【作者】 黄雪琴；

【导师】 魏尔清；

【作者基本信息】 浙江大学 ， 药理学， 2012， 博士

【摘要】 研究背景：脑缺血后的炎症反应在其病理生理发生过程中起了重要作用。脑缺血损伤发生后,促进外周和中枢神经系统的促炎症因子表达并分泌至细胞外,诱导脑内炎症变化,其结果之一是激活星形胶质细胞并逐渐形成胶质疤痕,研究炎症因子激活星形胶质细胞,有助于了解胶质疤痕形成机制和干预途径。脑缺血后脑内炎症因子包括转化生长因子β1(transforming growth factor β1, TGF-β1)以及半胱氨酰白三烯类(cysteinyl leukotrienes, CysLTs)。TGF-β1被广泛认为是一种与损伤相关的细胞因子,CysLTs是一类重要的炎症介质,两者在脑缺血、脑外伤等中枢神经系统损伤性疾病发病中均起重要作用。TGF-β1在正常脑组织中表达水平不高。脑缺血后,TGF-β1mRNA在脑组织中早期(缺血后6h)表达增加,缺血后2-4天达高峰,持续14-26天；缺血早期在全脑弥散表达,7-14天后主要表达于星形胶质细胞,与脑缺血后胶质疤痕的形成有关；体外实验表明,TGF-β1能够诱导星形胶质细胞增生肥大；促进星形胶质细胞迁移。CysLTs由5-脂氧合酶(5-lipoxygenase,5-LOX)催化花生四烯酸而形成,其作用通过半胱氨酰白三烯受体(cysteinyl leukotriene receptor, CysLT受体)起作用,该类受体主要包括CysLT1和CysLT2受体两种亚型。CysLT1受体在正常脑组织中表达水平不高。脑缺血后,缺血中心区CysLT1受体mRNA在缺血再灌注3、6、12h和7、14天表达上调；缺血周边区,再灌注后3天内CysLT1受体mRNA水平没有变化,7、14天明显增高,主要表达于增生的星形胶质细胞。药理学研究表明,CysLT1受体抑制剂pranlukast抑制小鼠局灶性脑缺血后诱导的胶质疤痕形成。在大鼠星形胶质细胞,轻度缺氧缺糖(oxygen-glucose deprivation, OGD)诱导CysLT1受体表达,并介导体外星形胶质细胞的增殖、激活；而CysLT2受体则介导中度OGD诱导的星形胶质细胞损伤。CysLT1受体介导星形胶质细胞激活的机制尚待阐明。由于星形胶质细胞激活受多种因素调节,CysLT1受体的效应可能与其他因素存在相互作用。在外周组织中,TGF-β1能上调支气管平滑肌细胞中CysLT1受体的表达,增强LTD4的促增殖作用；TGF-β1能上调胎儿肺成纤维细胞中CysLT1受体的表达；TGF-β1能增加树突状细胞中5-LOX和5-LOX激活蛋白(FLAP)的表达,增加白三烯的合成。而在中枢神经系统中,TGF-β1与CysLTs及其受体之间是否有相互调节的作用?如何调节?尚待阐明。尤其对星形胶质细胞激活,TGF-β1与CysLT1受体的相互作用,是本文拟阐明的问题。研究目的：本文拟观察半胱氨酰白三烯及CysLT1受体与TGF-β1在调节星形胶质细胞中的相互作用,进行以下几个方面的研究：1.首先,阐明CysLT1受体是否与TGF-β1相互作用。观察TGF-β1以及CysLT受体相关药物对细胞活性的影响,包括5-LOX抑制剂、CysLT1和CysLT2受体激动剂和拮抗剂。2.其次,进一步阐明TGF-β1与CysLT1受体之间的可能相互作用。重点观察TGF-β1与CysLT受体激动剂、拮抗剂及siRNA对星形胶质细胞激活的作用,包括对增殖和迁移作用的影响。3.然后,为了阐明TGF-β1与CysLT1受体之间的相互作用特点,从两个方面进行解析,一方面分析TGF-β1对CysLT1受体及其内源性激动剂CysLTs合成酶5-LOX的调节作用；另一方面,分析CysLT,受体激动剂对TGF-β1的调节作用。4.最后,为了阐明TGF-β1与CysLT1受体之间的相互作用的意义,在以往轻度OGD(1h)增强CysLT1受体表达及效应结果的基础上,观察轻度OGD是否增加TGF-β1释放。研究方-法：选用原代培养的大鼠大脑皮层星形胶质细胞作为细胞模型,细胞划痕方法检测细胞迁移；MTT还原试验法检测细胞活性；流式细胞术检测细胞的增殖；细胞免疫化学法检测细胞中5-LOX的核膜移位；ELISA法检测CysLTs释放;RT-PCR、 Western blot和细胞免疫荧光法检测细胞中5-LOX、CysLT1受体和CysLT2受体表达。以CysLT受体非选择性激动剂LTD4、CysLT2受体激动剂NMLTC4、TGF-β1信号抑制剂repsox,5-LOX抑制剂齐留通(zileuton), CysLT,受体选择性拮抗剂孟鲁司特(montelukast)知CysLT2受体选择性拮抗剂Bay cysLT2进行药理学干预处理,CysLT,受体siRNA进行基因干扰处理。在大鼠大脑皮层星形胶质细胞中,以OGD1h和4h诱导缺血性损伤。以MTT法、LDH法检测OGD/恢复(OGD/R)后星形胶质细胞的活性及损伤程度,并以ELISA法检测TGF-β1释放。研究结果：1. TGF-β1与CysLT受体对星形胶质细胞活性的影响原代培养的大鼠星形胶质细胞中,MTT还原试验法表明,TGF-β1和CysLT受体非选择性激动剂LTD4可增强细胞活性,并且,两者有相互增强；而CysLT2受体激动剂NMLTC4则无作用。TGF-β1的作用可被5-LOX抑制剂zileuton、CysLT1受体拮抗剂montelukast减弱,而CysLT2受体拮抗剂Bay cysLT2则无作用。这一结果初步表明TGF-β1与CysLT1受体的相互作用。2.TGF-β1与CysLT1受体对星形胶质细胞增殖和迁移功能的影响为了进一步阐明TGF-β1与CysLT1受体的相互作用,观察了TGF-β1与CysLT受体相关药物对星形胶质细胞增殖及迁移的作用。流式细胞术检测结果显示,不同浓度的TGF-β1与LTD4在24h内对星形胶质细胞的增殖均没有显著影响。细胞划痕法检测显示,TGF-β1能够促进星形胶质细胞的迁移活性(划痕愈合),并呈一定的时间剂量依赖性；低浓度LTD4(0.1-10nM)也能促进星形胶质细胞的迁移,两者合用具有协同作用。NMLTC4(0.01～100nM)对星形胶质细胞迁移没有影响。TGF-β1信号抑常剂repsox能够完全逆转TGF-β1的作用；5-LOX抑制剂齐留通(zileuton), CysLT1受体选择性拮抗剂孟鲁司特(montelukast)及CysLT1受体siRNA处理后均能减弱TGF-β1的作用；但CysLT2受体选择性拮抗剂BaycysLT2对TGF-β1的作用没有显著影响。这一结果表明,CysLT1受体及其内源性配体合成酶5-LOX介导TGF-β1促进星形胶质细胞激活(迁移增强)的作用。3. TGF-β1与5-LOX/CysLT,受体的相互调节作用为了阐明TGF-β1与CysLT1受体相互作用的特点,观察了两者间的相互调节。RT-PCR、Western blot和细胞免疫荧光法检测细胞显示,TGF-β1上调5-LOX和CysLT1受体表达,对CysLT2受体表达没有显著影响。细胞免疫化学法检测显示,TGF-β1作用于星形胶质细胞6h后发生5-LOX明显的移位,从细胞浆转移至细胞核膜上,24h后恢复。ELISA法检测显示,TGF-β1作用于星形胶质细胞1.5h后细胞培养基中的CysLTs含量显著增加,12h时达到高峰,维持到24h。另一方面,RT-PCR和ELISA法检测显示,不同浓度的LTD4或NMLTC4(1-100nM)作用于星形胶质细胞,对细胞中TGF-β1的表达和释放均没有显著影响。这一结果表明,TGF-β1与CysLT1受体相互作用的形式,是TGF-β1增加CysLT1受体内源性配体的产生,并增加CysLT1受体表达,从而增强CysLT1受体的效应；但是,CysLT1受体对TGF-β1没有明显影响。4.轻度OGD诱导TGF-β1释放以往报道,轻度OGD增强CysLT1受体表达并激活星形胶质细胞,在此验证了这一现象与TGF-β1之间的关系。结果显示,OGD1h恢复不同时间(48h和72h)后,细胞的活性依次增强,LDH释放没有显著变化；OGD1h后恢复12h,TGF-β1释放显著增高。但是,中度OGD(4h)损伤诱导星形胶质细胞损伤,而TGF-β1释放没有改变。这表明轻度缺血性损伤后星形胶质细胞激活与TGF-β1/CysLT1受体调节密切相关。结论：1.星形胶质细胞活性的药理学实验中,初步表明TGF-β1与CysLT1受体及5-LOX存在相互作用。2.对星形胶质细胞激活(增殖和迁移)的作用表明,CysLT1受体及其内源性配体合成酶5-LOX介导TGF-β1促进星形胶质细胞迁移增强的作用,但它们对细胞增殖无明显影响。3.相互作用特点的研究结果表明,TGF-β1与CysLT1受体相互作用的形式是TGF-β1增加CysLT1受体内源性配体的产生,并增加CysLT1受体表达,从而增强CysLT1受体的效应；但是,CysLT1受体对TGF-β1没有明显影响。4.轻度OGD(1h)可增加TGF-β1释放,表明轻度缺血性损伤后星形胶质细胞激活与TGF-β1/CysLT1受体调节密切相关。更多还原

【Abstract】 BackgroundsInflammation after cerebral ischemia plays an important role in the pathophysiological process. After ischemia, inflammatory responses are activated in the central nervous system, pro-inflammatory molecules are expressed and secreted, and intracerebral inflammation occurs. One of the consequences is activation of astrocytes that gradually form a glial scar. Study of activation of astrocytes by inflammatory factors will contribute to understanding of the mechanism of glial scar formation and the relevant interventions. The inflammatory factors induced by erebral ischemia include transforming growth factor beta1(TGF-β1) and cysteinyl leukotrienes (CysLTs). TGF-β1is widely considered as an injury-related cytokine or growth factor, and CysLTs are a kind of important inflammatory mediators. Both of them play important roles in ischemia and traumatic brain injury as well as in other central nervous system diseases.In normal brain tissue, TGF-β1expression level is low. After cerebral ischemia, TGF-β1mRNA expression in the brain is increased in early phase (6h after ischemia), which reaches peak levels in days2to4, continues for14-26days. In the early phase of ischemia, its expression is diffusive overall whole brain, and mainly expressed in astrocytes at7-14days. This is associated with glial scar formation after brain injury. The in vitro experiments indicate that TGF-β1is able to induce astrocyte hypertrophy and promote astrocyte migration.CysLTs are5-lipoxygenase (5-LOX) products generated from arachidonic acis, and their actions are mediated by cysteinyl leukotriene receptors (CysLT receptors), mainly including two subtypes, i.e. CysLT1and CysLT2receptors. CysLT1receptor expression level is low in normal brain tissue. In rats with focal cerebral ischemia, its expression is significantly up-regulated in the ischemic core at3,6,12h and7-14days after reperfusion. In the boundary zone, CysLT1receptor expression does not significantly change during3days, but significantly increases7-14days after reperfusion, which is primarily localized in proliferated astrocytes. Pharmacological study indicates that the CysLT1receptor antagonist pranlukast can inhibit glial scar formation after focal cerebral ischemia in mice.In the primary cultures of rat astrocytes, mild ischemic injury induced by1h of oxygen-glucose deprivation (OGD) enhances CysLT1receptor expression, which mediates astrocyte proliferation and activation. Whereas, CysLT2receptor mediates astrocyte injury induced by moderate OGD (4h). The mechanisms underlying CysLT1receptor-mediated astrocyte activation remains to be clarified. Because astrocyte activation is regulated by a variety of factors, it is possible that the effects of CysLT1receptor may result from interactions with other factors.In the peripheral tissues, TGF-β1up-regulates the expression of CysLT1receptor in bronchial smooth muscle cells, and enhanced the effect of LTD4on cell proliferation. TGF-β1also up-regulates CysLT1receptor expression in fetal lung fibroblasts. Furthermore, TGF-β1increases the expression of5-LOX and5-LOX activating protein (FLAP) as well as increases leukotriene synthesis in dendritic cells. In the central nervous system, whether and how TGF-β1interacts with CysLTs and its receptors in regulation of astrocyte activation remains to be elucidated. Especially, the interaction between TGF-β1and CysLT1receptor is the key point that will be investigated in this study.AimsIn the present study, we aimed to investigate the interactions of TGF-β1with CysLTs and CysLT1receptor in regulation of astrocyte activation in following aspects.1. At first, to preliminarily reveal the possible interaction, we determined the effects of TGF-β1as well as the drugs related to CysLT receptors on astrocyte viability; these drugs include the5-LOX inhibitor zileuton, and the agonists and antagonist of CysLT1and CysLT2receptors.2. Next, to further clarify the interaction beween TGF-β1and CysLT1receptor, we determined astrocyte activation, including proliferation and migration, induced by TGF-β1as well as CysLT receptor agonists, antagonists and specific siRNA.3. Then, to explore the properties of the interaction beween TGF-β1and CysLT1receptor, we analyzed the following two aspects. On one hand, we analyzed the actions of TGF-β1on the activation of CysLT1receptor and5-LOX, the key synthetic enzyme of the endogenous ligands CysLTs. On the other hand, we analyzed the actions of CysLTi receptor agonist on TGF-β1production.4. Finally, to elucidate the implications of the interaction beween TGF-β1and CysLT1receptor, we assessed whether mild OGD induces TGF-β1release based on previously reported findings that mild OGD enhances the expression and effects of CysLT1receptor. MethodsIn primarily cultured of rat cortical astrocytes, cell scratching was performed as a model of cell migration; MTT reduction assay was done to detect cell viability; flow cytometry was to assess cell proliferation; immunohistochemistry was to detect5-LOX nuclear translocation; ELISA was to detect CysLTs release. RT-PCR, Western blotting analysis and cellular immunofluorescence assay were performed to detect the expression of5-LOX, CysLT1receptor and CysLT2receptor. The non-selective CysLT receptor agonist LTD4, the CysLT2receptor selective agonist NMLTC4, the TGF-β1signaling inhibitor repsox, the5-LOX inhibitor zileuton, the CysLT1receptor selective antagonist montelukast and the CysLT2receptor selective antagonist Bay cysLT2were used in pharmacological treatments; and CysLT1receptor siRNA was used for gene interference of CysLT1receptor.In primary cultures of rat cortical astrocytes, ischemic injury was induced by OGD for1or4h. MTT reduction assay and LDH release were used to detect the viability and damage of astrocytes after OGD/recovery (OGD/R). ELISA was assessed to analyze the release of TGF-β1.Results1. Effects of TGF-β1and CysLT receptors on astrocyte viabilityIn the primary cultures of rat astrocytes, the results of MTT reduction assay showed that TGF-β1and the non-selective CysLT receptor agonist LTD4increased astrocyte viability, and they potentiated the effects each other. However, the CysLT2receptor agonist NMLTC4did not show any effect. Moreover, the effect of TGF-β1was attenuated by the5-LOX inhibitor zileuton and the CysLT1receptor antagonist montelukast, but not by the CysLT2receptor antagonist Bay cysLT2. These results suggested a possible interaction between TGF-β1and CysLT1receptor. 2. Effects of TGF-β1and CysLT1receptor on proliferation and migration of astrocytesTo further clarify the interaction between TGF-β1and CysLT1receptor in astrocyte activation, we assessed the effects of TGF-β1and the drugs related to CysLT1receptor on the proliferation and migration of astrocytes. Flow cytometric results showed that TGF-β1and LTD4at various concentrations did not affect astrocyte proliferation24h after exposure. Cell healing test showed that TGF-(31promoted astrocyte migration24h after scratching in a time and concentration-dependent manner. LTD4at lower concentrations (0.1-10nM) also promoted the migration, and potentiated the effect of TGF-β1on astrocyte migration. The TGF-(31signaling inhibitor repsox completely reversed the effect of TGF-β1. The5-LOX inhibitor zileuton and the CysLT1receptor antagonist montelukast as well as CysLT1receptor siRNA attenuated the effect of TGF-β1. However, the CysLT2receptor antagonist Bay cysLT2did not affect the effect of TGF-β1. These results indicated that the effect of TGF-β1on astrocyte activation (migration) can be mediated by activation of CysLT1receptor and5-LOX, the synthetic enzyme of endogenous CysLTs.3. The regulatory interactions between TGF-β1and5-LOX/CysLT1receptorTo characterize the interactions between TGF-β1and CysLT1receptor, we analyzed the regulatory interactions between them. RT-PCR, Western blotting analysis and cell immunofluorescence showed that TGF-β1up-regulated the expression of5-LOX and CysLT1receptor, but not that of CysLT2receptor. Cell immunofluorescence showed that5-LOX translocation from the cytosol to the nuclear membrane6h after exposure to TGF-β1, and the translocation disappeared24h after the exposure. ELISA results showed that TGF-β1increased the content of CysLTs in the culture media from1.5h, reached a peak at12h, and maintained until24h after exposure. On the other hand, RT-PCR and ELISA results showed that LTD4and NMLTC4at1-100nM did not affect the expression and release of TGF-β1. These results suggested that the interactions between TGF-β1and CysLT1receptor might be characterized by that TGF-β1potentiates the production of endogenous ligands of CysLT1receptor and the expression of CysLT1receptor, but not by potentiation of TGF-β1by CysLT1receptor.4. Mild OGD induces TGF-β1releaseIt has previously reported that mild OGD enhances CysLT1receptor expression and potentiates its effects. Here, we confirmed the relation of this phenomenon to TGF-β1. We found that following recovery for48and72h after1-h OGD, astrocyte viability was increased but LDH release did not change. At12h of recovery after1-h OGD, TGF-β1release was significantly increased. However, moderate OGD (4h) induced astrocyte injury, and did not affect TGF-β1release. Thses results suggested that astrocyte activation induced by mild ischemic injury might be associated with the regulation by TGF-β1/CysLT1receptor system.Conclusions:1. The pharmacological experiments of astrocyte viability preliminarily show the possible interactions of TGF-β1with CysLT1receptor and5-LOX activation.2. In the experiments of astrocyte activation (proliferation and migration), the effect of TGF-β1on astrocyte migration is mediated by activation of CysLT1receptor and5-LOX, a key synthetic enzyme of endogenous CysLTs. However, these agents do not affect astrocyte proliferation.3. The production of endogenous ligands due to5-LOX activation and the expression of CysLT1receptor are enhanced by TGF-β1, but TGF-β1is not regulated by CysLTi receptor. Namely, the interactions are characterized by potentiation of the signaling system of CysLT1receptor by TGF-β1, in other wards, the effect of TGF-β1is mediated by the CysLT1receptor signaling..4. Mild OGD induces TGF-β1release, suggesting that mild ischemic injury may enhance astrocyte activation through regualting TGF-β1/CysLT1receptor system.更多还原