几种中药活性成分对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制研究
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摘要
阿尔茨海默病(Alzheimer’s disease, AD)、帕金森病(Parkinson’sdisease, PD)、肌萎缩侧索硬化症(amyotrophic lateral sclerosis, ALS)等是严重危害人类健康的一类神经退行性疾病(neurodegenerative diseases，NDD)，是由脑中特定区域神经元发生退变而引起的慢性进行性神经系统疾病。近年来，NDD发病率急剧上升，特别是在老龄化人口中。由于其发病机制复杂，因此迄今尚无理想的防治药物问世。导致NDD发生的确切的病理生理学机理尚未明确，目前认为由多种原因引起，并提出了多种假说，其中代表性的有神经递质失衡学说，氧化应激学说、线粒体功能障碍学说、兴奋性神经毒性学说、异常蛋白聚积学说、基因突变、炎症过程、免疫异常及细胞凋亡学说等。近年来越来越多的资料证明，线粒体功能障碍和氧化应激与其他几种机制相互作用，氧化应激和线粒体参与可能是主要的触发因素。细胞内的活性氧(reactive oxygen species，ROS)在各种细胞内不断产生和清除，当体内氧化水平超过了抗氧化作用时，氧化应激就会发生。ROS在细胞信号传导中扮演了重要角色，参与多种生理和病理过程。
过氧化氢（H_2O_2）是体内代谢的产物,同时也是一种ROS，在诱导神经元细胞的损伤乃至死亡中起了关键作用。H_2O_2造成的细胞氧化损伤已成为研究神经细胞氧化损伤的重要工具之一。
赤芍和土木香均为临床中常用的药物。赤芍为毛茛科植物芍药或川赤芍的干燥根，具有清热凉血、化瘀止痛、消肿通经等功效。土木香是菊科植物土木香Inula helenium L.或藏木香Inula racemosa Hook.f.的干燥根，广泛分布于欧洲中南部和亚洲的喜马拉雅山脉，具有多种药理作用，具有祛痰、止咳等作用，常被用于治疗多种呼吸系统和消化系统疾病。现代药理实验证实，赤芍对神经系统具有保护作用，文献报道赤芍对于PD、AD等NDD的治疗具有一定的作用，但涉及其中的分子机制有待深入系统的研究。ROS在许多疾病包括神经退行性疾病的发生、发展中扮演了重要角色，我们推断具有增加细胞抗氧化能力、具有抑制ROS对细胞损伤的药物可能有助于NDD的防治，所以探讨具有清除ROS和具有神经保护潜能的天然物质对细胞氧化损伤保护作用和机制的研究具有重要意义。赤芍和土木香具有抗氧化作用，体外实验证实赤芍和土木香具有清除DPPH自由基、羟自由基等活性，我们推断赤芍对神经系统的保护作用和赤芍的抗氧化特性有关，赤芍发挥神经保护作用的分子机制可能与清除ROS和阻断细胞凋亡的信号通路有关。目前认为线粒体通路、死亡受体通路和内质网通路是三个主要的凋亡信号通路，caspase3是这些凋亡信号通路的执行者。
关于土木香总黄酮对SH-SY5Y氧化应激损伤的影响未见相关报道。黄酮类化合物是一类次生代谢产物广泛分布在不同植物中，具有各种生物活性如抗氧化、抗动脉粥样硬化、抗炎等;也有助于预防心血管疾病和抗血小板聚集。
基于以上研究背景，本研究以H_2O_2诱导的神经细胞损伤和凋亡为模型，探讨赤芍相关的三种活性单体芍药苷（paeoniflorin，PF）、没食子酸（gallic acid，GA）和赤芍801（propyl gallate，PG）对H_2O_2引起的人神经母细胞瘤株（SH-SY5Y）细胞损伤和凋亡的影响及其机制。具体内容如下：
1H_2O_2诱导SH-SY5Y细胞氧化损伤模型的建立
目的：建立H_2O_2诱导SH-SY5Y细胞氧化损伤模型。
方法：不同浓度H_2O_2(12.5、25.0、50.0、100、200、400、800、1600、3200、6400μmol·L~(-1))作用SH-SY5Y细胞不同时间（1、2、4、8、12、24小时）诱导SH-SY5Y细胞氧化损伤，通过相差显微镜观察SH-SY5Y细胞形态变化，利用CCK-8法测定细胞存活率，筛选H_2O_2致SH-SY5Y细胞氧化应激损伤模型的剂量；Heochest33258染色观察凋亡细胞的形态学特征；Annexin V-FITC/PI双染，流式检测细胞凋亡率；碘化丙啶（propidiumiodide，PI）染色流式细胞仪检测细胞周期构成比；硫辛酰胺脱氢酶（diaphorase）催化的INT （2-p-iodophenyl-3-nitrophenyl-tetrazoliumchloride）显色反应检测乳酸脱氢酶（lactic dehydrogenase，LDH）释放量，2',7'-Dichlorofluorescein diacetate（DCFH-DA）检测细胞内活性氧（ROS）；ELISA (Enzyme-linked Immuno Sorbent Assay)方法检测8-羟基脱氧鸟苷（8-hydroxy deoxyguanosine，8-OHdG）；JC-1染色检测细胞线粒体膜电位（mitochondrial membrane potential，MMP）；Fura-2AM荧光探针检测细胞内钙离子浓度；利用caspase3可以催化底物acetyl-Asp-Glu-Val-Aspp-nitroanilide (Ac-DEVD-pNA)的反应检测caspase3活性；应用caspase9催化特异性底物acetyl-Leu-Glu-His-Asp p-nitroanilide (Ac-LEHD-pNA)检测caspase9活性。
结果：过氧化氢组与正常对照组相比，H_2O_2剂量依赖、时间依赖地造成SH-SY5Y细胞氧化损伤。相对于正常组，终浓度为200μmol L~(-1)H_2O_2作用细胞24h后，细胞活力下降为65%，具有统计学差异(P<0.01)，细胞凋亡率上升(P<0.01)；明显出现了细胞凋亡的形态学特征；增殖指数（proliferation index, PI%）降低(P<0.05)；8-OHdG含量上升(P<0.01)；LDH释放量和细胞内ROS增加(P<0.01)，线粒体膜电位下降(P<0.01)，细胞内钙离子浓度上升(P<0.01)，caspase3和caspase9活性增强(P<0.01)。
结论：200μmol L~(-1)H_2O_2作用细胞24h，细胞活力显著下降，明显造成细胞氧化损伤，造模成功。
2没食子酸对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制研究
目的：以H_2O_2诱导SH-SY5Y细胞氧化损伤为模型，探讨没食子酸对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制。
方法：筛选没食子酸（gallic acid，GA）对SH-SY5Y细胞无毒剂量范围，以此剂量范围研究没食子酸对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制。将细胞分为5组。正常对照组（Control）， H_2O_2模型组（Model），GA5μmol L~(-1)组（GA1）、GA10μmol L~(-1)组（GA2）、GA25μmol L~(-1)组（GA3）。GA组先加入GA预处理1h后再加入终浓度为200μmol L~(-1)H_2O_2。Heochest33258染色观察凋亡细胞的形态学特征；Annexin V-FITC/PI双染，流式检测细胞凋亡率； PI染色流式细胞仪检测细胞周期构成比； diaphorase催化的INT显色反应检测LDH释放量；DCFH-DA检测细胞内ROS；ELISA法检测8-OHdG；JC-1染色检测细胞线粒体膜电位；Fura-2AM荧光探针检测细胞内钙离子浓度；利用caspase3可以催化底物Ac-DEVD-pNA的反应检测caspase3活性；应用caspase9催化特异性底物Ac-LEHD-pNA检测caspase9活性。
结果：过氧化氢组与正常对照组相比，终浓度为200μmol L~(-1)H_2O_2作用细胞24h后，细胞活力下降为65%，具有统计学差异(P<0.01)，细胞凋亡率上升(P<0.01)；明显出现了细胞凋亡的形态学特征；增殖指数（proliferation index, PI%）降低(P<0.05)；8-OHdG含量上升(P<0.01)；LDH释放量和细胞内ROS增加(P<0.01)，线粒体膜电位下降(P<0.01)，细胞内钙离子浓度上升(P<0.01)，caspase3和caspase9活性增强(P<0.01)。与H_2O_2损伤组相比，终浓度为5-25μmol·L~(-1)GA能显著改善上述指标的变化(P<0.05)。
结论：GA在一定剂量范围内对H_2O_2诱导的SH-SYSY神经细胞损伤具有明显的保护作用，其保护效应可能与清除ROS，减轻DNA氧化损伤，抑制线粒体通路介导的细胞凋亡有关。
3芍药苷对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制研究
目的：以H_2O_2诱导SH-SY5Y细胞氧化损伤为模型，探讨芍药苷对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制。
方法：筛选芍药苷（paeoniflorin，PF）对SH-SY5Y细胞无毒剂量范围，以此剂量范围研究芍药苷对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制。将细胞分为5组。正常对照组（Control）， H_2O_2模型组（Model），PF10μmol·L~(-1)组（PF1）、PF20μmol·L~(-1)组（PF2）、PF40μmol·L~(-1)组（PF3）。PF组先加入PF预处理1h后再加入终浓度为200μmol·L~(-1)H_2O_2。Heochest33258染色观察凋亡细胞的形态学特征；Annexin V-FITC/PI双染，流式检测细胞凋亡率；PI染色流式细胞仪检测细胞周期构成比；diaphorase催化的INT显色反应检测LDH释放量；DCFH-DA检测细胞内ROS；ELISA法检测8-OHdG；JC-1染色检测细胞线粒体膜电位；Fura-2AM荧光探针检测细胞内钙离子浓度；利用caspase3可以催化底物Ac-DEVD-pNA的反应检测caspase3活性；应用caspase9催化特异性底物Ac-LEHD-pNA检测caspase9活性。
结果：过氧化氢组与正常对照组相比，终浓度为200μmol·L~(-1)H_2O_2作用细胞24h后，细胞活力下降为65%，具有统计学差异(P<0.01)，细胞凋亡率上升(P<0.01)；明显出现了细胞凋亡的形态学特征；增殖指数（proliferation index, PI%）降低(P<0.05)；8-OHdG含量上升(P<0.01)；LDH释放量和细胞内ROS增加(P<0.01)，线粒体膜电位下降(P<0.01)，细胞内钙离子浓度上升(P<0.01)，caspase3和caspase9活性增强(P<0.01)。与H_2O_2损伤组相比，20-40μmol·L~(-1)PF能显著改善过氧化氢诱导的上述指标的改变(P<0.05)；终浓度10μmol·L~(-1)PF组与H_2O_2组相比PI%无显著变化（P>0.05），LDH和8-OHdG含量无显著变化（P>0.05），但能显著改善过氧化氢诱导的细胞凋亡的形态学特征，提高过氧化氢诱导的细胞活力下降(P<0.05)，抑制H_2O_2诱导的ROS上升(P<0.05)，抑制线粒体膜电位的下降(P<0.05)，抑制细胞内钙离子浓度的升高(P<0.05)，抑制caspase3和caspase9活性增强(P<0.05)，减轻H_2O_2诱导的细胞凋亡率的上升(P<0.05)。
结论：PF在一定剂量范围内对H_2O_2诱导的SH-SYSY神经细胞损伤具有明显的保护作用，其保护效应可能与清除ROS，减轻DNA氧化损伤，抑制线粒体通路介导的细胞凋亡有关。
4赤芍801对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制研究
目的：以H_2O_2诱导SH-SY5Y细胞氧化损伤为模型，探讨赤芍801对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制。
方法：筛选赤芍801（propyl gallate，PG）对SH-SY5Y细胞无毒剂量范围，以此剂量范围研究赤芍801对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制。将细胞分为5组。正常对照组（Control）， H_2O_2模型组（Model），PG20μmol·L~(-1)组（PG1）、PG40μmol·L~(-1)组（PG2）、PG80μmol·L~(-1)组（PG3）。PG组先加入PG预处理1h后再加入终浓度为200μmol·L~(-1)H_2O_2。Heochest33258染色观察凋亡细胞的形态学特征；Annexin V-FITC/PI双染，流式检测细胞凋亡率； PI染色流式细胞仪检测细胞周期构成比；diaphorase催化的INT显色反应检测LDH释放量；DCFH-DA检测细胞内ROS；ELISA法检测8-OHdG；JC-1染色检测细胞线粒体膜电位；Fura-2AM荧光探针检测细胞内钙离子浓度；利用caspase3可以催化底物Ac-DEVD-pNA的反应检测caspase3活性；应用caspase9催化特异性底物Ac-LEHD-pNA检测caspase9活性。
结果：过氧化氢组与正常对照组相比，终浓度为200μmol·L~(-1)H_2O_2作用细胞24h后，细胞活力下降为65%，具有统计学差异(P<0.01)，细胞凋亡率上升(P<0.01)；明显出现了细胞凋亡的形态学特征；增殖指数（proliferation index, PI%）降低(P<0.05)；8-OHdG含量上升(P<0.01)；LDH释放量和细胞内ROS增加(P<0.01)，线粒体膜电位下降(P<0.01)，细胞内钙离子浓度上升(P<0.01)，caspase3和caspase9活性增强(P<0.01)。与H_2O_2损伤组相比，40-80μmol L-PG均能显著改善过氧化氢诱导的上述指标的改变(P<0.05)；终浓度20μmol·L~(-1)PG组与H_2O_2组相比PI%无显著变化（P>0.05），LDH和8-OHdG含量无显著变化（P>0.05），但能显著改善过氧化氢诱导的细胞凋亡的形态学特征，提高过氧化氢诱导的细胞活力下降(P<0.05)，抑制H_2O_2诱导的ROS上升(P<0.05)，抑制线粒体膜电位的下降(P<0.05)，抑制细胞内钙离子浓度的升高(P<0.05)，抑制caspase3和caspase9活性增强(P<0.05)，减轻H_2O_2诱导的细胞凋亡率的上升(P<0.05)。
结论：PG在一定剂量范围内对H_2O_2诱导的SH-SYSY神经细胞损伤具有明显的保护作用，其保护效应可能与清除ROS，减轻DNA氧化损伤，抑制线粒体通路介导的细胞凋亡有关。
5土木香总黄酮对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制研究
目的：以H_2O_2诱导SH-SY5Y细胞氧化损伤为模型，探讨土木香总黄酮对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响。
方法：筛选土木香总黄酮（total flavonoids，TF）对SH-SY5Y细胞无毒剂量范围，以此剂量范围研究土木香总黄酮对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制。将细胞分为5组。正常对照组（Control），H_2O_2模型组（Model），TF5mg·L~(-1)组（TF1）、TF10mg·L~(-1)组（TF2）、TF20mg·L~(-1)组（TF3）。TF组先加入TF预处理1h后再加入终浓度为200μmol·L~(-1)H_2O_2。DCFH-DA检测细胞内ROS。
结果：过氧化氢组与正常对照组相比，终浓度为200μmol·L~(-1)H_2O_2作用细胞24h后，细胞活力显著下降(P<0.01)；与H_2O_2损伤组相比，5-20mg·L~(-1)TF能显著抑制过氧化氢诱导的凋亡(P<0.05)，抑制H_2O_2诱导的ROS上升(P<0.05)。
结论：TF在一定剂量范围内对H_2O_2诱导的SH-SYSY神经细胞损伤具有明显的保护作用，其保护机制有待进一步研究。
Neurodegenerative disease (NDD) is chronic progressive diseases of thenervous system caused by the specific brain areas in degeneration, includingAlzheimer’s disease (AD), Parkinson’s disease (PD),amyotrophielateralselerosis (ALS), etc. The incidence of NDD surges,especially in the aging population. Because the pathogenesis of NDD iscomplicated, there is still no ideal drug for preventing and curing NDD tillnow. Although the definite etiology and pathogenesis are not very clear up tonow, many studies show that NDD is caused by many different things. Thereare many views and hypothesis about the cause of NDD, among them,imbalance in neurotransmitters, oxidative stress, mitochondrial dysfunction,excitatory neural toxicity, anomalous proteins accumulation, gene mutation,inflammatory process, dysimmunity and apoptosis theory, etc. Increasingamounts of data show that mitochondrial dysfunction and oxidative stress hasbeen implicated in many neurological diseases and interacted with otherseveral mechanisms. Intracellular reactive oxygen species are continuouslyproduced and cleared. When the level of oxidation exceeds these antioxidantdefenses, oxidative stress occurs. ROS plays an important role in cell signaltransduction, and involves in many pathologic and physiological processes.
Hydrogen peroxide (H_2O_2) had been considered to be important signalingmolecules and natural products in the course of biological metabolism. H_2O_2plays an important role in neuron damage and even death. H_2O_2is one of themost important implements for studying neurons oxidative damage.
Radix Paeoniae Rubra (R. paeoniae) and Inula helenium is clinicallywidely used. R. paeoniae (Family：Ranunculaceae) is the root of PaeoniaRadix or Paeonia Veitchii Lynch, which is widely applicable to heat-clearing and blood-cooling, dispersing blood stasis and relieving pain, detumescence,stimulating the menstrual flow. Inula helenium (elecampane), a member of thecomposite family, is widely distributed throughout central and southernEurope as far as the Himalayas, in Asia. In the Chinese Pharmacopoeia, I.helenium is described as an expectorant, diaphoretic, and antitussive that isoften used to treat a variety of respiratory and digestive diseases. Recentresearch has demonstrated that the extracts of I. helenium exhibit many effectsincluding antibacterial, antitumor, and antiproliferative. At present, effect ofthe total flavonoids of elecampane on H_2O_2-induced SH SY5Y oxidative stresshas not been described.Flavonoid compounds are diverse class of secondaryplant metabolites that are widely distributed throughout the plant kingdom andhave been reported to possess a variety of biological activities such asantioxidant, anti-atherosclerotic, anti-inflammatory, and antithrombogenic;they also aid in cardiovascular disease prevention and anti-platelet aggregation.It has been reported that Paeonia Lactifiora has a protective effect for nervoussystem, Paeonia Lactifiora has certain influence on treatmenting of AD, PD,and its mechanism is required to further study systematically. ROS plays animportant role in disease (including NDD) onset and progression. We deducedthat the drug with antioxidation and inhibiting ROS may be useful forpreventing NDD. It is important for studying the protective effects andmechanism of natural product on the cellular oxidative damage. There isevidence that Paeonia Lactifiora has antioxidant properties, PaeoniaLactifiora can eliminate DPPH and hydroxyl radicals in vitro. There are closeconnection between the antioxidant properties of Paeonia Lactifiora and itsneuroprotection. The molecular mechanism of the neuroprotective effects of R.paeoniae may be related to removing ROS and blocking the apoptosissignaling pathway. The basic apoptotic pathway includes mitochondrialpathway, death receptor pathway and endoplasmic reticulum pathway.Caspase-3is the executor of the apoptotic signaling pathways.
Based on the above research background, this study was conducted todetermine the neuroprotective effects of three active monomers (gallic acid, paeoniflorin and propyl gallate) related to R. paeoniae on SH-SY5Y cellsinjured by H_2O_2and the molecular mechanisms underlying theseneuroprotective effects. The specific contents as follows:
1Establishment of the model of oxidative damage in SH-SY5Y cells inducedby H_2O_2
Objective：To establish the model of oxidative damage in SH-SY5Y cellsinduced by H_2O_2.
Method: Cultured human neuroblastoma SH-SY5Y cells were subjectedto oxidative damage with H_2O_2at different concentrations (12.5,25.0,50.0,100,200,400,800,1600,3200,6400μmol·L~(-1)) and at different times (1,2,4,8,12,24h). The cell viability was analyzed by CCK-8assay. The cellmorphologic changes were observed by inverted optical microscope. Typicalmorphological features of apoptotic cells were detected using Hoechst33258staining, futher, flow cytometric (FCM) was used to analysis the cell apoptosisand cell cycle alteration using Annexin V-FITC/PI and propidium iodidestaining; the releasing rate of lactic dehydrogenase (LDH) was determined bythe colour reaction of diaphorase-INT. Reactive oxygen species (ROS)production was determined by2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA) fluorescence.8-OHdG production was determined byenzyme-linked immunosorbent assay (ELISA). Mitochondria membranepotential was determined by JC-1staining; Intracellular Ca2+concentrationwas determined by Fura-2AM fluorescent probes. Caspase-3activity wasdetermined by caspase-3catalyze the substrate specificityacetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA). Caspase-9activitywas determined by caspase-9catalyze the substrate specificityacetyl-Leu-Glu-His-Asp p-nitroanilide (Ac-LEHD-pNA).
Results: H_2O_2treatment produced dose-dependent and time-dependentcytotoxicity compared with that of the normal control. In comparison tocontrol cells, exposure of cells to200μmol·L~(-1)H_2O_2for24h resulted inapproximately65%cell viability of control (P<0.01), the typical change ofcell apoptosis occurred, proliferation index decreased (P<0.05), apoptosis index increased (P<0.01). H_2O_2induced significantly the increase of LDHrelease, intracellular ROS, Ca2+and8-OHdG (P<0.01), and the decrease ofmitochondrial membrane potential (P<0.01). H_2O_2-induced oxidative stressincreased caspase-3activity and caspase-9activity (P<0.01).
Conclusion:200μmol·L~(-1)H_2O_2treatment produced cell activitydecreased remarkably. The model of oxidative damage in SH-SY5Y cellsinduced by H_2O_2is successfully established.
2Effects of Gallic Acid on Hydrogen Peroxide-induced Oxidative Damage inSH-SY5Y Cells
Objective：This study was conducted to determine the neuroprotectiveeffects of GA on SH-SY5Y cells injured by H_2O_2and the molecularmechanisms underlying these neuroprotective effects.
Method: CCK-8assay was performed to select non-toxic dose of gallicacid in SH SY5Y cells in treating SH SY5Y cells. Cultured humanneuroblastoma SH-SY5Y cells were subjected to oxidative damage with H_2O_2in the presence and absence of non-toxic dose of GA. Cells can be divided into5groups: normal, model,5μmol·L~(-1)GA,10μmol·L~(-1)GA and25μmol·L~(-1)GAgroup. For group GA, GA was added to the cells1h before treatment withH_2O_2. The cell viability was analyzed by CCK-8assay. The cell morphologicchanges were observed by inverted optical microscope. Typical morphologicalfeatures of apoptotic cells were detected using Hoechst33258staining, futher,FCM was used to analysis the cell apoptosis and cell cycle alteration usingAnnexin V-FITC/PI and propidium iodide staining; the releasing rate of LDHwas determined by the colour reaction of diaphorase-INT. ROS productionwas determined by DCFH-DA fluorescence.8-OHdG production wasdetermined by ELISA. Mitochondria membrane potential was determined byJC-1staining; Intracellular Ca2+concentration was determined by Fura-2AMfluorescent probes. Caspase-3activity was determined by caspase-3catalyzethe substrate specificityAc-DEVD-pNA. Caspase-9activity was determinedby caspase-9catalyze the substrate specificity Ac-LEHD-pNA.
Results: H_2O_2treatment produced dose-dependent and time-dependent cytotoxicity compared with that of the normal control. In comparison tocontrol cells, exposure of cells to200μmol·L~(-1)H_2O_2for24h resulted inapproximately65%cell viability of control (P<0.01), the typical change ofcell apoptosis occurred, proliferation index decreased (P<0.05), apoptosisindex increased (P<0.01). H_2O_2induced significantly the increase of LDHrelease, intracellular ROS, Ca2+and8-OHdG (P<0.01), and the decrease ofmitochondrial membrane potential (P<0.01). H_2O_2-induced oxidative stressincreased caspase-3activity and caspase-9activity (P<0.01). Finalconcentration of5-25μmol·L~(-1)GA significantly ameliorated the results ofmentioned indices as above markedly in comparison to model cells (P<0.05).
Conclusion: Our study revealed that GA provided neuroprotectionagainst H_2O_2-induced oxidative damage under a moderate dose giving amoderate dose GA in advance，which was related with eliminating ROS,attenuating DNA oxidative damage and inhibiting mitochondria mediatedapoptosis.
3Effects of Paeoniflorin on Hydrogen Peroxide-induced Oxidative Damage inSH-SY5Y Cells
Objective：This study was conducted to determine the neuroprotectiveeffects of PF on SH-SY5Y cells injured by H_2O_2and the molecularmechanisms underlying these neuroprotective effects.
Method: CCK-8assay was performed to select non-toxic dose of PF inSH SY5Y cells in treating SH SY5Y cells. Cultured human neuroblastomaSH-SY5Y cells were subjected to oxidative damage with H_2O_2in the presenceand absence of non-toxic dose of PF. Cells can be divided into5groups:normal, model,10μmol·L~(-1)PF,20μmol·L~(-1)PF and40μmol·L~(-1)PF group. Forgroup PF, PF was added to the cells1h before treatment with H_2O_2. The cellviability was analyzed by CCK-8assay. The cell morphologic changes wereobserved by inverted optical microscope. Typical morphological features ofapoptotic cells were detected using Hoechst33258staining, futher, FCM wasused to analysis the cell apoptosis and cell cycle alteration using AnnexinV-FITC/PI and propidium iodide staining; the releasing rate of LDH was determined by the colour reaction of diaphorase-INT. ROS production wasdetermined by DCFH-DA fluorescence.8-OHdG production was determinedby ELISA. Mitochondria membrane potential was determined by JC-1staining; Intracellular Ca2+concentration was determined by Fura-2AMfluorescent probes. Caspase-3activity was determined by caspase-3catalyzethe substrate specificityAc-DEVD-pNA. Caspase-9activity was determinedby caspase-9catalyze the substrate specificity Ac-LEHD-pNA.
Results: H_2O_2treatment produced dose-dependent and time-dependentcytotoxicity compared with that of the normal control. In comparison tocontrol cells, exposure of cells to200μmol·L~(-1)H_2O_2for24h resulted inapproximately65%cell viability of control (P<0.01), the typical change ofcell apoptosis occurred, proliferation index decreased (P<0.05), apoptosisindex increased (P<0.01). H_2O_2induced significantly the increase of LDHrelease, intracellular ROS, Ca2+and8-OHdG (P<0.01), and the decrease ofmitochondrial membrane potential (P<0.01). H_2O_2-induced oxidative stressincreased caspase-3activity and caspase-9activity (P<0.01). Finalconcentration of20-40μmol·L~(-1)PF significantly ameliorated the results ofmentioned indices as above markedly in comparison to model cells. Finalconcentration of10μmol·L~(-1)PF did not significantly change proliferationindex, LDH and8-OHdG (P>0.05), while10μmol·L~(-1)PF, significantlyameliorated morphological features of apoptotic cells, enhanced cell vitality(P<0.05), inhibited the increase of ROS (P<0.05), inhibited the decrease ofmitochondrial membrane potential (P<0.05), restrained the increase ofintracellular Ca2+(P<0.05), down-regulated the caspase-3activity andcaspase-9activity and ameliorated apoptosis increased induced by H_2O_2incomparison to model cells(P<0.05).
Conclusion: Our study revealed that PF provided neuroprotection againstH_2O_2-induced oxidative damage under a moderate dose giving a moderatedose PF in advance，which was related with eliminating ROS, attenuatingDNA oxidative damage and inhibiting mitochondria mediated apoptosis.4Effects of Propyl Gallate on Hydrogen Peroxide-induced Oxidative Damage in SH-SY5Y Cells
Objective：This study was conducted to determine the neuroprotectiveeffects of PG on SH-SY5Y cells injured by H_2O_2and the molecularmechanisms underlying these neuroprotective effects.
Method: CCK-8assay was performed to select non-toxic dose of PG inSH SY5Y cells in treating SH SY5Y cells. Cultured human neuroblastomaSH-SY5Y cells were subjected to oxidative damage with H_2O_2in the presenceand absence of non-toxic dose of PG. Cells can be divided into5groups:normal, model,20μmol·L~(-1)PG,40μmol·L~(-1)PG and80μmol·L~(-1)PG group. Forgroup PG, PG was added to the cells1h before treatment with H_2O_2. The cellviability was analyzed by CCK-8assay. The cell morphologic changes wereobserved by inverted optical microscope. Typical morphological features ofapoptotic cells were detected using Hoechst33258staining, futher, FCM wasused to analysis the cell apoptosis and cell cycle alteration using AnnexinV-FITC/PI and propidium iodide staining; the releasing rate of LDH wasdetermined by the colour reaction of diaphorase-INT. ROS production wasdetermined by DCFH-DA fluorescence.8-OHdG production was determinedby ELISA. Mitochondria membrane potential was determined by JC-1staining; Intracellular Ca2+concentration was determined by Fura-2AMfluorescent probes. Caspase-3activity was determined by caspase-3catalyzethe substrate specificityAc-DEVD-pNA. Caspase-9activity was determinedby caspase-9catalyze the substrate specificity Ac-LEHD-pNA.
Results: H_2O_2treatment produced dose-dependent and time-dependentcytotoxicity compared with that of the normal control. In comparison tocontrol cells, exposure of cells to200μmol·L~(-1)H_2O_2for24h resulted inapproximately65%cell viability of control (P<0.01), the typical change ofcell apoptosis occurred, proliferation index decreased (P<0.05), apoptosisindex increased (P<0.01). H_2O_2induced significantly the increase of LDHrelease, intracellular ROS, Ca2+and8-OHdG (P<0.01), and the decrease ofmitochondrial membrane potential (P<0.01). H_2O_2-induced oxidative stressincreased caspase-3activity and caspase-9activity (P<0.01). Final concentration of40-80μmol·L~(-1)PG significantly ameliorated the results ofmentioned indices as above markedly in comparison to model cells. Finalconcentration of20μmol·L~(-1)PG did not significantly change proliferationindex, LDH and8-OHdG (P>0.05), while20μmol·L~(-1)PG, significantlyameliorated morphological features of apoptotic cells, enhanced cell vitality(P<0.05), inhibited the increase of ROS (P<0.05), inhibited the decrease ofmitochondrial membrane potential (P<0.05), restrained the increase ofintracellular Ca2+(P<0.05), down-regulated the caspase-3activity andcaspase-9activity and ameliorated apoptosis increased induced by H_2O_2incomparison to model cells(P<0.05).
Conclusion: Our study revealed that PG provided neuroprotectionagainst H_2O_2-induced oxidative damage under a moderate dose giving amoderate dose PG in advance，which was related with eliminating ROS,attenuating DNA oxidative damage and inhibiting mitochondria mediatedapoptosis.
5Effects of total flavonoids from Inula helenium on HydrogenPeroxide-induced Oxidative Damage in SH-SY5Y Cells
Objective：This study was conducted to determine the neuroprotectiveeffects of total flavonoids (TF) from Inula helenium on SH-SY5Y cells injuredby H_2O_2.
Method: CCK-8assay was performed to select non-toxic dose of TF inSH-SY5Y cells in treating SH SY5Y cells. Cultured human neuroblastomaSH-SY5Y cells were subjected to oxidative damage with H_2O_2in the presenceand absence of non-toxic dose of TF. Cells can be divided into5groups:normal, model,5mg·L~(-1)TF,10mg·L~(-1)TF and20mg·L~(-1)TF group. For groupTF, TF was added to the cells1h before treatment with H_2O_2. The cell viabilitywas analyzed by CCK-8assay. ROS production was further determined byDCFH-DA fluorescence.
Results: H_2O_2treatment produced dose-dependent and time-dependentcytotoxicity compared with that of the normal control; and in comparison tocontrol group, cell viability was evidently declined(P<0.01). H_2O_2induced significantly the increase of intracellular ROS (P<0.01). Final concentration of5-20mg·L~(-1)PG significantly inhibited the increase of ROS induced by H_2O_2(P<0.05).
Conclusion: Our study revealed that TF provided neuroprotection againstH_2O_2-induced oxidative damage under a moderate dose giving a moderatedose TF in advance，which may be related with eliminating ROS. Furtherstudies will be needed to explore the mechanism of action for the protectiveeffects of TF.
过氧化氢（H_2O_2）是体内代谢的产物,同时也是一种ROS，在诱导神经元细胞的损伤乃至死亡中起了关键作用。H_2O_2造成的细胞氧化损伤已成为研究神经细胞氧化损伤的重要工具之一。
赤芍和土木香均为临床中常用的药物。赤芍为毛茛科植物芍药或川赤芍的干燥根，具有清热凉血、化瘀止痛、消肿通经等功效。土木香是菊科植物土木香Inula helenium L.或藏木香Inula racemosa Hook.f.的干燥根，广泛分布于欧洲中南部和亚洲的喜马拉雅山脉，具有多种药理作用，具有祛痰、止咳等作用，常被用于治疗多种呼吸系统和消化系统疾病。现代药理实验证实，赤芍对神经系统具有保护作用，文献报道赤芍对于PD、AD等NDD的治疗具有一定的作用，但涉及其中的分子机制有待深入系统的研究。ROS在许多疾病包括神经退行性疾病的发生、发展中扮演了重要角色，我们推断具有增加细胞抗氧化能力、具有抑制ROS对细胞损伤的药物可能有助于NDD的防治，所以探讨具有清除ROS和具有神经保护潜能的天然物质对细胞氧化损伤保护作用和机制的研究具有重要意义。赤芍和土木香具有抗氧化作用，体外实验证实赤芍和土木香具有清除DPPH自由基、羟自由基等活性，我们推断赤芍对神经系统的保护作用和赤芍的抗氧化特性有关，赤芍发挥神经保护作用的分子机制可能与清除ROS和阻断细胞凋亡的信号通路有关。目前认为线粒体通路、死亡受体通路和内质网通路是三个主要的凋亡信号通路，caspase3是这些凋亡信号通路的执行者。
关于土木香总黄酮对SH-SY5Y氧化应激损伤的影响未见相关报道。黄酮类化合物是一类次生代谢产物广泛分布在不同植物中，具有各种生物活性如抗氧化、抗动脉粥样硬化、抗炎等;也有助于预防心血管疾病和抗血小板聚集。
基于以上研究背景，本研究以H_2O_2诱导的神经细胞损伤和凋亡为模型，探讨赤芍相关的三种活性单体芍药苷（paeoniflorin，PF）、没食子酸（gallic acid，GA）和赤芍801（propyl gallate，PG）对H_2O_2引起的人神经母细胞瘤株（SH-SY5Y）细胞损伤和凋亡的影响及其机制。具体内容如下：
1H_2O_2诱导SH-SY5Y细胞氧化损伤模型的建立
目的：建立H_2O_2诱导SH-SY5Y细胞氧化损伤模型。
方法：不同浓度H_2O_2(12.5、25.0、50.0、100、200、400、800、1600、3200、6400μmol·L~(-1))作用SH-SY5Y细胞不同时间（1、2、4、8、12、24小时）诱导SH-SY5Y细胞氧化损伤，通过相差显微镜观察SH-SY5Y细胞形态变化，利用CCK-8法测定细胞存活率，筛选H_2O_2致SH-SY5Y细胞氧化应激损伤模型的剂量；Heochest33258染色观察凋亡细胞的形态学特征；Annexin V-FITC/PI双染，流式检测细胞凋亡率；碘化丙啶（propidiumiodide，PI）染色流式细胞仪检测细胞周期构成比；硫辛酰胺脱氢酶（diaphorase）催化的INT （2-p-iodophenyl-3-nitrophenyl-tetrazoliumchloride）显色反应检测乳酸脱氢酶（lactic dehydrogenase，LDH）释放量，2',7'-Dichlorofluorescein diacetate（DCFH-DA）检测细胞内活性氧（ROS）；ELISA (Enzyme-linked Immuno Sorbent Assay)方法检测8-羟基脱氧鸟苷（8-hydroxy deoxyguanosine，8-OHdG）；JC-1染色检测细胞线粒体膜电位（mitochondrial membrane potential，MMP）；Fura-2AM荧光探针检测细胞内钙离子浓度；利用caspase3可以催化底物acetyl-Asp-Glu-Val-Aspp-nitroanilide (Ac-DEVD-pNA)的反应检测caspase3活性；应用caspase9催化特异性底物acetyl-Leu-Glu-His-Asp p-nitroanilide (Ac-LEHD-pNA)检测caspase9活性。
结果：过氧化氢组与正常对照组相比，H_2O_2剂量依赖、时间依赖地造成SH-SY5Y细胞氧化损伤。相对于正常组，终浓度为200μmol L~(-1)H_2O_2作用细胞24h后，细胞活力下降为65%，具有统计学差异(P<0.01)，细胞凋亡率上升(P<0.01)；明显出现了细胞凋亡的形态学特征；增殖指数（proliferation index, PI%）降低(P<0.05)；8-OHdG含量上升(P<0.01)；LDH释放量和细胞内ROS增加(P<0.01)，线粒体膜电位下降(P<0.01)，细胞内钙离子浓度上升(P<0.01)，caspase3和caspase9活性增强(P<0.01)。
结论：200μmol L~(-1)H_2O_2作用细胞24h，细胞活力显著下降，明显造成细胞氧化损伤，造模成功。
2没食子酸对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制研究
目的：以H_2O_2诱导SH-SY5Y细胞氧化损伤为模型，探讨没食子酸对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制。
方法：筛选没食子酸（gallic acid，GA）对SH-SY5Y细胞无毒剂量范围，以此剂量范围研究没食子酸对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制。将细胞分为5组。正常对照组（Control）， H_2O_2模型组（Model），GA5μmol L~(-1)组（GA1）、GA10μmol L~(-1)组（GA2）、GA25μmol L~(-1)组（GA3）。GA组先加入GA预处理1h后再加入终浓度为200μmol L~(-1)H_2O_2。Heochest33258染色观察凋亡细胞的形态学特征；Annexin V-FITC/PI双染，流式检测细胞凋亡率； PI染色流式细胞仪检测细胞周期构成比； diaphorase催化的INT显色反应检测LDH释放量；DCFH-DA检测细胞内ROS；ELISA法检测8-OHdG；JC-1染色检测细胞线粒体膜电位；Fura-2AM荧光探针检测细胞内钙离子浓度；利用caspase3可以催化底物Ac-DEVD-pNA的反应检测caspase3活性；应用caspase9催化特异性底物Ac-LEHD-pNA检测caspase9活性。
结果：过氧化氢组与正常对照组相比，终浓度为200μmol L~(-1)H_2O_2作用细胞24h后，细胞活力下降为65%，具有统计学差异(P<0.01)，细胞凋亡率上升(P<0.01)；明显出现了细胞凋亡的形态学特征；增殖指数（proliferation index, PI%）降低(P<0.05)；8-OHdG含量上升(P<0.01)；LDH释放量和细胞内ROS增加(P<0.01)，线粒体膜电位下降(P<0.01)，细胞内钙离子浓度上升(P<0.01)，caspase3和caspase9活性增强(P<0.01)。与H_2O_2损伤组相比，终浓度为5-25μmol·L~(-1)GA能显著改善上述指标的变化(P<0.05)。
结论：GA在一定剂量范围内对H_2O_2诱导的SH-SYSY神经细胞损伤具有明显的保护作用，其保护效应可能与清除ROS，减轻DNA氧化损伤，抑制线粒体通路介导的细胞凋亡有关。
3芍药苷对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制研究
目的：以H_2O_2诱导SH-SY5Y细胞氧化损伤为模型，探讨芍药苷对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制。
方法：筛选芍药苷（paeoniflorin，PF）对SH-SY5Y细胞无毒剂量范围，以此剂量范围研究芍药苷对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制。将细胞分为5组。正常对照组（Control）， H_2O_2模型组（Model），PF10μmol·L~(-1)组（PF1）、PF20μmol·L~(-1)组（PF2）、PF40μmol·L~(-1)组（PF3）。PF组先加入PF预处理1h后再加入终浓度为200μmol·L~(-1)H_2O_2。Heochest33258染色观察凋亡细胞的形态学特征；Annexin V-FITC/PI双染，流式检测细胞凋亡率；PI染色流式细胞仪检测细胞周期构成比；diaphorase催化的INT显色反应检测LDH释放量；DCFH-DA检测细胞内ROS；ELISA法检测8-OHdG；JC-1染色检测细胞线粒体膜电位；Fura-2AM荧光探针检测细胞内钙离子浓度；利用caspase3可以催化底物Ac-DEVD-pNA的反应检测caspase3活性；应用caspase9催化特异性底物Ac-LEHD-pNA检测caspase9活性。
结果：过氧化氢组与正常对照组相比，终浓度为200μmol·L~(-1)H_2O_2作用细胞24h后，细胞活力下降为65%，具有统计学差异(P<0.01)，细胞凋亡率上升(P<0.01)；明显出现了细胞凋亡的形态学特征；增殖指数（proliferation index, PI%）降低(P<0.05)；8-OHdG含量上升(P<0.01)；LDH释放量和细胞内ROS增加(P<0.01)，线粒体膜电位下降(P<0.01)，细胞内钙离子浓度上升(P<0.01)，caspase3和caspase9活性增强(P<0.01)。与H_2O_2损伤组相比，20-40μmol·L~(-1)PF能显著改善过氧化氢诱导的上述指标的改变(P<0.05)；终浓度10μmol·L~(-1)PF组与H_2O_2组相比PI%无显著变化（P>0.05），LDH和8-OHdG含量无显著变化（P>0.05），但能显著改善过氧化氢诱导的细胞凋亡的形态学特征，提高过氧化氢诱导的细胞活力下降(P<0.05)，抑制H_2O_2诱导的ROS上升(P<0.05)，抑制线粒体膜电位的下降(P<0.05)，抑制细胞内钙离子浓度的升高(P<0.05)，抑制caspase3和caspase9活性增强(P<0.05)，减轻H_2O_2诱导的细胞凋亡率的上升(P<0.05)。
结论：PF在一定剂量范围内对H_2O_2诱导的SH-SYSY神经细胞损伤具有明显的保护作用，其保护效应可能与清除ROS，减轻DNA氧化损伤，抑制线粒体通路介导的细胞凋亡有关。
4赤芍801对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制研究
目的：以H_2O_2诱导SH-SY5Y细胞氧化损伤为模型，探讨赤芍801对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制。
方法：筛选赤芍801（propyl gallate，PG）对SH-SY5Y细胞无毒剂量范围，以此剂量范围研究赤芍801对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制。将细胞分为5组。正常对照组（Control）， H_2O_2模型组（Model），PG20μmol·L~(-1)组（PG1）、PG40μmol·L~(-1)组（PG2）、PG80μmol·L~(-1)组（PG3）。PG组先加入PG预处理1h后再加入终浓度为200μmol·L~(-1)H_2O_2。Heochest33258染色观察凋亡细胞的形态学特征；Annexin V-FITC/PI双染，流式检测细胞凋亡率； PI染色流式细胞仪检测细胞周期构成比；diaphorase催化的INT显色反应检测LDH释放量；DCFH-DA检测细胞内ROS；ELISA法检测8-OHdG；JC-1染色检测细胞线粒体膜电位；Fura-2AM荧光探针检测细胞内钙离子浓度；利用caspase3可以催化底物Ac-DEVD-pNA的反应检测caspase3活性；应用caspase9催化特异性底物Ac-LEHD-pNA检测caspase9活性。
结果：过氧化氢组与正常对照组相比，终浓度为200μmol·L~(-1)H_2O_2作用细胞24h后，细胞活力下降为65%，具有统计学差异(P<0.01)，细胞凋亡率上升(P<0.01)；明显出现了细胞凋亡的形态学特征；增殖指数（proliferation index, PI%）降低(P<0.05)；8-OHdG含量上升(P<0.01)；LDH释放量和细胞内ROS增加(P<0.01)，线粒体膜电位下降(P<0.01)，细胞内钙离子浓度上升(P<0.01)，caspase3和caspase9活性增强(P<0.01)。与H_2O_2损伤组相比，40-80μmol L-PG均能显著改善过氧化氢诱导的上述指标的改变(P<0.05)；终浓度20μmol·L~(-1)PG组与H_2O_2组相比PI%无显著变化（P>0.05），LDH和8-OHdG含量无显著变化（P>0.05），但能显著改善过氧化氢诱导的细胞凋亡的形态学特征，提高过氧化氢诱导的细胞活力下降(P<0.05)，抑制H_2O_2诱导的ROS上升(P<0.05)，抑制线粒体膜电位的下降(P<0.05)，抑制细胞内钙离子浓度的升高(P<0.05)，抑制caspase3和caspase9活性增强(P<0.05)，减轻H_2O_2诱导的细胞凋亡率的上升(P<0.05)。
结论：PG在一定剂量范围内对H_2O_2诱导的SH-SYSY神经细胞损伤具有明显的保护作用，其保护效应可能与清除ROS，减轻DNA氧化损伤，抑制线粒体通路介导的细胞凋亡有关。
5土木香总黄酮对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制研究
目的：以H_2O_2诱导SH-SY5Y细胞氧化损伤为模型，探讨土木香总黄酮对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响。
方法：筛选土木香总黄酮（total flavonoids，TF）对SH-SY5Y细胞无毒剂量范围，以此剂量范围研究土木香总黄酮对H_2O_2诱导SH-SY5Y细胞氧化损伤的影响及其机制。将细胞分为5组。正常对照组（Control），H_2O_2模型组（Model），TF5mg·L~(-1)组（TF1）、TF10mg·L~(-1)组（TF2）、TF20mg·L~(-1)组（TF3）。TF组先加入TF预处理1h后再加入终浓度为200μmol·L~(-1)H_2O_2。DCFH-DA检测细胞内ROS。
结果：过氧化氢组与正常对照组相比，终浓度为200μmol·L~(-1)H_2O_2作用细胞24h后，细胞活力显著下降(P<0.01)；与H_2O_2损伤组相比，5-20mg·L~(-1)TF能显著抑制过氧化氢诱导的凋亡(P<0.05)，抑制H_2O_2诱导的ROS上升(P<0.05)。
结论：TF在一定剂量范围内对H_2O_2诱导的SH-SYSY神经细胞损伤具有明显的保护作用，其保护机制有待进一步研究。
Neurodegenerative disease (NDD) is chronic progressive diseases of thenervous system caused by the specific brain areas in degeneration, includingAlzheimer’s disease (AD), Parkinson’s disease (PD),amyotrophielateralselerosis (ALS), etc. The incidence of NDD surges,especially in the aging population. Because the pathogenesis of NDD iscomplicated, there is still no ideal drug for preventing and curing NDD tillnow. Although the definite etiology and pathogenesis are not very clear up tonow, many studies show that NDD is caused by many different things. Thereare many views and hypothesis about the cause of NDD, among them,imbalance in neurotransmitters, oxidative stress, mitochondrial dysfunction,excitatory neural toxicity, anomalous proteins accumulation, gene mutation,inflammatory process, dysimmunity and apoptosis theory, etc. Increasingamounts of data show that mitochondrial dysfunction and oxidative stress hasbeen implicated in many neurological diseases and interacted with otherseveral mechanisms. Intracellular reactive oxygen species are continuouslyproduced and cleared. When the level of oxidation exceeds these antioxidantdefenses, oxidative stress occurs. ROS plays an important role in cell signaltransduction, and involves in many pathologic and physiological processes.
Hydrogen peroxide (H_2O_2) had been considered to be important signalingmolecules and natural products in the course of biological metabolism. H_2O_2plays an important role in neuron damage and even death. H_2O_2is one of themost important implements for studying neurons oxidative damage.
Radix Paeoniae Rubra (R. paeoniae) and Inula helenium is clinicallywidely used. R. paeoniae (Family：Ranunculaceae) is the root of PaeoniaRadix or Paeonia Veitchii Lynch, which is widely applicable to heat-clearing and blood-cooling, dispersing blood stasis and relieving pain, detumescence,stimulating the menstrual flow. Inula helenium (elecampane), a member of thecomposite family, is widely distributed throughout central and southernEurope as far as the Himalayas, in Asia. In the Chinese Pharmacopoeia, I.helenium is described as an expectorant, diaphoretic, and antitussive that isoften used to treat a variety of respiratory and digestive diseases. Recentresearch has demonstrated that the extracts of I. helenium exhibit many effectsincluding antibacterial, antitumor, and antiproliferative. At present, effect ofthe total flavonoids of elecampane on H_2O_2-induced SH SY5Y oxidative stresshas not been described.Flavonoid compounds are diverse class of secondaryplant metabolites that are widely distributed throughout the plant kingdom andhave been reported to possess a variety of biological activities such asantioxidant, anti-atherosclerotic, anti-inflammatory, and antithrombogenic;they also aid in cardiovascular disease prevention and anti-platelet aggregation.It has been reported that Paeonia Lactifiora has a protective effect for nervoussystem, Paeonia Lactifiora has certain influence on treatmenting of AD, PD,and its mechanism is required to further study systematically. ROS plays animportant role in disease (including NDD) onset and progression. We deducedthat the drug with antioxidation and inhibiting ROS may be useful forpreventing NDD. It is important for studying the protective effects andmechanism of natural product on the cellular oxidative damage. There isevidence that Paeonia Lactifiora has antioxidant properties, PaeoniaLactifiora can eliminate DPPH and hydroxyl radicals in vitro. There are closeconnection between the antioxidant properties of Paeonia Lactifiora and itsneuroprotection. The molecular mechanism of the neuroprotective effects of R.paeoniae may be related to removing ROS and blocking the apoptosissignaling pathway. The basic apoptotic pathway includes mitochondrialpathway, death receptor pathway and endoplasmic reticulum pathway.Caspase-3is the executor of the apoptotic signaling pathways.
Based on the above research background, this study was conducted todetermine the neuroprotective effects of three active monomers (gallic acid, paeoniflorin and propyl gallate) related to R. paeoniae on SH-SY5Y cellsinjured by H_2O_2and the molecular mechanisms underlying theseneuroprotective effects. The specific contents as follows:
1Establishment of the model of oxidative damage in SH-SY5Y cells inducedby H_2O_2
Objective：To establish the model of oxidative damage in SH-SY5Y cellsinduced by H_2O_2.
Method: Cultured human neuroblastoma SH-SY5Y cells were subjectedto oxidative damage with H_2O_2at different concentrations (12.5,25.0,50.0,100,200,400,800,1600,3200,6400μmol·L~(-1)) and at different times (1,2,4,8,12,24h). The cell viability was analyzed by CCK-8assay. The cellmorphologic changes were observed by inverted optical microscope. Typicalmorphological features of apoptotic cells were detected using Hoechst33258staining, futher, flow cytometric (FCM) was used to analysis the cell apoptosisand cell cycle alteration using Annexin V-FITC/PI and propidium iodidestaining; the releasing rate of lactic dehydrogenase (LDH) was determined bythe colour reaction of diaphorase-INT. Reactive oxygen species (ROS)production was determined by2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA) fluorescence.8-OHdG production was determined byenzyme-linked immunosorbent assay (ELISA). Mitochondria membranepotential was determined by JC-1staining; Intracellular Ca2+concentrationwas determined by Fura-2AM fluorescent probes. Caspase-3activity wasdetermined by caspase-3catalyze the substrate specificityacetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA). Caspase-9activitywas determined by caspase-9catalyze the substrate specificityacetyl-Leu-Glu-His-Asp p-nitroanilide (Ac-LEHD-pNA).
Results: H_2O_2treatment produced dose-dependent and time-dependentcytotoxicity compared with that of the normal control. In comparison tocontrol cells, exposure of cells to200μmol·L~(-1)H_2O_2for24h resulted inapproximately65%cell viability of control (P<0.01), the typical change ofcell apoptosis occurred, proliferation index decreased (P<0.05), apoptosis index increased (P<0.01). H_2O_2induced significantly the increase of LDHrelease, intracellular ROS, Ca2+and8-OHdG (P<0.01), and the decrease ofmitochondrial membrane potential (P<0.01). H_2O_2-induced oxidative stressincreased caspase-3activity and caspase-9activity (P<0.01).
Conclusion:200μmol·L~(-1)H_2O_2treatment produced cell activitydecreased remarkably. The model of oxidative damage in SH-SY5Y cellsinduced by H_2O_2is successfully established.
2Effects of Gallic Acid on Hydrogen Peroxide-induced Oxidative Damage inSH-SY5Y Cells
Objective：This study was conducted to determine the neuroprotectiveeffects of GA on SH-SY5Y cells injured by H_2O_2and the molecularmechanisms underlying these neuroprotective effects.
Method: CCK-8assay was performed to select non-toxic dose of gallicacid in SH SY5Y cells in treating SH SY5Y cells. Cultured humanneuroblastoma SH-SY5Y cells were subjected to oxidative damage with H_2O_2in the presence and absence of non-toxic dose of GA. Cells can be divided into5groups: normal, model,5μmol·L~(-1)GA,10μmol·L~(-1)GA and25μmol·L~(-1)GAgroup. For group GA, GA was added to the cells1h before treatment withH_2O_2. The cell viability was analyzed by CCK-8assay. The cell morphologicchanges were observed by inverted optical microscope. Typical morphologicalfeatures of apoptotic cells were detected using Hoechst33258staining, futher,FCM was used to analysis the cell apoptosis and cell cycle alteration usingAnnexin V-FITC/PI and propidium iodide staining; the releasing rate of LDHwas determined by the colour reaction of diaphorase-INT. ROS productionwas determined by DCFH-DA fluorescence.8-OHdG production wasdetermined by ELISA. Mitochondria membrane potential was determined byJC-1staining; Intracellular Ca2+concentration was determined by Fura-2AMfluorescent probes. Caspase-3activity was determined by caspase-3catalyzethe substrate specificityAc-DEVD-pNA. Caspase-9activity was determinedby caspase-9catalyze the substrate specificity Ac-LEHD-pNA.
Results: H_2O_2treatment produced dose-dependent and time-dependent cytotoxicity compared with that of the normal control. In comparison tocontrol cells, exposure of cells to200μmol·L~(-1)H_2O_2for24h resulted inapproximately65%cell viability of control (P<0.01), the typical change ofcell apoptosis occurred, proliferation index decreased (P<0.05), apoptosisindex increased (P<0.01). H_2O_2induced significantly the increase of LDHrelease, intracellular ROS, Ca2+and8-OHdG (P<0.01), and the decrease ofmitochondrial membrane potential (P<0.01). H_2O_2-induced oxidative stressincreased caspase-3activity and caspase-9activity (P<0.01). Finalconcentration of5-25μmol·L~(-1)GA significantly ameliorated the results ofmentioned indices as above markedly in comparison to model cells (P<0.05).
Conclusion: Our study revealed that GA provided neuroprotectionagainst H_2O_2-induced oxidative damage under a moderate dose giving amoderate dose GA in advance，which was related with eliminating ROS,attenuating DNA oxidative damage and inhibiting mitochondria mediatedapoptosis.
3Effects of Paeoniflorin on Hydrogen Peroxide-induced Oxidative Damage inSH-SY5Y Cells
Objective：This study was conducted to determine the neuroprotectiveeffects of PF on SH-SY5Y cells injured by H_2O_2and the molecularmechanisms underlying these neuroprotective effects.
Method: CCK-8assay was performed to select non-toxic dose of PF inSH SY5Y cells in treating SH SY5Y cells. Cultured human neuroblastomaSH-SY5Y cells were subjected to oxidative damage with H_2O_2in the presenceand absence of non-toxic dose of PF. Cells can be divided into5groups:normal, model,10μmol·L~(-1)PF,20μmol·L~(-1)PF and40μmol·L~(-1)PF group. Forgroup PF, PF was added to the cells1h before treatment with H_2O_2. The cellviability was analyzed by CCK-8assay. The cell morphologic changes wereobserved by inverted optical microscope. Typical morphological features ofapoptotic cells were detected using Hoechst33258staining, futher, FCM wasused to analysis the cell apoptosis and cell cycle alteration using AnnexinV-FITC/PI and propidium iodide staining; the releasing rate of LDH was determined by the colour reaction of diaphorase-INT. ROS production wasdetermined by DCFH-DA fluorescence.8-OHdG production was determinedby ELISA. Mitochondria membrane potential was determined by JC-1staining; Intracellular Ca2+concentration was determined by Fura-2AMfluorescent probes. Caspase-3activity was determined by caspase-3catalyzethe substrate specificityAc-DEVD-pNA. Caspase-9activity was determinedby caspase-9catalyze the substrate specificity Ac-LEHD-pNA.
Results: H_2O_2treatment produced dose-dependent and time-dependentcytotoxicity compared with that of the normal control. In comparison tocontrol cells, exposure of cells to200μmol·L~(-1)H_2O_2for24h resulted inapproximately65%cell viability of control (P<0.01), the typical change ofcell apoptosis occurred, proliferation index decreased (P<0.05), apoptosisindex increased (P<0.01). H_2O_2induced significantly the increase of LDHrelease, intracellular ROS, Ca2+and8-OHdG (P<0.01), and the decrease ofmitochondrial membrane potential (P<0.01). H_2O_2-induced oxidative stressincreased caspase-3activity and caspase-9activity (P<0.01). Finalconcentration of20-40μmol·L~(-1)PF significantly ameliorated the results ofmentioned indices as above markedly in comparison to model cells. Finalconcentration of10μmol·L~(-1)PF did not significantly change proliferationindex, LDH and8-OHdG (P>0.05), while10μmol·L~(-1)PF, significantlyameliorated morphological features of apoptotic cells, enhanced cell vitality(P<0.05), inhibited the increase of ROS (P<0.05), inhibited the decrease ofmitochondrial membrane potential (P<0.05), restrained the increase ofintracellular Ca2+(P<0.05), down-regulated the caspase-3activity andcaspase-9activity and ameliorated apoptosis increased induced by H_2O_2incomparison to model cells(P<0.05).
Conclusion: Our study revealed that PF provided neuroprotection againstH_2O_2-induced oxidative damage under a moderate dose giving a moderatedose PF in advance，which was related with eliminating ROS, attenuatingDNA oxidative damage and inhibiting mitochondria mediated apoptosis.4Effects of Propyl Gallate on Hydrogen Peroxide-induced Oxidative Damage in SH-SY5Y Cells
Objective：This study was conducted to determine the neuroprotectiveeffects of PG on SH-SY5Y cells injured by H_2O_2and the molecularmechanisms underlying these neuroprotective effects.
Method: CCK-8assay was performed to select non-toxic dose of PG inSH SY5Y cells in treating SH SY5Y cells. Cultured human neuroblastomaSH-SY5Y cells were subjected to oxidative damage with H_2O_2in the presenceand absence of non-toxic dose of PG. Cells can be divided into5groups:normal, model,20μmol·L~(-1)PG,40μmol·L~(-1)PG and80μmol·L~(-1)PG group. Forgroup PG, PG was added to the cells1h before treatment with H_2O_2. The cellviability was analyzed by CCK-8assay. The cell morphologic changes wereobserved by inverted optical microscope. Typical morphological features ofapoptotic cells were detected using Hoechst33258staining, futher, FCM wasused to analysis the cell apoptosis and cell cycle alteration using AnnexinV-FITC/PI and propidium iodide staining; the releasing rate of LDH wasdetermined by the colour reaction of diaphorase-INT. ROS production wasdetermined by DCFH-DA fluorescence.8-OHdG production was determinedby ELISA. Mitochondria membrane potential was determined by JC-1staining; Intracellular Ca2+concentration was determined by Fura-2AMfluorescent probes. Caspase-3activity was determined by caspase-3catalyzethe substrate specificityAc-DEVD-pNA. Caspase-9activity was determinedby caspase-9catalyze the substrate specificity Ac-LEHD-pNA.
Results: H_2O_2treatment produced dose-dependent and time-dependentcytotoxicity compared with that of the normal control. In comparison tocontrol cells, exposure of cells to200μmol·L~(-1)H_2O_2for24h resulted inapproximately65%cell viability of control (P<0.01), the typical change ofcell apoptosis occurred, proliferation index decreased (P<0.05), apoptosisindex increased (P<0.01). H_2O_2induced significantly the increase of LDHrelease, intracellular ROS, Ca2+and8-OHdG (P<0.01), and the decrease ofmitochondrial membrane potential (P<0.01). H_2O_2-induced oxidative stressincreased caspase-3activity and caspase-9activity (P<0.01). Final concentration of40-80μmol·L~(-1)PG significantly ameliorated the results ofmentioned indices as above markedly in comparison to model cells. Finalconcentration of20μmol·L~(-1)PG did not significantly change proliferationindex, LDH and8-OHdG (P>0.05), while20μmol·L~(-1)PG, significantlyameliorated morphological features of apoptotic cells, enhanced cell vitality(P<0.05), inhibited the increase of ROS (P<0.05), inhibited the decrease ofmitochondrial membrane potential (P<0.05), restrained the increase ofintracellular Ca2+(P<0.05), down-regulated the caspase-3activity andcaspase-9activity and ameliorated apoptosis increased induced by H_2O_2incomparison to model cells(P<0.05).
Conclusion: Our study revealed that PG provided neuroprotectionagainst H_2O_2-induced oxidative damage under a moderate dose giving amoderate dose PG in advance，which was related with eliminating ROS,attenuating DNA oxidative damage and inhibiting mitochondria mediatedapoptosis.
5Effects of total flavonoids from Inula helenium on HydrogenPeroxide-induced Oxidative Damage in SH-SY5Y Cells
Objective：This study was conducted to determine the neuroprotectiveeffects of total flavonoids (TF) from Inula helenium on SH-SY5Y cells injuredby H_2O_2.
Method: CCK-8assay was performed to select non-toxic dose of TF inSH-SY5Y cells in treating SH SY5Y cells. Cultured human neuroblastomaSH-SY5Y cells were subjected to oxidative damage with H_2O_2in the presenceand absence of non-toxic dose of TF. Cells can be divided into5groups:normal, model,5mg·L~(-1)TF,10mg·L~(-1)TF and20mg·L~(-1)TF group. For groupTF, TF was added to the cells1h before treatment with H_2O_2. The cell viabilitywas analyzed by CCK-8assay. ROS production was further determined byDCFH-DA fluorescence.
Results: H_2O_2treatment produced dose-dependent and time-dependentcytotoxicity compared with that of the normal control; and in comparison tocontrol group, cell viability was evidently declined(P<0.01). H_2O_2induced significantly the increase of intracellular ROS (P<0.01). Final concentration of5-20mg·L~(-1)PG significantly inhibited the increase of ROS induced by H_2O_2(P<0.05).
Conclusion: Our study revealed that TF provided neuroprotection againstH_2O_2-induced oxidative damage under a moderate dose giving a moderatedose TF in advance，which may be related with eliminating ROS. Furtherstudies will be needed to explore the mechanism of action for the protectiveeffects of TF.
引文
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