【作者】 孙瑜；

【导师】 夏照帆； 路卫； 朱世辉； 唐洪泰； 贲道锋； 王广庆； 王光毅；

【作者基本信息】 第二军医大学 ， 外科学， 2009， 博士

【摘要】 第一部分巨噬细胞移动抑制因子负向调节糖皮质激素抗炎作用的机制研究炎症是人体对感染、体内抗原抗体结合以及机械损伤的一种自身反应。适度的炎症反应对人体是有益的,而过度的或持续的炎症反应却会引起组织损伤和诱发疾病。糖皮质激素(glucocorticoid, GC)可以纠正炎症反应的失调,在临床中常被用来治疗包括脓毒性休克、全身炎症反应综合征、急性呼吸窘迫综合征等急性炎症反应引起的疾病和哮喘、类风湿关节炎、系统性红斑狼疮、肾小球肾炎、多发性硬化、银屑病等慢性炎症反应异常引起的疾病。GC主要是通过其与糖皮质激素受体(GR)的结合,经不同途径发挥作用。然而这种多效性作用也会产生不良反应,引起儿童生长迟缓,免疫抑制,高血压,抑制伤口修复,骨质疏松以及代谢紊乱等。Annexin 1作为一种磷脂酶A2(PLA2)的抑制蛋白,可以影响许多炎症反应复合物的形成,例如花生四烯酸代谢后的脂质炎症介质。体内PLA2存在cPLA2(胞浆型)和sPLA2(血清型)2种形式。cPLA2是脂质二十烷类炎症介质合成释放的关键酶,其活性的控制是调节炎症过程的中心。最近研究表明内源性Annexin 1特异性作用于cPLA2,全长的Annexin 1和羧基末端区域都可以抑制cPLA2活性,而外源性Annexin 1及其氨基末端片断与其膜受体相互作用抑制cPLA2活性。Annexin 1也抑制其他一些参与炎症的酶的表达和/或活性,如巨噬细胞中诱导型一氧化氮合酶(iNOS)和激活的小胶质细胞中诱导型环氧合酶(COX-2)。在巨噬细胞,Annexin 1诱导IL-10释放,IL-10是一个潜在的抗炎细胞因子,能够抑制细胞因子对COX和NOS表达的诱导。此外,糖皮质激素能诱导并激活Annexin I,通过抑制cPLA2α阻止花生四烯酸的产生和向类花生酸类物质的转变(如,前列腺素,血栓素,前列环素和白三烯),从而发挥其强大的抗炎作用。巨噬细胞移动抑制因子(macrophage migration inhibitory factor, MIF)是一种主要由巨噬细胞、激活的T细胞及垂体前叶促肾上腺皮质素细胞等合成的细胞因子。细菌、毒素及一些细胞因子刺激可诱导MIF释放,从而限制巨噬细胞的迁移。GC在全身免疫炎症反应中具有广泛的作用。外源性GC可抑制整个免疫炎症系统,包括促炎因子的表达,T细胞的激活,粘附分子的表达,细胞迁移与效应分子的生成等;内源性GC以一种双相、浓度依赖性的方式调节免疫炎症系统。迄今为止,MIF是唯一被认为能负向调节GC抗炎作用的细胞因子,它可能与局部或全身炎症、自身免疫性疾病等密切相关,亦可能是GC不能有效发挥抗炎作用的关键因子。MIF是如何对抗糖皮质激素的抗炎作用的,其机制至今仍未完全阐明。基于以上研究现状,我们提出这样的问题:既然糖皮质激素能诱导Annexin 1的表达来发挥抗炎作用,那么MIF负向调节糖皮质激素的抗炎作用是否与Annexin 1有关?我们将针对这一问题深入探讨,从而进一步明确MIF负向调节糖皮质激素抗炎作用的分子机制。我们首先观察了MIF是否具有负向调节糖皮质激素抑制脂质炎症介质释放的作用。用脂多糖(LPS)、MIF、地塞米松(Dex)分别或相继刺激小鼠腹腔巨噬细胞系RAW264.7后用ELISA方法检测细胞培养的上清液中前列腺素E2(PGE2)和白三烯B4(LTB4)的含量。在此基础上,进一步观察MIF负向调节Dex抑制脂质炎症介质释放的作用是否与Annexin 1相关,用Western blotting的方法检测了RAW264.7细胞胞浆中Annexin 1的蛋白表达变化。从反向证明的角度,我们用RNA干扰(RNAi)的方法阻断细胞内MIF的表达,再观察Dex是否能增强Annexin 1的表达。在明确了MIF和Annexin 1之间的关系后,我们进一步用Western blotting方法检测了在抑制内源性MIF表达或给予外源性重组MIF刺激后Annexin 1的下游信号蛋白胞浆内磷脂酶A2α(cPLA2α)的表达变化。最后在抑制内源性MIF表达的情况下,用ELISA方法检测了Dex刺激后细胞上清液中PGE2和LTB4的含量。实验结果显示:①RAW264.7用MIF和LPS刺激以后,细胞中脂质炎症介质PGE2和LTB4的产生明显增加,Dex能明显减少PGE2和LTB4的产生,然而加入外源性重组的MIF以后就可以逆转Dex的作用。②LPS刺激能减少内源性Annexin 1的表达,加入Dex后能明显增加Annexin 1的表达,但是加入外源性重组的MIF以后,Dex增强Annexin 1表达的作用就被逆转。随后,我们用RNA干扰的方法抑制内源性MIF的表达,发现RAW264.7细胞不表达MIF以后能增强Dex对Annexin 1的促表达作用,而且这个效应呈剂量依赖关系,即经Dex处理后,抑制内源性MIF表达的细胞和不抑制MIF表达的细胞相比,其上调Annexin 1表达的作用更明显。③Annexin 1作为内源性磷酸酶的抑制剂,在MIF调节Dex的作用中,是否也进一步影响胞浆内磷脂酶A2(α)(cPLA2α)的作用。研究证实,外源性MIF能够逆转Dex对cPLA2α活化的抑制作用,而内源性MIF不表达的细胞能够增强Dex抑制cPLA2α磷酸化的效果。④最后证实抑制内源性MIF表达还能增强Dex对PGE2和LTB4产生的抑制作用。也就从反面证实了结果①的内容。从以上结果我们得出结论:在小鼠腹腔巨噬细胞系RAW264.7中,MIF通过抑制抗炎蛋白Annexin 1的表达负向调节糖皮质激素抑制脂质炎症介质PGE2和LTB4的产生。Annexin 1作为MIF和糖皮质激素之间作用的关键性蛋白,有望成为治疗炎症性疾病的新靶点。第二部分巨噬细胞移动抑制因子参与内皮细胞胰岛素抵抗的机制研究血管内皮细胞在调节血管内环境稳定方面具有举足轻重的作用。血管内皮细胞维持着血管收缩与舒张、抑制与刺激血管平滑肌细胞增殖与移行、凝血与纤溶活性等多方面的平衡。已知血管内皮细胞是胰岛素的效应细胞,胰岛素与内皮细胞上的胰岛素受体结合后,通过促使内皮细胞合成及释放一氧化氮(NO)增加而实现其血管舒张的生理功能。NO是迄今所知最强有力的内源性血管舒张因子,在维持正常血管张力方面起主要作用。若机体内出现胰岛素抵抗的状态,如肥胖、2型糖尿病、高血压等情况发生,胰岛素介导的内皮细胞依赖性血管舒张功能受损,内皮源性NO产生减少,从而诱发内皮细胞胰岛素抵抗。多年的研究认为内皮功能紊乱是诱发内皮细胞胰岛素抵抗的基础,而炎症则是导致内皮功能紊乱的触发因素。已证实IL-6和TNF-α这两个炎症因子均能影响内皮功能从而诱发内皮细胞胰岛素抵抗,且是通过干扰胰岛素信号转导通路实现的。本论文的第一部分内容中介绍的巨噬细胞移动抑制因子(MIF)是一个强大的促炎细胞因子,它不仅能负向调节糖皮质激素的抗炎作用,且近年来大量研究发现MIF与动脉粥样硬化、糖尿病、肥胖、高血压等心血管及代谢系统疾病密切相关。鉴于胰岛素抵抗、炎症和动脉粥样硬化的密切关系,我们提出了这样的假设,MIF是否参与了内皮细胞胰岛素抵抗的过程,如果参与,那么其具体机制是怎样的?对于上述假设的研究目前国内外未见报道,即作为本部分的研究内容。我们以人的脐静脉血管内皮细胞HUVEC为研究对象,首先观察了胰岛素对HUVEC细胞产生和释放NO的影响。在此基础上,进一步观察MIF和胰岛素联合刺激对HUVEC细胞产生和释放NO的作用,从而明确MIF对内皮细胞功能的影响。在明确MIF对内皮细胞释放NO的影响后,我们从胰岛素介导NO产生和释放的胞内信号转导途径PI 3-kinase入手,主要针对了胰岛素受体底物-1(IRS-1)、磷脂酰肌醇三激酶(PI 3-kinase)亚基p85和内皮型一氧化氮合成酶(eNOS)这几个胰岛素信号转导通路上的关键性蛋白,通过检测IRS-1的磷酸化、PI 3-kinase亚基p85的活化和eNOS的磷酸化来反应胰岛素信号转导的变化(是否受阻或减弱)。研究MIF是否作用于该条信号通路上的几个关键性蛋白,旨在揭示MIF影响内皮细胞释放NO的分子机制。实验结果显示,胰岛素能以剂量依赖和时间依赖方式促使内皮细胞释放NO,但如果MIF联合胰岛素刺激,则大大减少内皮细胞产生和释放NO,用MIF拮抗剂则能基本恢复胰岛素释放NO的能力。从效应来看,MIF和胰岛素之间存在相互拮抗的作用。在此基础上,我们以胰岛素信号转导通路中的一条途径(PI 3-kinase途径)为研究对象,发现MIF可直接作用于内皮细胞的胰岛素信号转导系统,MIF增强IRS-1的丝氨酸(位点312和616)磷酸化并降低IRS-1的酪氨酸(位点612)磷酸化;另一方面,MIF显著降低了IRS蛋白与下游转导途径PI 3-kinase中活性亚基p85的结合,继而减少了eNOS的活化,从而减少NO合成和释放。且MIF拮抗剂能部分抵消上述MIF对胰岛素信号转导通路中几个关键性蛋白的影响。我们的研究结果证实了MIF干扰了胰岛素介导的NO释放,影响了内皮细胞维持正常的血管舒张,导致内皮细胞功能紊乱,从而参与了内皮细胞胰岛素抵抗的发生。这个发现不仅补充和丰富了现有的胰岛素抵抗的发病机制,而且进一步证实了炎症、胰岛素抵抗之间的密切关系。此外,研究还发现MIF主要是通过作用于胰岛素介导的PI 3-kinase信号转导途径上的几个关键性蛋白(IRS-1、PI 3-kinase p85、eNOS)来影响内皮细胞功能的。上述结果为临床防治胰岛素抵抗的发生提供了可靠的实验依据和可干预的靶点。更多还原

【Abstract】 PartⅠMacrophage Migration Inhibitory Factor Counter-regulates Anti-inflammatory Effects of GlucocorticoidsInflammation is a reflexive response to infection, the binding of antibodies to antigens within the body, mechanical irritation, or injury. Whereas restricted inflammation is beneficial, excessive or persistent inflammation incites tissue destruction and disease. Glucocorticoids (GC) are indicated for the treatment of acute inflammations including septic shock, systemic inflammatory response syndrome, acute respiratory distress syndrome and chronic ones including asthma, rheumatoid arthritis, systemic lupus erythematosus, glomerular nephritis, multiple sclerosis and psoriasis. The efficacy of GCs in alleviating inflammatory disorders results from the pleiotropic effects of the glucocorticoid receptor (GR) on multiple signaling pathways. Pleiotropy can, however, also have adverse effects: growth retardation in children, immunosuppression, hypertension, inhibition of wound repair, osteoporosis, and metabolic disturbances.Annexin 1 as an inhibitor of phospholipase A2 (PLA2) is responsible for its anti-inflammatory actions including the production of arachadonic acid. It is now apparent that a cytosolic form of PLA2 (cPLA2) as well as a secretory form (sPLA2) exist. cPLA2 exhibits partiality for the arachidonyl-containing phospholipids. cPLA2 is predominantly involved in the production of inflammatory lipid mediators, and arachadonic. Annexin has now been shown to inibit PLA2 activity directly, rather than by substrate depletion. Annexin 1 is also found to inhibit the activity of cycloocygenase 2 (COX-2). Annexin 1 expression can be up-regulated with GC treatment through the GR, which contributes to its anti-inflammatory activity.Macrophage migration inhibory factor (MIF) is a cytokine which is synthesized by macrophages, activated T cells and anterior pituitary gland corticotropic cells. MIF is released by innate immune cells such as monocytes and macrophages when exposed to microbial products and pro-inflammatory mediators and has the ability of inhibiting migration of macrophage. TNF-α, IL-1 as well as MIF compose the first line of defense of innate immune. GCs regulate a wide variety of systemic immunologic and inflammatory response. Exogenous GCs suppress the whole immune system including activation of T cells, expression of inflammatory cytokine and adhesion molecules, migration of cells and productioin of effector molecules. Endogenous GCs regulate immune system in a concentration and diphase-dependent manner. Until recently, no endogenous neuroendocrine or immune mediator had been identified that would function as a counter-regulatory partner of GCs to modulate immune response except MIF. MIF is closely related with local and systemic inflammation and plays a pathogenic role in antoimmune diseases. However, very little is known about the molecular basis of MIF-GC interactions within the immune system.Based on the backgrounds of above researches, we address the question that whether the counter-regulatory effect of MIF on GCs is related with Annexin 1, as which is induced by GCs to exert an anti-inflammatory response.Prostaglandin E2 (PGE2) and Leukotriene B4 (LTB4) production were measured by ELISA. Protein expression of Annexin 1, cytosolic phospholipase A2 alpha (cPLA2α) and phospho-cPLA2αwere evaluated by Western blotting with recombinant MIF or without endogenous MIF expression using ribonucleic acid interference (RNAi) method.The results showed that recombinant MIF counter-regulated the inhibition of dexamethasone (Dex) on PGE2 and LTB4 production in RAW 264.7 macrophages stimulated with lipopolysaccharides (LPS) in a dose-dependent manner. Stimulation of RAW 264.7 macrophages with LPS resulted in a down-regulation of Annexin 1, while Dex or Dex plus LPS led to a significant up-regulation of Annexin 1 expression. The effect of Dex on Annexin 1 was counter-regulated by the administration of recombinant MIF. RNAi-mediated knockdown of the intracellular MIF further increased Annexin 1 expression of RAW 264.7 macrophages after incubation with Dex, and accordingly resulted in a low production of PGE2 and LTB4 through inhibiting the activation of cPLA2α.Annexin 1 plays a key role in MIF counter-regulation of Dex inhibition on eicosanoids production, which indicates that exogenous Annexin 1 may partially reduce the effects of MIF on GCs, thus becoming a potentially effective steroid sparing therapy on inflammatory diseases. PartⅡMacrophage Migration Inhibitory Factor Affects Insulin Resistace of Endothelial CellsVascular endothelial cells have a key role of stabilizing the internal environment of blood vessels. They keep the balance of many functions of endothelium including contraction or dilation of vessels, stimulating or inhibiting proliferation and migration of vascular smooth muscle cells, blood coagulation or fibronolysis and so on. Vascular endothelial cell is one of the target organs of insulin. Insulin exerts its biological effect of vasodilation by binding to the cognate cell surface receptor on the endothelium and promoting the release of nitrogen monoxidum. Nitric oxide (NO), the most powerful endothelium-derived relaxing factor, has the major role of maintaining angiotasis. Insulin-resistant conditions including obesity, type 2 diabetes, and hypertension, a feature of endothelial dysfunction, is followed by endothelial insulin resistance due to reduction of NO.It has been presumed that endothelial dysfunction is the basis on induction of endothelial insulin resistance, and inflammation is the triggering factor of endothelial dysfunction. It has been demonstrated that interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), these two inflammatory cytokines, could induce endothelial insulin resistance by affecting endothelial function. They achived this effects through interacting with certain proteins which belong to insulin-mediated signaling pathway.Macrophage migration inhibitory factor (MIF) addressed in the front part of this thesis is a powerful inflammatory cytokine. It not only has the ability of counter-regulating the anti-inflammatory effects of glucocorticoids, but also has been found to have the close relationship with cardiovascular and metabolic diseases in recent years including atherosclerosis, diabetic mellitus, obesity and hypertension. Based on the close relationship of inflammation, insulin resistance and atherosclerosis, we addressed the hypothesis whether MIF would be involved in the pathogenesis of endothelial insulin resistance, and if involved, what the mechanism would be. We have not found similar research work at home and abroad, so we studied this subject in the following experiments.Firstly, we evaluated the effects of insulin on production and release of NO in human umbilical vein endothelial cells (HUVEC). Secondly, we examined the effects of both the MIF and insulin on NO in HUVEC cells in order to identify the synergistic or antagonistic action of them. Thirdly, we evaluated the effects of MIF on insulin-mediated signaling pathway by studying the phosphorylation of insulin receptor substrate-1 and endothelial nitric oxide synthase and the activation of PI 3-KINASE p85 subunit to elucidate the molecular mechanisms of interaction of MIF and insulin.The results showed that insulin increased the production and release of NO in HUVEC cells in a dose- and time-dependent fashion. However, the production and release of NO significantly reduced after stimulation with both MIF and insulin. However, antagonism of MIF almost restored the production and release of NO by insulin. So there is an antagonistic but synergistic action between MIF and insulin, that is to say exposure of HUVEC cells to MIF resulted in inhibition of insulin-stimulated production of NO. Next study showed that MIF affects insulin signaling involved in NO production in HUVEC cells. This event was associated with impaired IRS-1 phosphorylation at Tyr612, one site essential for engaging the p85 subunit of PI 3-KINASE, resulting in defective activation of PI 3-KINASE and eNOS. This inhibitory effect of MIF was reversed by the MIF antagonist ISO-1. MIF increased IRS-1 phosphorulation at Ser312 and Ser616, which could inactivate the IRS-1.Our data suggest that MIF increased IRS-1 phosphorylation at Ser312 and Ser616, thus impairing the vasodilator effects of insulin mediated by the IRS-1/PI 3-kinase/eNOS pathway. MIF induced insulin resistance in endothelial cells may play an important role in the pathophysiology of cardiovascular disease associated with hypertension and insulin resistance. The characterization of the molecular mechanism involved in MIF-induced insulin resistance in the endothelium may help to design efficacious pharmacological molecules to treat endothelial dysfunction associated with insulin resistance states.更多还原