农药鱼藤酮对表达α-突触核蛋白细胞的作用

本研究利用农药鱼藤酮作用于神经细胞 ,观察鱼藤酮对神经细胞的损伤作用以及线粒体功能障碍与α- synuclein积聚之间的关系。方法 :本实验选用人神经母细胞瘤细胞株SH-SY5Y ,实验组为α-synuclein GFP基因转染的细胞 ,鱼藤酮处理的转基因细胞和非转基因细胞 ,对照组为未处理的SH-SY5Y细胞。通过RT-PCR检测α-synuclein基因转染细胞的基因表达情况。荧光显微镜观察细胞内α-synuclein GFP表达产物绿色荧光蛋白。MTT法检测各组细胞活性。DCF检测细胞氧应激。HE染色、免疫组化检测α-synuclein在细胞中的状态。电镜观察细胞超微结构的改变。结果 :RT-PCR显示转基因细胞α-synuclein基因的表达。荧光显微镜观察显示细胞浆内可见绿色荧光蛋白 ,绿色荧光分布不均匀 ,可见蛋白积聚。MTT检测结果显示 ,与对照组相比 ,鱼藤酮处理的细胞增殖速度明显减小 (P <0. 01 )。鱼藤酮的浓度为 75nmol/ L、1 0 0nmol/ L时 ,未转基因与转基因的细胞活性相比较 ,前者的细胞活性低于后者 (P <0 .0 5 )。HE染色 ,鱼藤酮处理的转基因细胞胞浆减少、转基因细胞胞浆内也可见自噬体。胞浆内有嗜酸性包涵体样结构。免疫组化可见鱼藤酮处理高表达α-synuclein细胞明显变成梭形并有很长的突起。电镜显示鱼藤酮处理的细胞线粒体肿胀 ,嵴断裂 ,胞浆内形成自噬体。DCF检测转基因细胞内存在明显的氧化应激 ,并随鱼藤酮处理加重。结论 :农药鱼藤酮对多巴胺能神经细胞有明显的损伤作用 ,转基因细胞显示对较高浓度的鱼藤酮损伤有一定的耐受作用。α-synuclein可引起神经细胞的氧化应激并随鱼藤酮处理加重。提示环境因素可能与α-synuclein表达相互作用使多巴胺能神经元氧化应激进行性加重 ,这可能是引起PD的主要原因。     

麦芽酚对活性氧损伤人神经瘤细胞的保护作用

以人神经瘤细胞株 (SH SY5Y)为材料 ,使用过氧化氢 (H2 O2 )产生过量活性氧诱导SH SY5Y细胞株进入氧化应激状态 .研究麦芽酚对过量活性氧造成的SH SY5Y细胞株氧化损伤的保护作用 .分析活性氧对细胞膜蛋白和DNA的损伤 ,细胞线粒体功能变化 ,白介素 6 (IL 6 )的表达变化以及细胞核因子κB(NF κB)的激活 .结果显示 ,2mmol L麦芽酚保护细胞 2h后 ,对细胞膜蛋白和DNA的损伤均有明显的保护作用 ,减少了膜蛋白的氧化和细胞DNA片段化的形成 ,细胞线粒体功能损伤减小 ,细胞表达的IL 6减少 ,被激活的NF κB水平同时降低 .结果证明 ,麦芽酚可以有效保护活性氧对神经细胞的氧化损伤 ,维持细胞的正常生理功能     

右美托咪啶通过抑制NADPH氧化酶2缓解氧化应激小鼠模型神经元的毒性和认知障碍的机制

摘要 目的：探讨右美托咪啶通过抑制NADPH氧化酶2缓解氧化应激小鼠模型神经元的毒性和认知障碍的机制。方法：10只野生型以及20只Sod1KO雄性BALB/c小鼠,12月龄,根据实验目的分为3组：对照组（野生型小鼠）,模型组（氧化应激小鼠模型）和DEX组（氧化应激小鼠模型+50 μg/kg DEX治疗）,每组10只。通过MWM 测试检测小鼠的空间学习和记忆能力。通过免疫染色检测海马中Neu-N+细胞数和PSD-95表达水平。通过蛋白质印迹检测海马中Neu-N、PSD-95、TH、总α-突触核蛋白和Ser129-磷酸化α-突触核蛋白表达水平。通过ROS、MDA和SOD检测试剂盒分别检测ROS、MDA和SOD水平。通过 ELISA试剂盒检测NOX2水平。通过RT-qPCR检测IL-1β、IL-6和TNF-α水平。结果：对照小鼠表现出正常的空间学习功能,与对照组小鼠相比,模型组小鼠逃避潜伏期和游泳距离增加（P<0.05）,而DEX治疗能够降低模型组小鼠逃避潜伏期和游泳距离（P<0.05）。三组小鼠平均游泳速度没有统计性差异（P>0.05）。与对照组小鼠相比,模型组小鼠小鼠海马中Neu-N+细胞数和PSD-95表达水平降低（P<0.05）,而DEX治疗能够增加小鼠海马中Neu-N+细胞数和PSD-95表达水平（P<0.05）。与对照组小鼠相比,模型组小鼠小鼠海马中Neu-N、PSD-95和TH蛋白表达水平降低（P<0.05）,总α-突触核蛋白和Ser129-磷酸化α-突触核蛋白表达水平升高（P<0.05）,而DEX治疗能够增加小鼠海马中Neu-N、PSD-95和TH蛋白表达水平（P<0.05）,降低总α-突触核蛋白和Ser129-磷酸化α-突触核蛋白表达水平（P<0.05）。与对照组小鼠相比,模型组小鼠ROS和MDA水平增加,SOD水平降低（P<0.05）,而DEX治疗能够降低ROS和MDA水平,增加SOD水平（P<0.05）。与对照组小鼠相比,模型组小鼠NOX2水平增加（P<0.05）,而DEX治疗能够降低NOX2水平（P<0.05）。与对照组小鼠相比,模型组小鼠IL-1β、IL-6和TNF-α水平增加（P<0.05）,而DEX治疗能够降低IL-1β、IL-6和TNF-α水平（P<0.05）。结论：DEX对NOX2的抑制可通过抑制小鼠模型中的氧化应激和神经炎症来阻断学习和记忆障碍以及海马神经变性。     

G蛋白偶联受体激酶5在稳定表达α-Synuclein的SHSY5Y细胞中的基因表达调控功能

目的观测G蛋白偶联受体激酶5（G protein-coupled receptor kinase,GRK5）在稳定表达hα-synuclein（人突触核蛋白）的SHSY5Y细胞的胞核、胞浆中的表达情况并对其在帕金森病中的可能作用进行研究。方法应用Western blotting、组蛋白去乙酰化酶（histone deacetylase,HDAC）活性检测技术以及shRNA干扰技术等对稳定表达hα-synuclein的SHSY5Y细胞中GRK5的表达及其亚细胞分布、胞核内GRK5蛋白的功能进行研究。结果发现GRK5蛋白在过表达hα-synuclein的细胞核以及细胞浆内均表达增加,胞核中的GRK5蛋白通过影响组蛋白去乙酰化酶的活性对bcl-2基因的转录和表达进行调控。结论帕金森模型中GRK5通过对bcl-2基因的表达进行调控发挥作用。     

丙戊酸钠对抗鱼藤酮诱导的SH-SY5Y细胞损伤的线粒体机制

研究丙戊酸钠（sodiumvalproate,VPA）对抗鱼藤酮（Rotenone）诱导的SH-SY5Y细胞损伤的作用及线粒体机制。以l,10μmol／LVPA预处理SH－SY5Y细胞3h,再加入400nmol／LRotenone作用24h。MTT法检测与相差显微镜观察相结合,分析VPA对抗Rotenone损伤的作用;JC－1染色法与Mito－Tracker染色法分析线粒体膜电位及线粒体数量的变化;Clark氧电极法检测细胞呼吸功能;DCFH-DA探针法检测细胞中Ros的含量;并在离体线粒体上观察VPA对Ca^2＋诱导的线粒体肿胀的影响。结果发现,1,10p．mol／LVPA预处理SH．SY5Y细胞3h可对抗400nmol／LRotenoneI起的细胞损伤,并且可以提高损伤细胞中线粒体的膜电位,增加线粒体的数量,此外,还可以增强损伤细胞的呼吸功能,降低细胞中ROS的含量,但VPA并不能直接作用于离体的线粒体发挥神经保护作用。由此,VPA具有良好的神经保护作用,其机制与增强线粒体功能和数量、从而改善细胞功能有关,这为其应用于帕金森病的预防与治疗提供了实验依据。     

SH—SY5Y细胞的钙缓冲研究

目的：研究SH－SY5Y神经杂交瘤细胞的钙缓冲能力。方法：通过膜片钳手段,测量未分化的SH－SY5Y细胞钙离子通道电流;并应用显微荧光测量游离钙离子浓度和高钾去极化的方法,研究胞内Ca^2 浓度上升后浓度恢复的动力学过程。结果：未分化的SH－SY5Y细胞存在钙离子通道电流,在刺激时间间隔较短时（＜150s）,胞内钙浓度的恢复过程会由于缓冲机制的饱和而变慢;而时间间隔＞150s时,缓冲物质则可以基本恢复使得胞内钙的恢复过程基本保持不变。结论：钙缓冲蛋白在细胞内钙浓度的调节中起重要作用。     

miR-29b对TNF—α诱导的人脐静脉内皮细胞增殖与凋亡的影响

目的探索过表达miR-29b对TNF-α 诱导的人脐静脉内皮细胞（HUVECs）增殖与凋亡的影响及初步作用机制。方法MTT 法筛选TNF—α诱导HUVECs的最佳浓度和时间,建立细胞凋亡模型;MTY法筛选miR-29bmimic转染HUVECs的最佳转染时间和浓度：MTr法检测过表达miR-29b对TNF-α诱导HUVECs增殖活力的影响;Hoechst33342荧光染色检测过表达miR-29b对TNF—α诱导HUVECs凋亡的影响：Western印迹技术检测过表达miR-29b对Akt磷酸化水平、Bcl-2蛋白表达的影响。结果TNF—α诱导的HUVECs凋亡的最佳浓度为10ng／ml,最佳时间是48h;miR-29bmimics转染HUVECs的最佳浓度为50nmol/L,最佳作用时间是48h;过表达miR-29b能显著降低TNF-α诱导的HUVECs的增殖活力（P〈0．001）;Heochst33342荧光染色结果显示。miR-29b过表达能促进TNF-α诱导的HUVECs的凋亡（P〈0．05）;过表达miR-29b能显著下调Akt磷酸化（p-Akt）与Bcl-2蛋白的表达（P〈0．001）。结论过表达miR-29b可降低TNF-α诱导的HUVECs的增殖活力并促进其凋亡,其机制可能与下调Akt磷酸化、Bd-2蛋白的表达相关。     

GABPα抑制鱼藤酮诱导的PC12细胞氧化应激及凋亡

[目的]明确GABPα在鱼藤酮诱导的PC12细胞氧化应激及凋亡中的作用。[方法]利用鱼藤酮制备PC12细胞氧化应激模型,通过脂质体转染的方法在PC12细胞中过表达GABPα。利用Western Blot检测GABPα的表达,通过可见分光光度法和微量法分析氧化应激标记物NO和MDA水平,抗氧化标记物GSH的水平和SOD的活性,利用流式细胞仪检测细胞凋亡。[结果]与对照组相比,鱼藤酮处理后,PC12细胞的活力明显降低(46.71%±1.7%vs 99.88%±0.649%),NO和MDA的水平明显增高(0.285±0.004 vs 0.151±0.003,0.115±0.003 vs 0.044±0.002),GSH的水平及SOD的活性显著下降(11.53±0.572 vs 22.86±1.338,0.161±0.008 vs 0.315±0.026),细胞的凋亡明显增加(30.26%±2.359%vs 3.037%±0.043%)。在PC12细胞过表达GABPα并用鱼藤酮处理后,与空载体组相比,PC12细胞的活力明细增高(62.21%±2.344%vs 47.65%±3.228%),氧化...     

脉络宁对SH-SY5Y细胞氧糖剥夺再恢复损伤的保护作用及其机制研究

目的：研究脉络宁注射液对SH—SY5Y细胞氧糖剥夺／再复氧糖（OGI）／R）损伤的保护作用,并探讨其可能的作用机制。方法：体外培养SH-SY5Y细胞,将细胞随机分为正常组、氧糖剥夺模型组和脉络宁组（1．0mL·L^-1）,建立体外OGD／R细胞模型。倒置显微镜观察细胞形态;MTT法测定细胞存活率;测定乳酸脱氢酶（LDH）漏出量;Western Blot检测凋亡相关蛋白Bcl-2、Bax蛋白表达的变化。结果：与模型组相比,脉络宁能减轻OGD／R引起的SH-SY5Y细胞的损伤,明显提高细胞存活率（P〈0．05）,减少LDH的释放量（P〈0．05）,有效抑制Bax蛋白的表达（P〈0．05）,上调Bcl-2的表达（P〈0．05）。结论：脉络宁注射液对OGD／R引起的SH-sY5Y细胞损伤有保护作用,其机制可能与影响凋亡相关基因Bcl-2、Bax的表达有关。     

心肌α肌球蛋白重链启动子驱动的CREG蛋白表达载体的构建及鉴定

目的：构建心肌特异性α-肌球蛋白重链（α—myosin heavy chain,α—MHC）启动子启动E1A基因阻遏子（cellular repressor of E1A—stimulated genes,CREG）和增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）融合的真核表达载体。绿色荧光蛋白作为报告基因,方便在心肌细胞中直接观察CREG蛋白的表达,为心肌特异性转CREG基因动物模型制备提供载体。方法：用BamHI和EcoRI双酶切pcDNA3．1myc—His／hCREG质粒得到CREG基因,亚克隆入增强绿色荧光蛋白表达质粒pEGFP-N1中,构建pCREG-EGFP-N1;根据Genebank中公布的α-MHC基因的启动子序列,人工合成pUC57-α-MHC启动子基因序列,经AseI和NheI双酶切得到启动子α—MHC,亚克隆入pCREG—EGFP-N1中替代原CMV启动子,构建pα-MHC-CREG—EGFP-N1,测序鉴定。用脂质体法将该质粒转染体外培养的小鼠原代心肌细胞,荧光显微镜下观测绿色荧光蛋白的表达;Western blot检测CREG蛋白的表达。结果：成功构建pα—MHC—CREG-EGFP-N1质粒,酶切及测序结果正确;成功转染入原代培养小鼠心肌细胞,在荧光显微镜下可见绿色荧光蛋白的表达,Westem blot检测到CREG蛋白的表达。结论：重组质粒pα-MHC—CREG—EGFP-N1体外转染入原代培养小鼠心肌细胞后,目的基因能够在心肌细胞中有效表达,检测方法简便可靠,为下一步建立心肌细胞特异性表达CREG的过表达转基因小鼠、深入探讨CREG在心肌疾病发生中的生物学功能研究奠定了基础。     

Rifampicin Prevents SH-SY5Y Cells from Rotenone-Induced Apoptosis via the PI3K/Akt/GSK-3β/CREB Signaling Pathway

In addition to its original application for treating tuberculosis, rifampicin has multiple potential neuroprotective effects in chronic neurodegenerative diseases including Parkinson’s disease (PD) and Alzheimer’s disease. Inflammatory reactions and the PI3K/Akt pathway are strongly implicated in dopaminergic neuronal death in PD. This study aims to investigate whether rifampicin protects rotenone-lesioned SH-SY5Y cells via regulating PI3K/Akt/GSK-3β/CREB pathway. Rotenone-treated SH-SY5Y cells were used as the cell model to investigate the neuroprotective effects of rifampicin. Cell viability and apoptosis of SH-SY5Y cells were determined by CCK-8 assay and flow cytometry, respectively. The expression of Akt, p-Akt, GSK-3β, p-GSK-3β, CREB and p-CREB were measured by Western blot. Our results showed that the cell viability and level of phospho-CREB significantly decreased in SH-SY5Y cells exposed to rotenone when compared to the control group. Both the cell viability and the expression of phospho-CREB in cells pretreated with rifampicin were higher than those of cells exposed to rotenone alone. Moreover, pretreatment of SH-SY5Y cells with rifampicin enhanced phosphorylation of Akt and suppressed activity of GSK-3β. The addition of LY294002, a PI3K inhibitor, could suppress phosphorylation of Akt and CREB and activate GSK-3β, resulting in abolishment of neuroprotective effects of rifampicin on cells exposed to rotenone. Rifampicin provides neuroprotection against dopaminergic degeneration, partially via the PI3K/Akt/GSK-3β/CREB signaling pathway. These findings suggest that rifampicin could be an effective and promising neuroprotective candidate for treating PD.     

Dual effects of α-synuclein on neurotoxicity induced by low dosage of rotenone are dependent on exposure time in dopaminergic neuroblastoma cells

This study was designed to investigate the effects of α-synuclein on toxicity induced by long-term exposure to relatively low concentrations of rotenone.Compared with the control groups,the inhibition of cell viability which overexpressed α-synuclein(SH-SY5Y-Syn) improved after 1 and 2 weeks of rotenone treatment.The complex I activity was greater and the mitochondrial membrane swelling intensity was reduced after 1 and 2 weeks of treatment,which indicated that α-synuclein,at least in part,resists the rotenone-induced oxidative stress.The results indicate that α-synuclein has a dual effect on toxicity of rotenone according to exposure time in human SH-SY5Y cells.     

SOD3 Ameliorates Aβ<Subscript>25–35</Subscript>-Induced Oxidative Damage in SH-SY5Y Cells by Inhibiting the Mitochondrial Pathway

This study was designed to investigate the protective effects of extracellular superoxide dismutase (SOD3) against amyloid beta (Aβ_25–35)-induced damage in human neuroblastoma SH-SY5Y cells and to elucidate the mechanisms responsible for this beneficial effect. SH-SY5Y cells overexpressing SOD3 were generated by adenoviral vector-mediated infection and Aβ_25–35 was then added to the cell culture system to establish an in vitro model of oxidative stress. Cell viability, the generation of intracellular reactive oxygen species (ROS), the expression and activity of antioxidant enzymes, the levels of lipid peroxidation malondialdehyde (MDA), the expression of mitochondrial apoptosis-related genes and calcium images were examined. Following Aβ_25–35 exposure, SOD3 overexpression promoted the survival of SH-SY5Y cells, decreased the production of ROS, decreased MDA and calcium levels, and decreased cytochrome c, caspase-3, caspase-9 and Bax gene expression. Furthermore, SOD3 overexpression increased the expression and activity of antioxidant enzyme genes and Bcl-2 expression. Together, our data demonstrate that SOD3 ameliorates Aβ_25–35-induced oxidative damage in neuroblastoma SH-SY5Y cells by inhibiting the mitochondrial pathway. These data provide new insights into the functional actions of SOD3 on oxidative stress-induced cell damage.     

α－synuclein对小剂量鱼藤酮导致的线粒体损伤的调控作用研究

建立稳定表达 -synuclein基因的人神经母细胞瘤细胞株SH-SY5Y细胞,,鱼藤酮作用1、2、4周后分别提取各组细胞线粒体,检测其complexⅠ功能、线粒体膜肿胀度和超氧阴离子的含量。结果显示在1、2周时过表达 -synuclein基因的细胞与对照组细胞相比,前者的complexⅠ的活性高,线粒体膜肿胀度轻,线粒体超氧阴离子的含量少。但4周后,前者的complexⅠ活性明显低于后者,且线粒体膜肿胀度及线粒体内超氧阴离子的含量均显示前者高于后者,也是前者多于后者。由此可见过表达 -synuclein基因的细胞在早期对鱼藤酮的损伤有一定的抵抗作用,但长期作用后可能有加重损伤的作用。提示PD患者多巴胺能神经元内 -synuclein的增高可能对小剂量鱼藤酮导致的线粒体损伤存在双重调节作用,初期可能由于 -synuclein上调引起的预适应改变起到一定的保护作用。而失代偿后,可能由于神经元内积累的 -synuclein聚变,对线粒体膜稳定性的损伤,导致神经元变性死亡。     

Effect of oxytocin on neuroblastoma cell viability and growth

Oxytocin, released in response to different physiological stimuli, could play a key role in reducing stress reaction. It was suggested that it has protective effect against inflammation and consequences of oxidative stress. Mechanisms how oxytocin effects mediated in the brain tissue are unclear. In this study, oxytocin effect on cell growth and neuronal viability was examined. Human neuroblastoma (SH-SY5Y and SK-N-SH) and glioblastoma (U87MG) cells were exposed to different concentrations of oxytocin for 12-96 h. Potential protective effect of oxytocin treatment was investigated after exposing cells to oxidative stress using hydrogen peroxide (50 mM, 2 h) or 6-hydroxydopamine (25 μM, 24 h). Cell proliferation was measured by cell counting and cell viability was examined by MTT assay. Protein expression of selected neurotrophic factors was measured as an additional parameter. Oxytocin (1 μM) significantly increased cell number in all three cell types. Viability of SH-SY5Y cells was increased in the presence of oxytocin without significant effect of dose (0.01-1 μM). Cell death induced by hydrogen peroxide was not prevented by incubation with oxytocin. Oxytocin pretreatment blunted neurotoxin 6-OHDA reduction of cell viability in SH-SY5Y cells. Oxytocin (1 μM, 12 h) elevated amount of total proteins without increasing levels of brain-derived neurotrophic factor and neurotrophic growth factor. In conclusion, oxytocin increases growth and viability of neuroblastoma and glioblastoma cells without activation of neurotrophic factors. Oxytocin does not have protective effect in oxidative stress; however, it might be important for neuroprotection to dopaminergic neurons. Its proliferative effect might be important in native cell life, euplastic processes, and tumor progression.     

鱼藤酮诱导线粒体轻度损伤细胞氧化应激时硫氧还蛋白转录水平降低

观察鱼藤酮诱导的线粒体轻度损伤细胞氧化应激时硫氧还蛋白转录水平的变化,探讨细胞氧化损伤的可能机制。通过荧光素发光法检测ATP生成、细胞内活性氧(ROS)水平的变化,流式细胞术检测线粒体膜电位,了解低剂量鱼藤酮对线粒体功能的影响;继而用H2O2诱导细胞氧化损伤,MTT法检测细胞活性,观察正常及线粒体缺陷细胞氧化应激时,胞内硫氧还蛋白(Trx)mRNA水平的变化。结果表明,鱼藤酮以剂量依赖方式抑制线粒体ATP的产生、降低线粒体膜电位,而细胞内ROS水平增高;当线粒体损伤细胞氧化应激时胞内Trx mRNA水平降低,提示鱼藤酮诱导线粒体轻度损伤细胞抗氧化能力降低与Trx转录受到抑制有关。     

山楂叶金丝桃苷对高糖诱导SH-SY5Y细胞损伤的保护作用及机制

为研究金丝桃苷对高糖诱导的人神经母细胞瘤(SH-SY5Y)细胞氧化损伤的保护作用及机制,用含100mmo L/L葡萄糖和分别为20、50、100μmo L/L金丝桃苷的培养基共同孵育SH-SY5Y细胞36 h,检测细胞活力、细胞培养液中乳酸脱氢酶(LDH)水平及半胱氨酸天冬氨酸蛋白酶-3(caspase-3)活性,细胞内活性氧(ROS)水平、丙二醛(MDA)、还原型谷胱甘肽(GSH)含量和超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性及SIRT1和NF-кB基因的mRNA水平和蛋白含量。结果显示金丝桃苷可提高高糖诱导后SH-SY5Y细胞的存活率,抑制细胞LDH释放,清除ROS,降低MDA含量与caspase-3活性,增强SOD、CAT活性和GSH含量;同时,金丝桃苷还能提高SIRT1基因的mR-NA表达及蛋白含量,降低NF-кB基因的mRNA水平和蛋白含量。结果表明金丝桃苷能通过激活SIRT1基因,抑制NF-кB基因保护高糖所致SH-SY5Y细胞的氧化损伤。     

Paeoniflorin attenuated bupivacaine-induced neurotoxicity in SH-SY5Y cells via suppression of the p38 MAPK pathway

Bupivacain, a common local anesthetic, can cause neurotoxicity and permanent neurological disorders. Paeoniflorin has been widely reported as a potential neuroprotective agent in neural injury models. However, the roles and molecular basis of paeoniflorin in bupivacaine-induced neurotoxicity are still undefined. In the current study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to detect cell viability. Apoptotic rate was measured through double-staining of Annexin V-FITC and propidium iodide on a flow cytometer. Western blot assay was carried out to examine the protein levels of p38 mitogen-activated protein kinase (p38 MAPK), phosphorylated-p38 MAPK (p-p38 MAPK), Bcl-2, and Bax. caspase-3 activity was determined using a caspase-3 activity assay kit. We found that paeoniflorin dose-dependently attenuated bupivacaine-induced viability inhibition and apoptosis in SH-SY5Y cells. Moreover, paeoniflorin inhibited bupivacaine-induced activation of p38 MAPK pathway in SH-SY5Y cells. Paeoniflorin alone showed no significant effect on cell viability, apoptosis and p38 MAPK signaling in SH-SY5Y cells. Inhibition of p38 MAPK signaling by SB203580 or small interfering RNA targeting p38 (si-p38) abated bupivacaine-induced viability inhibition and apoptosis in SH-SY5Y cells. In conclusion, paeoniflorin alleviated bupivacaine-induced neurotoxicity in SH-SY5Y cells via suppression of the p38 MAPK pathway, highlighting the potential values of paeoniflorin in relieving bupivacaine-induced neurotoxicity.     

Superoxide dismutase as a target of clioquinol-induced neurotoxicity

Subacute myelo-optico-neuropathy (SMON) is a progressive neurological disorder affecting the spinal cord, peripheral nerves and optic nerves. Although it has been assumed that SMON was caused by intoxication of clioquinol, the mechanism underlying clioquinol-induced neurotoxicity is not fully understood. This study aimed to clarify the relevance of oxidative stress to clioquinol-induced neurotoxicity and the cause of the enhanced oxidative stress. Clioquinol induced cell death in human-derived neuroblastoma cell line, SH-SY5Y, in a dose-dependent manner. This process was accompanied by activation of caspase-3 and enhanced production of reactive oxygen species (ROS). We examined whether clioquinol inhibited the activity of superoxide dismutase-1 (SOD1), based on its metal chelating properties. Clioquinol inhibited activities of purified SOD1 in a dose-dependent manner. Cytosolic SOD activities were also inhibited in SH-SY5Y cells treated with clioquinol. Finally, addition of exogenous SOD1 to the culture significantly reduced enhanced ROS production and cell death induced by clioquinol in SH-SY5Y cells. These findings suggested that enhanced oxidative stress caused by inhibition of SOD1 undelay clioquinol-induced neurotoxicity and was relevant to the pathogenesis of SMON.     

Upregulation of miR-1306-5p decreases cerebral ischemia/reperfusion injury in vitro by targeting BIK

ABSTRACT

MiR-1306-5p is involved in the progression of acute heart failure, but its role in ischemic stroke remains unclear. Here, SH-SY5Y cells were exposed to oxygen–glucose deprivation (OGD) for 4, 8, and 12 h, respectively, and then reoxygenation for 12 h to construct OGD/R induced cell injury model. Cell viability, cell death, and cell apoptosis were assessed with CCK-8 assay, LDH assay, ?ow cytometry, and caspase-3 activity assay. The target gene of miR-1306-5p was confirmed by luciferase reporter assay. We found miR-1306-5p expression was significantly down-regulated in OGD/R-induced SH-SY5Y cell model. Moreover, miR-1306-5p protected SH-SY5Y cell against OGD/R-induced injury. Mechanistically, Bcl2-interacting killer (BIK) was the direct target gene of miR-1306-5p. Furthermore, BIK knockdown mimicked, while overexpression reversed the protective effects of miR-1306-5p against OGD/R induced injury. Our findings thus provide an experimental basis miR-1306-5p targeting BIK-based therapy for cerebral I/R injury.