Rifampicin inhibits apoptosis in rotenone-induced differentiated PC12 cells by ameliorating mitochondrial oxidative stress

BACKGROUND： Previous studies have shown that rifampicin exhibits neuroprotective effects, but the precise mechanisms remain unclear. Rifampicin is thought to exert the neuroprotective effect as a hydroxyl free radical scavenger. OBJECTIVE： To investigate the protective effects of rifampicin pretreatment on rotenone-induced mitochondrial oxidative stress in differentiated PC12 cells.DESIGN, TIME AND SETrlNG： A repeated measure, cell-based study was performed at the Department of Neurology, Second Affiliated Hospital, Sun Yat-sen University, China between December 2007 and November 2008. MATERIALS： PC12 cells were a kind gift from the Physiology Laboratory of Zhongshan Medical School, Sun Yat-sen University, China. Rotenone and rifampicin were purchased from Sigma, USA. METHODS： PC12 cells were differentiated by culturing with 100 ng/mL 7S nerve growth factor for 9 days in Dulbecco＇s modified Eagle＇s medium/Nutrient Mix F12 （DMEM/F12） supplemented with 10% fetal bovine serum. The cells were assigned to six groups according to various treatment conditions： control, cultured with normal media; rifampicin group, treated with 300 pmol/L rotenone for 26 hours; rotenone group, treated with 2.5 pmol/L rotenone for 24 hours; rifampicin pretreatment groups, pretreated with 100, 200, and 300 pmol/L rifampicin for 2 hours, respectively, followed by 2.5 μmol/L rotenone for 24 hours.MAIN OUTCOME MEASURES： Mitochondrial membrane potential was measured by fluorescence microscopy and flow cytometry, respectively, using rhodamine123 staining. Intracellular reactive oxygen species formation was analyzed by flow cytometry using 2＇, 7＇-dichlorofluorescin-diacetate staining, and intracellular reduced glutathione was measured with a microplate reader. Cell viability was determined by 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide assay. Cell apoptosis was detected by Hoechst 33342 staining and flow cytometry. RESULTS： Increased apoptosis in rotenone-induced, differentiated, PC12 cells was accompanied by the loss of mitochondrial transmembrane potential, the formation of reactive oxygen species, and reduced glutathione depletion （P 〈 0.01）. Rotenone-induced mitochondrial dysfunction was blocked in a dose-dependent manner by rifampicin （P 〈 0.05 or P 〈 0.01）, CONCLUSION： Pretreatment of differentiated PC12 cells with rifampicin blocked rotenone-induced apoptosis by ameliorating mitochondrial dysfunction and oxidative stress.

Humulus japonicus extract alleviates oxidative stress and apoptosis in 6-hydroxydopamine-induced PC12 cells

Objective:To explore the possible neuroprotective activities of Humulus japonicus extract against Parkinson's disease(PD)in a cellular model.Methods:PD was modeled in PC12 cells using 6-hydroxydopamine(6-OHDA).The cell activity,intracellular levels of reactive oxygen species(ROS),anti-oxidative and anti-apoptotic effects,and other related indicators and related signaling pathways were evaluated to elucidate the neuroprotective effects of Humulus japonicus extract.Results:Humulus japonicus extract exhibited anti-oxidative and anti-apoptotic effects in 6-OHDA-stimulated PC12 cells.It also reduced oxidative stress-induced ROS accumulation;upregulated antioxidant enzymes,such as glutathione,catalase,heme oxidase-1,and 8-oxguanine glycosylase 1;promoted cell survival by decreasing BAX and increasing Bcl-2 and sirtuin 1 expression via the MAPK and/or Nrf2 signaling pathways.Conclusions:Humulus japonicus extract has antioxidative and anti-apoptotic effects and could be developed as a promising candidate for preventing and treating oxidative stress-related neurodegenerative diseases.

Antioxidative effects of berberine pre-treatment on hydrogen peroxide-induced PC12 cell toxicity

Oxidative stress has been implicated in the pathogenesis of Alzheimer＇s disease. Oxidative damage could be prevented by augmenting the endogenous defense capacity against oxidative stress by antioxidant intake. As an effective alkaloid component of Chinese herbal medicine Rhizoma coptidis extract, berberine exhibits antioxidative properties and ameliorates memory impairment in a rat model of Alzheimer＇s disease. The present study investigated the protective effects of berberine on H2O2-induced PC12 cell toxicity. Results demonstrated that berberine protects PC12 cells from H2O2-induced apoptosis and increases PC12 cell viability. Lactate dehydrogenase release, reactive oxygen content, and malonyl dialdehyde levels were significantly decreased （P 〈 0.01）. The protective effects of berberine on H2O2-induced PC12 cell toxicity were achieved via the antioxidative effects of berberine.

Effects of serum containing natural cerebrolysin on glucose-regulated protein 78 and CCAAT enhancer-binding protein homologous protein expression in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress

BACKGROUND： Glucose-regulated protein 78 （GRP78）, a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein （CHOP）, a transcription factor specific for endoplasmic reticulum stress, can cause cell cycle arrest and cell apoptosis. OBJECTIVE： To study the protective effects of serum containing natural cerebrolysin on endoplasmic reticulum stress in tunicamycin-induced neuronal PC12 cells, and analyze the influence on GRP78 and CHOP expressions. DESIGN, TIME AND SETTING： A parallel controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital, Southern Medical University, between March 2006 and August 2008. MATERIALS： Adult Sprague-Dawley rats were perfused with natural Cerebrolysin aqueous extract （0.185 g/kg/d） to produce serum containing natural Cerebrolysin. Physiological saline was used to produce blank serum. PC12 cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Science. Tunicamycin was provided by Sigma （St. Louis, USA）, and natural Cerebrolysin, containing ginseng, rhizoma gastrodiae, and gingko leaf （1：2：2）, by Shengzhen Institute of Integrated Western and Traditional Chinese Medicine. METHODS： PC12 cells were treated with DMEM culture media containing 10% blank serum （normal control group）, tunicamycin （1 μg/mL; model group）, and 5%, 10%, and 15% serum containing natural cerebrolysin and tunicamycin （1 μ g/mL; low-, moderate-, and high-dose serum containing natural cerebrotysin groups）, for 2 hours. MAIN OUTCOME MEASURES： PC12 cells were treated with tunicamycin for 48 hours after which apoptosis was measured using the TUNEL method to calculate apoptotic index. GRP78 expression was detected using immunocytochemistry. After 24 hours of treatment with tunicamycin, GRP78 and CHOP mRNA expressions were measured using RT-PCR. RESULTS： The apoptotic index and CHOP mRNA expression were in the model group and three cerebrolysin groups were significantly increased when compared to the normal control group （P 〈 0.05）. In contrast, GRP78 mRNA and protein expressions were significantly decreased （P 〈 0.05）. CONCLUSION： Serum containing natural cerebrolysin significantly reduced apoptosis in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress. These results may be related to an up-regulation of GRP78 expression and down-regulation of CHOP expression, both of which displayed dose-dependent effects.

Isoflavone Attenuates the Nuclear Transcription Factor Kappa B (NF-<i>κ</i>B) Activation on MPP⁺-Induced Apoptosis of PC12 Cells

Objective: To explore the underlying molecular mechanisms of cellular response to the challenge by 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis of PC12 cells, an in vitro cell model for Parkinson’s disease, and the effect of NF-κB activation on the protection of Parkinson’s disease by Isoflavone (I). Methods: PC12 cells were used to establish the cell model of Parkinson’s disease, and are divided into five groups: control group;MPP+ group;I (Isoflavone) + MPP+ group;I group;SN-50 + MPP+ group. The content of NF-κB in PC12 cells was determined by immunocytochemistry;The viability of PC12 cells after treated with cell-permeable NF-κB inhibitor SN-50 and cell viability were measured by MTT assay;the expression levels of NF-κB p65 in cytoplasm and nuclear fractions were evaluated by western blot analysis;the mRNA expression of NF-κB p65 was analyzed by in situ hybridization (ISH). Results: Compared with the control group, the protein of NF-κB p65 both in cytoplasm and in nuclei was significantly higher than in I + MPP+ and MPP+ groups;similarly, the mRNA expression level of NF-κB p65 gene was also significantly higher;moreover, the protein expression of NF-κB p65 was much lower in I group (P + group, the protein of NF-κB p65 was significantly lower in I + MPP+ group, the mRNA expression level of NF-κB p65 gene was also significantly lower, and the protein expression level of NF-κB p65 was much lower in I + MPP+ group (P + group (P > 0.05). Conclusion: NF-κB activation is essential to MPP+-induced apoptosis in PC12 cells;but Isoflavone can inhibit the cell damage to some extent to execute its protective function, which may be involved in nigral neurodegeneration in patients with Parkinson’s disease.

Protective effect of erythropoietin against 1-methyl-4-phenylpyridinium-induced neurodegenaration in PC12 cells

Objective The neuroprotective effect of erythropoietin （EPO） against 1-methyl-4-phenylpyridinium （MPP^＋）- induced oxidative stress in cultured PC12 cells, as well as the underlying mechanism, were investigated. Methods PC12 ceils impaired by MPP^＋ were used as the cell model of Parkinson＇s disease. Methyl thiazolyl tetrazolium （MTT） was used to assay the viability of the PC12 cells exposed to gradient concentrations of EPO, and the terminal deoxynucleotidyl transferase dUTP nick end labelling （TUNEL） assay was used to analyze the apoptosis ratio of PC 12 cells. The expression of Bcl-2 and Bax in PC 12 cells were examined by Western blot, and the reactive oxygen species （ROS）, the mitochondrial transmembrane potential and the activity of caspase-3 in each group were detected by spectrofluorometer. Results Treatment of PC12 cells with MPP^＋ caused the loss of cell viability, which may be associated with the elevation in apoptotic rate, the formation of ROS and the disruption of mitochondrial transmembrane potential. It was also shown that MPP＋ significantly induced the upregulation of Bax/Bcl-2 ratio and the activation of caspase-3. In contrast, EPO significantly reversed these responses and had the maximum protective effect at 1 U/mL. Conclusion The inhibitive effect of EPO on the MPP^＋ -induced cytotoxicity may be ascribed to its anti-oxidative property and anti-apoptotic activity, and EPO may provide a useful therapeutic strategy for treatment of neurodegenerative diseases such as Parkinson＇s disease.

Knocking down TRPM2 expression reduces cell injury and NLRP3 inflammasome activation in PC12 cells subjected to oxygen-glucose deprivation

Transient receptor potential melastatin 2(TRPM2) is an important ion channel that represents a potential target for treating injury caused by cerebral ischemia. However, it is unclear whether reducing TRPM2 expression can help repair cerebral injury, and if so what the mechanism underlying this process involves. This study investigated the protective effect of reducing TRPM2 expression on pheochromocytoma(PC12) cells injured by oxygen-glucose deprivation(OGD). PC12 cells were transfected with plasmid encoding TRPM2 shRNAS, then subjected to OGD by incubation in glucose-free medium under hypoxic conditions for 8 hours, after which the cells were allowed to reoxygenate for 24 hours. Apoptotic cells, mitochondrial membrane potentials, reactive oxygen species levels, and cellular calcium levels were detected using flow cytometry. The relative expression of C-X-C motif chemokine ligand 2(CXCL2), NACHT, LRR, and PYD domain–containing protein 3(NALP3), and caspase-1 were detected using fluorescence-based quantitative reverse transcription-polymerase chain reaction and western blotting. The rates of apoptosis, mitochondrial membrane potentials, reactive oxygen species levels, and cellular calcium levels in the TRPM2-shRNA + OGD group were lower than those observed in the OGD group. Taken together, these results suggest that TRPM2 knockdown reduces OGD-induced neuronal injury, potentially by inhibiting apoptosis and reducing oxidative stress levels, mitochondrial membrane potentials, intracellular calcium concentrations, and NLRP3 inflammasome activation.

Effect of insulin on 1-methyl-4-phenylpyridinium ion-induced apoptosis of PC12 cells

BACKGROUND： Insulin receptor （IR） expression in the substantia nigra of patients with Parkinson disease （PD） is not only significantly lower than that in the substantia nigra of normal persons of the same age, but also significantly lower than that in other regions in brain of himself/herself. It suggests that the abnormal effect of insulin receptor-mediated insulin, as a neurotrophic factor, is very possibly related to the loss of dopaminergic neurons in the substantia nigra and striatum in patients with Parkinson disease. OBJECTIVE ： TO observe the interventional effect of insulin on 1-methyl-4-phenylpyridinium ion （MPP^＋）-induced apoptosis of PC12. DESIGN： Controlled observation SETTINGS： Department of Neurology, Beijing China-Japan Friendship Hospital; Department of Neurology Huashan Hospital Affiliated to Fudan University. MATERIALS： PC12 cells were provided by the Cell Bank, Shanghai Institute of Cell Biology, Chinese Academy of Science. MPP^＋, MTT, HOECHST 33258 （Invitrogen Life Technologies）, reverse transcription-polymerase chain reaction （RT-PCR） reagent （Takara Shuzo Co., Ltd.）, flow cytometer （Bacton Dickionson, San Jose, CA）, enzyme labelling instrument （Bio-Tek, Winooski, VT） and PCR circulation instrument （Takara Shuzo Co., Ltd） were used in this study. METHODS ： This study was carried out in the Department of Neurology, Huashan Hospital Affiliated to Fudan University during June 2003 to August 2004. （1） Cell culture and experimental grouping： PC12 cells were cultured according to the method from Peng et al, then were randomized into 3 groups; blank control group, MPP^＋ group and insulin group. （2） Detection of relative survival rate of cells： The relative survival rate of cells at different MPP^＋ final concentrations （0, 50, 100, 200, 300, 1 000 μmol/L） and at different culture time （0, 4, 8, 12, 18, 24 hours） in the 300 Fmol/L MPP^＋ group and different concentrations of insulin （0, 15, 50, 100 nmol/L） in the insulin group was detected with MTT method according to the method from Hansen et al. （3） Observation of cell apoptosis： After stained by HOECHST 33258, the apoptotic cells were observed under the fluorescence miscroscope with the method from Chen et al. （4） Dection of apoptotic rate of cells： Apoptotic rate of cells was detected with flow cytometry according to the method from Zhang et al. （5） The expression of tyrosine hydroxylase （TH） mRNA in PC12 cells was detected with RT-PCR methods according to the modified method from Peng et al. MAIN OUTCOME MEASURES ： Comparison of relative survival rate, apoptosis rate, the expression of IR mRNA and TH mRNA and cell apoptosis. RESULTS： （1） After 12-hour incubation of 100, 200, 300 and 1 000 μmol/L MPP^＋, the relative survival rate of PC12 cells was （72.88±2.91）%, （60.64±0.81）%, （54.56±0.76）% and （16.89±2.83）%, respectively, which was significantly lower than that of blank control group （100%, P 〈 0.05）; After 12, 18 and 24-hour incubation, the relative survival rate of PC12 cells was （54.56±0.76）%, （42.43±0.16）% and （23.56±0.17）% respectively, which was significantly lower than that of blank control group （100%, P〈 0.05）; When 15, 50 and 100 nmol/L insulin was pre-added to cells, the relative survival rate was （70.10±0.16）%, （78.01 ±2.43）% and （83.55±1.43）%, respectively, which was significantly higher than MPP^＋ alone [（54.56±0.76）%, P 〈 0.05]. （2） Appototic bodies were rarely seen in the blank control group, but densely gathered in the MPP^＋ group and were significantly decreased in the insulin group. （3） Apoptosis rate of PC12 cells in the MPP^＋ group was significantly higher than that in the blank control group [（36.56±0.89）% vs. （2.34±0.23）%, P〈 0.05], and that in the 15, 50, 100 nmol/L insulin group [（30.01±0.04）%, （24.23±0.37）%, （20.01 ±1.01）%, respectivelyl was significantly lower than that in MPP^＋ group （P 〈 0.05）. （4） The TH mRNA expression in PC12 cells in MPP^＋ group was significantly lower than that in blank control group; The expression of TH mRNA in insulin group was gradually increased in an insulin dose-dependent manner. There were no significant changes in the expression of IR mRNA under different experimental conditions. CONCLUSION： Insulin can resist MPP^＋-induced apoptosis of PC12 cells, lessen the damage of PC12 cells, but does not change the gene expression of target cell insulin receptor.

Protective effects of Ginkgo biloba extract on 6-hydroxydopamine-induced apoptosis in PC12 cells

The present study analyzed the protective effects of Ginkgo biloba extract against 6-hydroxydopamine-induced PC12 cell apoptosis in a model of Parkinson＇s disease. The results showed that Ginkgo biloba extract had a potent cytoprotective action and inhibited apoptosis of PC12 cells induced by 6-hydroxydopamine. Ginkgo biloba extract decreased the ratio of Bax to Bcl-2 and markedly inhibited the activation of p53 and caspase-3. These experimental findings indicate that Ginkgo biloba extract may significantly reduce the effects of oxidative stress induced by 6-hydroxydopamine in PC12 cells and suppress cell apoptosis. The potential effects of Ginkgo biloba extract might be greater than those of levodopa in the treatment of Parkinson＇s disease.

Acanthopanax senticosus aqueous extract protects PC12 cells against hypoxic injury

Acanthopanax senticosus aqueous extract, at 10, 5 and 2.5 μg/mL, lessened hypoxia-induced PC12 cell injury. Pretreatment with the extract upregulated Bcl-2 expression, downregulated Bax expression, elevated superoxide dismutase activity, diminished malondialdehyde content, relieved Ca2＋ overloading, and suppressed apoptosis of PC12 cells. These effects were most pronounced at 10 μg/mL. These results suggest that Acanthopanax senticosus aqueous extract protects PC12 cells from hypoxia-induced injury in a dose-dependent manner.

α-Linolenic acid alleviates aluminium chloride-induced toxicity in PC12 cells by activation of PKA-CREB-BDNF signaling pathway

Aluminum has been associated with neurodegenerative diseases.ALA(α-linolenic acid),an essential dietary component for human health,possesses prominent biological activities.Herein,we aim to explore the neuroprotective effects of ALA on aluminum toxicity and reveal the underlying mechanism.Results show that aluminum chloride(denoted as Al)enabled cell viability decline and apoptosis with oxidative stress and mitochondrial damage in differentiated rat pheochromocytoma cells(PC12)for 24 h incubation.Compared with Al(10 mmol/L)treatment alone,ALA(50μmol/L)pretreatment for 24 h significantly enhanced cell viability by 28.40%,and hindered cell apoptosis by 12.35%,together with recovering redox state balance and alleviating mitochondrial damage.It was measured that ALA treatment upregulated Bcl-2 expression and down-regulated Bax level,accompanied with an expression decline of caspase-3 and caspase-9.Meanwhile,ALA pretreatment was proved to increase protein kinase A(PKA)expression and to promote phosphorylation of cAMP response element-binding protein(p-CREB),resulting in elevation on the level of brain-derived neurotrophic factor(BDNF).The above results showed that ALA attenuated Al toxicity in PC12 cells by mediating the PKA-CREBBDNF signaling pathway.

The neuroprotective effect of walnut-derived peptides against glutamate-induced damage in PC12 cells: mechanism and bioavailability

In our previous study, defatted walnut meal hydrolysate(DWMH) could attenuate D-galactose-induced acute memory deficits in vivo, and six potent active peptides including WSREEQ, WSREEQE, WSREEQEREE, ADIYTE, ADIYTEEAG and ADIYTEEAGR were identified. The aim of this study was to investigate the possible mechanism underlying their neuroprotective effects on glutamate-induced apoptosis in PC12 cells and their digestive stability. Results showed that all these peptides could attenuate the reduction of cell viability caused by glutamate in PC12 cells, especially WSREEQEREE and ADIYTEEAGR. The addition of Arg residue in WSREEQEREE and ADIYTEEAGR might be the potential reason for their stronger protective effects. Additionally, these two peptides possibly protected PC12 cells against glutamate-induced apoptosis via activating intracellular antioxidant defence(superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px)) through Kelch-like ECH-associated protein 1(Keap1) inhibition, inhibiting ROS production, Ca;influx and mitochondrial membrane potential(MMP) collapse as well as regulating the expression of apoptosis-related proteins(Bax and Bcl-2). This might be due to the presence of Trp, Tyr and Arg in these two peptides. However, encapsulation of WSREEQEREE and ADIYTEEAGR should be considered based on their digestive sensibility during in vitro gastrointestinal digestion.

The p38-MAPK Pathway Is Involved in Neuroprotection of Artemisinin against H2O2-induced Apoptosis in PC12 Cells

Oxidative stress, owing to the excessive production of ROS （reactive oxygen species）, is one of the leading causes for the progression of AD （Alzheimer＇s disease）. Increasing evidences suggested that oxidative stress insult impaired the physiological functioning of neuronal cells by inducing cell apoptosis. The search for drug candidates that can effectively protect neurons from oxidative stress insult might hold therapeutic potential for AD. In the present study, we tested the neuroprotective effects and the related action mechanisms of artemisinin, a FDA-approved anti-malarial drug, against H2O2 induced oxidative damage in PC12 cells. It was found that artemisinin reduced cell viability loss caused by hydrogen peroxide （H2O2） in PC12 cells. In addition, data from Flow cytometry displayed that artemisinin significantly decreased the apoptosis of PC 12 cells induced by H2O2. Furthermore Western blot analysis displayed that artemisinin stimulated the p38MAPK signaling, while treatment of PC 12 cells with specific p38MAPK pathway inhibitor SB203580 blocked the neuroprotective effect of artemisinin. These results together indicated that artemisinin is a potential protectant, and it protects PC12 cells against H2O2 injury through activation of the p38MAPK pathway.

Apoptosis-inducing activity of endocrine-disrupting chemicals in cultured PC12 cells

Endocrine-disrupting chemicals (EDCs) are known to exert estrogen-like effects that are similar to those made by naturally produced hormones or by inhibition of the receptors in the cell receiving the hormones. Recently, several reports have indicated that EDCs can affect the developing central nervous system. In our current study, we report that some EDCs induce apoptosis in cultured PC12 cells and can be classified into three groups. Bisphenol A (BPA), p-nonylphenol (NP) and tributyltin chloride (TBT) were found to induce endoplasmic reticulum (ER) stress-associated apoptosis and activate the unfolded protein response (UPR) system, whereas benomyl (beno) induced non-ER stress-associated apoptosis. The half-maximal apoptosis-inducing concentrations (IC50) of these EDCs were 160 μM for BPA, 25.6 μM for NP, 640 nM for TBT and 48 μM for beno. Although these concentrations are higher than those found in the environment, some EDCs may have apoptotic effects on various cells in the body, including neurons, through their accumulation in the body over time or condensation through the food chain. On the other hand, benzopyrene, fenvalerate, styrene monomer and bis(2-ethylhexyl)phthalate did not induce apoptosis in PC12 cells. We analyzed also whether apoptosis-inducing EDCs had an estrogen-like effect on cultured PC12 cells transfected with a luciferase reporter plasmid, the activity of which is dependent on estrogen receptor α. We found that BPA had an estrogen-like effect (EC50 = 5.9 μM) but that NP, TBT and beno did not in transfected PC12 cells. These results suggest that BPA may predomi-nantly exert estrogenic effects, but others may pre-dominantly have apoptosis-inducing effects on cells in the body exposed to a polluted environment.

二苯乙烯苷通过抑制氧化应激对MPP^+诱导的PC12细胞凋亡的保护作用

为了探讨2,3,5,4’-四羟基二苯乙烯-2-ο-β-D-葡萄糖苷(2,3,5,4’-tetrahydroxystibene-2-ο-β-D-glucoside,TSG)对1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium,MPP+)诱导PC12细胞凋亡的影响及其可能机制,本实验分为对照组,MPP+处理组和TSG(1、5和10μmol/L)预处理组。用Hoechst33258染色法和流式细胞术测定细胞凋亡,对活性氧(reactive oxygen species,ROS)敏感的荧光探针2,7-dichlorofluorescin dictate(DCF-DA),丙二醛(malondialdehyde,MDA)和总抗氧化能力(total anti-oxida-tion competence,T-AOC)检测试剂盒测定细胞的氧化应激的变化。结果显示:TSG三个浓度预处理后,对比MPP+处理组,观察到TSG在一定范围内以剂量依赖方式,使细胞核凝聚明显减少,细胞凋亡率降低。另外,TSG预处理后,PC12细胞中增高的ROS和MDA水平较MPP+处理组明显有所减低,T-AOC有所增强。以上结果提示,TSG可抑制MPP+诱导的PC12细胞的凋亡,其机制可能与TSG抑制氧化应激有关。

MPP^+诱导PC12细胞氧化应激损伤的实验研究

目的:考察MPP+诱导PC12细胞氧化应激损伤作用,并探讨其机制。方法:以不同浓度、不同作用时间的MPP+作用于PC12细胞,MTT法检测细胞存活率,流式细胞仪测细胞凋亡率,紫外可见分光光度计测LDH、NO、NOS、MDA含量和SOD活性。结果:MPP+终浓度达到300μmol/L时,作用48h后可明显降低PC12细胞存活率(P<0.001),提高细胞凋亡率(P<0.01),增强LDH、NO、NOS、MDA的含量和降低SOD活性,有统计学意义(P<0.05)。结论:MPP+能诱导PC12细胞氧化应激损伤,其作用机制可能是通过增加PC12细胞NO和NOS含量,引起胞内SOD的活性降低,从而引起脂质过氧化物增加来损伤多巴胺能神经细胞的。

Nrf2活化剂SFP对MPP^+所致PC12细胞氧化应激损伤的保护作用

目的探讨激活核因子红系2相关因子2(nuclear factor erythroid-derived 2-related factor 2,Nrf2)对1-甲基-4-苯基吡啶离子(MPP+)诱导PC12细胞氧化应激损伤的保护作用及其机制。方法以MPP+损伤PC12细胞作为帕金森病(PD)细胞模型,采用四甲基偶氮唑盐(MTT)法检测细胞存活率,流式细胞术检测细胞凋亡率,双氯荧光黄乙酸乙酯(DCF-DA)染色检测细胞内活性氧簇(ROS)的生成量,罗丹明123(Rh123)染色检测细胞线粒体膜电位(ΔΨm),免疫印迹法检测磷酸化的蛋白激酶B(pAkt)表达水平,评价Nrf2激动剂莱菔硫烷(SFP)对该损伤模型的影响并探讨其相关机制。结果①500μmol/L的MPP+可以导致PC12细胞的存活率下降,凋亡率增加,0.1、0.5、1.0、5.0、10.0μmol/L浓度的SFP可以使PC12细胞的存活率增加、凋亡率降低,并在5.0μmol/L时达到最大保护效果;②MPP+导致PC12细胞ROS生成增加、ΔΨm降低,SFP可以减轻该氧化应激损伤;③SFP增加Akt磷酸化水平,该效应及其抗氧化损伤作用可被PI3K抑制剂LY294002所拮抗。结论 Nrf2活化剂SFP能抑制MPP+对PC12细胞的氧化应激损伤,其机制是通过PI3K/Akt通路实现的。

硫化氢对MPP^+诱导PC12细胞氧化应激损伤的保护作用

目的探讨硫化氢对MPP+诱导PC12细胞氧化应激损伤的保护作用。方法以MPP+损伤PC12细胞作为帕金森病的细胞模型,甲氮甲唑蓝(MTT)法检测细胞存活率;丙酮酸二硝基苯腙比色法检测细胞上清液乳酸脱氢酶(LDH)漏出量;硫代巴比妥法检测细胞上清液中丙二醛(MDA)浓度;双氢罗丹明123染色FCM检测细胞内活性氧水平变化;应用硫化氢钠(sodium hydrosulfide,NaHS)作为H2S的供体。结果 200μmol/L和400μmol/L硫氢化钠呈浓度依赖性阻断MPP+引起PC12细胞存活率的降低;400μmol/L硫氢化钠能明显抑制400μmol/L MPP+诱导的PC12细胞LDH的漏出,以及抑制MDA和活性氧的产生。结论硫化氢对MPP+诱导PC12细胞的氧化应激损伤具有保护作用。

女贞子对MPP^+诱导的PC12细胞氧化应激损伤保护作用研究

目的:探讨女贞子是否对MPP+诱导的PC12细胞氧化应激损伤具有保护作用。方法:采用MPP+诱导的PC12细胞作为帕金森病体外模型,同时给予不同浓度的女贞子醇提液、水提液。通过MTT法测定细胞存活率,通过超氧化物歧化酶(SOD)、一氧化氮(NO)、丙二醛(MDA)及乳酸脱氢酶(LDH)试剂盒来检测女贞子对细胞氧化应激损伤的影响。结果:与正常组相比,模型组细胞的细胞存活率显著下降,SOD抑制率、NO含量、MDA含量及LDH水平显著升高(P<0.01);与模型组相比,浓度为1mg/mL的女贞子水提液显著提高细胞存活率,显著降低SOD抑制率、NO含量、MDA含量及LDH水平。结论:女贞子水提液对细胞的氧化应激损伤具有一定的保护作用。

Protective effects of MCI-186 on oxidative damage in a cell model of Alzheimer's disease

Oxidative stress has an important role in the development of Alzheimer＇s disease （AD）. Beta amyloid protein 25-35 （Aβ25-35） can generate oxygen free radicals, and MCI-186 （3-methyl-l-phenyl-2-pyrazolin-5-one, edaravone） can specifically eliminate hydroxyl radicals. The present study introduced Aβ25-35 into PC12 cells to establish a cell model of AD, and investigated the neuroprotective effects of MCI-186 on AD. Results showed that MCI-186 had a positive effect on the prevention and treatment of AD by inhibiting protein oxidative products, advanced glycation end products, lipid oxidative end products and DNA oxidative damage in PC12 cells induced by Aβ25-35.