单唾液酸神经节苷脂钠对血管内皮细胞氧化损伤的保护作用及机制

发布时间：2018-01-21 17:57

本文关键词： 神经节苷脂钠内皮细胞 PI3K GSK-3 NF-κb　出处：《吉林大学》2014年博士论文　论文类型：学位论文

【摘要】：目的大脑中动脉栓塞（middle cerebral artery oclusion,MCAO）法复制大鼠局灶性脑缺血模型，探讨单唾液酸神经节苷脂钠（monosialotetrahexosylganglioside,GM-1）对缺血性脑损伤大鼠的保护作用及机制，并通过H2O2介导的体外人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVEC）损伤模型，从体外实验角度探讨GM-1对血管内皮细胞的保护作用，初步明确磷脂酰肌醇3激酶（phosphatidylinositol3kinase,PI3K）/糖原合成酶激酶-3（glycogen synthasekinase3,GSK-3）信号通路在脑缺血性损伤中的作用。方法本研究从体内实验和体外细胞培养2个方面进行探讨，主要方法如下： 1.动物实验将大鼠随机分为假手术组（Sham组）、脑缺血组（Model组）及GM-1治疗组3组，每组10只，除Sham组外，其余2组大鼠均采用改良线栓法阻塞大鼠右侧大脑中动脉复制局灶性脑缺血模型，GM-1治疗组于造模成功当天腹腔注射GM-110mg/kg体重，每天两次，连续给药7d。Sham组及脑缺血组腹腔注射生理盐水1ml/kg体重，每天两次，连续给药7d。3组大鼠自手术清醒后每日采用改良NSS评分标准（16分制）进行神经功能评分；术后第8天处死大鼠，取脑组织行常规HE染色及免疫组化检测各组大鼠脑组织CD31、PI3K及GSK-3表达，结果用Image J图像分析软件进行半定量分析。 2.体外实验将常规培养的HUVEC分为对照组（CTRL组），H2O2处理组及GM-1低、中、高剂量（1、5、10mg/l）处理组，分别用CCK-8试剂盒检测细胞增殖能力，用流式细胞术进行细胞周期分析，Bradford法测定样本总蛋白浓度，以免疫荧光法、Western blot法检测各组培养细胞PI3K、Akt及GSK-3蛋白表达情况；并分别检测各组HUVEC细胞浆与细胞核中NF-κb的表达。进一步将HUVEC分为对照组（CTRL组），H2O2处理组，CHIR-99021处理组及LY294002处理组，用Western blot法和Real-time PCR法检测各组培养细胞细胞浆与细胞核中PI3K、Akt及GSK-3蛋白及mRNA含量。结果 1. GM-1对MCAO致大鼠脑缺血损伤的保护作用 1.1各组大鼠一般状况及神经功能评分比较动物实验结果显示，假手术组大鼠精神状态良好，活动正常，皮毛光泽，术后无神经功能障碍表现，未见任何反射异常；脑缺血组大鼠精神状态较差，活动减少，出现不同程度行为学障碍，随着饲养时间的延长，上述症状有不同程度的恢复；GM-1组大鼠于造模初期，上述症状与脑缺血组相似，随给药时间延长，，脑缺血症状改善明显，自给药第4天起，与脑缺血组比较，差异有统计学意义（P0.01）。 1.2各组大鼠脑组织HE染色结果 Sham组大鼠脑组织神经元及胶质细胞数量、形态分布正常，细胞膜完整，胞浆均匀，细胞核位于中央，染色质分布较均匀；脑缺血组大鼠脑组织在光镜下的主要改变是不同程度的神经元变性、坏死和胶质细胞反应。镜下可见缺血灶中心区以神经元变性、坏死为主，缺血灶周围胶质细胞增生活跃，并逐渐向缺血灶中心移动，缺血区胶原成分增加。GM-1治疗组大鼠脑组织神经变性及坏死情况较脑缺血组有不同程度的改善。 1.3各组大鼠脑组织CD31免疫组化结果 CD31阳性细胞呈棕色或棕黄色。在Sham组大鼠，CD31阳性细胞呈单个散在分布或排列成细条索状排列。与Sham组比较，脑缺血组大鼠缺血区周围CD31表达明显增加，GM-1组阳性信号增加，与脑缺血组比较，差异具有显著性（P0.01）。 2. GM-1对PI3K/GSK-3信号转导通路的影响 2.1各组大鼠脑组织PI3K免疫组化结果在Sham组大鼠，PI3K主要在正常神经元细胞浆中表达，胶质细胞及内皮细胞中亦有少量表达；在脑缺血组，脑缺血7d后，缺血灶中PI3K表达量减少，阳性信号主要集中在血管内皮细胞及增生的胶质细胞细胞浆，与Sham组比较，差异有统计学意义（P0.05）；GM-1组大鼠缺血灶中胶质细胞增生明显，PI3K仍主要表达于增生的胶质细胞及内皮细胞胞浆中，与脑缺血组比较，差异具有显著性（P0.01）。 2.2各组大鼠脑组织GSK-3免疫组化结果 Sham组大鼠脑组织中GSK-3表达量为6.42±0.58%，GSK-3阳性信号主要分布神经元、胶质细胞及内皮细胞细胞浆中；在脑缺血组，脑缺血7d后，缺血灶中GSK-3表达量明显增加，且主要集中在血管内皮细胞及增生的胶质细胞细胞浆中，残存的神经元细胞中亦有表达，与Sham组比较，GSK-3表达量增加，差异有显著性（P0.05）；GM-1组大鼠缺血灶GSK-3仍主要表达于增生的胶质细胞及内皮细胞胞浆中，与脑缺血组比较，其表达量明显降低，差异具有显著性（P0.01）。 3. H2O2与血管内皮细胞氧化损伤 3.1CCK-8检测结果以对照组细胞为100%，H2O2处理组细胞增殖率为75.97%，明显低于对照组（P0.001）；GM-1中、高剂量细胞增殖能力明显高于H2O2处理组，差异显著（P0.001，0.05），而与H2O2处理组比较，GM-1低剂量组细胞增殖能力差异不显著。 3.2流式细胞术结果对照组50.87%细胞处于G2/M期和S期，与H2O2处理组（13.52%）比较，组间差异显著（P0.0001）。GM-1低、中、高剂量组处于G2/M期和S期的细胞比例分别为33.95%，30.70%和28.68%，与H2O2处理组比较，差异显著（P0.001，0.01）。 4. PI3K/GSK-3信号转导途径与内皮细胞氧化损伤 4.1PI3K及GSK-3Western blot检测结果与对照组比较，H2O2处理组HUVEC PI3K表达量明显降低（P0.01），与H2O2模型组比较，GM-1中、高剂量组PI3K表达明显增加（P0.05）；与对照组比较，H2O2处理组细胞GSK-3/β表达明显升高（P0.01）；在GM-1各处理组，随着GM-1应用浓度降低，GSK-3/β蛋白表达量逐渐升高，与H2O2损伤组比较，GM-1高剂量组GSK-3/β蛋白表达量明显下降，差异有显著性（P0.05）。与H2O2处理组比较，CHIR-99021组及CHIR-99021+H2O2组PI3K和p-Akt蛋白表达量明显增加（P0.01），GSK-3表达量则明显降低（P0.01）；LY294002+H2O2组PI3K和p-GSK-3表达量显著降低（P0.01）；LY294002组和LY294002+H2O2组p-Akt表达量明显增加（P0.01）。 4.2Real-time PCR结果与对照组比较，H2O2处理组PI3K、Akt mRNA表达明显降低，差异有显著性（P0.01），而GSK-3mRNA表达量明显升高（P0.01）。与H2O2组比较，CHIR-99021组及CHIR-99021+H2O2组PI3K、Akt mRNA表达量明显增加（P0.01），GSK-3表达量则明显降低（P0.01）；LY294002+H2O2组PI3K和GSK-3表达量显著降低（P0.01）；LY294002组和LY294002+H2O2组Akt mRNA表达量明显增加（P0.01）。 5. NF-κb与内皮细胞损伤 5.1NF-κb Western blot检测结果各组HUVEC细胞胞浆中NF-κb表达量差异无显著性，而各组细胞NF-κb在细胞核中表达量差异有显著性，H2O2处理组NF-κb核/浆比值为12.07±2.96，与对照组比较，差异有显著性（P0.05）。与H2O2处理组比较，GM-1中、高剂量NF-κb核/浆比明显升高（P0.01）。 5.2免疫荧光双重染色结果 NF-κb在体外培养的HUVEC中均有表达，但各组细胞NF-κb表达部位有差异，H2O2处理组NF-κb主要在HUVEC胞浆表达，而在胞核有较低表达。图像分析结果显示，与H2O2处理组比较，GM-1高剂量组NF-κb在细胞核中表达明显增加，差异具有显著性（P0.05）。结论 1．GM-1可有效保护大脑缺血性损伤，血管内皮细胞可能为其实现此作用的一个重要治疗靶点。 2．GM-1刺激血管内皮细胞增生的作用可能与其激活PI3K/GSK-3信号转导通路及NF-κb的核转位有关。创新点 1．本研究从内皮细胞损伤修复角度探讨GM-1对脑缺血性损伤的保护作用，国内外未见报道。 2．GM-1保护脑损伤的机制相关研究较多，但本研究首次探讨其对PI3K/GSK-3信号通路及NF-κb的影响。
[Abstract]:objective
Middle cerebral artery occlusion (middle cerebral artery oclusion, MCAO) rat model of focal cerebral ischemia was established to investigate single sialic acid ganglioside sodium (monosialotetrahexosylganglioside, GM-1) on the protective effect and mechanism of ischemic brain injury in rats, and mediated by H2O2 in human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVEC) damage model, to investigate the protective effect of GM-1 on vascular endothelial cells in vitro, initially identified phosphatidylinositol 3 kinase (phosphatidylinositol3kinase, PI3K) / glycogen synthase kinase -3 (glycogen synthasekinase3 GSK-3) signaling pathway in brain ischemic injury.
Method
This study is discussed from 2 aspects: in vivo experiment and in vitro cell culture. The main methods are as follows:
1. animal experiments
The rats were randomly divided into sham operation group (group Sham), cerebral ischemia group (Model group) and GM-1 treatment group, 3 groups, 10 rats in each group, except Sham group, the other 2 rats were treated with modified artery occlusion model of focal cerebral ischemia was copied on the right side of rat brain, GM-1 in the treatment group rats by intraperitoneal injection of GM-110mg/kg on weight, two times a day, continuous administration of 7d.Sham group and cerebral ischemia group by intraperitoneal injection of saline 1ml/kg weight, two times a day, continuous administration of 7d.3 group rats awake after modified NSS daily operation standard for evaluation (16 points) neurological score big; rats were sacrificed eighth days after operation, take the brain tissue by HE staining and immunohistochemistry in brain tissue of rats with CD31, the expression of PI3K and GSK-3, the results of semi quantitative analysis using Image J image analysis software.
2. in vitro experiment
The cultured HUVEC were divided into control group (CTRL group), H2O2 treatment group and GM-1 low, high dose treatment group (1,5,10mg/l), respectively with CCK-8 assay for cell proliferation, cell cycle was analyzed by flow cytometry, the determination of total protein sample concentration Bradford, immunofluorescence Western blot, detected and cultured PI3K cells, the expression of Akt and GSK-3 protein were detected; and the expression of NF- kappa B were HUVEC cytoplasm and nucleus.
HUVEC was further divided into control group (group CTRL), H2O2 treatment group, CHIR-99021 treatment group and LY294002 treatment group. Western, blot and Real-time PCR methods were used to detect the contents of PI3K, Akt and protein and Western in cytoplasm and nucleus of cultured cells.
Result
Protective effect of 1. GM-1 on cerebral ischemia injury in rats induced by MCAO
1.1 comparison of general state and neurological function score of rats in each group
Animal experimental results showed that the rats in the sham operation group were in good spirits, normal activities, shiny coat, no postoperative nerve dysfunction, no abnormal reflex; cerebral ischemia rats, poor mental state, reduced activity, with varying degrees of behavioral disorder, with the feeding time, the symptoms have different degrees of recovery GM-1 group; rats in early stage, the symptoms and cerebral ischemia were similar, with the prolongation of injection, cerebral ischemia symptoms improved significantly, self medicine fourth days, compared with ischemia group, the difference was statistically significant (P0.01).
1.2 HE staining results of brain tissue of rats in each group
The number of neurons and glial cells in brain tissue of rats in group Sham, normal distribution, cell membrane integrity, uniform cytoplasm, the nucleus is located in the central, chromatin distribution is uniform; the main change of brain tissue in rats with cerebral ischemia group under light microscope is the degeneration of neurons in different degree, necrosis and glial cell reaction under the microscope. Visible focal ischemia in the central area of neuronal degeneration, necrosis, active focal ischemia surrounding glial cell proliferation, and gradually move to the center of focal ischemia, ischemia area of collagen increased in.GM-1 treatment group rat brain nerve degeneration and necrosis compared with ischemic group have different degrees of improvement.
1.3 immunohistochemical results of CD31 in rat brain tissue
CD31 positive cells showed brown or brown yellow. The rats in the Sham group, CD31 positive cells were scattered in a single strip or arranged in cords. Compared with group Sham, significantly increased the expression of CD31 around ischemic area of rats with cerebral ischemia group, GM-1 group, the positive signal increased, compared with ischemic group, the difference was significant (of P0.01).
Effect of 2. GM-1 on PI3K/GSK-3 signal transduction pathway
2.1 immunohistochemical results of PI3K in rat brain tissue
The rats in the Sham group, PI3K expressed mainly in the cytoplasm of normal neurons, glial cells and endothelial cells also expressed a little; in cerebral ischemia group, 7d after cerebral ischemia, reduce the amount of PI3K expression in focal ischemia, glial cell cytoplasm positive signals mainly in vascular endothelial cells and hyperplasia, compared with in Sham group, the difference was statistically significant (P0.05); glial cell hyperplasia of focal ischemia in rats of GM-1 group, PI3K is mainly expressed on the proliferation of glial cells and endothelial cells in the cytoplasm, compared with ischemia group, the difference was significant (P0.01).
2.2 immunohistochemical results of GSK-3 in rat brain tissue
The expression of GSK-3 was 6.42 + 0.58% in brain tissue in Sham rats, GSK-3 positive signals were mainly distributed in neurons, glial cells and endothelial cells in the cytoplasm; in the cerebral ischemia group, 7d after cerebral ischemia, increased the expression of GSK-3 in focal ischemia, glial cells and mainly in vascular endothelial cells and hyperplasia in the remaining neurons also expressed, compared with Sham group, the expression of GSK-3 increased significantly (P0.05); group GM-1 rat focal ischemia GSK-3 is still mainly expressed on the proliferation of glial cells and endothelial cells, and cerebral ischemia group, the expression decreased obviously the difference was significant (P0.01).
3. H2O2 and oxidative damage of vascular endothelial cells
3.1CCK-8 detection results
The cells in control group was 100%, H2O2 group cell proliferation rate was 75.97%, significantly lower than the control group (P0.001); high dose GM-1, cell proliferation was significantly higher than that in H2O2 group, significant difference (P0.001,0.05), while the treatment group compared with H2O2 group, low dose GM-1 cell proliferation ability was not significant difference.
Results of 3.2 flow cytometry
The 50.87% cells in control group were in G2/M phase and S stage. Compared with H2O2 treatment group (13.52%), there was significant difference between groups (P0.0001),.GM-1 was low, and the proportion of cells in G2/M phase and S phase was 33.95%, 30.70% and 28.68%, respectively, which was significantly different from H2O2 treatment group (P0.001,0.01).
4. PI3K/GSK-3 signal transduction pathway and oxidative damage of endothelial cells
4.1PI3K and GSK-3Western blot detection results
Compared with the control group, H2O2 treatment group HUVEC PI3K expression significantly decreased (P0.01), compared with the H2O2 model in the GM-1 group, high dose group significantly increased expression of PI3K (P0.05); compared with the control group, H2O2 treatment group GSK-3/ beta cell expression was significantly increased (P0.01); GM-1 in all treatment group, with GM-1 application of lower concentration, the expression of GSK-3/ beta protein was gradually increased, compared with the H2O2 model group, the expression of GM-1 in high dose group GSK-3/ beta protein significantly decreased, there was significant difference (P0.05).
Compared with H2O2 treatment, the expression of CHIR-99021 and CHIR-99021+H2O2 groups of PI3K and p-Akt protein were significantly increased (P0.01), the expression of GSK-3 was significantly decreased (P0.01); the expression of LY294002+H2O2 PI3K and p-GSK-3 group was significantly decreased (P0.01); the expression of LY294002 in group LY294002+H2O2 and group p-Akt were significantly increased (P0.01).
4.2Real-time PCR results
Compared with the control group, H2O2 treatment group PI3K, Akt mRNA expression was significantly reduced, there were significant differences (P0.01), and the expression of GSK-3mRNA was significantly increased (P0.01). Compared with H2O2 group, CHIR-99021 group and CHIR-99021+H2O2 PI3K group, Akt mRNA expression was significantly increased (P0.01), the expression of GSK-3 decreased significantly (P0.01 the expression of LY294002+H2O2 and PI3K); group GSK-3 was significantly decreased (P0.01); the expression of LY294002 group and LY294002+H2O2 group Akt mRNA were significantly increased (P0.01).
5. NF- kappa B and endothelial cell injury
Detection results of 5.1NF- kappa B Western blot
HUVEC was in the cytoplasm of NF- kappa B expression had no significant difference, while the cells were in the nucleus NF- kappa B expression had significantly difference, H2O2 group NF- kappa B nuclear / cytoplasm ratio was 12.07 + 2.96, compared with the control group, there was significant difference (P0.05) in GM-1 group. And with H2O2, high dose of NF- kappa B nuclear / cytoplasm ratio was significantly increased (P0.01).
5.2 double immunofluorescence double staining results
Showed the expression of NF- K B in HUVEC cultured in vitro, but each cell NF- kappa B expression sites between H2O2 treatment group NF- kappa B mainly in HUVEC cytoplasm, and in nuclei with low expression. Image analysis results showed that compared with H2O2, GM-1 NF- K B in high dose group the nucleus was significantly increased, the difference was significant (P0.05).
conclusion
1.GM-1 can effectively protect ischemic brain damage, and vascular endothelial cells may be an important therapeutic target for this effect.
The effect of 2.GM-1 on the proliferation of vascular endothelial cells may be related to the activation of the PI3K/GSK-3 signal transduction pathway and the nuclear translocation of NF- kappa B.
innovation point
1. this study explored the protective effect of GM-1 on ischemic brain damage from the angle of repair of endothelial cell injury, and no reports have been reported at home and abroad.
There are many studies on the mechanism of 2.GM-1 for the protection of brain injury. However, this study is the first to discuss the effect of PI3K/GSK-3 signaling pathway and NF- kappa B.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743

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