N-乙酰血清素的一种新型衍生物在大鼠蛛网膜下腔出血模型中的神经保护作用

发布时间：2018-05-10 06:27

  本文选题：蛛网膜下腔出血 + 早期脑损伤　； 参考：《浙江大学》2015年博士论文

【摘要】：背景与目的： 蛛网膜下腔出血(subarachnoid hemorrhage, SAH)仅占所有脑卒中病例的5%,但是具有较高的死亡率和致残率,因此其诊治仍然是每一个神经外科医师所面临的最棘手的问题之一。 早期脑损伤(early brain injury, EBI)是指SAH后急性期(0-3天)脑组织发生的一系列病理生理改变导致的继发性脑损伤。越来越多的研究已表明EBI的发生与严重程度和SAH的预后密切相关。而神经元的凋亡作为EBI中的关键环节,近年来已成为SAH研究中的热点。相关实验性研究已证明在SAH动物模型中抗神经元凋亡可有效地改善神经功能缺失。 原肌球蛋白相关激酶B(Tropomyosin-related kinase B, TrkB)是体内多种神经营养因子的高亲和性受体。近年来大量研究表明,TrkB相关信号通路与多种中枢神经系统疾病的病理生理机制密切相关。在缺血性脑卒中动物模型中,激活TrkB可有效地减轻早期脑损伤并改善预后。然而,TrkB在SAH中的作用却从未进行过深入的研究。 N-乙酰血清素(N-acetyl serotonin, NAS)是体内由血清素转化为褪黑素时的中间产物。N-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-2-oxopiperidine-3-carboxamide (HIOC)作为NAS的一种新型衍生物,相对于NAS来说,能够更有效地选择性激活TrkB受体。然而HIOC却从未用于卒中动物模型的研究。 因此,本研究的目的是为了揭示HIOC在实验性SAH模型中通过激活TrkB受体,从而减轻SAH后早期脑损伤并改善预后的潜在神经保护作用。 材料与方法： 本实验共使用156只成年雄性Sprague-Dawley (SD)大鼠。采用血管内穿刺法诱导蛛网膜下腔出血模型。SAH造模前24小时经侧脑室注射TrkB小干扰RNA(small interfering RNA, siRNA)和对照小干扰RNA (scramble siRNA)。SAH造模后3小时经侧脑室注射HIOC和脑源性神经营养因子(brain-derived neurotrophic factor, BDNF),并比较两组间疗效。在预后研究中评估SAH出血量评分和神经功能学评分；在机制研究中,通过免疫蛋白印记法(western blot)检测穿刺侧大脑半球ΓrkB,磷酸化TrkB (p-TrkB),磷酸化细胞外信号调节激酶(phosphorylated extracellular signal regulated kinase, p-ERK), B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)和裂解半胱氨酸天冬氨酸蛋白酶3(cleaved caspase-3, CC3)等蛋白水平的表达；通过免疫荧光共染验证TrkB在神经元上表达；采用末端标记法(Terminal deoxynucleotidyl transferase-mediated uridine5'-triphosphate-biotin nick end-labeling, TUNEL)染色观察神经元凋亡水平。 结果： 1.SAH前24小时侧脑室注射TrkB siRNA下调了大脑半球TrkB蛋白的表达,减少了SAH后24小时TrkB的激活水平和抗凋亡蛋白Bcl-2的表达,并加重了神经功能缺失。 2.SAH后经侧脑室注射HIOC可有效地减轻神经功能缺失。使用高剂量(30ug)的HIOC治疗比低剂量(6ug)更好地改善了预后评分。3.SAH造模后经侧脑室注射HIOC可通过激活TrkB/ERK信号通路,减少神经元凋亡。造模前24小时侧脑室注射TrkB siRNA后阻断了该信号通路的激活从而抵消了HIOC的神经保护作用,对照siRNA的使用不会影响HIOC的神经保护作用。 4.在SAH后侧脑室注射HIOC和BDNF均可有效地激活TrkB。在SAH后6小时,两者的效用无显著差别；而在SAH后24小时,HIOC比BDNF具有更强的抗凋亡作用。 结论： 在SAH模型后注射HIOC可通过激活TrkB/ERK信号通路减少SAH后的早期脑损伤。HIOC可能成为今后脑卒中治疗的一种具有应用前景的药物。
[Abstract]:Background and purpose:
Subarachnoid hemorrhage (subarachnoid hemorrhage, SAH) accounts for only 5% of all cerebral apoplexy cases, but has a high mortality and disability rate, so the diagnosis and treatment of the subarachnoid hemorrhage is still one of the most difficult problems facing every neurosurgeon.
Early brain injury (EBI) refers to secondary brain damage caused by a series of pathophysiological changes in the brain tissue after SAH (0-3 days). More and more studies have shown that the occurrence and severity of EBI are closely related to the prognosis of SAH. The withering of neurons, as a key link in EBI, has become a SAH in recent years. Experimental studies have demonstrated that anti neuronal apoptosis in SAH animal models can effectively improve neurological deficits.
The promyosin related kinase B (Tropomyosin-related kinase B, TrkB) is a high affinity receptor for various neurotrophic factors in the body. In recent years, a large number of studies have shown that TrkB related signaling pathways are closely related to the pathophysiological mechanism of various central nervous system diseases. In the animal model of ischemic stroke, the activation of TrkB can be effectively reduced. Mild early brain injury and improved prognosis. However, the role of TrkB in SAH has not been thoroughly studied.
N- acetyl serotonin (N-acetyl serotonin, NAS) is a new derivative of.N-[2- (5-hydroxy-1H-indol-3-yl) ethyl]-2-oxopiperidine-3-carboxamide (HIOC), a intermediate product of the transformation from serotonin to melatonin, as a new derivative of NAS, which is more effective than NAS for selectively activating TrkB receptors. However, HIOC has never been used for death. The study of the animal model in the middle.
Therefore, the aim of this study is to reveal the potential neuroprotective effect of HIOC in the experimental SAH model by activating the TrkB receptor, thus reducing the early brain damage after SAH and improving the prognosis.
Materials and methods:
156 adult male Sprague-Dawley (SD) rats were used in this experiment. The intravascular puncture method was used to induce the subarachnoid hemorrhage model.SAH to be injected with TrkB small interference RNA (small interfering RNA, siRNA) and control small interference RNA (scramble siRNA) 24 hours before the model of the model of the subarachnoid hemorrhage, and 3 hours after the injection of the lateral ventricle and brain source God. Brain-derived neurotrophic factor (BDNF) and comparison of the efficacy between the two groups were compared. In the prognostic study, the SAH hemorrhage score and the neurologic score were evaluated. In the mechanism study, the immunoglobulin imprinting (Western blot) was used to detect the cerebral hemisphere in the cerebral hemisphere of gamma rkB, phosphorylated TrkB (p-TrkB), and phosphorylated extracellular signal regulation. Kinase (phosphorylated extracellular signal regulated kinase, p-ERK), the expression of B lymphocytoma -2 (B-cell lymphoma-2, Bcl-2) and Lysic cysteine aspartic proteinase 3, expressed on the neuron by immunofluorescence CO staining; Ynucleotidyl transferase-mediated uridine5'-triphosphate-biotin nick end-labeling (TUNEL) staining was used to observe neuronal apoptosis.
Result锛，

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