Effects Of Active Ingredients Of Chinese Medicine On Hydrogen Peroxide-induced Oxidative Damage In SH-SY5Y Cells

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 （H₂O₂） had been considered to be important signalingmolecules and natural products in the course of biological metabolism. H₂O₂plays an important role in neuron damage and even death. H₂O₂is 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 H₂O₂-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 H₂O₂and the molecular mechanisms underlying theseneuroprotective effects. The specific contents as follows:1Establishment of the model of oxidative damage in SH-SY5Y cells inducedby H₂O₂Objective：To establish the model of oxidative damage in SH-SY5Y cellsinduced by H₂O₂.Method: Cultured human neuroblastoma SH-SY5Y cells were subjectedto oxidative damage with H₂O₂at 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: H₂O₂treatment produced dose-dependent and time-dependentcytotoxicity compared with that of the normal control. In comparison tocontrol cells, exposure of cells to200μmol·L^-1H₂O₂for24h 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）. H₂O₂induced significantly the increase of LDHrelease, intracellular ROS, Ca2+and8-OHdG （P<0.01）, and the decrease ofmitochondrial membrane potential （P<0.01）. H₂O₂-induced oxidative stressincreased caspase-3activity and caspase-9activity （P<0.01）.Conclusion:200μmol·L^-1H₂O₂treatment produced cell activitydecreased remarkably. The model of oxidative damage in SH-SY5Y cellsinduced by H₂O₂is successfully established.2Effects of Gallic Acid on Hydrogen Peroxide-induced Oxidative Damage inSH-SY5Y CellsObjective：This study was conducted to determine the neuroprotectiveeffects of GA on SH-SY5Y cells injured by H₂O₂and 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 H₂O₂in the presence and absence of non-toxic dose of GA. Cells can be divided into5groups: normal, model,5μmol·L^-1GA,10μmol·L^-1GA and25μmol·L^-1GAgroup. For group GA, GA was added to the cells1h before treatment withH₂O₂. 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: H₂O₂treatment produced dose-dependent and time-dependent cytotoxicity compared with that of the normal control. In comparison tocontrol cells, exposure of cells to200μmol·L^-1H₂O₂for24h 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）. H₂O₂induced significantly the increase of LDHrelease, intracellular ROS, Ca2+and8-OHdG （P<0.01）, and the decrease ofmitochondrial membrane potential （P<0.01）. H₂O₂-induced oxidative stressincreased caspase-3activity and caspase-9activity （P<0.01）. Finalconcentration of5-25μmol·L^-1GA 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 H₂O₂-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 CellsObjective：This study was conducted to determine the neuroprotectiveeffects of PF on SH-SY5Y cells injured by H₂O₂and 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 H₂O₂in the presenceand absence of non-toxic dose of PF. Cells can be divided into5groups:normal, model,10μmol·L^-1PF,20μmol·L^-1PF and40μmol·L^-1PF group. Forgroup PF, PF was added to the cells1h before treatment with H₂O₂. 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: H₂O₂treatment produced dose-dependent and time-dependentcytotoxicity compared with that of the normal control. In comparison tocontrol cells, exposure of cells to200μmol·L^-1H₂O₂for24h 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）. H₂O₂induced significantly the increase of LDHrelease, intracellular ROS, Ca2+and8-OHdG （P<0.01）, and the decrease ofmitochondrial membrane potential （P<0.01）. H₂O₂-induced oxidative stressincreased caspase-3activity and caspase-9activity （P<0.01）. Finalconcentration of20-40μmol·L^-1PF significantly ameliorated the results ofmentioned indices as above markedly in comparison to model cells. Finalconcentration of10μmol·L^-1PF did not significantly change proliferationindex, LDH and8-OHdG （P>0.05）, while10μmol·L^-1PF, 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 H₂O₂incomparison to model cells（P<0.05）.Conclusion: Our study revealed that PF provided neuroprotection againstH₂O₂-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 CellsObjective：This study was conducted to determine the neuroprotectiveeffects of PG on SH-SY5Y cells injured by H₂O₂and 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 H₂O₂in the presenceand absence of non-toxic dose of PG. Cells can be divided into5groups:normal, model,20μmol·L^-1PG,40μmol·L^-1PG and80μmol·L^-1PG group. Forgroup PG, PG was added to the cells1h before treatment with H₂O₂. 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: H₂O₂treatment produced dose-dependent and time-dependentcytotoxicity compared with that of the normal control. In comparison tocontrol cells, exposure of cells to200μmol·L^-1H₂O₂for24h 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）. H₂O₂induced significantly the increase of LDHrelease, intracellular ROS, Ca2+and8-OHdG （P<0.01）, and the decrease ofmitochondrial membrane potential （P<0.01）. H₂O₂-induced oxidative stressincreased caspase-3activity and caspase-9activity （P<0.01）. Final concentration of40-80μmol·L^-1PG significantly ameliorated the results ofmentioned indices as above markedly in comparison to model cells. Finalconcentration of20μmol·L^-1PG did not significantly change proliferationindex, LDH and8-OHdG （P>0.05）, while20μmol·L^-1PG, 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 H₂O₂incomparison to model cells（P<0.05）.Conclusion: Our study revealed that PG provided neuroprotectionagainst H₂O₂-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 CellsObjective：This study was conducted to determine the neuroprotectiveeffects of total flavonoids （TF） from Inula helenium on SH-SY5Y cells injuredby H₂O₂.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 H₂O₂in the presenceand absence of non-toxic dose of TF. Cells can be divided into5groups:normal, model,5mg·L^-1TF,10mg·L^-1TF and20mg·L^-1TF group. For groupTF, TF was added to the cells1h before treatment with H₂O₂. The cell viabilitywas analyzed by CCK-8assay. ROS production was further determined byDCFH-DA fluorescence.Results: H₂O₂treatment 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）. H₂O₂induced significantly the increase of intracellular ROS （P<0.01）. Final concentration of5-20mg·L^-1PG significantly inhibited the increase of ROS induced by H₂O₂（P<0.05）.Conclusion: Our study revealed that TF provided neuroprotection againstH₂O₂-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.