內(nèi)質(zhì)網(wǎng)應激PERK通路在蛛網(wǎng)膜下腔出血早期腦損傷中的作用及機制研究
發(fā)布時間：2018-08-13 15:12
【摘要】：研究背景蛛網(wǎng)膜下腔出血(subarachnoid hemorrhage,SAH)是一種臨床常見的神經(jīng)外科急癥,具有高死亡率、高致殘率的特點,據(jù)流行病學調(diào)查,人群年患病率約為1‰,死亡率高達30%。遲發(fā)性腦血管痙攣曾被認為是SAH致傷機制中的主要因素,一直是傳統(tǒng)研究的熱點領(lǐng)域。然而近年來多項臨床隨機雙盲對照研究顯示,緩解腦血管痙攣的治療并不能改善SAH患者的預后。某些藥物雖可有效改善患者預后,但其并無對抗腦血管痙攣的作用。因此近年來,本領(lǐng)域的權(quán)威學者提出了 SAH后早期腦損傷這一概念,即SAH發(fā)病后72小時內(nèi)所引發(fā)的早期腦損傷對SAH后神經(jīng)功能的康復發(fā)揮著決定性的影響作用。早期腦損傷是一個復雜的病理生理學過程,主要包括顱內(nèi)壓升高、腦血流量降低、腦灌注壓下降、神經(jīng)細胞死亡、血腦屏障破壞和腦水腫等。然而其具體損傷機制及上游調(diào)控信號目前尚不明確,需進一步研究探索。內(nèi)質(zhì)網(wǎng)是細胞內(nèi)重要的細胞器,它是蛋白質(zhì)合成、加工以及貯存Ca2+的主要場所。它對內(nèi)外環(huán)境刺激極為敏感,當其功能紊亂時導致錯誤折疊與未折疊蛋白在腔內(nèi)聚集的狀態(tài),被稱為內(nèi)質(zhì)網(wǎng)應激(endoplasmic reticulum stress)。內(nèi)質(zhì)網(wǎng)應激繼而引發(fā)未折疊蛋白反應(unfolded protein response,UPR),包括暫停蛋白質(zhì)合成、激活分子伴侶和折疊酶的轉(zhuǎn)錄活性、誘導內(nèi)質(zhì)網(wǎng)相關(guān)性降解,以應對內(nèi)外環(huán)境各種刺激因素。當刺激因素持續(xù)存在時,內(nèi)質(zhì)網(wǎng)應激將過度增強,進而激活一系列信號分子,引發(fā)細胞死亡等嚴重后果。眾多研究表明內(nèi)質(zhì)網(wǎng)應激在神經(jīng)退行性疾病、神經(jīng)系統(tǒng)炎癥的發(fā)生發(fā)展過程中發(fā)揮著重要作用,是該研究領(lǐng)域的熱點方向。但關(guān)于內(nèi)質(zhì)網(wǎng)應激在SAH后的作用及其具體機制,目前尚缺乏深入的研究。因此,本研究將圍繞內(nèi)質(zhì)網(wǎng)應激PERK-eIF2α-ATF4通路,觀察其在SAH后的表達水平,應用干預手段探索其在早期腦損傷中的作用及具體機制,為SAH的治療提供新思路與新靶點。實驗方法第一部分1.采用線栓穿刺法制作蛛網(wǎng)膜下腔出血(SAH)模型,成年雄性SD大鼠被隨機分為假手術(shù)組(Sham組),蛛網(wǎng)膜下腔出血組(SAH組);SAH組按取材時間不同又分為蛛網(wǎng)膜下腔出血后3小時組(SAH3h組)、蛛網(wǎng)膜下腔出血后6小時組(SAH6h組)、蛛網(wǎng)膜下腔出血后12小時組(SAH12h組)、蛛網(wǎng)膜下腔出血24后小時組(SAH 24 h組)和蛛網(wǎng)膜下腔出血后72小時組(SAH 72 h組)。應用Garcia量表評估大鼠神經(jīng)功能障礙狀況,利用干濕重法測定腦水腫程度,通過Western blot檢測內(nèi)質(zhì)網(wǎng)應激PERK-eIF2α-ATF4通路相關(guān)蛋白表達水平(PERK、p-PERK、eIF2α、p-eIF2α與ATF4),采用免疫熒光雙標染色法觀察表達內(nèi)質(zhì)網(wǎng)應激相關(guān)蛋白的神經(jīng)細胞類型。第二部分1.成年雄性SD大鼠SAH模型建立后1小時,應用立體定位下側(cè)腦室注射法分別給予溶劑、低劑量PERK特異性抑制劑GSK2606414(30μg)或高劑量PERK特異性抑制劑GSK2606414(90μg),隨機分為假手術(shù)+溶劑組、SAH+溶劑組、SAH+GSK2606414(30μg)組與 SAH+GSK2606414(90μg)組。2.應用Garcia量表評估大鼠神經(jīng)功能障礙狀況,采用TUNEL染色法測定神經(jīng)細胞凋亡狀況,應用Western blot檢測內(nèi)質(zhì)網(wǎng)應激相關(guān)蛋白(PERK、p-PERK、eIF2α、p-eIF2α),凋亡相關(guān)蛋白(Akt、Bcl-2、Bax、Caspase-3)的表達水平。根據(jù)前述研究結(jié)果,確定GSK2606414抗神經(jīng)細胞凋亡的適宜劑量。應用Akt抑制劑Wortmannin作為干預手段進行預處理,探索內(nèi)質(zhì)網(wǎng)應激PERK通路的下游作用機制。第三部分1.成年雄性SD大鼠SAH模型建立后,應用立體定位下側(cè)腦室注射法給予適宜劑量PERK特異性抑制劑GSK2606414,并加用Akt抑制劑Wortmannin作為預處理,隨機分為假手術(shù)+溶劑組、SAH+溶劑組、SAH+GSK2606414組、SAH+Wortmannin 組、SAH+GSK2606414+Wortmannin 組。2.應用Garcia量表評估大鼠神經(jīng)功能障礙狀況,利用干濕重法測定腦水腫程度,通過Evans blue法評估血腦屏障破壞程度,應用Western blot檢測內(nèi)質(zhì)網(wǎng)應激相關(guān)蛋白(PERK、p-PERK)、以及血腦屏障緊密連接相關(guān)蛋白(ZO-1、Occludin、Claudin5)的表達水平。探索內(nèi)質(zhì)網(wǎng)應激抑制劑對SAH后血腦屏障破壞的保護作用,以及其可能的下游作用機制。結(jié)果第一部分1.蛛網(wǎng)膜下腔出血(SAH)模型建立后72h內(nèi)大鼠的腦水含量程度與神經(jīng)功能障礙持續(xù)加重;Western blot檢測顯示SAH模型建立后,p-PERK、p-eIF2α及ATF4的表達水平顯著升高,于72小時達到高峰;免疫熒光雙重染色提示,SAH后72h,腦組織中PERK陽性細胞數(shù)量顯著增加,PERK與神經(jīng)元標記蛋白NeuN共表達;此外,PERK表達于部分星形膠質(zhì)細胞標記蛋白GFAP(glial fibrillary acidic protein)陽性的神經(jīng)細胞中。第二部分1.應用PERK特異性抑制劑GSK2606414可顯著抑制內(nèi)質(zhì)網(wǎng)應激相關(guān)蛋白p-PERK、p-eIF2α在SAH模型大鼠中的表達水平,顯著緩解SAH大鼠的神經(jīng)功能障礙;2.SAH發(fā)生后,大鼠腦組織中抗凋亡蛋白Bcl-2表達水平顯著降低,促凋亡蛋白Bax、Cleaved Caspase-3表達水平顯著升高,應用GSK2606414可顯著提高Bcl-2表達水平,減少Bax、Cleaved Caspase-3表達水平;TUNEL染色顯示,SAH大鼠腦組織中可觀察到大量TUNEL陽性細胞,通過GSK2606414治療可顯著減少TUNEL陽性細胞的數(shù)量,并且高劑量GSK2606414(90μg)具有更好的治療效果。靜脈注射Akt特異性抑制劑Wortmannin預處理可顯著抑制GSK2606414的神經(jīng)保護作用,加重神經(jīng)細胞凋亡水平。第三部分1.應用GSK2606414處理可顯著緩解SAH大鼠腦水腫程度,減少腦組織中Evans blue染料滲出,提高血腦屏障緊密連接蛋白ZO-1、Occludin、Claudin5的表達水平。2.應用Akt抑制劑Wortmannin預處理會加重腦水腫程度,增加腦組織中Evans blue染料滲出,降低緊密連接蛋白ZO-1、Occludin、Claudin5的表達水平,顯著阻斷PERK抑制劑GSK2606414對SAH大鼠的血腦屏障保護作用。結(jié)論1.蛛網(wǎng)膜下腔出血后早期腦損傷中,內(nèi)質(zhì)網(wǎng)應激PERK-eIF2α-ATF4通路顯著激活,并廣泛分布于神經(jīng)元與星形膠質(zhì)細胞,提示內(nèi)質(zhì)網(wǎng)應激可能在蛛網(wǎng)膜下腔出血后早期腦損傷中發(fā)揮重要作用。2.應用PERK特異性抑制劑GSK2606414可顯著降低蛛網(wǎng)膜下腔出血后內(nèi)質(zhì)網(wǎng)應激水平,改善蛛網(wǎng)膜下腔出血大鼠的神經(jīng)功能障礙,抑制神經(jīng)細胞凋亡,保護血腦屏障完整性,減輕腦水腫。3.Akt特異性抑制劑Wortmannin預處理可顯著阻斷GSK2606414的抗凋亡、保護血腦屏障作用,提示GSK2606414主要通過抑制內(nèi)質(zhì)網(wǎng)應激,激活Akt信號通路以發(fā)揮神經(jīng)保護作用。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R743.35
本文編號：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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R743.35
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