抑制miR-181a对鱼藤酮诱导的SH-SY5Y多巴胺能细胞损伤的保护作用

目的:通过抑制mi R-181a表达研究其对鱼藤酮诱导SH-SY5Y的细胞损伤、内质网应激以及未折叠蛋白反应(unfolded protein response,UPR)的影响,为mi R-181a在帕金森病治疗方面提供初步的实验依据。方法:不同浓度的鱼藤酮诱导SH-SY5Y作用12、24、48、72 h;mi R-181a类似物和mi R-181a抑制剂及0.4μmol/L鱼藤酮诱导SH-SY5Y作用24 h;采用CCK-8法检测细胞存活率;Annexin V/PI双染检测细胞凋亡;双荧光素酶报告基因检测mi R-181a与葡萄糖调节蛋白78(glucose regulated protein 78 k D,GRP78)的靶标关系;q RT-PCR检测mi R-181a和GRP78 m RNA表达水平;Western Blot检测GRP78、肌醇需酶1α(inositol-requiring enzyme 1,IRE1α)、X-盒结合蛋白1(X box-binding protein 1,XBP1)和CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein-homologous protein,CHOP)的蛋白表达水平。结果:(1)CCK-8结果显示:随着鱼藤酮浓度(0.1μmol/L)增加,SH-SY5Y存活率降低,并存在浓度和时间依赖性;其中0.4μmol/L作用24 h后存活率已降为0.6(P0.05),加入mi R-181a抑制剂后细胞存活率升高达0.8(P0.05);(2)Annexin V/PI双染结果显示:抑制mi R-181a表达,细胞的凋亡降低至0.23(P0.05);(3)双荧光素酶报告基因检测显示:mi R-181a与GRP78存在直接的靶标关系;(4)q RT-PCR结果显示:0.4μmol/L鱼藤酮作用24 h后细胞mi R-181a呈高表达(P0.05),抑制mi R-181a表达可上调GRP78 m RNA水平(P0.05);(5)Western Blot结果显示:抑制mi R-181a表达可上调GRP78和下调p IRE1α、XBP1和CHOP的蛋白表达(P0.05)。结论:抑制mi R-181a表达可减轻鱼藤酮诱导的SH-SY5Y细胞损伤,其机制可能通过上调GRP78进而调节内质网应激以及抑制UPR相关信号通路,从而发挥对神经元细胞的保护作用。     

STCH减弱Aβ_(25-35)对SH-SY5Y培养细胞神经毒性作用的初步研究

目的:研究应激和分子伴侣蛋白(stress and chaperon,STCH)拮抗β淀粉样蛋白(beta-amyloid25-35,Aβ25-35)在体外对人神经母细胞瘤细胞系SH-SY5Y毒性的机制。方法:用Aβ25-35(20μmol/L)在不同时间点分别处理SH-SY5Y培养细胞,然后采用逆转录-聚合酶链式反应(RT-PCR)和蛋白印记(Western Blot)法检测内质网应激相关分子的表达和c-Jun amino-terminal kinase(JNK)的磷酸化,并采用负载钙荧光探针(acetoxymethyl ester ofFura-2,Fura-2AM)钙成像技术检测过表达STCH对100nmol/L星孢霉素(staurosporin)引起的钙超载的情况。结果:Aβ25-35处理SH-SY5Y细胞后引起STCHmRNA表达上调,在3h开始升高,12h达到高峰,然后下降;而葡萄糖调节蛋白94(glucose-regulated protein94,Grp94)则于处理后6h表达上调,9h达到高峰,然后快速下降。Aβ25-35处理SH-SY5Y细胞能够激活JNK通路,致其磷酸化增加,过表达STCH,能够抑制JNK的磷酸化。过表达的STCH能够显著拮抗stauroposrine引起的游离钙增加。结论:STCH可能通过拮抗游离钙增加,抑制JNK通路而发挥保护神经元的作用,这将为我们寻找神经保护的策略提供有益的线索。     

奥氮平对鱼藤酮诱导PC12细胞凋亡的保护作用

探讨非典型抗精神病药物奥氮平对鱼藤酮诱导神经元凋亡的可能保护作用及其机制。以NGF诱导后的PC12细胞作为细胞模型,采用鱼藤酮诱导细胞凋亡。用不同浓度奥氮平、氟哌啶醇预处理后,观察鱼藤酮对PC12细胞的作用,分别用噻唑蓝(MTT)法检测细胞活性、流式细胞仪检测细胞凋亡率、Hoechst33342染色观察细胞形态学改变,并对二者的结果进行比较。鱼藤酮以剂量依赖的方式杀死PC12细胞。鱼藤酮(6μmol/L)作用后,奥氮平(50μmol/L)组预处理48h的细胞活力显著高于对照组(无药物预处理)(P<0.05);鱼藤酮(4、6、8μmol/L)作用后,氟哌啶醇组(20、40、60μmol/L)的细胞活力均低于对照组。流式细胞仪检测结果显示,对照组、氟哌啶醇(20μmol/L)组、奥氮平(50μmol/L)组、空白对照组(无药物预处理以及鱼藤酮处理)的细胞凋亡率依次为(32.2±1.3)%、(42.1±1.0)%、(14.0±1.0)%和(1.3±0.3)%。Hoechst33342染色结果显示,对照组每个视野多见凋亡细胞,细胞核裂解为碎块,氟哌啶醇组更明显;奥氮平组凋亡细胞数量较氟哌啶醇组及对照组为少。结果提示奥氮平对鱼藤酮诱导PC12细胞的凋亡具有保护作用,这可能是奥氮平和氟哌啶醇在精神分裂症病人应用中有不同的治疗效果和副作用的部分机制。     

分子伴侣功能的研究进展

分子伴侣是一类能够协助其他多肽进行正常折叠、组装、转运、降解的蛋白 ,并在 DNA的复制、转录、细胞骨架功能、细胞内的信号转导等广泛的领域都发挥着重要的生理作用 ,其功能异常会导致多种相关的疾病     

转染α-突触核蛋白的SH-SY5Y细胞增强对鱼藤酮毒性的耐受

目的探讨α-突触核蛋白(α-synuc le in)在鱼藤酮处理的人类多巴胺能SH-SY5Y细胞内的作用。方法用脂质体转染的方法将-αsynuc le in基因转染入SH-SY5Y细胞内,并筛选稳定表达-αsynuc le in的细胞。经过不同浓度的鱼藤酮处理后,测定细胞活力、细胞内氧化应激和抗氧化能力。结果经鱼藤酮处理后,所有的细胞均表现为细胞活力下降和细胞内氧化应激增强。与正常SH-SY5Y细胞相比,过表达-αsynuc le in的细胞表现为较高的细胞活力和抗氧化能力(P<0.01)。结论α-synuc le in过表达可能通过增强SH-SY5Y细胞活力和抗氧化能力,来部分对抗鱼藤酮的毒性作用。     

RNA伴侣分子的研究进展

RNA只有折叠成天然构像才能具有正常的功能。RNA不是孤立存在的 ,它总是与蛋白质结合成复合物发挥作用。在动力学和热力学上 ,RNA易于形成错误折叠。RNA伴侣分子可以帮助RNA正确折叠或抑制RNA错误折叠 ,可以使错误折叠的RNA构像解链并形成正确的空间折叠。文章对RNA伴侣分子的分子特性和作用机制进行了探讨 ,介绍了几种常见RNA伴侣分子活性鉴定的策略 ,列举了几种重要的RNA伴侣分子并论述了RNA伴侣分子的意义及当前研究中存在的问题。     

利多卡因对SH-SY5Y细胞的损伤作用

目的: 采用SH-SY5Y细胞体外培养模型,通过观察细胞形态、测定细胞活力和细胞凋亡率,探讨不同浓度利多卡因对SH-SY5Y细胞的损伤作用。方法: SH-SY5Y细胞离体培养, 分为4组, 即: 正常培养组(对照组, 未经药物处理); 0.5%、1%、2%利多卡因组(L1、L2、L3组), 分别用相应浓度的利多卡因处理SH-SY5Y细胞10 min。在药物处理10 min后观察细胞形态, 并分别在药物处理后5 min (T1)、10 min (T2)、药物处理结束后15 min (T3)、药物处理结束后12 h (T4)测定细胞活力及细胞凋亡率。结果: 正常培养SH-SY5Y细胞胞体粗大, 呈多角形, 出现树突状突起, 神经突起, 多而粗大, 分布成网络, 各实验组SH-SY5Y细胞则变圆, 回缩, 轴突渐消失。各实验组不同浓度的利多卡因对SH-SY5Y细胞活力均有明显影响, 利多卡因处理5 min后, 各组细胞活力明显下降, 且随剂量增加, 细胞活力下降明显增加。对照组细胞凋亡率在各时点保持在5.9%~6.3%之间, 实验组细胞在利多卡因处理后各时点细胞凋亡率明显增加, 且随着浓度的增加,细胞凋亡率也增加。结论: 0.5%、1%、2%利多卡因对SH-SY5Y细胞均有损伤作用, 且随浓度的增加,损伤程度加重。     

咖啡酸苯乙酯对鱼藤酮诱导帕金森细胞模型的保护

BACKGROUND: Caffeic acid phenethyl ester (CAPE) can inhibit lipid peroxidation after rat brain injury. However, the trend of 5-lipoxygenaseis (5-LOX) and cysteinyl leukotrienes (CysLTs) in model of Parkinson’s disease, and whether CAPE protects against rotenone-induced cellular injuries by inhibiting the levels of 5-LOX and CysLTs still need further research.     

抗坏血酸对癫痫大鼠海马分子伴侣介导的自噬激活的影响

目的: 探讨癫痫大鼠海马氧化应激反应和分子伴侣介导的自噬(chaperone-mediated autophagy,CMA)活性的变化,以及抗氧化剂抗坏血酸 (ascorbic acid, AA)神经保护作用的可能机制。方法: 实验大鼠分为空白对照组、癫痫24 h组、AA预处理癫痫组和AA对照组。应用RT-PCR和免疫印迹法检测海马组织2a型溶酶体相关膜蛋白(lysosome-associated membrane protein type 2a,LAMP2a)mRNA和蛋白的变化,采用化学法检测海马组织丙二醛(malondialdehyde, MDA)和超氧化物歧化酶(superoxide dismutase,SOD)的水平。结果: 癫痫组大鼠海马组织LAMP2a转录和合成明显高于空白对照组,MDA含量明显升高,而SOD的水平显著下降。AA预处理可显著减低癫痫大鼠海马LAMP2a的转录和合成,并明显降低MDA的含量,却显著提高SOD的水平。结论: 癫痫发作导致的海马损伤时存在CMA激活现象和显著的氧化应激反应;抗氧化剂AA通过抑制CMA活性和降低氧化应激反应来减轻癫痫发作中的海马损伤,具有神经保护作用。     

DADLE对SH-SY5Y细胞缺氧复氧损伤的保护作用

目的：探讨[D-Ala2,D-Leu5] enkephalin（ DADLE）对人神经母细胞瘤细胞SH-SY5Y 细胞的缺氧复氧的保 护作用。方法：将体外培养的SH-SY5Y 细胞分别缺氧6 、12、24 h 和48 h,均复氧4 h 后观察细胞形态改变,采用 MTT法检测细胞生存率,评估细胞损伤程度。在此基础上,于缺氧12 h 期间首先给予DADLE 100 pmol/L 作用于 细胞,评价DADLE能否减轻神经细胞缺氧复氧损伤;继而分别用10 pmol/L、100 pmol/L、1 nmol/L、10 nmol/L 和100 nmol/L 不同浓度的DADLE溶液处理细胞,研究DADLE保护作用是否具有剂量效应关系。结果：SH-SY5Y 细胞缺氧6、12、24 h 或48 h,复氧4 h 后,细胞生存率分别为（68.1±4.3）％、（52.6±2.8）％、（26.7±1.5）％ 或（3.5±1.7）％。SH-SY5Y 细胞缺氧12 h 期间给予DADLE 100 pmol/L 处理可使细胞生存率上升到（58.7±0.46）%。 5 个给药浓度中,10 pmol/L 和100 pmol/L DADLE可使神经细胞生存率上升,而1、10 nmol/L 和100 nmol/L 则变 化不明显。结论：DADLE对SH-SY5Y 的缺氧复氧损伤具有保护作用,且其保护效果呈现出较小剂量优于较大剂量 的量效关系,本实验中最佳保护剂量为10 pmol/L。     

人参皂苷Re对人神经母细胞瘤SH-SY5Y细胞缺氧复氧损伤的保护作用

目的：探讨人参皂苷Re 对人神经母细胞瘤SH-SY5Y 细胞缺氧复氧损伤的保护作用。 方法：采用缺氧复 氧法制备SH-SY5Y 细胞损伤模型,分为对照组、模型组、Re 6.25 μg/mL 组和Re 12.50 μg/mL 组。采用MTT 法测定细胞存活率,四甲基罗丹明甲酯（TMRM）染色评估细胞线粒体膜电位变化,DAPI 染色观察细胞凋亡 率,并进一步采用免疫印迹检测各组细胞核因子NF-E2 相关因子2（Nrf-2）、Bax 和p53 的蛋白表达。 结果：人 参皂苷Re 对SH-SY5Y 细胞活力无显著影响;缺氧复氧条件下,细胞存活率显著降低,而人参皂苷Re 在6.25、 12.50 μg/mL 浓度下预保护可显著提高细胞存活率。缺氧复氧损伤后,人参皂苷Re 6.25、12.50 μg/mL 能显著提 高SH-SY5Y 细胞的线粒体膜电位水平和抑制SH-SY5Y 细胞的凋亡率。人参皂苷Re 能够提高Nrf-2 的蛋白表达, 抑制Bax 和p53 蛋白的表达。结论：人参皂苷Re 可以抑制缺氧复氧损伤细胞的凋亡和氧化损伤,对缺氧复氧诱导 的神经细胞具有显著的保护作用。     

蛇床子素对转染APP595/596基因的SH-SY5Y细胞的保护作用

目的:研究蛇床子素对转染APP595/596基因的SH-SY5Y细胞的作用,以探讨其可能的作用机制。方法:体外培养SH-SY5Y细胞,并转染APP595/596基因,体外构建研究Aβ致病作用的细胞模型。采用CCK-8法检测细胞存活率;检测LDH评价细胞的损伤程度;流式细胞术检测细胞凋亡;用RT-PCR和Western blot法检测β-分泌酶(又称β位点APP裂解酶1,β-site APP cleaving enzyme 1,BACE 1)的mRNA及蛋白的表达;采用免疫荧光细胞化学和Western blot法检测Aβ的表达。结果:蛇床子素对转染APP595/596基因的SH-SY5Y神经细胞有保护作用,可增加细胞的存活率,降低LDH的释放,抑制细胞的凋亡,减少BACE1的mRNA与蛋白的表达,抑制Aβ的生成。结论:蛇床子素可保护转染APP595/596基因的SH-SY5Y神经细胞,其保护机制可能与减少BACE l的mRNA与蛋白表达有关。     

Protective effects of quercetin against hydrogen peroxide-induced apoptosis in human neuronal SH-SY5Y cells

Hydrogen peroxide (H₂O₂) is a major reactive oxygen species that has been implicated in various neurodegenerative diseases. Quercetin, one of the plant flavonoids, has been reported to harbor various physiological properties including antioxidant activity. In this study, we investigated the neuroprotective effects of quercetin against H₂O₂-induced apoptosis in human neuronal SH-SY5Y cells. H₂O₂-mediated cytotoxicity and lactate dehydrogenase release were suppressed in a quercetin concentration-dependent manner. In addition, quercetin repressed the expression of the pro-apoptotic Bax gene and enhanced that of the anti-apoptotic Bcl-2 gene in SH-SY5Y cells. Moreover, quercetin effectively inhibited the activation of the caspase cascade that leads to DNA fragmentation, a key feature of apoptosis, and subsequent cell death. These results indicate the importance of quercetin in protecting against H₂O₂-mediated neuronal cell death. Thus, quercetin might potentially serve as an agent for prevention of neurodegenerative diseases caused by oxidative stress and apoptosis.     

Resistance to geldanamycin-induced apoptosis in differentiated neuroblastoma SH-SY5Y cells

Geldanamycin (GA) is a specific inhibitor of the 90 kDs heat shock protein (Hsp90) in the cytoplasm of mammalian cells, which binds directly to Hsp90 and promotes proteolytic degradation of its client proteins. As an antitumor drug, GA antagonizes the protecting effects of Hsp90 on cell survival, while its mechanisms remain unclear. Here, we show that GA induces apoptosis in a human neuroblastoma cell line, SH-SY5Y. Treatment of the cells with all trans retinoic acid (RA) generates a neuron-like, morphological change of differentiation, and results in the activation of ERK and Akt pathways, an inhibition of the nuclear translocation of p53 induced by GA, and induces higher resistance to the GA-induced apoptosis. These results provide the first evidence for the requirement of p53 nucleation in SH-SY5Y cells to counteract GA in neuron survival.     

Baicalin protects against thrombin induced cell injury in SH-SY5Y cells

Baicalin, an extract from the dried root of Scutellaria baicalensis Georgi, was shown to be neuroprotective. However, the precise mechanisms are incompletely known. In this study, we determined the effect of baicalin on thrombin induced cell injury in SH-SY5Y cells, and explored the possible mechanisms. SH-SY5Y cells was treated with thrombin alone or pre-treated with baicalin (5, 10, 20 μM) for 2 h followed by thrombin treatment. Cells without thrombin and baicalin treatment were used as controls. Cell viability was detected by MTT assay. Cell apoptosis was analyzed by flow cytometry. Real-time PCR was performed to determine the mRNA expression of protease-activated receptor-1 (PAR-1). Western blotting was conducted to determine the protein expression of PAR-1, Caspase-3 and NF-κB. Baicalin reduced cell death following thrombin treatment in a dose-dependent manner, with concomitant inhibition of NF-κB activation and suppression of PAR-1 expression. In addition, baicalin reduced Caspase-3 expression. The above findings indicated that baicalin prevents against cell injury after thrombin stimulation possibly through inhibition of PAR-1 expression and NF-κB activation.     

Cytoprotective response induced by electromagnetic stimulation on SH-SY5Y human neuroblastoma cell line

It is well known that physiological functions and pathological conditions of cells and tissues can be influenced not only by chemical molecules, but also by physical stimuli such as electromagnetic waves. In particular, epidemiological studies suggest possible associations between exposure to electromagnetic fields and an increased risk of tumors and neurodegenerative disorders, such as Alzheimer's disease. However, depending on the dose and on the length of treatment, the electromagnetic stimuli can be harmful or induce a cytoprotective cellular response, suggesting a possible application in medical therapy. In this study, under a tissue engineering viewpoint, we investigated the effects of an electromagnetic wave (magnetic field intensity, 2 mT; frequency, 75 Hz) on a neuronal cellular model characterized by the overexpression of the amyloid precursor protein (APP). After a prolonged electromagnetic treatment, lower mitochondrial activity and proliferation rate, resulting in a higher cellular quiescence, were observed. Focusing on the stress and oxidative pathways, we detected an overall increase of two fundamental proteins, the chaperone heat shock protein HSP70 and the free radical scavenger superoxide dismutase-1 enzyme (SOD-1). Interestingly, we found that the electromagnetic stimulation promotes the nonamyloidogenic processing of APP through an increased expression of the α-secretase ADAM10 and an enhanced release of the soluble neurotrophic factor sAPPα (a product of the ADAM10-mediated cleavage of APP). In conclusion, these findings suggest that the electromagnetic stimulus, if properly administered in terms of dose and timing, is able to induce a cytoprotective response in the cell. Moreover, these results suggest a possible use of this particular physical stimulation to improve the functional capability of the cells to face noxae.     

Modulation of tyrosine hydroxylase expression by melatonin in human SH-SY5Y neuroblastoma cells

We have previously reported in vivo preservation of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, following treatment with physiological doses of melatonin, in a 6-hydroxydopamine model of Parkinson's disease. Based on these findings, we postulated that melatonin would similarly modulate the expression of TH in vitro. Therefore, using human SH-SY5Y neuroblastoma cells which can differentiate into dopaminergic neurons following treatment with retinoic acid, we first examined whether these cells express melatonin receptors. Subsequently, the physiological dose-dependent effects of melatonin on TH expression were examined in both undifferentiated and differentiated cells. The novel detection of the G protein-coupled melatonin MT(1) receptor in SH-SY5Y cells by RT-PCR was confirmed by sequencing and Western blotting. In addition, following treatment of SH-SY5Y cells with melatonin (0.1-100 nM) for 24h, Western analysis revealed a significant increase in TH protein levels. A biphasic response, with significant increases in TH protein at 0.5 and 1 nM melatonin and a reversal at higher doses was seen in undifferentiated cells; whereas in differentiated cells, melatonin was effective at doses of 1 and 100 nM. These findings suggest a physiological role for melatonin in modulating TH expression, possibly via the MT(1) receptor.