪氨酸磷酸酶（protein tyrosine phosphatase，PTP）、IκBα、NF-κB、COX-2-mRNA和细胞培养上清中前列腺素E2（prostaglandin E2，PGE2）水平的变化，以及单独或联合应用阿斯匹林、PTP抑制剂－过氧钒酸钠（peroxovanadium，POV）、蛋白激酶C抑制剂－Calphostin C干预处理30min后上述检测指标的变化。研究结果显示：(1) LPS刺激后，猪肺泡巨噬细胞内PKC活性、胞核提取物NF-κB、COX-2 mRNA和细胞培养上清PGE2水平呈一过性升高，胞浆PTP活性和IκBα水平则表现为一过性降低，且PKC、PTP活性变化最先出现；(2) 非甾体类抗炎药物阿斯匹林在抑制LPS刺激诱导的s-AM PKC活性、NF-κB活化、COX-2 mRNA表达和PGE2合成与释放的同时，可抑制LPS刺激诱导的IκBα降解和PTP活性的下调；(3) 联合抑制试验，PKC非特异性抑制剂Calphostin C可增强阿斯匹林对LPS刺激诱导的s-AM炎症介质的合成和释放，而PTP抑制剂过氧钒酸钠则表现相反的作用。研究结果表明：LPS刺激猪肺泡巨噬细胞产生炎症介质的病理生理过程中，蛋白<WP=9>激酶活性增强和磷酸酶活性降低，引起蛋白激酶-磷酸酶系统失去平衡，导致IκBα的酸化和泛素化降解增强，从而启动和增强NF-κB信号的转导，前炎症介质（如COX-2）基因转录和表达增强，引起炎症介质过度释放。上述研究证实，阿斯匹林可抑制PKC活性，联合应用阿斯匹林和蛋白激酶抑制剂可有效降低肺泡巨噬细胞LPS刺激后过度释放炎症介质。上述研究结果似为临床治疗急性肺损伤提供了新的思路。更多还原

【Abstract】 Acute lung injury (ALI) is a pulmonary manifestation of uncontrolled systemic inflammatory response syndrome (SIRS), in which activation of numerous inflammatory effector cells such as polymorphonuclear leukocytes and macrophages triggers excessive release of inflammatory mediators and cytokines. Mortality is still high in the patients with severe ALI ，especially in association with multiple organ dysfunction (MOD) or multiple organ failure (MOF). Unfortunately, the mechanisms of the pathologic processes remain unclear. It has been found, however, that nuclear factor-kappa B (NF-kappa B) might play a central role in the cellular signaling of pathological processes including ALI in which the activation of NF-kappa B could lead to the synthesis and secretion of numerous pre-inflammatory cytokines. The phosphorylation and degradation of inhibitory kappa B alpha is the most important step in the activation of NF-kappa B. Resolution of an excessive inflammatory response, therefore, should be achieved by restoring the dynamic balance of phosphorylation and dephosphorylation in inflammatory cells such as macrophages. The balance of phosphorylation and dephosphorylation in inflammatory cells would prevent sustained activation of NF-kappa B and excessive release of inflammatory mediators involved in the pathological process of ALI. In the present study, the methods as fluorescence assay, Western blot assay, electrophoretic mobility shift assay (EMSA), reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbant assay (ELISA) were used to determine the dynamic changes of the activity of protein kinase C (PKC) and protein tyrosine phosphatase (PTP), the levels of inhibitory kappa B alpha and cyclooxygenase-2 (COX-2) mRNA expression in swine alveolar macrophages (s-AM) stimulated with lipopolysaccharide (LPS). The concentrations of prostaglandin E2 in the s-AM culture supernatant were measured. After pretreatment with aspirin, peroxovanadium and calphostin C alone or in different combinations for 30 minutes, all the above mentioned parameters were also measured. A summary of the main results are as follows: 1) After stimulation of the alveolar<WP=7>macrophages with LPS, the activity of PKC, the activated NF-kappa B in the nuclear extract, the level of COX-2 mRNA expression and the concentration of prostglandin E2 reached peak levels, while the activity of PTP and the inhibitory kappa B alpha level in the cytoplasm decreased to their basal levels. The changes in PKC and PTP activity appeared much earlier than the other changes. 2) The non-steroidal anti-inflammatory drug aspirin can inhibited the increase in PKC activity, the activation level of NF-kappa B, the expression level of COX-2 mRNA and the concentration of prostaglandin E2 in the cytoplasm or culture supernatant of alveolar macrophages stimulated with LPS. Conversely, it has a negative effect on the degradation of inhibitory kappa B alpha and the down-regulation of PTP induced by the stimulation with lipopolysaccharde in alveolar macrophages. 3) The non-specific inhibitor of PKC, calphostin C, enhanced the inhibitory effects of aspirin on inflammatory responses induced by lipopolysaccharide in alveolar macrophages, while the PTP inhibitor, peroxovanadium, exerted the oppsite effects. Our results suggest that there is a disturbance of the PKC and PTP system in swine alveolar macrophages stimulated with lipopolysaccharide, and aspirin is effective in restoring the balance of the system, which might be useful as a reference in clinical therapy of ALI.更多还原