Small molecule inhibitor DDQ-treated hippocampal neuronal cells show improved neurite outgrowth and synaptic branching

The process of neurite outgrowth and branching is a crucial aspect of neuronal development and regeneration.Axons and dendrites,sometimes referred to as neurites,are extensions of a neuron's cellular body that are used to start networks.Here we explored the effects of diethyl(3,4-dihydroxyphenethylamino)(quinolin-4-yl)methylphosphonate(DDQ)on neurite developmental features in HT22 neuronal cells.In this work,we examined the protective effects of DDQ on neuronal processes and synaptic outgrowth in differentiated HT22cells expressing mutant Tau(mTau)cDNA.To investigate DDQ chara cteristics,cell viability,biochemical,molecular,western blotting,and immunocytochemistry were used.Neurite outgrowth is evaluated through the segmentation and measurement of neural processes.These neural processes can be seen and measured with a fluorescence microscope by manually tracing and measuring the length of the neurite growth.These neuronal processes can be observed and quantified with a fluorescent microscope by manually tracing and measuring the length of the neuronal HT22.DDQ-treated mTau-HT22 cells(HT22 cells transfected with cDNA mutant Tau)were seen to display increased levels of synaptophysin,MAP-2,andβ-tubulin.Additionally,we confirmed and noted reduced levels of both total and p-Tau,as well as elevated levels of microtubule-associated protein 2,β-tubulin,synaptophysin,vesicular acetylcholine transporter,and the mitochondrial biogenesis protein-pe roxisome prolife rator-activated receptor-gamma coactivator-1α.In mTa u-expressed HT22 neurons,we observed DDQ enhanced the neurite characteristics and improved neurite development through increased synaptic outgrowth.Our findings conclude that mTa u-HT22(Alzheimer's disease)cells treated with DDQ have functional neurite developmental chara cteristics.The key finding is that,in mTa u-HT22 cells,DDQ preserves neuronal structure and may even enhance nerve development function with mTa u inhibition.

Potassium bisperoxo(1,10-phenanthroline) oxovanadate suppresses proliferation of hippocampal neuronal cell lines by increasing DNA methyltransferases

Bisperoxo(1,10-phenanthroline) oxovanadate(BpV) can reportedly block the cell cycle. The present study examined whether BpV alters gene expression by affecting DNA methyltransferases(DNMTs), which would impact the cell cycle. Immortalized mouse hippocampal neuronal precursor cells(HT_(22)) were treated with 0.3 or 3 μM BpV. Proliferation, morphology, and viability of HT_(22) cells were detected with an IncuCyte real-time video imaging system or inverted microscope and 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, respectively. mRNA and protein expression of DNMTs and p21 in HT_(22) cells was detected by real-time polymerase chain reaction and immunoblotting, respectively. In addition, DNMT activity was measured with an enzyme-linked immunosorbent assay. Effects of BpV on the cell cycle were analyzed using flow cytometry. Results demonstrated that treatment with 0.3 μM BpV did not affect cell proliferation, morphology, or viability; however, treatment with 3 μM BpV decreased cell viability, increased expression of both DNMT3B mRNA and protein, and inhibited the proliferation of HT_(22) cells; and 3 μM BpV also blocked the cell cycle and increased expression of the regulatory factor p21 by increasing DNMT expression in mouse hippocampal neurons.

HT22细胞氧糖剥夺再灌注及Grasp65过表达干预后高尔基体的形态变化及其可能机制研究

目的探讨HT22细胞氧糖剥夺再灌注及Grasp65过表达干预后高尔基体的形态变化及其可能机制。方法利用小鼠海马神经元细胞系HT22为研究对象,HT22细胞经氧糖剥夺再灌注损伤及Grasp65过表达干预后,采用MTT法检测细胞存活率;Hoechest33258荧光染色法评估细胞凋亡;并应用细胞免疫荧光技术观察高尔基体的形态;应用Western blot技术检测GM130和GAAP蛋白的表达。结果氧糖剥夺再灌注可导致HT22细胞的活性显著降低(P<0.05),凋亡率显著增高(P<0.05);并可导致高尔基体形态的异常,随着再灌注时间的延长,高尔基体逐渐发生碎裂,尤其以再灌注12 h和24 h最为明显;GM130、GAAP的表达水平在氧糖剥夺再灌注后出现下降,特别是在再灌注12 h、24 h后出现了显著下降(P<0.05)。过表达Grasp65后,HT22细胞在氧糖剥夺再灌注所致高尔基体碎裂出现减少(P<0.05),碎裂程度减轻,同时GM130和GAAP的表达均显著增加(P<0.05),HT22细胞的存活率大大提高(P<0.05),凋亡率显著降低(P<0.05)。结论缺血再灌注损伤的细胞模型中,同样发生了高尔基体的碎裂;过表达Grasp65可以减轻氧糖剥夺再灌注损伤所致的高尔基体碎裂,并可以减少HT22细胞的凋亡,其机制可能与上调GM130和GAAP的表达有关。

蛋白酶抑制剂H89对神经元细胞氧糖剥夺再灌注后高尔基体形态及细胞凋亡的影响

目的探讨蛋白酶抑制剂H89对神经元细胞经氧糖剥夺再灌注损伤后高尔基体形态和细胞凋亡的影响。方法将体外常规培养的小鼠海马神经元细胞HT22分为对照组、模型组和H89干预组。模型组与H89干预组按再灌注时间点分6h、12h和24h三个亚组。采用MTT法检测细胞存活率;Hoechest33258荧光染色法评估细胞凋亡;细胞免疫荧光技术观察高尔基体形态;Western blot技术检测GM130与GAAP蛋白表达。结果 H89干预组较模型组细胞活力有所上升,其中12h时间点(OD值0.1467±0.0090)与同期模型组比较,差异有统计学意义(P<0.05)。H89干预组较模型组细胞凋亡率有所下降,但差异均无统计学意义(P>0.05)。H89干预组在6h和12h时间点高尔基体碎裂程度较同期模型组稍有减轻。H89干预组GM130表达水平较模型组无显著升高(P>0.05)。GAAP表达水平仅在24h时间点(灰度比值0.4066±0.0288)显著升高(P<0.05)。结论 H89干预不能减轻神经元细胞经氧糖剥夺再灌注损伤后的高尔基体碎裂和细胞凋亡。

7,8-二羟-9,10-环氧苯并[a]芘诱发小鼠海马神经元HT22细胞铁死亡及其机制

[背景]苯并[a]芘(BaP)具有神经毒性,可诱发人和动物海马神经元死亡从而导致空间学习记忆功能障碍,但其机制尚不清楚。[目的]观察BaP活性代谢物7,8-二羟-9,10-环氧苯并[a]芘(BPDE)诱发小鼠海马神经元HT22细胞铁死亡的发生规律,初步探讨其潜在机制,为BaP神经毒性机制研究提供依据。[方法]选用小鼠海马神经元HT22细胞,分为溶剂对照组和低、中、高浓度BPDE染毒组(0.25、0.50、0.75μmol·L^(-1))。CCK8法测定细胞存活率。光镜和电镜观察细胞形态及超微结构。荧光探针法检测细胞内活性氧(ROS)水平和Fe^(2+)水平。试剂盒检测细胞内铁、4-羟基壬烯酸(4-HNE)、丙二醛(MDA)、谷胱甘肽(GSH)和谷胱甘肽过氧化物酶(GSH-PX)水平。Western blotting法检测铁死亡特征蛋白酰基辅酶A合成长链家族成员4(ACSL4)、环氧合酶2(COX2)、溶质载体家族7成员11(SLC7A11)、谷胱甘肽过氧化物酶4(GPX4)蛋白表达水平。用铁死亡抑制剂20μmol·L^(-1)去铁胺(DFO)和10μmol·L^(-1)3-氨基-4-环己基氨基苯甲酸乙酯(Fer-1)进行干预,观察对各浓度BPDE染毒组细胞存活率和相关铁死亡特征指标和蛋白表达水平的影响。[结果]随着BPDE染毒浓度的增加,HT22细胞存活率逐渐下降,各BPDE染毒组细胞存活率均明显低于溶剂对照组(P<0.01)。光镜下可见高浓度BPDE组细胞数量明显减少,形态萎缩变形,突触减少;电镜下可见高浓度BPDE组HT22细胞表现出线粒体皱缩,嵴减少,线粒体膜密度增加。与溶剂对照组相比,高浓度染毒组细胞内脂质ROS、Fe^(2+)、4-HNE及MDA水平明显增加(P<0.01);GSH、GSH-PX水平明显降低(P<0.01),ASCL4、COX2蛋白表达水平明显增加(P<0.01),SLC7A11、GPX4蛋白表达水平明显下降(P<0.01)。铁死亡抑制剂DFO、Fer-1可明显逆转高浓度BPDE组细胞的存活率(P<0.01)、铁死亡特征指标(ROS、Fe^(2+)、4-HNE、MDA、GSH、GSH-PX水平)(P<0.01)以及铁死亡相关蛋白水平的表达(ACSL4、COX2、SLC7A11、GPX4)(P<0.01)。[结论]BPDE可诱发小鼠海马神经元HT22细胞铁死亡,其机制可能与抑制SLC7A11/GSH/GPX4轴及诱发细胞铁离子代谢紊乱有关。