基于RIP1-RIP3-MLKL信号通路探讨三七总皂苷对拟缺血再灌注脑微血管内皮细胞Necroptosis的干预作用

发布时间：2018-04-27 03:23

本文选题：脑微血管内皮细胞 + necroptosis　；参考：《北京中医药大学》2017年硕士论文

【摘要】：近些年,国内外学者发现了一种新的细胞死亡形式"Necroptosis",它是由死亡受体介导的,被一系列信号传导通路所调控的caspase非依赖性的细胞死亡方式,同时具有坏死和凋亡的特征。这种细胞死亡形式最早在小鼠大脑中动脉缺血再灌注损伤模型中发现,随后多个实验证实necroptosis参与了缺血缺氧造成的脑组织损害。三七是我国著名的传统中药材,临床广泛应用于缺血性脑卒中的治疗。三七总皂苷(total saponins ofpanax notoginseseng,PNS)是中药三七的主要药效组分,前期研究发现三七总皂苷能明显降低实验性脑缺血大鼠的神经功能评分,减少脑梗死体积,减轻脑水肿,其发挥脑保护的作用与阻抑脑缺血后血管炎症损伤、促进血管修复和保护血管内皮细胞密切相关。因此,在前期实验研究的基础上,本实验利用缺氧缺糖复氧复糖法联合caspase抑制剂z-VAD-FMK干预制备了缺血再灌注损伤脑微血管内皮细胞necroptosis模型;观察了三七总皂苷对缺血再灌注损伤诱导的脑微血管内皮细胞发生necroptosis及其RIP1-RIP3-MLKL信号通路相关因子表达的影响,以深入揭示三七总皂昔在脑缺血再灌注损伤中发挥血管内皮保护作用的分子机制。目的:探讨三七总皂苷对缺血再灌注损伤诱导的脑微血管内皮细胞necroptosis的影响,以及对RIP1-RIP3-MLKL信号转导通路和线粒体损伤的调节作用,以揭示三七总皂苷在脑缺血再灌注损伤中发挥抗脑微血管内皮细胞necroptosis的分子机制,为三七总皂苷治疗缺血性脑中风的药理机制提供新的科学诠释。方法:1.缺血再灌注损伤脑微血管内皮细胞necroptosis模型的建立:利用原代培养的大鼠脑微血管内皮细胞(brain microvascular endothelial cells,BMECs),首先采用缺氧缺糖复氧复糖的方法筛选了最佳损伤时间点,制备拟缺血再灌注损伤(ischemia-reperfusion injury,IRI)模型。在此基础上利用caspase抑制剂z-VAD-FMK进行干预,分别采用CCK-8检测、透射电镜观察、Annexin V-FITC/PI双染色法进行流式细胞分析,观察造模后细胞的死亡特征,建立necroptosis细胞模型。2.观察三七总皂苷对缺血再灌注损伤脑微血管内皮细胞necroptosis的影响:将培养传至第3代的大鼠脑微血管内皮细胞随机分成4组:正常组、IRI+z-VAD-FMK组、IRI+z-VAD-FMK+PNS组和IRI+z-VAD-FMK+Nec-1组。除正常组外,其余三组均按照上述方法进行造模。IRI+z-VAD-FMK+PNS组,细胞在造模前3h和造模过程中给药,三七总皂苷的给药浓度是22μg/ml。IRI+z-VAD-FMK+Nec-1组,细胞在造模前30min及造模过程中按10μmol/L浓度加入necroptosis特异性阻断剂necstatin-1(Nec-1)。造模及处理结束后,采用CCK-8检测各组细胞活性,透射电镜观察细胞超微结构和形态变化,Annexin V-FITC/PI双染色法利用流式细胞仪检测细胞死亡方式。3.三七总皂苷对缺血再灌注损伤脑微血管内皮细胞RIP1-RIP3-MLKL信号通路的影响:将传至第3代的大鼠脑微血管内皮细胞和7代以后的人脑微血管内皮细胞随机分成 4 组:正常组、IRI+z-VAD-FMK 组、IRI+z-VAD-FMK+PNS 组和 IRI+z-VAD-FMK+Nec-1组。给药方法同以上。采用荧光定量PCR(RT-PCR)和免疫印迹(Western Blotting)法检测RIP1-RIP3-MLKL信号通路中RIP1、RIP3、MLKL的mRNA及蛋白磷酸化水平。4.三七总皂苷对缺血再灌注损伤脑微血管内皮细胞necroptosis线粒体膜电位变化的影响:细胞的分组同上,采用RT-PCR和Western Blotting法检测各组细胞PGAM5、Drp1的mRNA及蛋白表达水平;通过JC-1染色,分别用流式细胞仪和荧光倒置相差显微镜观察各组细胞线粒体膜电位的变化。结果:1.制备了拟缺血再灌注损伤脑微血管内皮细胞necroptosis模型:缺氧缺糖2h复氧复糖8h时,OD值降低明显(P0.01),细胞损伤明显且损伤主要发生在再灌后4-8h,比较符合缺血再灌注损伤特点,因此确定该时间点作为模拟缺血再灌注损伤的最佳时间点。加入z-VAD-FMK后细胞具有明显坏死细胞特征,necroptosis特异性抑制剂Nec-1可显著降低Q2象限细胞百分比率,提高细胞活性(P0.05),提示细胞在这一过程中发生了 necroptosis。2.三七总皂苷可减轻缺血再灌注损伤脑微血管内皮细胞发生necroptosis:与IRI+z-VAD-FMK 组相比,IRI+z-VAD-FMK+PNS 组与 IRI+z-VAD-FMK+Nec-1 组细胞活性明显上升(P0.01,P0.01),细胞结构损伤有所改善,细胞核和细胞膜较完整,线粒体损伤改善;Q2象限细胞比率显著降低(P0.05),Q3象限比率显著升高(P0.01),具有统计学意义。3.三七总皂苷对缺血再灌注损伤脑微血管内皮细胞RIP1-RIP3-MLKL信号通路有下调作用:造模后,RIP1-RIP3-MLKL信号转导通路被激活,RIP1、RIP3、MLKLmRNA表达和蛋白磷酸化水平明显升高,与IRI+z-VAD-FMK组相比较,三七总皂苷能够降低三种信号因子蛋白磷酸化及mRNA的表达,与Nec-1显示出相似作用。4.三七总皂苷可降低缺血再灌注损伤脑微血管内皮细胞necroptosis线粒体损伤:造模后,大鼠脑微血管内皮细胞PGAM5、Drp1的mRNA和蛋白表达明显升高,线粒体膜电位下降;与IRI+z-VAD-FMK组相比,IRI+z-VAD-FMK+PNS组和IRI+z-VAD-FMK+Nec-1组细胞PGAM5、Drp1mRNA和蛋白表达均下降,线粒体膜电位上升。结论:1.缺氧缺糖复氧复糖法联合z-VAD-FMK干预后脑微血管内皮细胞的死亡特征符合necroptosis的特点,提示该方法可制备necroptosis模型。2.三七总皂苷可有效降低上述方法诱导的脑微血管内皮细胞necroptosis的发生,其内在机制与抑制RIP1-RIP3-MLKL信号通路活化,继而下调下游PGAM5、Drp1的表达,减轻线粒体损伤有关,这可能是三七总皂苷在脑缺血再灌注损伤中发挥血管内皮保护作用的分子机制之一。该结论为中药三七的"通络"作用提供了现代生物学基础。
[Abstract]:In recent years, domestic and foreign scholars have discovered a new form of cell death "Necroptosis", which is a death receptor mediated, caspase non dependent cell death mode regulated by a series of signal transduction pathways, and has the characteristics of necrosis and apoptosis. This form of cell death was the earliest in the middle cerebral artery ischemia and reperfusion in mice. It was found in the damage model that the subsequent experiments confirmed that necroptosis was involved in the brain tissue damage caused by ischemia and hypoxia. 37 it was a famous traditional Chinese medicine in China, and was widely used in the treatment of ischemic stroke. 37 total saponins (total saponins ofPanax Notoginseseng, PNS) were the main components of Chinese medicine 37, the preliminary study It is found that 37 total saponins can obviously reduce the neurological function score of experimental cerebral ischemia rats, reduce the volume of cerebral infarction and reduce the brain edema. The effect of the total saponins can be closely related to the inhibition of vascular inflammation after cerebral ischemia, the promotion of vascular repair and the protection of vascular endothelial cells. The necroptosis model of cerebral microvascular endothelial cells damaged by ischemia-reperfusion injury was prefabricated by hypoxic and glucose deficient reoxygenation combined with caspase inhibitor z-VAD-FMK, and the effects of 37 total saponins on the expression of necroptosis and RIP1-RIP3-MLKL signaling pathway related factors in cerebral microvascular endothelial cells induced by ischemia-reperfusion injury were observed. To explore the molecular mechanism of vascular endothelium protection of 37 total soap shake in cerebral ischemia reperfusion injury. Objective: To explore the effect of 37 total saponins on the necroptosis of cerebral microvascular endothelial cells induced by ischemia-reperfusion injury, and the regulation of RIP1-RIP3-MLKL signal transduction pathway and mitochondrial damage to reveal three Seven total saponins play the molecular mechanism of anti cerebral microvascular endothelial cell necroptosis in cerebral ischemia reperfusion injury, and provide a new scientific interpretation for the pharmacological mechanism of 37 total saponins in the treatment of ischemic stroke. Method: the establishment of the necroptosis model of cerebral microvascular endothelial cells in 1. ischemia reperfusion injury: using the primary culture of rat brain microsphere Brain microvascular endothelial cells (BMECs), first of all, the optimal time point of injury time was screened by the method of hypoxia and glucose deficiency complex carbohydrate. The model of pseudo ischemia-reperfusion injury (ischemia-reperfusion injury, IRI) was prepared. On the basis of this, the caspase inhibitor z-VAD-FMK was used to intervene with CCK-8 detection and transmission, respectively. Electron microscope observation, Annexin V-FITC/PI double staining method for flow cytometry, observed the death characteristics of the cells after the model, and established the necroptosis cell model.2. to observe the effect of 37 total saponins on the cerebral microvascular endothelial cell necroptosis in the ischemic reperfusion injury: the cultured rat cerebral microvascular endothelial cells were divided into 4 groups randomly. The normal group, the IRI+z-VAD-FMK group, the IRI+z-VAD-FMK+PNS group and the IRI+z-VAD-FMK+Nec-1 group. Except the normal group, the other three groups were made the model.IRI+z-VAD-FMK+PNS group according to the above methods. The cells were given in the 3H and the mold making process before the model, and the concentration of the 37 total saponins was 22 Mu g/ ml.IRI+z-VAD-FMK+Nec-1 group. The cells were in the 30min and the model before making the mold. The necroptosis specific blocking agent necstatin-1 (Nec-1) was added to the concentration of 10 micron mol/L. After the model and treatment, the cell activity was detected by CCK-8, the ultrastructure and the morphological changes were observed by transmission electron microscopy. The Annexin V-FITC/PI double staining method was used to detect the cell death of.3. 37 total saponins by flow cytometry. The effect of perfusion injury on RIP1-RIP3-MLKL signal pathway of cerebral microvascular endothelial cells: third generations of rat brain microvascular endothelial cells and 7 generations of human brain microvascular endothelial cells were randomly divided into 4 groups: normal group, IRI+z-VAD-FMK group, IRI+z-VAD-FMK+PNS group and IRI+z-VAD-FMK+ Nec-1 group. PCR (RT-PCR) and immunoblotting (Western Blotting) assay were used to detect the effects of RIP1, RIP3, MLKL, and protein phosphorylation level.4. 37 total saponins on the changes in the necroptosis mitochondrial membrane potential of cerebral microvascular endothelial cells in the RIP1-RIP3-MLKL signaling pathway. The mRNA and protein expression levels of PGAM5 and Drp1 were measured in each group. The changes in mitochondrial membrane potential of each cell were observed by flow cytometry and fluorescence inversion phase contrast microscope by JC-1 staining. Results: 1. the necroptosis model of cerebral microvascular endothelial cells with ischemic reperfusion injury was prepared. The decrease of OD value was reduced when hypoxia and glucose deficient 2H reoxygenate 8h was reduced. Obviously (P0.01), the cell damage is obvious and the damage mainly occurs after reperfusion 4-8h, which is more in line with the characteristics of ischemia-reperfusion injury. Therefore, the time point is the best time point for simulating the ischemia reperfusion injury. After the addition of z-VAD-FMK, the cells have obvious necrotic cell characteristics, and the necroptosis specific inhibitor Nec-1 can significantly reduce the Q2 quadrant. The percentage of cells increased cell activity (P0.05), suggesting that necroptosis.2. 37 total saponins in this process could reduce the incidence of necroptosis: in cerebral microvascular endothelial cells from ischemia reperfusion injury and IRI+z-VAD-FMK group, and the cell activity of IRI+z-VAD-FMK+PNS group and IRI+ z-VAD-FMK+Nec-1 group increased significantly (P0.01, P0.01). Cell structure damage improved, nuclear and cell membrane were more complete, mitochondrial damage improved, Q2 quadrant cell ratio decreased significantly (P0.05), Q3 quadrant ratio increased significantly (P0.01), and.3. 37 total saponins had a downregulation effect on RIP1-RIP3-MLKL signaling pathway of cerebral microvascular intravascular cells after ischemia-reperfusion injury: after modeling, RI The P1-RIP3-MLKL signal transduction pathway was activated, and the expression of RIP1, RIP3, MLKLmRNA and protein phosphorylation increased significantly. Compared with the IRI+z-VAD-FMK group, 37 total saponins could reduce the phosphorylation of three signal factor proteins and the expression of mRNA. The similar effect of.4. 37 total saponins to Nec-1 could reduce the cerebral microvascular injury of ischemia reperfusion injury. Necroptosis mitochondrial damage in the skin cells: after the model, the rat brain microvascular endothelial cells PGAM5, Drp1 mRNA and protein expression significantly increased, the mitochondrial membrane potential decreased. Compared with the IRI+z-VAD-FMK group, IRI+z-VAD-FMK+PNS and IRI+z-VAD-FMK+Nec-1 group PGAM5, Drp1mRNA and protein expression decreased, mitochondrial membrane potential increased. Conclusion: 1. deficiency The characteristics of the death of cerebral microvascular endothelial cells after z-VAD-FMK intervention with oxygen glucose deficiency combined with carbohydrate and reoxygenation method are consistent with the characteristics of necroptosis. It is suggested that the preparation of necroptosis model.2. 37 total saponins can effectively reduce the occurrence of necroptosis in the cerebral microvascular endothelial cells induced by the above method, and its intrinsic mechanism and the suppression of RIP1-RIP3-MLKL signal The pathway activation, then down down downstream PGAM5, Drp1 expression, alleviates mitochondrial damage, which may be one of the molecular mechanisms of 37 total saponins playing the protective role of vascular endothelium in cerebral ischemia reperfusion injury. This conclusion provides a biological basis for the "collaterals" of Chinese medicine 37.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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