【作者】 张荣建；

【导师】 李胜男；

【作者基本信息】 南京医科大学 ， 药理学， 2009， 硕士

【摘要】 血栓闭塞性脉管炎(thromboangitis obilterans, TAO)是一种炎症性、闭塞性和周期发作的慢性周围血管病,多发于青壮年男性,主要侵犯四肢远端的中小动静脉,尤其是下肢血管,是青壮年男性肢体缺血及致残的主要原因之一。病理变化为血管壁节段性、非化脓性炎症伴腔内血栓形成,阻塞管腔引起肢体缺血、疼痛,呈周期性发作,最终引起肢端坏疽。目前只知TAO发病诱因为寒冻、吸烟以及性激素等,而其发病机理尚未完全阐明,还没有哪一种观点可以解释TAO的所有临床表现;同时该病的治疗方法和使用的药物不尽相同,疗效报道不一,仍为医学领域的难题之一。因此,探索TAO发病机理有着重大的理论与实际意义。Urocortin(Ucn)是一个40个氨基酸的多肽,属于促肾上腺皮质激素释放因子(corticotrophin-releasing factor, CRF)肽类家族。Ucn通过结合CRF受体(corticotrophin-releasing factor receptors, CRFRs)发挥多种生物学效应。近年来包括本实验室在内的研究表明,外周的Ucn作为一个自分泌或旁分泌的促炎因子参与很多炎性疾病,如风湿性关节炎、骨性关节炎、溃疡性结肠炎等。而且在很多炎症状态下,Ucn能促进炎性介质的释放,加重炎症。另外,Ucn还被报道参与了血管内皮功能紊乱。环加氧酶2 (cyclooxygenase-2, COX-2),作为一种参与花生四烯酸代谢的限速酶,被广泛报道参与各种炎症,包括血管炎症(动脉粥样硬化、TAO)。有研究表明,和正常人相比,TAO病人肱动脉内皮依赖性舒张能力下降了近50%,而选择性COX-2抑制剂则明显改善血管内皮功能。同时,Fiessinger等人多次临床病例对照试验结果也支持COX-2和TAO的关系。另一方面,近年来的很多文献支持Ucn和COX-2在炎症中的关系。Ucn能促进COX-2的表达从而参与了一系列的炎性疾病,如风湿性关节炎、血管炎等。而且,COX-2途径参与了Ucn诱导的炎症介质(如TNF-α、IL-6)的释放。更为重要的是,选择性COX-2抑制剂能取消Ucn引起的上述效应,缓解炎症。综上所述,鉴于TAO的血管炎本质和Ucn的促炎作用(包括参与血管内皮功能紊乱),我们推测Ucn与TAO可能有着非常密切的联系。那么究竟Ucn对TAO的发生发展有无影响?有何影响?COX-2是否参与了Ucn的上述作用?至今还未见报道。本文第一部分采用月桂酸钠注射大鼠股动脉复制TAO大鼠模型,研究Ucn、COX-2在TAO大鼠病变血管和血液中的表达,探索Ucn对TAO发生发展的影响,评价COX-2在TAO中的作用;第二部分利用体外分离培养的大鼠主动脉内皮细胞(RAECs),旨在细胞水平上研究Ucn对大鼠血管内皮炎症(LPS预先刺激造模)中COX-2表达的影响,并探讨相应受体和细胞内信号通路机制。第一部分Urocortin在大鼠血栓闭塞性脉管炎中的作用及对血管COX-2表达的影响目的:研究Ucn对大鼠TAO发生发展的影响,评价COX-2在TAO中的作用,探索TAO的发病机理。方法:月桂酸钠注射大鼠股动脉复制TAO动物模型,并以第十二天各组大鼠患肢的外观进行病变分级。H-E染色用来观察大鼠股动脉的形态学改变;大鼠的血液学指标(包括血常规、凝血和血液流变学指标)用于生化检测;ELISA用于检测血浆Ucn、TXB2和PGE2的水平;RT-PCR和Western blotting用于检测大鼠股动脉Ucn、CRFR1/2和COX-2的表达。结果:在造模后的第十二天大多数大鼠呈典型的TAO症状和体征。和假手术大鼠相比,TAO模型大鼠血液呈高凝状态,血浆内源性Ucn、PGE2水平显著升高,股动脉Ucn、CRFR1、CRFR1α和COX-2表达明显增加。给予外源性Ucn则进一步加重了高凝状态,促进了COX-2和PGE2的表达,恶化了TAO。Ucn的上述效应能被选择性CRFR1拮抗剂NBI-27914或非选择性CRFRs拮抗剂astressin阻断,然而选择性CRFR2拮抗剂antisauvagine-30则对Ucn组大鼠无明显作用。结论: Ucn能促进TAO大鼠血液高凝状态、COX-2的表达,加重脉管炎。上述作用是由CRFR1介导的。而血小板和COX-2可能是Ucn对TAO影响的潜在靶点。第二部分Urocortin对大鼠主动脉内皮细胞COX-2表达影响及机制的研究目的:研究Ucn对大鼠血管内皮炎症(LPS预先刺激造模)中COX-2表达的影响,并探讨相应的受体和细胞内信号通路机制。方法:大鼠主动脉内皮细胞(RAECs)的分离、培养和鉴定。实验均采用生长稳定的5-8代RAECs。RT-PCR和Western blotting分别用于检测COX-2 mRNA和蛋白的表达。ELISA用于检测细胞上清液中PGE2的含量;Western blotting检测MAPKs (p38MAPK、ERK1/2、JNK)、Akt和NF-κB的磷酸化水平;免疫荧光细胞化学法检测RAECs内NF-κB的核转位情况。结果:Ucn呈时间和浓度依赖性地增强LPS刺激下的RAECs中COX-2的表达;而且上调的COX-2 mRNA和蛋白分别在4h和8h达到峰值。Ucn的上述作用能被CRFR2拮抗剂antisauvagine-30完全阻断,而CRFR1拮抗剂NBI-27914却没有明显的作用。ELISA检测各组细胞上清液PGE2的结果也进一步证实了上述的发现。更为重要的是p38MAPK和NF-κB参与了RAECs上CRFR2介导的COX-2表达,而ERK1/2、JNK和Akt无明显改变。结论:Ucn通过CRFR2增强了LPS刺激下的RAECs中COX-2的表达从而参与了血管炎症,而且p38MAPK和NF-κB参与了CRFR2介导的上述效应。综上所述,本文工作的主要创新之处在于:1.整体实验揭示了Ucn对TAO大鼠血管炎有显著地促进作用,其机制与加重血液高凝和上调COX-2等相关,为探索TAO的发病机理积累了学术基础。2.离体实验表明Ucn上调了RAECs中COX-2的表达从而参与了血管炎症,其机制是由CRFR2介导的,与p38MAPK、NF-κB信号通路的激活相关,为血管炎症的药物治疗提供了新的靶标。3.整体和离体实验结果提示COX-2途径是Ucn参与血管炎症的重要机制之一。研究结果为血管炎症的防治提供了理论依据。更多还原

【Abstract】 Thromboangiitis obliterans (TAO) is a nonatherosclerotic, segmental, inflammatory disease that most commonly affects the small and medium-sized arteries, veins and nerves of the extremities. In the characteristic acute phase lesion, in association with occlusive cellular thrombosis, the acute inflammation which involves all layers of the vessel wall leads TAO to be classified as a vasculitis. Cold, tobacco and sexual hormone are reported to have strong associations with TAO. However, the exact etiology for TAO has not been well defined. Furthermore, treatments of TAO are diverse while curative effects of TAO are controversial. Taken together, it is greatly necessary and meaningful to explore the mechanisms of TAO.Urocortin (Ucn), a 40 aa corticotropin-releasing factor (CRF) family peptide, has been demonstrated to be widely expressed in peripheral tissues including cardiovascular system, gastrointestinal tract, immune system, et al. Recently, Ucn has been considered to be a potent locally expressed autocrine or paracrine pro-inflammatory factor in a series of inflammatory diseases, such as rheumatoid arthritis & osteoarthritis and ulcerative colitis. Moreover, it can stimulate the release of pro-inflammatory mediators under inflammatory conditions. In addition, Ucn may have a negative impact on the normal function of ECs by suppressing vascularization.COX-2, the rate-limiting enzyme in metabolism of arachidonic acid, has been abundantly reported to take part in inflammatory diseases including artherosclerosis and TAO. In case-control studies, prostacycline derivatives have shown to be more effective than placebo for the therapy of. Moreover, blockade COX-2 could ameliorate capability of endothelium-dependent vasodilation of brachial arteroy in TAO patients. On the other hand, CRF family peptides could induce COX-2 expression under a series of inflammatory conditions, such as human arthritis, vasculitis and so on. Furthermore, COX-2 pathway is involved in regulation of Ucn-mediated pro-inflammatory cytokine productions, such as TNF-α, IL-6 and so on. COX-2 inhibitor application could abolish the above effects induced by Ucn. Taken together, CRF family peptides may participate in the pathophysiology of many inflammatory conditions via COX-2 pathway.Taken together, it is reasonable to believe that Ucn, which can be synthesized and secreted by systemic vasculature, may be a potent locally expressed pro-inflammatory factor in TAO, which is classified as a vasculitis with endothelial dysfunction. However, Ucn’s function on peripheral vasculitis and potential role in modulating TAO progression has rarely been illustrated. In this study, we investigated and first reported the role of Ucn in sodium laurate induced TAO rat model and COX-2 expression. Furthermore, we examined the effects of Ucn on COX-2 expression in lipopolysaccharide (LPS)-induced rat aortic endothelial cells (RAECs) and explore the relevant mechanisms.Part 1 Effects of urocortin on the development of vasculitis and COX-2 expression in thromboangiitis obliterans rat modelBackground and purpose: The present study was performed to examine the effects of Ucn on sodium laurate induced peripheral arterial occlusive disease in rats, exploring the mechanisms of thromboangiitis obliterans (TAO).Experimental approach: Sodium laurate induced peripheral vasculitic rat model was made. The degree of the disease on the 12th day was macroscopically graded. Histopathology and transmission electron microscopy observation were used to investigate the histological changes of rat femoral arteries. The parameters of blood routine, blood rheology, blood coagulation and plasma Ucn, TXB2 and PGE2 levels were measured. The expressions of Ucn, CRFR1/2 and COX-2 at both mRNA and protein levels were determined by RT-PCR and Western blotting.Key results: Most rats showed TAO signs and symptoms on the 12th day after sodium laurate injection. In model group, the blood was in a hypercoagulable state; plasma Ucn and PGE2 levels were elevated; and the expressions of Ucn, CRFR1, CRFR1αsubtype and COX-2 from rat femoral arteries were markedly increased. Ucn application aggravated the hypercoagulable state and augmented COX-2 expression. Furthermore, these effects could be abolished by CRFR1 antagonist, NBI-27914, or nonselective CRFRs antagonist, astressin, while CRFR2 antagonist, antisauvagine-30, could not influence these effects of Ucn. Conclusion and implications: These findings suggest that Ucn can aggravate the hypercoagulable state and vasculitis in sodium laurate induced TAO model rats via CRFR1. COX-2 may participate in this aggravating process.Part 2 Urocortin induced COX-2 expression via corticotrophin releasing factor type 2 receptor in rat aortic endothelial cellsBackground and purpose: The present study was performed to examine the effects of Ucn on COX-2 expression in lipopolysaccharide (LPS)-induced rat aortic endothelial cells (RAECs) and explore the relevant mechanisms.Experimental approach: RAECs were isolated from adult male Wistar rats and identified at the first passage. Experiments were performed on cells at passages 5 through 8 from primary culture. The expression of COX-2 at both mRNA and protein levels were determined by semi-quantitative RT-PCR and Western blotting analysis. Levels of PGE2 in culture medium were measured by ELISA. Furthermore, phosphorylation status of p38MAPK, ERK1/2, JNK, Akt and NF-κB was analyzed by Western blotting; nuclear translocation of NF-κB was observed by immunofluorescence.Key results: Ucn augmented LPS-induced RAECs COX-2 expression in a time- and concentration-dependent manner. Ucn application increased PGE2 levels. These effects could be abolished by CRFR2 antagonist, antisauvagine-30, but not by CRFR1 antagonist, NBI-27914. Moreover, Ucn2 application activated p38MAPK and augmented NF-κB nuclear translocation while ERK1/2, JNK and Akt pathways were not involved in this process.Conclusions and implications: These findings suggest that Ucn exerted a pro-inflammatory function by augmenting LPS-induced RAECs COX-2 expression via CRFR2, in which p38MAPK and NF-κB pathways were involved.The major contributions of the present study lie in:1. The in vivo study suggests that Ucn can aggravate the hypercoagulable state and vasculitis in sodium laurate induced TAO model rats via CRFR1. COX-2 may participate in this aggravating process. These works provide a novel mechanism for the development of TAO.2. The in vitro study suggests that Ucn exerts a pro-inflammatory function by augmenting LPS-induced RAECs COX-2 expression via CRFR2, in which p38MAPK and NF-κB pathways were involved. Targeting CRFR2 may be a novel approach to treat vasculitis. 3. Taken together, the in vivo and in vitro studies strongly indicate that COX-2 is one of the important target of Ucn’participating in vasculitis, which provides a therapeutic approach for vasculitis.更多还原