P2X7受体参与炎性刺激大鼠硬脑膜三叉神经节中NALP3炎性小体的激活
发布时间：2018-09-03 07:38
【摘要】：背景：偏头痛是一种严重的脑功能紊乱性疾病,其全球患病率在12%左右,严重影响患者的日常工作和生活。其临床表现为单侧的搏动性头痛,日常活动可加剧疼痛程度,常伴有恶心、呕吐等症状。三叉神经血管系统(trigemino vascular system, TGVS)的激活是偏头痛发生发展过程中最重要的事件,在此过程中三叉神经末梢释放血管活性神经肽类物质,后者导致硬脑膜上发生神经源性炎症,从而使硬脑膜伤害性感受器长时程敏化,临床表现为严重的偏侧头痛,现在认为这种现象系初级痛觉神经元发生了周围敏化。炎性小体系组织细胞在受到内外源性刺激时组装合成的一类多蛋白复合体,能够促进细胞因子IL-1p和IL-18的成熟和分泌。成熟的IL-1β通过激活三叉神经节卫星胶质细胞,引起一系列炎症反应,进而导致偏头痛外周敏化的发生。炎性小体有4种类型:NALP1,NALP3, NLRC4和AIM2。其中,NALP3炎性小体是最重要的一种类型。研究已经证实激活炎性小体主要通过P2X7受体、活性氧及组织蛋白酶三条通路。但是目前罕有关于NALP3炎性小体参与偏头痛外周敏化的发生及其激活途径方面的研究。因此我们通过建立炎性刺激大鼠硬脑膜模型(常用的偏头痛模型)提出假设：炎性刺激大鼠硬脑膜模型中可以观察到活化的NALP3炎性小体,此外,P2X7受体可能参与其激活。 目的：通过采用炎性刺激大鼠硬脑膜疼痛模型,探讨嘌呤受体P2X配体门控性离子通道7(P2X7R)是否参与偏头痛发作过程中三叉神经节内NALP3炎性小体的激活。 方法：健康成年雄性SD大鼠42只,免疫印迹实验取27只随机分为9组,每组3只,分别为空白对照组、致炎剂3h组、致炎剂6h组、致炎剂1d组、致炎剂2d组、致炎剂3d组、生理盐水对照组、致炎剂2d对照组和亮蓝G(BBG)治疗组。免疫荧光双重染色实验取15只随机分为3组,每组5只,分别为空白对照组、生理盐水对照组和致炎剂2d组。观察各组大鼠P2X7R和NALP3在三叉神经节中的表达情况,并且运用P2X7受体特异性拮抗剂亮蓝G(Brilliant Blue G, BBG)干预后,观察各组大鼠P2X7R. NALP3的表达。 结果：①免疫荧光双重染色结果：NALP3蛋白定位表达于三叉神经元细胞内,而P2X7R主要表达于卫星胶质细胞。致炎剂组NALP3及P2X7R荧光强度明显高于空白对照组和生理盐水对照组,说明致炎剂刺激大鼠硬脑膜能成功诱导三叉神经节中NALP3及P2X7R蛋白的表达。②免疫印迹结果：与空白对照组相比,各致炎剂组NALP3、P2X7R蛋白表达量均明显升高,具有统计学意义(P0.05)；各蛋白表达量随给药次数及药物作用时间基本呈正相关。BBG能有效抑制P2X7R和NALP3的蛋白表达,与空白组和生理盐水对照组相比,差异具有统计学意义(P0.05)。 结论：在炎性刺激大鼠硬脑膜模型中可以观察到三叉神经节内P2X7R和NALP3炎性小体的激活,此外,P2X7R特异性拮抗剂BBG能有效地抑制NALP3蛋白的表达,说明P2X7受体参与炎性小体的激活。
[Abstract]:Background: migraine is a severe disorder of brain function. The global prevalence rate of migraine is about 12%, which seriously affects the daily work and life of patients. Its clinical manifestation is unilateral pulsatile headache, daily activities can aggravate the degree of pain, often accompanied by nausea, vomiting and other symptoms. The activation of (trigemino vascular system, TGVS) in the trigeminal vascular system is the most important event in the occurrence and development of migraine, during which the trigeminal nerve endings release vasoactive neuropeptides, which lead to neurogenic inflammation on the dura mater. So that the dura nociceptive receptors were sensitized for a long time, and the clinical manifestations were severe hemiheadache. It is now believed that this phenomenon is caused by peripheral sensitization of primary nociceptive neurons. A class of polyprotein complexes assembled by inflammatory small tissue cells stimulated by exogenous and exogenous stimuli can promote the maturation and secretion of cytokines IL-1p and IL-18. Mature IL-1 尾 activates satellite glial cells in the trigeminal ganglion, causing a series of inflammatory reactions, which leads to peripheral sensitization of migraine. Inflammatory corpuscles have four types: NALP1, NALP3, NLRC4 and AIM2. The inflammatory corpuscles of NALP3 are the most important type. It has been demonstrated that the activation of inflammatory corpuscles is mainly via three pathways: P2X7 receptor, reactive oxygen species and cathepsin. However, there are few studies on the role of NALP3 inflammatory corpuscles in the pathogenesis and activation of peripheral sensitization of migraine. Therefore, we hypothesized that activated NALP3 inflammatory corpuscles can be observed in the inflammatory stimulation rat dural model (the commonly used migraine model), in addition, P2X7 receptor may be involved in its activation. Aim: to investigate whether purine receptor P2X ligand gated ion channel 7 (P2X7R) is involved in the activation of NALP3 inflammatory corpuscles in trigeminal ganglion during migraine attack. Methods: Forty-two healthy adult male SD rats were randomly divided into 9 groups with 3 rats in each group: the blank control group, the inflammatory agent group for 3 hours, the inflammatory agent group for 6 hours, the inflammatory agent group for 1 day, the inflammatory agent group for 2 days, and the inflammatory agent group for 3 days. Normal saline control group, inflammatory agent 2 days control group and bright blue G (BBG) treatment group. Fifteen rats were randomly divided into 3 groups: blank control group, normal saline control group and inflammatory agent group for 2 days. The expression of P2X7R and NALP3 in trigeminal ganglion was observed, and P2X7R was observed after treated with brilliant blue G (Brilliant Blue G, BBG), a specific antagonist of P2X7 receptor. Expression of NALP3. Results the results of double immunofluorescence staining showed that the protein was localized in trigeminal neurons, while P2X7R was mainly expressed in satellite glial cells. The fluorescence intensity of NALP3 and P2X7R in the inflammatory group was significantly higher than that in the blank control group and the saline control group. The results showed that the expression of NALP3 and P2X7R protein in trigeminal ganglion could be induced successfully by stimulation of dural with inflammatory agent. The results showed that compared with the blank control group, the expression of NALP3,P2X7R protein in each group was significantly increased (P0.05). The expression of each protein was positively correlated with the times of administration and the time of drug action. BBG could effectively inhibit the protein expression of P2X7R and NALP3 compared with the blank group and normal saline control group the difference was statistically significant (P0.05). Conclusion: the activation of P2X7R and NALP3 inflammatory corpuscles in trigeminal ganglion can be observed in the rat dural model of inflammatory stimulation. In addition, BBG, a specific antagonist of P2X7R, can effectively inhibit the expression of NALP3 protein, indicating that P2X7 receptor is involved in the activation of inflammatory corpuscles.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R747.2
本文编号：2219341
[Abstract]:Background: migraine is a severe disorder of brain function. The global prevalence rate of migraine is about 12%, which seriously affects the daily work and life of patients. Its clinical manifestation is unilateral pulsatile headache, daily activities can aggravate the degree of pain, often accompanied by nausea, vomiting and other symptoms. The activation of (trigemino vascular system, TGVS) in the trigeminal vascular system is the most important event in the occurrence and development of migraine, during which the trigeminal nerve endings release vasoactive neuropeptides, which lead to neurogenic inflammation on the dura mater. So that the dura nociceptive receptors were sensitized for a long time, and the clinical manifestations were severe hemiheadache. It is now believed that this phenomenon is caused by peripheral sensitization of primary nociceptive neurons. A class of polyprotein complexes assembled by inflammatory small tissue cells stimulated by exogenous and exogenous stimuli can promote the maturation and secretion of cytokines IL-1p and IL-18. Mature IL-1 尾 activates satellite glial cells in the trigeminal ganglion, causing a series of inflammatory reactions, which leads to peripheral sensitization of migraine. Inflammatory corpuscles have four types: NALP1, NALP3, NLRC4 and AIM2. The inflammatory corpuscles of NALP3 are the most important type. It has been demonstrated that the activation of inflammatory corpuscles is mainly via three pathways: P2X7 receptor, reactive oxygen species and cathepsin. However, there are few studies on the role of NALP3 inflammatory corpuscles in the pathogenesis and activation of peripheral sensitization of migraine. Therefore, we hypothesized that activated NALP3 inflammatory corpuscles can be observed in the inflammatory stimulation rat dural model (the commonly used migraine model), in addition, P2X7 receptor may be involved in its activation. Aim: to investigate whether purine receptor P2X ligand gated ion channel 7 (P2X7R) is involved in the activation of NALP3 inflammatory corpuscles in trigeminal ganglion during migraine attack. Methods: Forty-two healthy adult male SD rats were randomly divided into 9 groups with 3 rats in each group: the blank control group, the inflammatory agent group for 3 hours, the inflammatory agent group for 6 hours, the inflammatory agent group for 1 day, the inflammatory agent group for 2 days, and the inflammatory agent group for 3 days. Normal saline control group, inflammatory agent 2 days control group and bright blue G (BBG) treatment group. Fifteen rats were randomly divided into 3 groups: blank control group, normal saline control group and inflammatory agent group for 2 days. The expression of P2X7R and NALP3 in trigeminal ganglion was observed, and P2X7R was observed after treated with brilliant blue G (Brilliant Blue G, BBG), a specific antagonist of P2X7 receptor. Expression of NALP3. Results the results of double immunofluorescence staining showed that the protein was localized in trigeminal neurons, while P2X7R was mainly expressed in satellite glial cells. The fluorescence intensity of NALP3 and P2X7R in the inflammatory group was significantly higher than that in the blank control group and the saline control group. The results showed that the expression of NALP3 and P2X7R protein in trigeminal ganglion could be induced successfully by stimulation of dural with inflammatory agent. The results showed that compared with the blank control group, the expression of NALP3,P2X7R protein in each group was significantly increased (P0.05). The expression of each protein was positively correlated with the times of administration and the time of drug action. BBG could effectively inhibit the protein expression of P2X7R and NALP3 compared with the blank group and normal saline control group the difference was statistically significant (P0.05). Conclusion: the activation of P2X7R and NALP3 inflammatory corpuscles in trigeminal ganglion can be observed in the rat dural model of inflammatory stimulation. In addition, BBG, a specific antagonist of P2X7R, can effectively inhibit the expression of NALP3 protein, indicating that P2X7 receptor is involved in the activation of inflammatory corpuscles.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R747.2
【二级参考文献】
相关期刊论文 前1条
1 刘婷;沈飞飞;韦俊超;万琪;;头痛宁对电刺激三叉神经节大鼠脑膜血流与CGRP的影响[J];中国疼痛医学杂志;2010年04期
，本文编号：2219341
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