IL-33促进Th2应答和抑制Th17应答的效应能够缓解小鼠缺血性脑损伤
发布时间：2018-08-07 13:53
【摘要】：白细胞介素(Interleukin, IL)-33是近年来发现的IL-1家族新成员,它作为细胞因子,可通过IL-33/ST2信号通路促进Th2型免疫反应,同时也是一种核蛋白,可参与调控基因转录。在炎性刺激下,IL-33还可以作为一种“警报素”警示组织损伤或应激。IL-33是ST2受体的唯一特异性配体,其生物学活性的发挥主要依赖与跨膜型ST2受体(ST2L)结合。IL-33调节免疫反应的作用具有多样性,取决于不同的疾病和模型。IL-33可促进Th2型炎症性疾病的发病,如哮喘、特应性皮炎和过敏反应等,然而在一些与Th2型免疫反应相关的疾病或模型,如动脉粥样硬化、多发性硬化和实验性肠炎等,IL-33可通过促进Th1/Th2平衡向Th2偏移而起到保护性作用。此外,新近研究还发现了IL-33可调节Th17型免疫应答。IL-33在多组织器官高表达,而脑和脊髓是IL-33表达量最高的器官之一,提示IL-33在调节中枢神经系统的病理生理学过程中可能起着重要作用。近年来,有关IL-33或IL-33/ST2信号通路在中枢神经系统疾病中的作用是神经免疫学的研究热点之一。 缺血性脑卒中是中老年人常见的脑血管疾病,可以导致脑组织损伤并伴随炎症反应,从而引发一系列症候群。研究表明,Th1/Th2介导的免疫应答以及Th17型免疫应答在缺血性脑损伤的炎性病理机制中发挥重要作用。IL-33作为Th2型免疫反应的关键活化分子,在缺血性脑卒中发病机制中的作用仍有待阐明。为了探讨IL-33在缺血性脑卒中发病过程中的作用及其机制,本研究利用缺血性脑卒中动物模型,观察缺血性脑损伤时IL-33及其受体ST2L表达的变化规律,并探讨IL-33对缺血性脑损伤程度的影响。论文共分为三个部分,主要内容和结果如下： 一、短暂性脑缺血后IL-33及其受体ST2L的表达 目的：检测IL-33及其受体ST2L在短暂性脑缺血后不同时程和不同脑区的表达情况,探讨缺血性脑损伤时IL-33及其受体ST2L表达的规律性。 方法：利用线栓法闭塞大脑中动脉(MCAO)30min诱导建立小鼠可逆性局灶性脑缺血再灌注损伤模型,通过半定量RT-PCR和Western blot检测缺血再灌注后超急性期(6h)、急性期(24h)和延迟期(3d)缺血侧脑组织IL-33mRNA和蛋白表达水平,并通过免疫组织化学染色观察了IL-33在不同缺血脑区(运动皮层、感觉皮层、海马和纹状体)各时间点的表达情况。此外,对IL-33的受体ST2L mRNA表达水平也进行了检测。 结果：与假手术组相比,IL-33mRNA和蛋白水平在MCAO6h和3d表达减少,ST2L mRNA表达水平在MCAO24h和3d表达减少,其它时间点IL-33和ST2L表达无显著改变。除了MCAO24h组运动皮层和纹状体阳性染色增加外,IL-33阳性细胞数在缺血后不同时程各脑区均有不同程度减少。 结论：MCAO模型小鼠缺血再灌注后IL-33和ST2L表达的总体趋势是下降的,提示短暂性脑缺血后IL-33/ST2L介导的Th2型免疫应答是下调的。 二、侧脑室注射IL-33对MCAO模型脑损伤的影响 目的：观察外源性IL-33作用于MCAO模型后,对缺血性脑损伤严重程度、脑组织炎症及脾脏免疫功能的影响。 方法：利用GST表达纯化系统构建小鼠IL-33成熟肽的重组质粒,并纯化制备具有生物学活性的IL-33蛋白。在小鼠MCAO模型前30min侧脑室注射IL-33(2μg,5μl)或PBS5μl,于缺血再灌注后不同时间点(6h,24h和3d)通过Zea Longa评分法评估神经行为学症状,TTC染色法评估脑梗死体积,HE染色观察缺血侧海马及皮层炎性细胞浸润情况,并观察脾脏大小的改变。 结果：与侧脑室注射PBS的MCAO对照组相比,侧脑室注射IL-33可减轻MCAO24h和MCAO3d的神经行为学症状(24h:2.0±0.302vs2.875±0.125;3d:1.833±0.167vs2.917±0.193；p0.05)和脑梗死体积(24h:21.89±4.8%vs36.27±1.6%;3d:8.52±4.175vs37.08±3.35%；p0.05).组织学染色显示侧脑室注射IL-33的MCAO24h和MCAO3d缺血侧海马和皮层炎性细胞浸润数量较少。此外,侧脑室注射IL-33也增加了MCAO24h和MCAO3d的脾脏指数(24h:0.36±0.02vs0.31±0.04;3d:0.30±0.05vs0.22±0.01;p0.05),脾脏萎缩不明显。与对照组相比,侧脑室注射IL-33对MCAO6h组的神经行为学症状、脑梗死体积、脑组织炎性浸润、脾脏指数及大小均无明显差异。 结论：侧脑室注射IL-33可减缓短暂性脑缺血再灌注后急性期和延迟期的脑损伤及炎症程度,并缓解了脾脏免疫功能的抑制。 三、IL-33减缓短暂性脑缺血后脑损伤的免疫学机制 目的：探讨侧脑室注射IL-33缓解MCAO模型缺血再灌注后急性期和延迟期脑损伤及炎症程度的免疫学机制。 方法：分离侧脑室注射IL-33的MCAO24h和MCAO3d组缺血脑和脾脏中的单个核细胞,通过流式细胞术检测Thl、Th2和Th17细胞亚群的改变,以及用ELISA检测血清中细胞因子IFN-γ、IL-4和IL-17的表达。 结果：与侧脑室注射PBS的MCAO对照组相比,侧脑室注射IL-33可减少MCAO24h和MCAO3d脑组织中IL-17+T细胞和IFN-γ+T细胞的百分比,并增加了IL-4+T细胞的阳性率,Th1/Th2比值下降。与此同时,侧脑室注射IL-33也减少了脾脏中IL-17+T细胞和IFN-y+T细胞的百分率,但对IL-4+T细胞的阳性率无明显影响。相应地,侧脑室注射IL-33也减少了血清中IL-17和IFN-γ的表达量,增加了IL-4的表达量。 结论：IL-33减缓短暂性脑缺血后急性期和延迟期损伤严重程度的作用可能与促进Th2型免疫反应和抑制Thl7型免疫反应有关。 本研究首次在小鼠MCAO模型的基础上检测了IL-33及其受体ST2L在缺血再灌注后超急性期到延迟期的表达情况,发现IL-33和ST2L在短暂性脑缺血后的总体表达趋势是下降的,通过侧脑室注射IL-33可显著改善MCAO模型小鼠缺血再灌注后急性期和延迟期的损伤严重程度,其可能机制是外源性IL-33促进Th1/Th2平衡向Th2偏移和抑制Th17型免疫应答有关。本研究为改善脑卒中预后和治疗提供了新思路。
[Abstract]:Interleukin (IL) -33 is a new member of the IL-1 family found in recent years. As a cytokine, it can promote Th2 type immune response by IL-33/ST2 signaling pathway. It is also a nuclear protein that can regulate gene transcription. Under inflammatory stimulation, IL-33 can also be a warning tissue damage or stress.IL as a "warning hormone" warning. -33 is the only specific ligand for the ST2 receptor. Its biological activity is mainly dependent on the diversity of the role of the transmembrane ST2 receptor (ST2L) combined with.IL-33 to regulate the immune response, depending on the different diseases and models.IL-33 can promote the pathogenesis of Th2 type inflammatory diseases, such as asthma, atopic dermatitis and allergic reactions, however, in some cases Diseases or models associated with type Th2 immunoreaction, such as atherosclerosis, multiple sclerosis, and experimental enteritis, IL-33 can play a protective role by promoting the migration of Th1/Th2 to Th2. In addition, the recent study also found that the IL-33 adjustable Th17 immune response.IL-33 is highly expressed in multiple tissues, while the brain and spinal cord are the IL-33 tables. One of the highest amounts of organs suggests that IL-33 may play an important role in the regulation of the pathophysiology of the central nervous system. In recent years, the role of IL-33 or IL-33/ST2 signaling pathways in central nervous system diseases is one of the hot topics in neuroimmunology.
Ischemic stroke is a common cerebrovascular disease in middle-aged and elderly people, which can lead to brain tissue damage and associated with inflammatory response, which leads to a series of syndrome. The study shows that Th1/Th2 mediated immune response and Th17 type immune response play an important role in the inflammatory pathological mechanism of ischemic brain damage as Th2 type immune response. The role of key activating molecules in the pathogenesis of ischemic stroke remains to be elucidated. In order to explore the role and mechanism of IL-33 in the pathogenesis of ischemic stroke, this study uses ischemic stroke animal model to observe the changes in the expression of IL-33 and its receptor ST2L in ischemic brain injury, and to explore the effect of IL-33 on ischemia. The paper is divided into three parts. The main contents and results are as follows:
1. Expression of IL-33 and its receptor ST2L after transient ischemic attack
Objective: to detect the expression of IL-33 and its receptor ST2L in different time history and different brain regions after transient ischemic stroke, and to explore the regularity of the expression of IL-33 and its receptor ST2L in ischemic brain injury.
Methods: the rat model of reversible focal cerebral ischemia reperfusion injury was induced by occlusion of the middle cerebral artery (MCAO) 30min by the method of thread thrombus, and the semi quantitative RT-PCR and Western blot were used to detect the hyperacute phase (6h) after ischemia-reperfusion, and the level of IL-33mRNA and protein expression in the cerebral tissue of the acute phase (24h) and delay period (3D), and through the immune group. The expression of IL-33 in different ischemic brain regions (motor cortex, sensory cortex, hippocampus and striatum) was observed at various time points in different ischemic brain regions. In addition, the expression level of IL-33 receptor ST2L mRNA was also detected.
Results: compared with the sham operation group, the expression of IL-33mRNA and protein decreased in MCAO6h and 3D, the expression level of ST2L mRNA decreased in MCAO24h and 3D, and there was no significant change in the expression of IL-33 and ST2L at other time points. Except for the positive staining of the motor cortex and striatum in the MCAO24h group, the number of IL-33 positive cells was all in the different brain regions after the ischemia. There is a decrease in different degrees.
Conclusion: the overall trend of IL-33 and ST2L expression in the MCAO model mice after ischemia-reperfusion was decreased, suggesting that the Th2 type immune response mediated by IL-33/ST2L was downregulated after transient ischemic stroke.
Two, the effect of intracerebroventricular injection of IL-33 on MCAO brain injury.
AIM: To observe the effects of exogenous IL-33 on the severity of ischemic brain injury, brain inflammation and spleen immune function in MCAO model.
Methods: the recombinant plasmid of mouse IL-33 mature peptide was constructed by GST expression and purification system, and the biological activity of IL-33 protein was purified. IL-33 (2 mu g, 5 U L) or PBS5 Mu l were injected into the ventricle of 30min side of the mouse MCAO model before the mice, and the neurobehavioral symptoms were evaluated at different time points after ischemia reperfusion. The volume of cerebral infarction was assessed by TTC staining. Inflammatory cell infiltration in hippocampus and cortex was observed by HE staining, and the size of spleen was also observed.
Results: compared with the MCAO control group injected with PBS in the lateral ventricle, the lateral ventricle injection of IL-33 could reduce the neurobehavioral symptoms of MCAO24h and MCAO3d (24h:2.0 + 0.302vs2.875 + 0.125; 3d:1.833 + 0.167vs2.917 + 0.193; P0.05) and the volume of cerebral infarction (24h:21.89 + 4.8%vs36.27 + 1.6%; 8.52 + 3.35%); histological staining showed In the lateral ventricle, the number of MCAO24h and MCAO3d in the hippocampal and cortical inflammatory cells in the ischemic side of the IL-33 was less. In addition, the injection of IL-33 in the lateral ventricle also increased the spleen index of MCAO24h and MCAO3d (24h:0.36 + 0.02vs0.31 + 0.04; 3d:0.30 + 0.05vs0.22 + 0.01; P0.05), and the spleen atrophy was unknown. There were no significant differences in neurobehavioral symptoms, infarct volume, inflammatory infiltration in the brain, spleen index and size.
Conclusion: the injection of IL-33 in the lateral ventricle can slow down the brain damage and inflammation in the acute and delayed period of transient cerebral ischemia and reperfusion, and alleviate the suppression of spleen immune function.
Three, IL-33 slowed down the immunological mechanism of brain injury after transient ischemic attack.
AIM: To investigate the immunological mechanism of intraventricular injection of IL-33 to alleviate acute and delayed brain injury and inflammation in MCAO model after ischemia-reperfusion.
Methods: the ischemic brain and spleen cells in group MCAO24h and MCAO3d of IL-33 were injected into the lateral ventricle, and the changes of Thl, Th2 and Th17 cell subgroups were detected by flow cytometry, and the expression of serum cytokines IFN- y, IL-4 and IL-17 in serum was detected by ELISA.
Results: compared with the MCAO control group injected with PBS in the lateral ventricle, the lateral ventricle injection of IL-33 could reduce the percentage of IL-17+T cells and IFN- gamma +T cells in the MCAO24h and MCAO3d brain tissues, and increased the positive rate of IL-4+T cells and the Th1/Th2 ratio decreased. Meanwhile, the lateral ventricle injection IL-33 also reduced the percentage of the spleen and the cells. There was no significant effect on the positive rate of IL-4+T cells. Accordingly, the injection of IL-33 in the lateral ventricle also reduced the expression of IL-17 and IFN- gamma in the serum, and increased the expression of IL-4.
Conclusion: the effect of IL-33 on reducing the severity of acute and delayed injury after transient ischemic stroke may be related to the promotion of Th2 type immune response and the inhibition of Thl7 type immune response.
This study was the first to detect the expression of IL-33 and its receptor ST2L in the hyperacute to delay period after ischemia reperfusion on the basis of the mouse MCAO model. It was found that the overall expression trend of IL-33 and ST2L was decreased after transient ischemic stroke. The acute phase after ischemia reperfusion in MCAO model mice could be improved significantly through the injection of IL-33 in the lateral ventricle. The possible mechanism of injury severity in the delay period is that exogenous IL-33 promotes the migration of Th1/Th2 balance to Th2 and inhibits the Th17 type immune response. This study provides a new idea to improve the prognosis and treatment of stroke.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.3
本文编号：2170225
[Abstract]:Interleukin (IL) -33 is a new member of the IL-1 family found in recent years. As a cytokine, it can promote Th2 type immune response by IL-33/ST2 signaling pathway. It is also a nuclear protein that can regulate gene transcription. Under inflammatory stimulation, IL-33 can also be a warning tissue damage or stress.IL as a "warning hormone" warning. -33 is the only specific ligand for the ST2 receptor. Its biological activity is mainly dependent on the diversity of the role of the transmembrane ST2 receptor (ST2L) combined with.IL-33 to regulate the immune response, depending on the different diseases and models.IL-33 can promote the pathogenesis of Th2 type inflammatory diseases, such as asthma, atopic dermatitis and allergic reactions, however, in some cases Diseases or models associated with type Th2 immunoreaction, such as atherosclerosis, multiple sclerosis, and experimental enteritis, IL-33 can play a protective role by promoting the migration of Th1/Th2 to Th2. In addition, the recent study also found that the IL-33 adjustable Th17 immune response.IL-33 is highly expressed in multiple tissues, while the brain and spinal cord are the IL-33 tables. One of the highest amounts of organs suggests that IL-33 may play an important role in the regulation of the pathophysiology of the central nervous system. In recent years, the role of IL-33 or IL-33/ST2 signaling pathways in central nervous system diseases is one of the hot topics in neuroimmunology.
Ischemic stroke is a common cerebrovascular disease in middle-aged and elderly people, which can lead to brain tissue damage and associated with inflammatory response, which leads to a series of syndrome. The study shows that Th1/Th2 mediated immune response and Th17 type immune response play an important role in the inflammatory pathological mechanism of ischemic brain damage as Th2 type immune response. The role of key activating molecules in the pathogenesis of ischemic stroke remains to be elucidated. In order to explore the role and mechanism of IL-33 in the pathogenesis of ischemic stroke, this study uses ischemic stroke animal model to observe the changes in the expression of IL-33 and its receptor ST2L in ischemic brain injury, and to explore the effect of IL-33 on ischemia. The paper is divided into three parts. The main contents and results are as follows:
1. Expression of IL-33 and its receptor ST2L after transient ischemic attack
Objective: to detect the expression of IL-33 and its receptor ST2L in different time history and different brain regions after transient ischemic stroke, and to explore the regularity of the expression of IL-33 and its receptor ST2L in ischemic brain injury.
Methods: the rat model of reversible focal cerebral ischemia reperfusion injury was induced by occlusion of the middle cerebral artery (MCAO) 30min by the method of thread thrombus, and the semi quantitative RT-PCR and Western blot were used to detect the hyperacute phase (6h) after ischemia-reperfusion, and the level of IL-33mRNA and protein expression in the cerebral tissue of the acute phase (24h) and delay period (3D), and through the immune group. The expression of IL-33 in different ischemic brain regions (motor cortex, sensory cortex, hippocampus and striatum) was observed at various time points in different ischemic brain regions. In addition, the expression level of IL-33 receptor ST2L mRNA was also detected.
Results: compared with the sham operation group, the expression of IL-33mRNA and protein decreased in MCAO6h and 3D, the expression level of ST2L mRNA decreased in MCAO24h and 3D, and there was no significant change in the expression of IL-33 and ST2L at other time points. Except for the positive staining of the motor cortex and striatum in the MCAO24h group, the number of IL-33 positive cells was all in the different brain regions after the ischemia. There is a decrease in different degrees.
Conclusion: the overall trend of IL-33 and ST2L expression in the MCAO model mice after ischemia-reperfusion was decreased, suggesting that the Th2 type immune response mediated by IL-33/ST2L was downregulated after transient ischemic stroke.
Two, the effect of intracerebroventricular injection of IL-33 on MCAO brain injury.
AIM: To observe the effects of exogenous IL-33 on the severity of ischemic brain injury, brain inflammation and spleen immune function in MCAO model.
Methods: the recombinant plasmid of mouse IL-33 mature peptide was constructed by GST expression and purification system, and the biological activity of IL-33 protein was purified. IL-33 (2 mu g, 5 U L) or PBS5 Mu l were injected into the ventricle of 30min side of the mouse MCAO model before the mice, and the neurobehavioral symptoms were evaluated at different time points after ischemia reperfusion. The volume of cerebral infarction was assessed by TTC staining. Inflammatory cell infiltration in hippocampus and cortex was observed by HE staining, and the size of spleen was also observed.
Results: compared with the MCAO control group injected with PBS in the lateral ventricle, the lateral ventricle injection of IL-33 could reduce the neurobehavioral symptoms of MCAO24h and MCAO3d (24h:2.0 + 0.302vs2.875 + 0.125; 3d:1.833 + 0.167vs2.917 + 0.193; P0.05) and the volume of cerebral infarction (24h:21.89 + 4.8%vs36.27 + 1.6%; 8.52 + 3.35%); histological staining showed In the lateral ventricle, the number of MCAO24h and MCAO3d in the hippocampal and cortical inflammatory cells in the ischemic side of the IL-33 was less. In addition, the injection of IL-33 in the lateral ventricle also increased the spleen index of MCAO24h and MCAO3d (24h:0.36 + 0.02vs0.31 + 0.04; 3d:0.30 + 0.05vs0.22 + 0.01; P0.05), and the spleen atrophy was unknown. There were no significant differences in neurobehavioral symptoms, infarct volume, inflammatory infiltration in the brain, spleen index and size.
Conclusion: the injection of IL-33 in the lateral ventricle can slow down the brain damage and inflammation in the acute and delayed period of transient cerebral ischemia and reperfusion, and alleviate the suppression of spleen immune function.
Three, IL-33 slowed down the immunological mechanism of brain injury after transient ischemic attack.
AIM: To investigate the immunological mechanism of intraventricular injection of IL-33 to alleviate acute and delayed brain injury and inflammation in MCAO model after ischemia-reperfusion.
Methods: the ischemic brain and spleen cells in group MCAO24h and MCAO3d of IL-33 were injected into the lateral ventricle, and the changes of Thl, Th2 and Th17 cell subgroups were detected by flow cytometry, and the expression of serum cytokines IFN- y, IL-4 and IL-17 in serum was detected by ELISA.
Results: compared with the MCAO control group injected with PBS in the lateral ventricle, the lateral ventricle injection of IL-33 could reduce the percentage of IL-17+T cells and IFN- gamma +T cells in the MCAO24h and MCAO3d brain tissues, and increased the positive rate of IL-4+T cells and the Th1/Th2 ratio decreased. Meanwhile, the lateral ventricle injection IL-33 also reduced the percentage of the spleen and the cells. There was no significant effect on the positive rate of IL-4+T cells. Accordingly, the injection of IL-33 in the lateral ventricle also reduced the expression of IL-17 and IFN- gamma in the serum, and increased the expression of IL-4.
Conclusion: the effect of IL-33 on reducing the severity of acute and delayed injury after transient ischemic stroke may be related to the promotion of Th2 type immune response and the inhibition of Thl7 type immune response.
This study was the first to detect the expression of IL-33 and its receptor ST2L in the hyperacute to delay period after ischemia reperfusion on the basis of the mouse MCAO model. It was found that the overall expression trend of IL-33 and ST2L was decreased after transient ischemic stroke. The acute phase after ischemia reperfusion in MCAO model mice could be improved significantly through the injection of IL-33 in the lateral ventricle. The possible mechanism of injury severity in the delay period is that exogenous IL-33 promotes the migration of Th1/Th2 balance to Th2 and inhibits the Th17 type immune response. This study provides a new idea to improve the prognosis and treatment of stroke.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.3
【参考文献】
相关期刊论文 前1条
1 苏革,黄荣芳,闻立时,成会明;采用热丝CVD法在多种基材上沉积金刚石薄膜[J];表面技术;1998年01期
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