内质网应激PERK通路在蛛网膜下腔出血早期脑损伤中的作用及机制研究
发布时间：2018-08-13 15:12
【摘要】：研究背景蛛网膜下腔出血(subarachnoid hemorrhage,SAH)是一种临床常见的神经外科急症,具有高死亡率、高致残率的特点,据流行病学调查,人群年患病率约为1‰,死亡率高达30%。迟发性脑血管痉挛曾被认为是SAH致伤机制中的主要因素,一直是传统研究的热点领域。然而近年来多项临床随机双盲对照研究显示,缓解脑血管痉挛的治疗并不能改善SAH患者的预后。某些药物虽可有效改善患者预后,但其并无对抗脑血管痉挛的作用。因此近年来,本领域的权威学者提出了 SAH后早期脑损伤这一概念,即SAH发病后72小时内所引发的早期脑损伤对SAH后神经功能的康复发挥着决定性的影响作用。早期脑损伤是一个复杂的病理生理学过程,主要包括颅内压升高、脑血流量降低、脑灌注压下降、神经细胞死亡、血脑屏障破坏和脑水肿等。然而其具体损伤机制及上游调控信号目前尚不明确,需进一步研究探索。内质网是细胞内重要的细胞器,它是蛋白质合成、加工以及贮存Ca2+的主要场所。它对内外环境刺激极为敏感,当其功能紊乱时导致错误折叠与未折叠蛋白在腔内聚集的状态,被称为内质网应激(endoplasmic reticulum stress)。内质网应激继而引发未折叠蛋白反应(unfolded protein response,UPR),包括暂停蛋白质合成、激活分子伴侣和折叠酶的转录活性、诱导内质网相关性降解,以应对内外环境各种刺激因素。当刺激因素持续存在时,内质网应激将过度增强,进而激活一系列信号分子,引发细胞死亡等严重后果。众多研究表明内质网应激在神经退行性疾病、神经系统炎症的发生发展过程中发挥着重要作用,是该研究领域的热点方向。但关于内质网应激在SAH后的作用及其具体机制,目前尚缺乏深入的研究。因此,本研究将围绕内质网应激PERK-eIF2α-ATF4通路,观察其在SAH后的表达水平,应用干预手段探索其在早期脑损伤中的作用及具体机制,为SAH的治疗提供新思路与新靶点。实验方法第一部分1.采用线栓穿刺法制作蛛网膜下腔出血(SAH)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3小时组(SAH3h组)、蛛网膜下腔出血后6小时组(SAH6h组)、蛛网膜下腔出血后12小时组(SAH12h组)、蛛网膜下腔出血24后小时组(SAH 24 h组)和蛛网膜下腔出血后72小时组(SAH 72 h组)。应用Garcia量表评估大鼠神经功能障碍状况,利用干湿重法测定脑水肿程度,通过Western blot检测内质网应激PERK-eIF2α-ATF4通路相关蛋白表达水平(PERK、p-PERK、eIF2α、p-eIF2α与ATF4),采用免疫荧光双标染色法观察表达内质网应激相关蛋白的神经细胞类型。第二部分1.成年雄性SD大鼠SAH模型建立后1小时,应用立体定位下侧脑室注射法分别给予溶剂、低剂量PERK特异性抑制剂GSK2606414(30μg)或高剂量PERK特异性抑制剂GSK2606414(90μg),随机分为假手术+溶剂组、SAH+溶剂组、SAH+GSK2606414(30μg)组与 SAH+GSK2606414(90μg)组。2.应用Garcia量表评估大鼠神经功能障碍状况,采用TUNEL染色法测定神经细胞凋亡状况,应用Western blot检测内质网应激相关蛋白(PERK、p-PERK、eIF2α、p-eIF2α),凋亡相关蛋白(Akt、Bcl-2、Bax、Caspase-3)的表达水平。根据前述研究结果,确定GSK2606414抗神经细胞凋亡的适宜剂量。应用Akt抑制剂Wortmannin作为干预手段进行预处理,探索内质网应激PERK通路的下游作用机制。第三部分1.成年雄性SD大鼠SAH模型建立后,应用立体定位下侧脑室注射法给予适宜剂量PERK特异性抑制剂GSK2606414,并加用Akt抑制剂Wortmannin作为预处理,随机分为假手术+溶剂组、SAH+溶剂组、SAH+GSK2606414组、SAH+Wortmannin 组、SAH+GSK2606414+Wortmannin 组。2.应用Garcia量表评估大鼠神经功能障碍状况,利用干湿重法测定脑水肿程度,通过Evans blue法评估血脑屏障破坏程度,应用Western blot检测内质网应激相关蛋白(PERK、p-PERK)、以及血脑屏障紧密连接相关蛋白(ZO-1、Occludin、Claudin5)的表达水平。探索内质网应激抑制剂对SAH后血脑屏障破坏的保护作用,以及其可能的下游作用机制。结果第一部分1.蛛网膜下腔出血(SAH)模型建立后72h内大鼠的脑水含量程度与神经功能障碍持续加重;Western blot检测显示SAH模型建立后,p-PERK、p-eIF2α及ATF4的表达水平显著升高,于72小时达到高峰;免疫荧光双重染色提示,SAH后72h,脑组织中PERK阳性细胞数量显著增加,PERK与神经元标记蛋白NeuN共表达;此外,PERK表达于部分星形胶质细胞标记蛋白GFAP(glial fibrillary acidic protein)阳性的神经细胞中。第二部分1.应用PERK特异性抑制剂GSK2606414可显著抑制内质网应激相关蛋白p-PERK、p-eIF2α在SAH模型大鼠中的表达水平,显著缓解SAH大鼠的神经功能障碍;2.SAH发生后,大鼠脑组织中抗凋亡蛋白Bcl-2表达水平显著降低,促凋亡蛋白Bax、Cleaved Caspase-3表达水平显著升高,应用GSK2606414可显著提高Bcl-2表达水平,减少Bax、Cleaved Caspase-3表达水平;TUNEL染色显示,SAH大鼠脑组织中可观察到大量TUNEL阳性细胞,通过GSK2606414治疗可显著减少TUNEL阳性细胞的数量,并且高剂量GSK2606414(90μg)具有更好的治疗效果。静脉注射Akt特异性抑制剂Wortmannin预处理可显著抑制GSK2606414的神经保护作用,加重神经细胞凋亡水平。第三部分1.应用GSK2606414处理可显著缓解SAH大鼠脑水肿程度,减少脑组织中Evans blue染料渗出,提高血脑屏障紧密连接蛋白ZO-1、Occludin、Claudin5的表达水平。2.应用Akt抑制剂Wortmannin预处理会加重脑水肿程度,增加脑组织中Evans blue染料渗出,降低紧密连接蛋白ZO-1、Occludin、Claudin5的表达水平,显著阻断PERK抑制剂GSK2606414对SAH大鼠的血脑屏障保护作用。结论1.蛛网膜下腔出血后早期脑损伤中,内质网应激PERK-eIF2α-ATF4通路显著激活,并广泛分布于神经元与星形胶质细胞,提示内质网应激可能在蛛网膜下腔出血后早期脑损伤中发挥重要作用。2.应用PERK特异性抑制剂GSK2606414可显著降低蛛网膜下腔出血后内质网应激水平,改善蛛网膜下腔出血大鼠的神经功能障碍,抑制神经细胞凋亡,保护血脑屏障完整性,减轻脑水肿。3.Akt特异性抑制剂Wortmannin预处理可显著阻断GSK2606414的抗凋亡、保护血脑屏障作用,提示GSK2606414主要通过抑制内质网应激,激活Akt信号通路以发挥神经保护作用。
[Abstract]:Background Subarachnoid hemorrhage (SAH) is a common clinical neurosurgical emergency with high mortality and disability. According to epidemiological investigation, the annual prevalence of SAH is about 1 and the mortality rate is as high as 30%. However, in recent years, many randomized, double-blind, controlled clinical studies have shown that the treatment of cerebral vasospasm can not improve the prognosis of SAH patients. Some drugs can effectively improve the prognosis of SAH patients, but they have no effect on the treatment of cerebral vasospasm. Early brain injury is a complex pathophysiological process, including elevated intracranial pressure, decreased cerebral blood flow, decreased cerebral perfusion pressure, death of nerve cells and destruction of blood-brain barrier. Endoplasmic reticulum is an important organelle in cells. It is the main site for protein synthesis, processing and storage of Ca2 +. It is extremely sensitive to stimuli from the internal and external environment, causing misfolded and unfolded eggs when its function is disordered. The state of white aggregation in the lumen is called endoplasmic reticulum stress. Endoplasmic reticulum stress is followed by unfolded protein response (UPR), which includes the suspension of protein synthesis, activation of molecular chaperone and folding enzyme transcriptional activity, and induction of ER-related degradation to cope with various spines in the internal and external environment. When stimuli persist, endoplasmic reticulum stress will increase excessively, and then activate a series of signaling molecules, leading to cell death and other serious consequences. Numerous studies have shown that endoplasmic reticulum stress plays an important role in the development of neurodegenerative diseases and inflammation of the nervous system. Therefore, this study will focus on the endoplasmic reticulum stress PERK-eIF2alpha-ATF4 pathway, observe its expression level after SAH, explore its role and specific mechanism in early brain injury by intervention means, and provide new ideas and new targets for the treatment of SAH. The adult male SD rats were randomly divided into sham operation group (Sham group) and subarachnoid hemorrhage group (SAH group). The SAH group was divided into subarachnoid hemorrhage 3 hours group (SAH 3 hours group) and subarachnoid hemorrhage 6 hours group (SAH group) according to the sampling time. Six hours after subarachnoid hemorrhage, 12 hours after subarachnoid hemorrhage (SAH 12 hours group), 24 hours after subarachnoid hemorrhage (SAH 24 hours group) and 72 hours after subarachnoid hemorrhage (SAH 72 hours group). The expression of endoplasmic reticulum stress-related protein (ER-SRP) was detected by immunofluorescence double-labeling staining. Part 2. One hour after the establishment of SAH model in adult male SD rats, the solvents were injected into the lateral ventricle of the adult male SD rats by stereotaxic method, and the low dose of PERK was used. Specific inhibitor GSK2606414 (30 ug) or high-dose PERK specific inhibitor GSK2606414 (90 ug) were randomly divided into sham-operation + solvent group, SAH + solvent group, SAH + GSK2606414 (30 ug) group and SAH + GSK2606414 (90 ug) group. 2. Garcia scale was used to evaluate the neurological dysfunction in rats, and TUNEL staining was used to detect the neuronal apoptosis. Western blot was used to detect the expression levels of ER stress-related proteins (PERK, p-PERK, eIF2alpha, p-eIF2alpha) and apoptosis-related proteins (Akt, Bcl-2, Bax, Caspase-3). According to the above results, the appropriate dose of GSK2606414 against neuronal apoptosis was determined. The downstream mechanism of ERK pathway. Part 3 1. Adult male SD rats were randomly divided into sham-operation + solvent group, SAH + GSK2606414 group, SAH + Wortmannin group, SAH + GSK2606414 group, SAH + Wortmannin group, SAH + Wortmannin group, SAH + Wortmannin group and Akt inhibitor Wortmannin group. Group B, SAH + GSK2606414 + Wortmannin group 2. Garcia scale was used to assess the status of neurological impairment, the degree of brain edema was measured by dry-wet weight method, the degree of blood-brain barrier damage was evaluated by Evans blue method, the stress-related proteins of endoplasmic reticulum (PERK, p-PERK) and the tight junction-related proteins of blood-brain barrier (ZO-1, Occl) were detected by Western blot. To explore the protective effect of endoplasmic reticulum stress inhibitors on the damage of blood-brain barrier after SAH and its possible downstream mechanism. The expression levels of p-PERK, p-eIF2alpha and ATF4 increased significantly after the establishment of SAH, reaching a peak at 72 hours. Immunofluorescence double staining indicated that the number of PERK-positive cells in the brain increased significantly 72 hours after SAH, and PERK co-expressed with neuron-labeled protein NeuN. In addition, PERK was expressed in some astrocyte-labeled protein GFAP (glial fibrillary acid). The expression of ER stress-related protein p-PERK and p-eIF2alpha in SAH model rats was significantly inhibited by the application of PERK-specific inhibitor GSK2606414, and the neurological dysfunction in SAH rats was significantly alleviated. 2. After SAH, the expression of anti-apoptotic protein Bcl-2 in rat brain tissue was significantly decreased. GSK2606414 significantly increased the expression of Bcl-2 and decreased the expression of Bax and Cleaved Caspase-3. TUNEL staining showed that a large number of TUNEL-positive cells could be observed in the brain of SAH rats, and the number of TUNEL-positive cells could be significantly reduced by GSK2606414 treatment. High dose of GSK2606414 (90 ug) had better therapeutic effect. Pretreatment with Wortmannin, a specific inhibitor of Akt, significantly inhibited the neuroprotective effect of GSK2606414 and aggravated the level of neuronal apoptosis. Part 3. GSK2606414 treatment significantly alleviated brain edema in SAH rats and decreased Evans blue dye leakage in brain tissue. Pretreatment with Akt inhibitor Wortmannin could aggravate the degree of brain edema, increase the effusion of Evans blue dye, decrease the expression of tight junction protein ZO-1, Occludin and Claudin-5, and significantly block the effect of PERK inhibitor GSK2606414 on SAH rats. Conclusion 1. Endoplasmic reticulum stress may play an important role in early brain injury after subarachnoid hemorrhage. 2. Application of PERK-eIF2 alpha-ATF4 inhibitor GSK260 may play an important role in early brain injury after subarachnoid hemorrhage. 6414 can significantly reduce the level of endoplasmic reticulum stress after subarachnoid hemorrhage, improve the neurological dysfunction, inhibit neuronal apoptosis, protect the integrity of blood-brain barrier, and reduce brain edema. 3. Pretreatment with Wortmannin, an Akt-specific inhibitor, can significantly block GSK2606414's anti-apoptosis and protect the blood-brain barrier. GSK2606414 plays a neuroprotective role by inhibiting ER stress and activating Akt signaling pathway.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R743.35
本文编号：2181357
[Abstract]:Background Subarachnoid hemorrhage (SAH) is a common clinical neurosurgical emergency with high mortality and disability. According to epidemiological investigation, the annual prevalence of SAH is about 1 and the mortality rate is as high as 30%. However, in recent years, many randomized, double-blind, controlled clinical studies have shown that the treatment of cerebral vasospasm can not improve the prognosis of SAH patients. Some drugs can effectively improve the prognosis of SAH patients, but they have no effect on the treatment of cerebral vasospasm. Early brain injury is a complex pathophysiological process, including elevated intracranial pressure, decreased cerebral blood flow, decreased cerebral perfusion pressure, death of nerve cells and destruction of blood-brain barrier. Endoplasmic reticulum is an important organelle in cells. It is the main site for protein synthesis, processing and storage of Ca2 +. It is extremely sensitive to stimuli from the internal and external environment, causing misfolded and unfolded eggs when its function is disordered. The state of white aggregation in the lumen is called endoplasmic reticulum stress. Endoplasmic reticulum stress is followed by unfolded protein response (UPR), which includes the suspension of protein synthesis, activation of molecular chaperone and folding enzyme transcriptional activity, and induction of ER-related degradation to cope with various spines in the internal and external environment. When stimuli persist, endoplasmic reticulum stress will increase excessively, and then activate a series of signaling molecules, leading to cell death and other serious consequences. Numerous studies have shown that endoplasmic reticulum stress plays an important role in the development of neurodegenerative diseases and inflammation of the nervous system. Therefore, this study will focus on the endoplasmic reticulum stress PERK-eIF2alpha-ATF4 pathway, observe its expression level after SAH, explore its role and specific mechanism in early brain injury by intervention means, and provide new ideas and new targets for the treatment of SAH. The adult male SD rats were randomly divided into sham operation group (Sham group) and subarachnoid hemorrhage group (SAH group). The SAH group was divided into subarachnoid hemorrhage 3 hours group (SAH 3 hours group) and subarachnoid hemorrhage 6 hours group (SAH group) according to the sampling time. Six hours after subarachnoid hemorrhage, 12 hours after subarachnoid hemorrhage (SAH 12 hours group), 24 hours after subarachnoid hemorrhage (SAH 24 hours group) and 72 hours after subarachnoid hemorrhage (SAH 72 hours group). The expression of endoplasmic reticulum stress-related protein (ER-SRP) was detected by immunofluorescence double-labeling staining. Part 2. One hour after the establishment of SAH model in adult male SD rats, the solvents were injected into the lateral ventricle of the adult male SD rats by stereotaxic method, and the low dose of PERK was used. Specific inhibitor GSK2606414 (30 ug) or high-dose PERK specific inhibitor GSK2606414 (90 ug) were randomly divided into sham-operation + solvent group, SAH + solvent group, SAH + GSK2606414 (30 ug) group and SAH + GSK2606414 (90 ug) group. 2. Garcia scale was used to evaluate the neurological dysfunction in rats, and TUNEL staining was used to detect the neuronal apoptosis. Western blot was used to detect the expression levels of ER stress-related proteins (PERK, p-PERK, eIF2alpha, p-eIF2alpha) and apoptosis-related proteins (Akt, Bcl-2, Bax, Caspase-3). According to the above results, the appropriate dose of GSK2606414 against neuronal apoptosis was determined. The downstream mechanism of ERK pathway. Part 3 1. Adult male SD rats were randomly divided into sham-operation + solvent group, SAH + GSK2606414 group, SAH + Wortmannin group, SAH + GSK2606414 group, SAH + Wortmannin group, SAH + Wortmannin group, SAH + Wortmannin group and Akt inhibitor Wortmannin group. Group B, SAH + GSK2606414 + Wortmannin group 2. Garcia scale was used to assess the status of neurological impairment, the degree of brain edema was measured by dry-wet weight method, the degree of blood-brain barrier damage was evaluated by Evans blue method, the stress-related proteins of endoplasmic reticulum (PERK, p-PERK) and the tight junction-related proteins of blood-brain barrier (ZO-1, Occl) were detected by Western blot. To explore the protective effect of endoplasmic reticulum stress inhibitors on the damage of blood-brain barrier after SAH and its possible downstream mechanism. The expression levels of p-PERK, p-eIF2alpha and ATF4 increased significantly after the establishment of SAH, reaching a peak at 72 hours. Immunofluorescence double staining indicated that the number of PERK-positive cells in the brain increased significantly 72 hours after SAH, and PERK co-expressed with neuron-labeled protein NeuN. In addition, PERK was expressed in some astrocyte-labeled protein GFAP (glial fibrillary acid). The expression of ER stress-related protein p-PERK and p-eIF2alpha in SAH model rats was significantly inhibited by the application of PERK-specific inhibitor GSK2606414, and the neurological dysfunction in SAH rats was significantly alleviated. 2. After SAH, the expression of anti-apoptotic protein Bcl-2 in rat brain tissue was significantly decreased. GSK2606414 significantly increased the expression of Bcl-2 and decreased the expression of Bax and Cleaved Caspase-3. TUNEL staining showed that a large number of TUNEL-positive cells could be observed in the brain of SAH rats, and the number of TUNEL-positive cells could be significantly reduced by GSK2606414 treatment. High dose of GSK2606414 (90 ug) had better therapeutic effect. Pretreatment with Wortmannin, a specific inhibitor of Akt, significantly inhibited the neuroprotective effect of GSK2606414 and aggravated the level of neuronal apoptosis. Part 3. GSK2606414 treatment significantly alleviated brain edema in SAH rats and decreased Evans blue dye leakage in brain tissue. Pretreatment with Akt inhibitor Wortmannin could aggravate the degree of brain edema, increase the effusion of Evans blue dye, decrease the expression of tight junction protein ZO-1, Occludin and Claudin-5, and significantly block the effect of PERK inhibitor GSK2606414 on SAH rats. Conclusion 1. Endoplasmic reticulum stress may play an important role in early brain injury after subarachnoid hemorrhage. 2. Application of PERK-eIF2 alpha-ATF4 inhibitor GSK260 may play an important role in early brain injury after subarachnoid hemorrhage. 6414 can significantly reduce the level of endoplasmic reticulum stress after subarachnoid hemorrhage, improve the neurological dysfunction, inhibit neuronal apoptosis, protect the integrity of blood-brain barrier, and reduce brain edema. 3. Pretreatment with Wortmannin, an Akt-specific inhibitor, can significantly block GSK2606414's anti-apoptosis and protect the blood-brain barrier. GSK2606414 plays a neuroprotective role by inhibiting ER stress and activating Akt signaling pathway.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R743.35
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相关期刊论文 前4条
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