糖基化终末产物通过氧化应激及内质网应激诱导SH-SY5Y细胞凋亡的研究

  本文关键词：糖基化终末产物通过氧化应激及内质网应激诱导SH-SY5Y细胞凋亡的研究，由笔耕文化传播整理发布。

        研究背景阿尔茨海默病(AD)是老年人常见的疾病之一,严重影响人类的身心健康,给患者家庭及社会带来了沉重的负担。近年来越来越多的研究显示,2型糖尿病与AD的发生及发展密切相关,Luchsinger等对1262名老年人群调查显示1/3的痴呆与糖尿病有关,相关流行病学调查发现,DM患者发生AD的危险性是非DM患者的2倍。糖尿病导致AD发生的机制与胰岛素细胞信号转导异常、高胆固醇血症及高血糖状态下非酶糖基化反应生成的糖基化终末产物(advanced glycation end products, AGEs)有关。糖的醛基和蛋白质的氨基经过复杂的非酶糖基化反应(Maillard反应)生成一类不可逆聚合物,即糖基化终末产物,AGEs在糖尿病视网膜病变、动脉硬化、糖尿病肾病等靶器官损害中发挥重要作用,而AD患者脑内老年斑和神经元纤维缠结中均发现有AGEs的聚积,Takeuchi和Maczurek等研究也指出,AGEs在糖尿病所致的认知功能障碍和AD病理变化中起着非常重要的作用,是引起老年人认知功能下降的危险因素之一。AGEs能够促进蛋白的聚积与交联,直接抑制蛋白的正常功能,并能够通过促进活性氧(reative oxygen species, ROS)的产生对机体造成生物损伤,除此之外还能与其特异性受体(主要是RAGE)结合进而通过一系列信号转导通路发挥其间接毒性作用。AD的典型病理改变是在特定脑区内出现老年斑(senile plaques, SPs)、神经元纤维缠结(neurofibrillarytangles, NFFs)、神经元及突触丢失。神经元进行性减少是AD的重要病理特征,其中,细胞凋亡起了主导作用。凋亡是程序性的细胞死亡过程,外源性死亡受体途径、内源性线粒体凋亡通路以及内质网应激介导的细胞凋亡过程目前是公认的细胞凋亡的主要通路,近年来研究发现内质网应激性凋亡途径在多种疾病如糖尿病、脑梗塞、阿尔茨海默病的发病中扮演了重要角色,我们以往研究提示AGEs参与APP异常代谢及Aβ的生成,而内质网是生成Aβ1-42的主要场所,在APP的代谢中起着重要作用。内质网内蛋白质折叠功能障碍、钙平衡的破坏及氧化反应的异常等都会触发内质网应激(ERS)。AGEs是否通过触发氧化应激及内质网应激进而导致细胞凋亡参与AD发生发展尚不清楚,为此,我们设计该课题,选择非常接近正常神经细胞形态及生理功能的人神经母细胞瘤细胞株SH-SY5Y,以此为研究对象,以非酶糖基化修饰的牛血清白蛋白(AGE-bovine serum albumin, AGE-BSA)处理细胞,并分别以抗RAGE中和抗体(RAGE-antibody, RAGE-Ab)、抗氧化剂α-硫辛酸(alpha lipoic acid, ALA)及NADPH氧化酶抑制剂二亚苯基碘(diphenyleneiodonium, DPI)预处理细胞探讨AGEs是否通过氧化应激、内质网应激促进细胞凋亡,揭示AGEs参与阿尔茨海默病发生发展的可能相关机制。目的本实验通过研究AGEs对体外培养的SH-SY5Y细胞凋亡的影响及氧化应激和内质网应激在该过程中的作用,探讨AGEs参与AD发生发展的可能机制。方法选择体外培养的SH-SY5Y细胞为模型,分别以不同浓度(0、50、100、200、300μg/mL)的糖基化修饰的牛血清白蛋白(AGE-BSA)处理SH-SY5Y细胞48h,选取AGE-BSA敏感浓度200μg/mL处理细胞不同时间(24、36、48h),流式细胞仪(FCM)分别检测各组细胞凋亡率,确定AGE-BSA诱导SH-SY5Y凋亡最佳浓度及时间；将细胞随机分为6组：正常对照组、BSA对照组(200μg/mL BSA)、AGE-BSA组(200μg/mLAGE-BSA)、AGE-BSA+RAGE-Ab组：预先用抗RAGE-Ab (10μg/mL)干预细胞1h,再加入AGE-BSA (200μg/mL); AGE-BSA+ALA组：预先用ALA(终浓度200μmol/L)干预细胞1h,再加入AGE-BSA(200gg/mL)；AGE-BSA+DPI组：先加DPI(30nmo1/L)干预1h,加入AGE-BSA(200gg/mL)。处理48h后FCM检测各组细胞凋亡率,Hoechst33258荧光染色观察凋亡细胞核形态变化；应用活性氧荧光探针DCFH-DA检测AGE-BSA干预SH-SY5Y细胞不同时间(0、12、24、48h)后活性氧(ROS)水平,及不同药物处理48h后细胞ROS水平；通过免疫细胞荧光化学及免疫蛋白印迹方法观察AGEs处理细胞不同时间(0、12、24、48h)后GRP78、p-eIF2α、Caspase-12的蛋白表达情况；选取各蛋白表达高峰时间点,应用免疫蛋白印迹方法检测各不同处理组SH-SY5Y细胞内GRP78、p-eIF2α、Caspase-12蛋白表达变化。结果1.FCM测细胞凋亡率1.1正常细胞凋亡率为(2.23±0.08)%,50、100、200、300μg/mLAGE-BSA作用48h后凋亡率分别为(2.98±0.67)%、(8.23±0.42)%、(16.8±1.27)%、(19.6±2.89)%；200μg/mL AGE-BSA作用24、36、48h后凋亡率分别为(7.54±1.08)%、(10.75±1.19)%、(16.8±1.27)%,提示AGE-BSA能诱导细胞凋亡,且凋亡率随AGE-BSA浓度增加及时间延长而增高。1.2分别用RAGE中和抗体、ALA、DPI预处理细胞后再加入AGE-BSA200μg/mL作用48h,细胞凋亡率分别为(5.18±0.16)%、(5.94±0.10)%、(7.26±1.22)%,与AGE-BSA组相比凋亡率分别下降69.1%、64.6%、56.8%,差异显著(P<0.01或P<0.05),BSA对照组与正常对照组比较差异无统计学意义。2. Hoechst33258荧光染色观察凋亡细胞核形态：不同处理组细胞经Hoechst33258染色在荧光显微镜下观察形态,正常对照组和BSA对照组细胞核呈弥散均匀的蓝色荧光,无核浓聚,AGE-BSA组可见大量散在凋亡样改变的细胞核,表现为亮蓝色,出现核碎裂,荧光强度比正常细胞明显增强,抗RAGE-Ab、ALA、DPI预处理组均可见细胞荧光强度比AGE-BSA组降低,凋亡细胞数明显减少。3. DCFH-DA检测SH-SY5Y细胞ROS水平3.1AGE-BSA干预0h的细胞内仅有少量ROS, AGE-BSA干预细胞12h可见细胞内ROS水平显著升高,接近0h组细胞的4倍,并且随AGE-BSA作用时间的延长,ROS水平不断增高,与0h组细胞比较差异具有统计学意义(P<0.01)。3.2AGE-BSA及不同预处理48h检测ROS水平：与正常组比较,AGE-BSA组细胞内ROS明显增多(P<0.01),是正常组的6.95倍,而NC组与BSA对照组比较无显著差异,RAGE-Ab组、ALA组、DPI组细胞内ROS水平较AGE-BSA组分别下降56.2%、46.5%、54.3%,差异均有统计学意义(P<0.01),提示AGEs可能通过与其受体RAGE结合进而激活NADPH氧化酶促进ROS的产生。4.免疫细胞荧光化学及免疫蛋白印迹方法观察SH-SY5Y细胞中的GRP78、p-eIF2α、Caspase-12蛋白表达并进行半定量分析4.1免疫细胞荧光化学方法观察GRP78、p-eIF2α、Caspase-12在SH-SY5Y细胞的表达,AGE-BSA处理后荧光染色阳性细胞数明显增多,GRP78主要在胞浆表达,而p-eIF2α全细胞都有表达,但胞核荧光强度更高,免疫蛋白印迹方法结果显示AGE-BSA分别处理细胞0、12、24、48h, GRP78、p-eIF2α、 Caspase-12蛋白表达水平呈时间依赖性增高,GRP78、p-eIF2α在AGEs处理24h后出现表达峰值,Caspase-12在48h出现表达高峰。4.2按分组要求给予细胞不同处理24h,免疫蛋白印迹结果提示AGEs组GRP78、p-eIF2α蛋白表达水平分别为正常对照组的3.56倍、6.39倍,而预先用抗RAGE-Ab、DPI、ALA分别处理细胞可以显著降低GRP78、p-eIF2α的表达,GRP78下降率分别为39.5%、14.1%、19.3%,p-eIF2α下降率分别为28%、41%、33%,差异有统计学意义(P<0.05或P<0.01)。处理48h后检测Caspase-12蛋白表达水平,Caspase-12在正常细胞和BSA组弱表达,AGE-BSA乍用于细胞可上调Caspase-12蛋白表达,与正常组和BSA组比较差异均有显著性(P<0.01),而应用抗RAGE-Ab、ALA和DPI分别预处理细胞均可部分阻断AGE-BSA导致的Caspase-12上调,下降率分别为35.9%、43.7%、50.5%,差异显著(P<0.01)。结论1. AGEs通过与其受体RAGE结合,进而激活SH-SY5Y的NADPH氧化酶促进促进ROS产生。2. AGEs可能通过促进ROS的产生引发GRP78表达增加及PERK-eIF2a途径的内质网应激,进而诱导神经细胞凋亡,参与AD的发病。3.阻断AGEs的受体RAGE及DPI、α-硫辛酸抗氧化干预能通过降低氧化应激及内质网应激减少神经细胞凋亡,可望成为防治AD的新的治疗靶点。

    Background Alzheimer’s disease is one of the frequently encountered disease, it seriously affected physical and mental health of humen, and it has brought a heavy burden to patients family and social. Recent studies have pointed out that T2DM closely associated with the occurrence and development of AD. Luchsinger investigated1262elderly people and the results demonstrate that1/3of dementia related to diabetes. Relevant epidemiological survey found that the risk of DM patients suffer from AD was2times of nondiabetic. Mechnisms about DM caused occurrence of AD related to abnormal insulin signal transduction, hypercholesterolemia and advanced glycation end products produced in non-enzymetic glycosylation. Advanced glycation end products (AGEs) are structurally diverse irreversible polymer to which the amino sugar and protein deposits through a series of complex non-enzymatic reaction (Maillard response). AGEs played an important part in diabetic retinopathy, arteriosclerosis and diabetic nephropathy and other target organ damage.There were accumulation of AGEs in senile plaque and neurofibrillary tangles in AD patients. Takeuchi and Maczurek’s research indicated that AGEs played an important part in cognitive dysfunction resulted from DM and AD, AGEs is one of the risk factors cause cognitive decline of elderly people. AGEs may promote protein crosslinking and accumulation, inhibit the functional proteins, stimulate the generation of reactive oxygen species (ROS) directly caused biological damage. AGEs can also bind to the receptors for AGEs (RAGE), further to play indirect toxic effects through a series of signal transduction pathways.Pathological characteristic of AD is the formation of senile plaques, neurofibrillary tangles and neuronal and synaptic loss in specific brain regions. An significant pathologic feature of AD is progressive and specific neurons loss, and apoptosis holds a prominent place in cells death. Apoptosis is programmed cell death, and it has been known that there are3pathways can result cell apoptosis, including death receptors pathway, mitochondria pathway and endoplasmic reticulum stress (ERS)-mediated apoptosis. Recent studies found that ERS-mediated apoptosis play an important role in occurrence of many diseases such as diabetes and AD et al. Our previous studies indicated AGEs participated in abnormal metabolism of APP and production of Ap, ER is the main place which produced Aβ1-42and play an important part in metabolism of APP. Dysfunction of protein folding in ER, destructed calcium balance and abnormal oxidation reaction both can trigger ERS. It is not very explicitly that whether AGEs lead to apoptosis.through triggering oxidative stress and ERS and whether it involved in occurrence and development of AD. SH-SY5Y cell is closely similar to normal nerve cell in morphology and physiological functions. Therefore, we design this subject and choose SH-SY5Y as research object. We use AGE-bovine serum albumin to treat SH-SY5Y cells, and respectively pretreat cells with RAGE-antibody, alpha lipoic acid (ALA) and diphenyleneiodonium (DPI), in order to explore whether AGEs cause cell apoptosis through triggering oxidative stress and ERS, reveal the possible mechanism of AGEs involved in the occurrence and development of AD.Objective To investigate the effect of AGEs on SH-SY5Y cells apoptosis and the role of oxidative stress and ERS in this process, further to explore the possible mechanism of AGEs cause to AD.Methods As object of this study, the cultured SH-SY5Y cells were treated with different concentrations of AGE-BSA for48h, or treated with AGE-BSA(200ug/ml) for different times, cells apoptosis detected by flow cytometry (FCM) to determine the best concentration and time of AGE-BSA.Divide SH-SY5Y cells into six groups randomly, the normal control group, the BSA group, the AGE-BSA group, the AGE-BSA+anti-RAGE-Ab group, the AGE-BSA+Alpha Lipoic acid(ALA) group, the AGE-BSA+Diphenyleneiodonium (DPI) group. To detect the cells apoptosis by FCM and Hoechst staining after treatment with AGE-BSA and other drugs for48h; The level of ROS evaluated by the2’,7’-dichlorofluorescein diacetate (DCFH-DA) method. The expression of GRP78、p-eIF2α、Caspase-12was analysed by western blotting and immunofluorescence after treatment with AGE-BSA for0-48hours. Then select the peak time of each protein, and use western blotting to estimate the expression of GRP78、p-eIF2α、Caspase-12in different groups.Results1. Cell apoptosis rate measured by FCM1.1Apoptosis rate of normal control group was(2.23±0.08)%, after treatment with50,100.200,300μg/mL AGE-BSA for48hours the rate rised to(2.98±0.67)%,(8.23±0.42)%,(16.8±1.27)%,(19.6±2.89)%, after treatment with200μg/mL AGE-BSA for24hours, the rate was (7.54±1.08)%, it was (10.75±1.19)%for36hours and it was (16.8±1.27)%for48hours. Therefore, AGE-BSA can induce apoptosis, moreover the growth of apoptosis rate was in a time and concentration-dependent manner.1.2After pretreatment with RAGE-Ab, ALA and DPI respectively, the apoptosis rates have declined to (5.18±0.16)%,(5.94±0.10)%,(7.26±1.22)%, the decline rate were69.1%、64.6%、56.8%, the differences were significant compared with AGE-BSA group(P<0.05).There was no significant difference between NC group and BSA group.2. Apoptosis nucleus were detected by Hoechst33258fluorescent staining.Cells of different groups stained with Hoechst33258were observed under fluorescence microscope, the nucleus appeared uniformly dispersed of blue fluorescence, and there are no pyknoticnucleus. There were bright blue apoptosis-like nucleus in AGE-BSA group cells, and nuclear fragmentation appears. Fluorescence intensity was significantly enhanced than the normal cells. The fluorescence intensity of RAGE-Ab group、ALA group and DPI group were lower and apoptotic cells were significantly reduced than AGE-BSA group.3. The level of ROS in SH-SY5Y was detected by DCFH-DA3.1There was a small amount of ROS in cells without any AGE-BSA, and after treatment with AGE-BSA for12h, we detected the level of ROS has increased significantly, it was4times what the Oh group. The level of ROS was increased constantly along with treatment time lasting, and the differences were significant (P<0.01).3.2The ROS level of AGE-BSA group was6.95times what the NC group was(P <0.01) and there was no significant difference between NC group and BSA group. The ROS level of RAGE-Ab group, ALA group and DPI group were significantly decreased than AGE-BSA group, the decline rate were56.2%.46.5%,54.3%respectively (P<0.01). The results indicated that AGEs promoted the generation of ROS via the pathway of AGEs-RAGE-NADPH oxidase.4. Expression of GRP78, p-eIF2α, Caspase-12protein detected by immunofluorescence and western blotting4.1Estimated by immunofluorescence and western blotting, treatment with AGEs for0,12,24,48hours, the increased levels of GRP78and p-eEF2α in a time-dependent manner, with peak at24h. The Caspase-12showed the highest level at48h.4.2The levels of GRP78and p-eIF2α were analyzed in the lysates treated with200μg/mL AGE-BSA for24h, the two proteins in AGE-BSA group were respectively3.56times and6.39times what the NC group was, and which could be significantly blocked by the pretreatments of RAGE-Ab, ALA or DPI. The decline rate of GRP78were respectively39.5%,14.1%,19.3%, and that of p-eIF2α were28%,41%,33%, the differences were significant (P<0.05or P<0.01). The level of Caspase-12was measured in the lysates treated with200 μg/mL ASA-BSA for48h and apoptotic initiator marker Caspase-12were similar between the control and BSA groups, however, the level of Caspase-12in AGE-BSA group increased significantly compared with the NC group and BSA group (P<0.01). After pretreatment with RAGE-Ab, ALA or DPI, the level of Caspase-12have fallen by35.9%.43.7%,50.5%respectively(P<0.01).Conclusion1. AGEs could promote the production of ROS through binding to RAGE, further to activate NADPH oxidase.2. AGEs could trigger ERS through stimulating the generation of ROS, and further to cause nerve cells apoptosis, it may be a mechanism of AGEs cause to AD.3. Blocking the binding of AGEs-RAGE, decreasing ROS by ALA or inhibiting NADPH oxidase activity can efficiently prevent oxidative stress and ERS, consequently reduced cell death. It may provide a new method for the treatment of AD.

          糖基化终末产物通过氧化应激及内质网应激诱导SH-SY5Y细胞凋亡的研究

目录4-5CONTENTS5-6中文摘要6-11ABSTRACT11-15符合说明16-17前言17-20材料与方法20-31实验结果31-34讨论34-42结论42-43附图43-48参考文献48-54致谢54-55攻读硕士学位期间参与课题及发表的论文55-56学位论文评阅及答辩情况表56

  本文关键词：糖基化终末产物通过氧化应激及内质网应激诱导SH-SY5Y细胞凋亡的研究，由笔耕文化传播整理发布。