硫化氢对蛛网膜下腔出血后早期脑损伤的影响及其机制的研究

发布时间：2018-08-04 09:09

【摘要】：第一部分大鼠蛛网膜下腔出血后内源性硫化氢的生成及硫化氢对早期脑损伤的保护作用目的:探讨蛛网膜下腔出血(Subarachnoid hemorrhage,SAH)大鼠内源性硫化氢(Hydrogen sulfide,H2S)主要生成途径及H2S对SAH后早期脑损伤(Early brain injury,EBI)的影响。方法:雄性SD大鼠96只,按随机数字表分为空白组、SAH组、SAH+Na HS低剂量组(SAH+Na HS-L 1.4mg/kg)、SAH+Na HS高剂量组(SAH+Na HS-H 5.6mg/kg),采用视交叉前池注血法制作SAH模型,48 h后处死。测定血浆、大脑皮层H2S含量,免疫组化双染色分别观察大脑皮层神经元胱硫醚-β-合酶(Cystathionine-β-synthase,CBS)、3-巯基丙酮酸转硫酶(3-merecaptopyruvate sulfurtransferase,MPST or 3-MST)定位,western blot检测CBS、3-MST表达,测定新鲜脑组织活性氧(ROS)、丙二醛(MDA)含量和超氧化物歧化酶(SOD)、谷胱甘肽过氧化酶(GSH-Px)活性,测定脑组织含水量、伊文氏蓝(Evans blue,EB)含量,TUNEL评价脑神经细胞凋亡,记录神经认知功能评分。结果:CBS、3-MST在大脑皮层神经元均有分布,SAH48h后CBS表达下调,3-MST表达上调,H2S含量下降,氧化应激指标ROS、MDA含量显著升高,SOD、GSH-Px活性显著下降,脑组织含水量、伊文氏蓝含量显著升高,Caspase 3蛋白表达显著上调,脑神经细胞凋亡显著增多,大鼠神经认知功能评分显著下降。高低剂量Na HS预处理可弥补SAH后内源性H2S不足,可促使CBS、3-MST表达上调,可使氧化应激指标ROS、MDA含量明显下降,SOD、GSH-Px活性明显增强,脑组织含水量、伊文氏蓝含量明显降低,脑神经细胞凋亡明显减少,大鼠神经认知功能评分明显改善,且高剂量效果优于低剂量。结论:CBS/H2S、3-MST/H2S均参与SAH后内源性H2S的生成,H2S对SAH后EBI具有保护作用,其作用具有剂量依赖性。第二部分硫化氢对大鼠蛛网膜下腔出血后急性脑血管痉挛的影响目的:探讨硫化氢(H2S)对大鼠蛛网膜下腔出血(SAH)后急性脑血管痉挛(a CVS)的影响,并对其可能机制初步探讨。方法:48只雄性SD大鼠随机均分为空白组、SAH组、Na HS低剂量组和高剂量组。造模48h后HE染色法、TUNEL与免疫组化双染色分别观察各组大鼠大脑前动脉A2段(ACA A2段)、大脑中动脉M1段(MCA M1段)部血管形态、管腔面积以及内皮细胞凋亡,western blot检测脑血管组织Caspase 3蛋白表达。结果:大鼠SAH模型,造模后ACAA2段管腔明显狭窄,血管内皮细胞凋亡明显增加,凋亡蛋白Caspase 3表达明显增强;Na HS干预可有效扩张血管、抑制内皮细胞凋亡,显著下调Caspase 3表达,且高剂量效果优于低剂量。结论:H2S可有效扩张痉挛的脑血管,其保护功能可能是通过抑制脑血管内皮细胞凋亡起作用,而且具有剂量依赖性。第三部分硫化氢对氧合血红蛋白诱导的体外蛛网膜下腔出血模型的影响目的:研究硫化氢(H2S)对体外蛛网膜下腔出血(SAH)的影响及其机制的初步探讨。方法:氧合血红蛋白(Oxygenated hemoglobin,Oxy Hb,10μΜ)模拟SAH刺激因素分别作用脑微血管内皮细胞(brain microvascular endothelial cells,BMVECs)和神经元(Neurons),以Na HS(15μM、30μM)预处理。24h后观察细胞形态,统计BMVECs、Neurons存活率,测定活性氧ROS含量,收集细胞western blot检测Caspase 3表达。结果:Oxy Hb干预后存活神经元显著减少,活性氧(ROS)含量显著升高,Caspase3表达显著上调;Na HS干预后存活神经元显著增多,ROS含量显著下降,Caspase 3表达显著下调。Oxy Hb干预后存活脑微血管内皮细胞显著减少,ROS含量显著升高,Caspase 3表达显著上调;Na HS干预后存活脑微血管内皮细胞显著增多,ROS含量显著下降,Caspase 3表达显著下调。结论:在体外H2S通过抑制ROS生成改善SAH后BMVECs、Neurons凋亡。
[Abstract]:Partial hydrogen sulfide production after subarachnoid hemorrhage and the protective effect of hydrogen sulfide on early brain damage in rats after subarachnoid hemorrhage: To explore the main pathway of endogenous hydrogen sulfide (Hydrogen sulfide, H2S) in Subarachnoid hemorrhage (SAH) rats and the early brain injury after SAH (Early brain injury, EBI). Methods: 96 male SD rats were divided into blank group, SAH group, SAH+Na HS low dose group (SAH+Na HS-L 1.4mg/kg) and SAH+Na HS high dose group (SAH+Na HS-H 5.6mg/kg). Cortical neurons cystthiether beta synthase (Cystathionine- beta -synthase, CBS), 3- mercapto pyruvate thiotransferase (3-merecaptopyruvate sulfurtransferase, MPST or 3-MST) localization, Western blot detection CBS, the determination of fresh brain tissue active oxygen, malonic two aldehyde content and superoxide dismutase, glutathione peroxidase H-Px) activity, determination of brain tissue water content, Evans blue (EB) content, TUNEL evaluation of neuronal apoptosis and recording neurocognitive function score. Results: CBS, 3-MST in the cerebral cortex neurons are distributed, SAH48h CBS expression down, 3-MST expression up-regulated, H2S content decreased, oxidative stress index ROS, significant increase The activity of Px decreased significantly, the content of brain water content, the content of Evans blue significantly increased, the expression of Caspase 3 protein was significantly up-regulated, the apoptosis of cerebral nerve cells increased significantly, and the neurocognitive function score of the rats decreased significantly. High and low dose Na HS preconditioning could make up the endogenous H2S deficiency after SAH, which could promote the up regulation of CBS and 3-MST, which could make the oxidative stress index RO The content of S, MDA decreased obviously, the activity of SOD, GSH-Px increased obviously, the water content of brain tissue, the content of Evans blue obviously decreased, the apoptosis of brain nerve cells decreased obviously, the neurocognitive function score of the rat was obviously improved, and the high dose effect was better than the low dose. Conclusion: CBS/H2S, 3-MST/ H2S are all involved in the production of endogenous H2S after SAH, H2S is guaranteed for SAH EBI. The effect of the second part of hydrogen sulfide on acute cerebral vasospasm after subarachnoid hemorrhage in rats: To explore the effect of hydrogen sulfide (H2S) on acute cerebral vasospasm (a CVS) after subarachnoid hemorrhage (SAH) in rats and to discuss its possible mechanism. Methods: 48 male SD rats were randomly distributed They were divided into blank group, SAH group, Na HS low dose group and high dose group. HE staining method was used to build model 48h, TUNEL and immunohistochemical double staining were used to observe the A2 segment of the anterior cerebral artery (ACA A2 segment), M1 segment of the middle cerebral artery (MCA M1 segment), the area of the lumen and the apoptosis of endothelial cells, and to detect the 3 protein of the cerebral vascular tissue. Results: SAH model of rats, the ACAA2 segment of the cavity was obviously narrowed, the apoptosis of vascular endothelial cells increased obviously and the expression of apoptotic protein Caspase 3 was obviously enhanced. Na HS intervention could effectively dilate blood vessels, inhibit apoptosis of endothelial cells, reduce the expression of Caspase 3 significantly, and the effect of high dose is better than low dose. Conclusion H2S can effectively dilate spasmodic. Cerebral vascular, its protective function may be mediated by inhibiting apoptosis of cerebral vascular endothelial cells, and is dose-dependent. The effect of the third part of hydrogen sulfide on the model of subarachnoid hemorrhage induced by oxygenated hemoglobin in vitro: the study of the effect of hydrogen sulfide (H2S) on subarachnoid hemorrhage (SAH) in vitro and its mechanism Methods: Oxygenated hemoglobin (Oxy Hb, 10 micron) simulated SAH stimulation factors in the brain microvascular endothelial cells (brain microvascular endothelial cells, BMVECs) and neurons (Neurons). Western blot was collected to detect the expression of Caspase 3. Results: after Oxy Hb intervention, the survival neurons were significantly decreased, the content of active oxygen (ROS) was significantly increased, the expression of Caspase3 was significantly up-regulated, the survival neurons increased significantly after Na HS intervention, the ROS content was significantly decreased, Caspase 3 table significantly downregulated the survival of the microvascular endothelial cells after.Oxy. The content of ROS increased significantly and the expression of Caspase 3 increased significantly. After Na HS intervention, the survival of the cerebral microvascular endothelial cells increased significantly, the content of ROS decreased significantly, and the expression of Caspase 3 decreased significantly. Conclusion: H2S can improve the BMVECs of SAH after SAH and Neurons withering in vitro.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.35

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