SIRT1在大鼠蛛网膜下腔出血后早期脑损伤模型的神经保护作用及机制研究

发布时间：2018-04-23 20:08

本文选题：蛛网膜下腔出血 + 早期脑损伤　；参考：《浙江大学》2016年博士论文

【摘要】：研究背景自发性蛛网膜下腔出血(spontaneous subarachnoid hemorrhage, SAH)是神经外科常见的危重急症,大约30%的病人在发病初期死亡,10%的病人死于其后续发生的各种并发症,其总体死亡率高达50%。自发性蛛网膜下腔出血大约占所有中风疾病的5-7%,全世界的发病率大约为10/10万人。大多数患者死亡发生于疾病早期,其中最主要原因是自发性蛛网膜下腔出血或者并发急性脑积水后导致的颅内压急剧增高。进一步的基础研究表明自发性蛛网膜下腔出血后早期脑损伤(Early brain injury, EBI)是导致高死亡率和致残率的主要原因。早期脑损伤是指从蛛网膜下腔出血开始至72小时这一时间窗之内的脑损伤,它是一个非常复杂的病理生理学过程。目前的研究认为早期脑损伤主要与颅内压升高、脑血流量降低、脑灌注压下降、血脑屏障破坏和脑水肿等有关,同时还有很多研究证据表明凋亡是早期脑损伤的主要病理改变因,神经元与内皮细胞的凋亡在蛛网膜下腔出血模型中均有报道,而且均与脑水肿密切相关。肿瘤抑制因子p53是非常重要的凋亡调控因子,蛛网膜下腔出血后发现p53的表达水平明显增高。而p53又是最早被发现能被乙酰化调节而且非组蛋白的蛋白质,当p53末端的赖氨酸被乙酰化,将激活p53增强其对下游分子的转录,比如Bax, Puma, Noxa, Bid等凋亡促进分子从而起到促进凋亡的作用。关于p53乙酰化进一步研究发现,乙酰化作用能在同一赖氨酸位点与其他蛋白修饰作用竞争比如泛素化和甲基化,从而调节其转录的活性甚并起到进一步调节生物功能的目的。以往的实验证据表明蛋白的乙酰化依赖于去乙酰化酶家族分子的作用,p53的去乙酰化调控就是依赖于去乙酰化酶的作用。Sirtuins家族属于Ⅲ类去乙酰化酶家族,它能够对组蛋白或非组蛋白去乙酰化。SIRT1是哺乳动物sirtuins家族成员中研究最多,在以往的研究中发现其在多种生理或者病理过程中起到重要的调控作用。除了能够对H1、H3和H4去乙酰化之外,它还被发现能对p53、NF-κB、FoxO等转录因子去乙酰化,因此在理论上SIRT1能够通过对上述蛋白乙酰化状态的调节而具有调节细胞周期,凋亡和肿瘤抑制的作用。很多体外或者体内研究证实SIRT1能够通过对重要分子去乙酰化的调节而对心脑等器官起到很好的保护作用。白藜芦醇是一种天然存在化合物,在多种植物中含量很高比如葡萄皮中,同时它也是SIRT1的一种激活剂,在许多研究中它被发现能够具有类似于热量限制(calorie restriction, CR)带来的生物体生命周期延长的作用。相反sirtinol是SIRT1的抑制剂,在多个研究中通过抑制SIRT1的活性来验证SIRT1在不同生物或生理活动中的作用。本研究将观察SIRT1在蛛网膜下腔出血早期脑损伤中后其表达调控情况以及其对神经元凋亡、脑水肿和神经功能的影响,然后通过激活与抑制SIRT1来进一步阐明SIRT1在蛛网膜下腔出血后早期脑损伤中的作用机制。实验方法第一部分首先采用颈内动脉线栓穿刺法制作大鼠蛛网膜下腔出血模型。成年雄性SD大鼠被随机分为：假手术组(Sham组),蛛网膜下腔出血组(SAH组),SAH组按取材时间不同又分为蛛网膜下腔出血后6h组(SAH 6h组),蛛网膜下腔出血后24h组(SAH 24h组),蛛网膜下腔出血48h组(SAH48h组)。通过神经功能缺陷评分,脑水含量测定检测脑损伤程度。采用Western blot检测自SIRT1的表达和凋亡相关蛋白(p53及其乙酰化状态)的表达；采用免疫荧光法检测神经元凋亡细胞数目。第二部分1.成年雄性SD大鼠被随机分为：假手术+溶剂组(Sham组)、蛛网膜下腔出血+溶剂组(SAH组)和蛛网膜下腔出血+白藜芦醇预处理组(SAH+RES组)(模型制作前48小时开始每日腹腔注射白藜芦醇(100mg/kg))。通过神经功能缺陷评分,不同部位脑组织的脑水含量测定检测早期脑损伤程度。2.成年雄性SD大鼠被随机分为：假手术组(Sham组),蛛网膜下腔出血组(SAH组),蛛网膜下腔出血+溶剂组(SAH+SOL),蛛网膜下腔出血+白藜芦醇组(SAH+RES),蛛网膜下腔出血+白藜芦醇+脑室DMSO组(SAH+RES+DMSO)和蛛网膜下腔出血+白藜芦醇+脑室sirtinol组(SAH+RES+SIR).白藜芦醇在蛛网膜下腔出血模型制作前48小时腹腔注射100mg/kg, sirtinol为脑室注射10ul, Sham组除不刺穿颈内动脉外其余操作与SAH组相同,(SAH+RES+DMSO)组脑室内注射与sirtinol等体积DMSO.然后在蛛网膜下腔出血模型制作完成后24小时采集数据：神经功能功能缺陷评分,不同部位脑组织的脑水含量测定,Evans blue评估血脑屏障破坏程度；采用Western blot法检测ZO-1、Occludin、cluadin5、SIRT1、凋亡相关蛋白p53及其乙酰化状态和凋亡相关蛋白(活化的caspase3)的表达；免疫荧光多重标记法检测神经元细胞凋亡情况；实时荧光定量PCR检测p53下游调控蛋白(Bax, Puma, Noxa, Bid)的mRNA表达情况。结果第一部分蛛网膜下腔出血后48h内大鼠的脑水含量上升程度和神经功能评分下降程度在24h时达到最大值,western blot结果显示蛛网膜下腔出血后SIRT1在24小时达到低谷,此时p53表达达高峰,其乙酰化水平达高峰。免疫组织化学结果提示,蛛网膜下腔出血后24h,脑组织TUNEL阳性标记的凋亡细胞数增力口,TUNEL和神经元标记物NeuN共荧光定位,提示凋亡在神经元细胞发生。第二部分1.蛛网膜下腔出血24小时后通过脑水含量及神经功能的评估发现白藜芦醇预处理缓解了左右脑半球脑水含量的上升和神经功能缺陷,白藜芦醇预处理降低了蛛网膜下腔出血后大鼠的死亡率。2.蛛网膜下腔出血24小时后观察发现白藜芦醇预处理缓解了左右脑半球脑水含量的上升和神经功能缺陷,而sirtinol逆转了白藜芦醇的作用。通过测量脑组织Evans Blue的含量发现,蛛网膜下腔出血后血脑屏障严重破坏,白藜芦醇预处理保护了血脑屏障(减少Evans blue渗出),而sirtinol作用以后原本减少的Evans blue又增多了,说明其逆转了白藜芦醇的血脑屏障保护作用。通过western blot检测发现,蛛网膜下腔出血后24小时ZO-1、Occludin. Claudin5等紧密链接蛋白表达下降是导致血脑屏障通透性增加的直接原因之一,白藜芦醇预处理能上调其表达水平从而减轻血脑屏障通透性的增加,但使用了sirtinol之后白藜芦醇上调上述紧密连接蛋白的作用被逆转。在SIRT1激活方面的作用,白藜芦醇预处理使蛛网膜下腔出血后SIRT1表达下降得到减轻,而sirtinol能够反转白藜芦醇对SIRT1表达上调的作用；同时白藜芦醇能够明显减少p53和乙酰化p53的水平,降低了激活的caspase3的表达,而Sirtinol则逆转了白藜芦醇下调p53、乙酰化p53和激活的caspase3的作用。进一步通过TUNEL和NeuN免疫荧光共定位方法检测凋亡神经元数目发现,白藜芦醇预处理可以明显减少凋亡神经元数目,sirtinol抑制了白藜芦醇的减轻神经元凋亡的作用。通过荧光定量PCR检测发现白藜芦醇预处理明显减少了BaxmRNA水平,其余几个凋亡相关蛋白的mRNA水平有下降的趋势但未发现统计学差异；而sirtinol明显抑制了白藜芦醇对Bax mRNA水平下调的作用。结论第一部分大鼠颈内动脉穿刺的蛛网膜下腔出血观察返现脑水肿、p53和乙酰化p53均在24小时达到高峰,神经功能缺陷明显,而SIRT1在此时最低,结合神经元凋亡情况,提示SIRT1可能参与了蛛网膜下腔出血早期脑损伤相关的后神经元凋亡的调控。第二部分白藜芦醇预处理能够改善蛛网膜下腔出血后大鼠神经功能缺陷,减轻脑水肿；通过白藜芦醇预处理和sirtinol逆转白藜芦醇的研究发现：白藜芦醇其血脑屏障保护可能是通过上调ZO-1、Occludin、Claudin5等紧密连接蛋白而起作用,同时白藜芦醇上调了SIRT1的表达水平,降低了p53和乙酰化p53的水平,激活的caspase3也下降了,免疫荧光检查也证实白藜芦醇预处理使凋亡的神经元细胞减少；相反使用SIRT1抑制剂sirtinol在各方面抑制了白藜芦醇的神经元保护作用,脑水肿减轻被逆转、减少的Evans blue渗出又开始增多、被白藜芦醇减轻的大鼠神经功能缺陷再次加重,分子检测方面ZO-1、Occludin、Claudin5等血脑屏障相关蛋白再次下调,SIRT1表达水平低于单独白藜芦醇预处理、p53和乙酰化p53水平转向增高,凋亡神经元数目增高。通过使用SIRT1增强剂和抑制剂从神经功能、脑水肿和血脑屏障检测,进而在信号通路等分子水平说明SIRT1信号通路在蛛网膜下腔出血后起到重要调节作用,进而减轻脑水肿、保护血脑屏障、减少神经元凋亡、改善神经功能。
[Abstract]:Spontaneous subarachnoid hemorrhage (SAH) is a common critical emergency in the Department of neurosurgery. About 30% of the patients died at the beginning of the onset, and 10% of the patients died of subsequent complications. The overall mortality rate was as high as 50%. spontaneous subarachnoid hemorrhage accounted for all apoplexy diseases. The incidence of 5-7% in the world is about 10/10 million. Most of the patients died in the early stages of the disease, the main cause of which was the spontaneous subarachnoid hemorrhage or the rapid increase of intracranial pressure following acute hydrocephalus. Further basic studies showed early brain injury (Early brain) after spontaneous subarachnoid hemorrhage. Injury, EBI) is the main cause of high mortality and disability. Early brain injury is a very complicated pathophysiological process from the beginning of subarachnoid hemorrhage to 72 hours. It is a very complicated pathophysiological process. The decline, blood brain barrier destruction and brain edema are related, and there are many research evidence that apoptosis is the main cause of early brain damage. Apoptosis of neurons and endothelial cells is reported in the subarachnoid hemorrhage model, and it is closely related to brain edema. Tumor suppressor factor p53 is a very important regulation of apoptosis. After subarachnoid hemorrhage, the expression level of p53 was significantly increased. And p53 was the earliest protein that was found to be acetylation and non histone. When lysine at the p53 terminal was acetylated, it activated p53 to enhance the transcription of its downstream molecules, such as Bax, Puma, Noxa, Bid and so on to promote apoptosis. Further studies on p53 acetylation have found that acetylation can compete with other proteins at the same lysine site to compete with other proteins such as ubiquitination and methylation, thus regulating its transcriptional activity and further regulating biological functions. Previous experimental evidence showed that acetylation of protein was dependent on deacetylation. The role of enzyme family molecules, p53 deacetylation regulation is dependent on the action of deacetylase, the.Sirtuins family belongs to class III deacetylase family. It can study histone or non histone.SIRT1 in mammalian sirtuins family members most. In the study, it is found in a variety of Physiology or pathology. In addition to the ability to deacetylation of H1, H3 and H4, it is also found to be able to deacetylation of p53, NF- kappa B, FoxO and other transcription factors, so in theory SIRT1 can regulate cell cycle, apoptosis and tumor inhibition by regulating the state of acetylation of these proteins. Many in vitro or in vivo SIRT1 Internal research has confirmed that SIRT1 can protect the heart and brain by regulating the deacetylation of important molecules. Resveratrol is a natural compound, which is very high in a variety of plants, such as grape skin, and is also an activator of SIRT1. In many studies it has been found to be similar to that. The effect of the life cycle prolongation of calorie restriction (CR). Reverse sirtinol is an inhibitor of SIRT1. In several studies, the role of SIRT1 in different biological or physiological activities is verified by inhibiting the activity of SIRT1. This study will observe the expression of SIRT1 in the early brain damage of subarachnoid hemorrhage. The effect of control and its effect on neuronal apoptosis, brain edema and nerve function, and then further elucidate the mechanism of the action of SIRT1 in early brain injury after subarachnoid hemorrhage by activating and inhibiting SIRT1. SD rats were randomly divided into sham operation group (group Sham), subarachnoid hemorrhage group (group SAH), group SAH was divided into group 6h (group SAH 6h) after subarachnoid hemorrhage, 24h group (SAH 24h group) after subarachnoid hemorrhage, 48h group of subarachnoid hemorrhage (SAH48h group) after subarachnoid hemorrhage, and the determination of brain water content by nerve function defect score. The expression of self SIRT1 and the expression of apoptosis related protein (p53 and its acetylation state) were detected by Western blot, and the number of apoptotic cells was detected by immunofluorescence. Second part 1. adult male SD rats were randomly divided into sham operation + solvent group (Sham group), subarachnoid hemorrhage + solvent group (SAH group) and spiders Subomentum hemorrhage + resveratrol preconditioning group (group SAH+RES) (group SAH+RES, 100mg/kg) was injected daily 48 hours before the model was made. Through the neurological deficit score, the brain water content in different parts of the brain was measured and the adult male SD rats were randomly divided into the sham operation group (group Sham) and the arachnoid arachnoid Lower hemorrhage group (group SAH), subarachnoid hemorrhage + solvent group (SAH+SOL), subarachnoid hemorrhage + resveratrol group (SAH+RES), subarachnoid hemorrhage + resveratrol + ventricle DMSO group (SAH+RES+DMSO) and subarachnoid hemorrhage + resveratrol + ventricle sirtinol group (SAH+RES+SIR). Resveratrol was 48 small before the subarachnoid hemorrhage model Intraperitoneal injection of 100mg/kg, sirtinol for intraventricular injection of 10ul, group Sham in addition to the internal carotid artery, the rest of the operation is the same as the SAH group, (SAH+RES+DMSO) group intracerebral injection and sirtinol volume DMSO. and then the subarachnoid hemorrhage model was completed 24 hours after the completion of the data: neural function defect score, different parts of the brain tissue Determination of brain water content and Evans blue to evaluate the damage of blood brain barrier; Western blot method was used to detect the expression of ZO-1, Occludin, cluadin5, SIRT1, apoptosis related protein p53 and its state of acetylation and apoptosis related protein (activated Caspase3); immunofluorescence multiple labeling method was used to detect neuronal apoptosis; real-time fluorescent quantitative PCR The expression of mRNA in p53 downstream regulatory proteins (Bax, Puma, Noxa, Bid) was detected. Results after subarachnoid hemorrhage, the level of brain water content and the decrease of neural function score in 48h rats reached the maximum at 24h, and Western blot showed that after subarachnoid hemorrhage, the p53 table was reached at 24 hours. At the peak of dada peak, its acetylation level reached the peak. The immunohistochemical results suggested that 24h, TUNEL positive apoptotic cells in the brain tissue increased the number of apoptotic cells after subarachnoid hemorrhage, TUNEL and neuron marker NeuN co fluorescence localization, suggesting that apoptosis occurred in the neuron cells. The second part of subarachnoid hemorrhage was contained in brain water after 24 hours of subarachnoid hemorrhage. Resveratrol pretreatment relieved the increase of brain water content and neural dysfunction in the left and right hemispheres. Resveratrol pretreatment reduced the mortality of rats after subarachnoid hemorrhage.2. after subarachnoid hemorrhage 24 hours after subarachnoid hemorrhage observed that resveratrol preconditioning relieved the brain water content in the left and right hemispheres Sirtinol reversed the effect of resveratrol on the rise and neural dysfunction. By measuring the content of Evans Blue in the brain tissue, the blood brain barrier was seriously damaged after subarachnoid hemorrhage, and resveratrol pretreated the blood brain barrier (reducing the Evans blue exudation), and the decreased Evans blue increased after the sirtinol action. It is shown that it reverses the protective effect of resveratrol on the blood brain barrier. Through Western blot detection, it is found that the decrease of the expression of close linked protein 24 hours after subarachnoid hemorrhage, ZO-1, Occludin. Claudin5, is one of the direct causes of the increase of blood brain barrier permeability, and resveratrol preconditioning can increase the expression level and reduce the blood brain. The barrier permeability increased, but the effect of resveratrol on up - regulation of the close connexin was reversed after the use of sirtinol. In the activation of SIRT1, resveratrol pretreated the decrease of SIRT1 expression after subarachnoid hemorrhage, while sirtinol could reverse the effect of resveratrol on the up regulation of SIRT1 expression; and resveratrol Resveratol significantly reduced the level of p53 and acetylation of p53 and reduced the expression of activated Caspase3, while Sirtinol reversed the effect of resveratrol on p53, acetylated p53 and activated Caspase3. The number of apoptotic neurons was detected by TUNEL and NeuN immunofluorescence method, and resveratrol pretreatment could be significantly reduced. The number of fewer apoptotic neurons, sirtinol inhibited the effect of resveratrol on the reduction of neuronal apoptosis. By fluorescence quantitative PCR detection, resveratrol pretreatment significantly reduced the level of BaxmRNA, and the mRNA level of the remaining apoptosis related proteins decreased but no statistical difference was found, while sirtinol significantly inhibited resveratrol. The effect of alcohol on the downregulation of Bax mRNA. Conclusion the first part of the subarachnoid hemorrhage of the internal carotid artery of the rat was observed to return to the brain edema. P53 and acetylated p53 reached the peak at 24 hours, and the nerve function defects were obvious, and SIRT1 was the lowest at this time, combining with the neuron apoptosis, suggesting that SIRT1 may be involved in the early subarachnoid hemorrhage. The second part of resveratrol pretreatment can improve the neural function defects and reduce the brain edema after subarachnoid hemorrhage. The study of resveratrol pretreatment and sirtinol reversal of resveratrol may be found that the protection of resveratrol on the blood brain barrier may be by up regulation of ZO-1, Occludin Claudin5 and other close connexin played a role, while resveratrol up-regulated the expression of SIRT1, reduced the level of p53 and acetylation of p53, and decreased the activation of Caspase3. Immunofluorescence also confirmed that resveratrol pretreated the apoptotic neuron cells, and SIRT1 inhibitor sirtinol was suppressed in all aspects. The neuroprotective effect of resveratrol was reversed, the decrease of brain edema was reversed, the decrease of Evans blue exudation began to increase, and the neural functional defects of rats reduced by resveratrol were aggravated again. The molecular detection of ZO-1, Occludin, Claudin5 and other blood brain barrier related proteins decreased again, and the expression level of SIRT1 was lower than that of resveratrol alone, p53 And the level of acetylated p53 increased and the number of apoptotic neurons increased. By using SIRT1 enhancers and inhibitors from neural function, brain edema and blood brain barrier detection, and then at the signal pathway, the SIRT1 signaling pathway was played an important role in the subarachnoid hemorrhage, thus alleviating brain edema and protecting the blood brain barrier, Reduce neuron apoptosis and improve nerve function.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.35

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