PXR激活在拮抗SH-SY5Y细胞凋亡中的作用

人神经母细胞瘤细胞SH-SY5Y细胞可以表达神经元特异性的酪氨酸羟化酶、多巴胺-β-羟化酶以及多巴胺转运体等,因此可用于建立帕金森病的体外模型。虽然帕金森综合症发病的确切机制至今尚不清楚,但众多的病理学资料证实该病患者存在中脑黑质多巴胺能神经元的凋亡。自由基、兴奋性     

Carnosic Acid Prevents Beta-Amyloid-Induced Injury in Human Neuroblastoma SH-SY5Y Cells via the Induction of Autophagy

Beta-amyloid (Aβ), the hallmark protein in Alzheimer’s disease (AD), induces neurotoxicity that involves oxidative stress and mitochondrial dysfunction, leading to cell death. Carnosic acid (CA), a polyphenolic diterpene isolated from the herb rosemary (Rosemarinus officinalis), was investigated in our study to assess its neuroprotective effect and underlying mechanism against Aβ-induced injury in human neuroblastoma SH-SY5Y cells. We found that CA pretreatment alleviated the Aβ_25–35-induced loss of cell viability, inhibited both Aβ_1–42 accumulation and tau hyperphosphorylation, reduced reactive oxygen species generation, and maintained the mitochondrial membrane potential. Moreover, CA increased the microtubule-associated protein light chain 3 (LC3)-II/I ratio and decreased SQSTM1(p62), indicating that CA could induce autophagy. Autophagy inhibitor 3-methyladenine (3-MA) attenuated the neuroprotective effect of CA, suggesting that autophagy was involved in the neuroprotection of CA. It was also observed that CA activated AMP-activated protein kinase (AMPK) but inhibited mammalian target of rapamycin (mTOR). Furthermore, blocking AMPK with si-AMPKα successfully inhibited the upregulation of LC3-II/I, prevented the downregulation of phosphorylation of mTOR and SQSTM1(p62), indicating that CA induced autophagy in SH-SY5Y cells via the activation of AMPK. These results suggested that CA might be a potential agent for preventing AD.     

Activation of Protein Kinase C in Permeabilized Human Neuroblastoma SH-SY5Y Cells

The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.     

Angelica polymorpha Maxim Induces Apoptosis of Human SH-SY5Y Neuroblastoma Cells by Regulating an Intrinsic Caspase Pathway

Angelica polymorpha Maxim root extract (APRE) is a popular herbal medicine used for treating stomachache, abdominal pain, stomach ulcers, and rheumatism; however the effect of APRE on cancer cells has not yet been explored. Here, we examined APRE cytotoxicity seen on target neuroblastoma cells (NB) using cell viability assays, DAPI visualization of fragmented DNA, and Western blotting analysis of candidate signaling pathways involved in proliferation and apoptosis. We demonstrated that APRE reduced cell viability in NB to a greater extent than in fibroblast cells. In addition, we found that APRE could inhibit the three classes of MAPK proteins and could also down-regulate the PI3K/AKT/GSK-3β activity all being relevant for proliferation and survival. APRE could also up-regulate Bax expression and down-regulate Bcl-2 and Mcl-1. With APRE treatment, depolarization of mitochondria membrane potential and activation of caspase-3 was demonstrated in the SH-SY5Y cells. We could not found increased activity of death receptor and caspase-8 as markers of the extrinsic apoptosis pathway for the APRE treated cells. In presence of a caspase-3 siRNA and a pan-caspase inhibitor, APRE could not reduce the viability of NB cells to a significant degree. So we predicted that with APRE, the intrinsic pathway was solely responsible for inducing apoptosis as we also showed that the non-caspase autophagy pathway or ER stress-ROS mediated pathways were not involved. These findings demonstrate that an intrinsic mitochondria-mediated apoptosis pathway mediates the apoptotic effects of APRE on SH-SY5Y cells, and that APRE shows promise as a novel agent for neuroblastoma therapy.     

Gas1 Knockdown Increases the Neuroprotective Effect of Glial Cell-Derived Neurotrophic Factor Against Glutamate-Induced Cell Injury in Human SH-SY5Y Neuroblastoma Cells

Growth arrest-specific 1 (Gas1) protein acts as an inhibitor of cell growth and a mediator of cell death in nervous system during development and is also re-expressed in adult neurons during excitotoxic insult. Due to its structural similarity to the glial cell-derived neurotrophic factor family receptors α (GFRα), Gas1 is likely to interfere with the neuroprotective effect of GDNF. In the present study, we investigated the expression profile of Gas1 during glutamate insults in human SH-SY5Y neuroblastoma cells as well as the influence of Gas1 inhibition on the protective effect of GDNF against glutamate-induced cell injury. Our data showed that Gas1 expression was significantly increased with the treatment of glutamate in SH-SY5Y cells. The silencing of Gas1 by small interfering RNA promoted the protective effect of GDNF against glutamate-induced cytotoxicity as well as cell apoptosis, which effect was likely mediated through activating Akt/PI3 K-dependent cell survival signaling pathway and inhibiting mitochondrial-dependent cell apoptosis signaling pathway via Bad dephosphorylation blockade. In summary, this study showed the synergistic effect of Gas1 inhibition and GDNF against glutamate-induced cell injury in human SH-SY5Y neuroblastoma cells, which information might significantly contribute to better understanding the function of Gas1 in neuronal cells and form the basis of the therapeutic development of GDNF in treating human neurodegenerative diseases in the future.     

MAPK磷酸酯酶-1通过抑制JNK和p38的磷酸化调节SH-SY5Y神经母细胞瘤的凋亡

目的研究MKP-1在SH-SY5Y神经母细胞瘤中的抗凋亡。方法建立稳定表达MKP-1的SH-SY5Y细胞,用H2O2诱导细胞凋亡,并通过Western blotting比较分析MKP-1的表达对JNK和p38磷酸化的调节。结果①H2O2诱导SH-SY5Y细胞表达MKP-1,同时导致JNK和p38的去磷酸化;②在稳定表达MKP-1的SH-SY5Y细胞中,MKP-1可以抑制JNK和p38的磷酸化。③稳定表达MKP-1的SH-SY5Y细胞抵抗H2O2诱导细胞凋亡的能力比对照细胞提高了1倍左右。结论MKP-1对神经细胞的凋亡具有重要的调节作用,提示MKP-1作为调节ERK、JNK和p38蛋白激酶信号途径的重要分子,可能对退行性神经系统疾病的发病机制和治疗有重要的作用。     

(−)-Deprenyl Protects Human Dopaminergic Neuroblastoma SH-SY5Y Cells from Apoptosis Induced by Peroxynitrite and Nitric Oxide

Abstract: In Parkinson's disease the cell death of dopamine neurons has been proposed to be mediated by an apoptotic death process, in which nitric oxide may be involved. This article reports the induction of apoptosis by nitric oxide and peroxynitrite in human dopaminergic neuroblastoma SH-SY5Y cells and the antiapoptotic activity of (−)-deprenyl. After the cells were treated with a nitric oxide donor, NOR-4, or a peroxynitrite donor, SIN-1, DNA damage was quantitatively studied using a single-cell gel electrophoresis (comet) assay. NOR-4 and SIN-1 induced DNA damage dose-dependently. Cycloheximide and alkaline treatment of the cells prevented the DNA damage, indicating that the damage is apoptotic and that it depends on the intracellular signal transduction. Superoxide dismutase and the antioxidants reduced glutathione and α-tocopherol protected the cells from the DNA damage. (−)-Deprenyl protected the cells from the DNA damage induced by nitric oxide or peroxynitrite almost completely. The protection by (−)-deprenyl was significant even after it was washed from the cells, indicating that (−)-deprenyl may activate the intracellular system against apoptosis. These results suggest that (−)-deprenyl or related compounds may be neuroprotective to dopamine neurons through its antiapoptotic activity.     

热量限制对SH-SY5Y细胞氧化损伤的影响

目的观察热量限制培养条件下,SH-SY5Y细胞抗氧化应激损伤的能力。方法建立过氧化氢诱导的SH-SY5Y细胞损伤模型。体外培养SH-SY5Y细胞,分为对照组、损伤组（50、100、250、500、1 000μmol/L H2O2）、低糖组（2 g/L）、低糖＋损伤组,进行细胞形态观察、测定各组细胞的噻唑蓝（MTT）代谢率、乳酸脱氢酶（LDH）漏出率。结果与对照组比较,（50、100、250、500、1 000）μmol/L H2O2损伤1 h后MTT代谢率测定细胞活力,50μmol/L组与对照组比较差异无统计学意义（P〉0.05）;其他组与对照组比较,随着H2O2浓度的增加,细胞活力呈递减趋势,差异具有显著性（P〈0.01）;选定250μmol/L H2O2组为损伤应激源。用低糖预处理细胞24 h,给与250μmol/L H2O2损伤1 h后测定MTT代谢率显示,与对照组比较,损伤组活力明显下降,低糖组活力上升（P〈0.01）;与损伤组比较,低糖＋损伤组活力明显上升（p〈0.01）;继续培养至7 h发现,与对照组比较,低糖组活力上升（P〈0.01）;与损伤组比较,低糖＋损伤组活力明显上升（P〈0.01）。进一步检测LDH漏出率显示,损伤1 h后结果显示,与对照组比较,损伤组漏出率明显增加（P〈0.05）,低糖组漏出率稍有减少（P〉0.05）;与损伤组比较,低糖＋损伤组漏出率明显减少（P〈0.01）;继续培养7h显示,低糖7h组与低糖1 h组比较,漏出稍有增多（P〉0.05）,低糖＋损伤组7 h组与低糖＋损伤组1 h比较漏出率稍有增加（P〈0.05）;细胞形态学观察显示,未加损伤之前,低糖组的细胞形态,与对照组比较无明显改变。加入损伤药物1h后的细胞形态与对照组比较无明显改变。加入损伤药物7 h后的细胞形态,低糖组和对照组细胞突起伸展良好细长,损伤组可见细胞数目明显减少,死细胞多,突起回缩,细胞明显变圆,贴壁性不好,透光性差。结论热量限制能提高神经细胞的抗氧化应激能力,增加细胞生存率,降低死亡率。     

Potentiation of Carbachol-Induced Ca2+ Release by Peroxynitrite in Human Neuroblastoma SH-SY5Y Cells

We have investigated the effect of 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, on carbachol-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in human neuroblastoma SH-SY5Y cells by means of single cell imaging of [Ca²⁺]_i. SIN-1 potentiated carbachol-induced [Ca²⁺]_i rise regardless of external Ca²⁺, and the potentiation was completely inhibited by superoxide dismutase, indicating that peroxynitrite may enhance Ca²⁺ release from intracellular stores. On the other hand, SIN-1 reduced carbachol-induced inositol 1,4,5-trisphosphate (IP₃) formation. Genistein, a tyrosine kinase inhibitor, potentiated carbachol-induced rise of [Ca²⁺]_i regardless of external Ca²⁺. These results suggest that peroxynitrite may potentiate the release of Ca²⁺ from intracellular stores through the perturbation of regulation in tyrosine phosphorylation-dephosphorylation system.     

Protective Effects of Fisetin Against 6-OHDA-Induced Apoptosis by Activation of PI3K-Akt Signaling in Human Neuroblastoma SH-SY5Y Cells

Neurochemical Research - 6-Hydroxydopamine (6-OHDA) induces the production of reactive oxygen species (ROS) that are associated with various neurodegenerative diseases such as...     

Nicotinic Receptor-Mediated Release of Noradrenaline in the Human Neuroblastoma SH-SY5Y

Abstract: Dimethylphenylpiperazinium iodide (a nicotinic agonist) evokes noradrenaline release from human neuroblastoma SH-SY5Y cells that have been pretreated with 12- O -tetradecanoylphorbol 13-acetate for 8 min. This effect of dimethylphenylpiperazinium iodide was inhibited by 1 μ M mecamylamine but not by 1 μ M atropine, which suggests that SH-SY5Y cells express nicotinic receptors coupled to the release of noradrenaline. Dimethylphenylpiperazinium iodide-evoked release was enhanced by 5 μ M Bay K 8644 (an L-type calcium agonist) and inhibited by 1 μ M nifedipine. Dimethylphenylpiperazinium iodide depolarised SH-SY5Y cells and enhanced the level of intracellular calcium in cells loaded with fura 2. The effects of dimethylphenylpiperazinium iodide on noradrenaline release, depolarisation, and intracellular calcium levels were all inhibited by 1 μ M desmethylimipramine. The results of this study show that nicotinic receptors in SH-SY5Y cells stimulate noradrenaline release by activation of L-type calcium channels.     

Neuroprotective Effects of Ugonin K on Hydrogen Peroxide-Induced Cell Death in Human Neuroblastoma SH-SY5Y Cells

Oxidative stress plays an important role in the pathological processes of various neurodegenerative diseases. Ugonin K, a flavonoid isolated from the rhizomes of Helminthostachys zeylanica, possesses potent antioxidant property. In this study, we investigate the neuroprotective effects of ugonin K on hydrogen peroxide (H₂O₂)-induced apoptosis in SH-SY5Y cells. Incubation of SH-SY5Y cells with H₂O₂ for 24 h induced cell death measured with MTT assay. Hoechst 33258 staining confirmed that the reduced cell viability by H₂O₂ was due to apoptosis. In addition, H₂O₂ increased the expression of 17-kDa cleaved fragment of caspase-3 which could be reversed by pretreatment with ugonin K. Pretreatment with ugonin K attenuated H₂O₂-induced cell death in a dose-dependent manner. Neuroprotective effect of ugonin K was abolished by ERK and PI3K inhibitors. Pretreatment with JNK kinase and p38 MAPK inhibitors had no effect on ugonin K-mediated protection against H₂O₂-induced apoptosis. Western blotting with anti-phospho-ERK1/2 and anti-phospho-Akt (pS473) antibodies showed that ugonin K increased both ERK1/2 and Akt phosphorylation. These results suggest that ugonin K by activation of ERK1/2 and PI3K/Akt signal pathways protects SH-SY5Y cells from H₂O₂-induced apoptosis.     

Neuroprotective Effects of Phenolic and Carboxylic Acids on Oxidative Stress-Induced Toxicity in Human Neuroblastoma SH-SY5Y Cells

An increase in oxidative stress is a key factor responsible for neurotoxicity induction and cell death leading to neurodegenerative diseases including Parkinson’s and Alzheimer’s diseases. Plant phenolics exert diverse bioactivities i.e., antioxidant, anti-inflammatory, and neuroprotective effects. Herein, phenolic compounds, namely protocatechuic aldehyde (PCA) constituents of Hydnophytum formicarum Jack. including vanillic acid (VA) and trans-ferulic acid (FA) found in Spilanthes acmella Murr., were explored for anti-neurodegenerative properties using an in vitro model of oxidative stress-induced neuroblastoma SH-SY5Y cells. Exposure of the neuronal cells with H₂O₂ resulted in the decrease of cell viability, but increasing in the level of reactive oxygen species (ROS) together with morphological changes and inducing cellular apoptosis. SH-SY5Y cells pretreated with 5 µM of PCA, VA, and FA were able to attenuate cell death caused by H₂O₂-induced toxicity, as well as decreased ROS level and apoptotic cells after 24 h of treatment. Pretreated SH-SY5Y cells with phenolic compounds also helped to upregulate H₂O₂-induced depletion of the expressions of sirtuin-1 (SIRT1) and forkhead box O (FoxO) 3a as well as induce the levels of antioxidant (superoxide dismutase (SOD) 2 and catalase) and antiapoptotic B-cell lymphoma 2 (Bcl-2) proteins. The findings suggest that these phenolics might be promising compounds against neurodegeneration.     

TIMP-1基因缄默削弱SH-SY5Y细胞的增殖潜能并诱导其凋亡增强

前期研究发现,人基质金属蛋白酶组织抑制剂-1(tissue inhibitors of metalloproteinases-1,TIMP-1)在唐氏综合征（Down’s syndrome ,DS)胎儿脑组织内表达下调．为了探讨TIMP-1表达下调参与DS脑病变发生的可能机制,本研究以人神经母细胞瘤细胞（SH-SY5Y）为模型,观察TIMP-1基因沉默后对其增殖和凋亡的影响．应用LipofectaminTM2000将TIMP-1特异性短发卡 RNA( short hairpin RNA,shRNA)导入SH-SY5Y细胞,经嘌呤霉素筛选获得稳定表达TIMP-1-shRNA细胞株;应用RT-PCR、real-time PCR和Western 印迹对干扰效率进行鉴定：与SH-SY5Y细胞相比,无论在mRNA水平还是蛋白水平,SH-SY5Y-TIMP-1-shRNA细胞中TIMP-1的表达显著下调（下调率接近100%）．结果显示,已成功构建了TIMP-1基因沉默的SH-SY5Y细胞模型．在此基础上,通过MTT检测发现,TIMP-1基因沉默后SH-SY5Y细胞增殖减慢;流式细胞仪和荧光显微镜凋亡检测显示,TIMP-1基因沉默后SH-SY5Y细胞凋亡明显增加．这些研究结果表明,TIMP-1基因沉默能削弱SH-SY5Y细胞的增殖能力并增强SH-SY5Y的凋亡效应,提示TIMP-1可能是通过影响神经细胞的增殖和凋亡参与DS智力低下的发病过程．     

SU5416 and EGCG Work Synergistically and Inhibit Angiogenic and Survival Factors and Induce Cell Cycle Arrest to Promote Apoptosis in Human Malignant Neuroblastoma SH-SY5Y and SK-N-BE2 Cells

Malignant neuroblastomas are solid tumors in children. Available therapeutic agents are not highly effective for treatment of malignant neuroblastomas. Therefore, new treatment strategies are urgently needed. We tested the efficacy of combination of SU5416 (SU), an inhibitor of the vascular endothelial growth factor receptor-2 (VEGFR-2), and (−)-epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, for controlling growth of human malignant neuroblastoma SH-SY5Y and SK-N-BE2 cells. Combination of 20 μM SU and 50 μM EGCG synergistically inhibited cell survival, suppressed expression of VEGFR-2, inhibited cell migration, caused cell cycle arrest, and induced apoptosis. Combination of SU and EGCG effectively blocked angiogenic and survival pathways and modulated expression of cell cycle regulators. Apoptosis was induced by down regulation of Bcl-2, activation of caspase-3, and cleavage of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP). Taken together, this combination of drugs can be a promising therapeutic strategy for controlling the growth of human malignant neuroblastoma cells.     

Involvement of Matrix Metalloproteinases on the Inhibition of Cells Invasion and Migration by Emodin in Human Neuroblastoma SH-SY5Y Cells

Emodin (1,3,8-trihydroxy-6-methylanthaquinone), an active component present in the root and rhizome of Rheum palmatum L. (Polygonaceae) has anti-bacterial, anti-tumor, diuretic and vasorelaxant effects. However, its mechanism of action on the cell migration and invasion of human neuroblastoma cancer SH-SY5Y cells is not fully understood. In this study, firstly, the effects of emodin on the percentage of viable cells were examined by using MTT assay and it was found that emodin induced dose-and time-dependent inhibition in human neuroblastoma SH-SY5Y cells. Second, the effects of emodin on the migration and invasion of SH-SY5Y cells were examined by using wound assay and matrigel counting and the results showed that emodin suppressed the migration and invasion of SH-SY5Y cells. Third, we examined the effect of emodin on the levels of associated proteins by using Western blotting and the results indicated that emodin inhibited the levels of GRB2, RhoA, HIF-1α, VEGF, FAK, iNOS, COX2, p-p38, p-c-jun, MMP2, MMP9 and MMP7 but promoted the levels of PKC, PI3K, MEKK3 and NF-κB p65 that led to the inhibition of migration and invasion of SH-SY5Y cells in vitro.