| BackgroundParkinson’s disease (PD) is one of the most common chronic neurodegenera -tive disorder, and is the second most common neurodegenerative disorder after Alzheimer’s disease (AD). PD affects approximately 2% of the population aged 65 or over.Clinical symptoms of PD including static tremor, bradykinesia, muscle rigidity and abnormal posture. characterized by a selective loss of dopaminergic neurons in the substania nigra and subsequent depletion of dopamine in the striatum. Up to now, the etiology and pathogenesis of PD are not fully elucidated. The participation of known factors such as oxidative stress and mitochondrial dysfunction.So if a drug resistance to oxidative stress or can inhibit the mitochondrial dysfunction, could become the new drug for the treatment of PD.Clarifing the mechanisms of PD onset and development of neuroprotective therapeutics are largely depend on establishment of disease model, especially model in vitro. MPTP is a neurotoxin that produces a parkinsonian syndrome in both humans and experimental animals. Its neurotoxic effects also appear to involve energy depletion and free radical generation. MPTP is converted to its metabolite MPP+ by MAO-B. MPP+ is selectively accumulated by high affinity dopamine transporters and taken up into the mitochondria of dopaminergic neurons, where it disrupts oxidative phosphorylation by inhibiting complex I of the mitochondrial electron transport chain. thereby exhibiting dopaminergic neurotoxicity.MPTP or MPP+ are widely used and are recognized a classic models of PD in the academic world.Saikosaponin-d (Ssd), one of the major components extracted from Bupleurum falcatum L exhibits a variety of pharmaceutical activities including anti-tumor, anti-inflammation and immunomodulatory properties. In this study, SH-SY5Y cell under optimized MPP+ exposure was selected in comparison to features of lesioned model. The aim is to study the neuroprotective effect of SSd as well as the molecular mechanism, on MPP+-induced cytotoxicity in SH-SY5Y cells, and to Provide a scientific basis for drug and the research mentality for PD treatment.Objective1. Observe the inhibition effects to oxidative stress and the cell apoptosis of Ssd on MPP+-induced cytotoxicity SH-SY5Y cells;2. To investigate the related mechanism of effects Ssd inhibit oxidative stress and the cell apoptosis. To investigated the molecular mechanism underlying the protective effects of Ssd on SH-SY5Y cells.Materials and methodsCell culture and reagentsSH-SY5Y cells were grown in Dulbecco’s modified Eagle medium Ham’s F12 (Gibco, CA,USA) supplemented with 10% fetal bovine serum,100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37℃ in a humidified 5% CO2/95% air incubator. Ssd was dissolved in culture medium at a concentration of 5 mM for a stock solution. For treatment of cells, Ssd was diluted in culture medium to the appropriate concentration. Ssd and MPP+ were purchased from Sigma (St. Louis,MO, USA).Cell viability assayThe cell viability was measured by a quantitative colorimetric assay with MTT. To be brief,exponentially growing cells at a density of 5×104 were seeded in a 96-well plate cells/well. After 24 h, SH-SY5Y cells were firstly subjected to a serial condition test with Ssd (15,30,45 μM) for 5 h and then with MPP+ for 24 h.The optical density was measured using a microplate reader at a wavelength of 450 nm. All readings were compared with the control,which represented 100% viability.Apoptosis assayCellular apoptosis was monitored by the fluorescein isothiocyanate (FITC)/Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose,CA) according to the manufacturer’s protocol.To be brief, SH-SY5Y cells at the density of 1×105 cells/well were stained with FITC/Annexin V and propidium iodide (PI). Then, cell apoptosis was measured using a flow cytometry analyzer (BD Biosciences, San Jose, CA).Caspase-3 activity assaySH-SY5Y cells at the density of 1 x 104 cells/well were treated with MPP+ and/or Ssd for 24 h, and caspase-3 activity was determined according to the instruction of caspase-3 colorimetric assay kit (St. Louis, MO, US), which is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase-3, resulting in the release of the p-nitroaniline (pNA) moiety. The proteolytic reaction was carried out in isolation buffer containing 50μg of cytosolic protein and 50 μM specific caspase substrates. The reaction was measured by changes in absorbance at 405nm using a 96-well plate reader.Measurement of ROS productionCells were incubated with 2,7-dichlorofluorescein diacetate (DCF-DA) (Sigma, St. Louis, MO, USA) for 1 h at 37℃ in the dark, and then resuspended in PBS. Intracellular ROS production was assessed using a fluorescence microscope (OLYMPUS, Germany).Western blotTotal cell extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on a nitrocellulose filter membrane. The membrane was incubated with primary antibodies against SIRT3 (1:1000 dilution) and GAPDH (1:5000 dilution).Horseradish peroxidase-conjugated secondary antibodies (1:10000) were applied on the membrane and the bound secondary antibody was detected with the enhanced chemiluminescence reagents (Pierce, Rockford, IL). All antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA.Statistical analysisData are expressed as mean ± SD. The difference between groups was analyzed by ANOVA using the SPSS 16.0 statistical software (SPSS, Inc., Chicago, IL, USA). A P value of< 0.05 was considered statistically significant. Results1. Effects of Ssd on MPP+-induced cytotoxicity in SH-SY5Y cells.To examine if Ssd affected the survival of SH-SY5Y cells, different concentrations of Ssd (0,15,30,45,60 μM) were added to the cells for 24 h. The result is low doses of Ssd (15-45μM) had little effect on cell proliferation, whereas Ssd at high dose (60 μM) obviously inhibited the growth of SH-SY5Y cells.Consistent with previous studies, we found that treatment with MPP+(0,0.1,0.5, 1 and 1.5mM) caused a significant decrease in cell viability in a concentration-dependent manner. Based on these data, we decided to use low doses of Ssd (15,30,45 μM) and MPP+ at 1 mM for the following experiments. To examine the protective effect of Ssd on MPP+-induced cell deaths, SH-SY5Y cells were exposed to different doses (15,30,45 μM) of Ssd 5 h prior to MPP+(1 mM) treatment. The results of MTT assay showed that Ssd significantly increased the viability of the SH-SY5Y cells after MPP+ administration for 24 h.2. Ssd inhibited MPP+-induced ROS accumulation in SH-SY5Y cells.Oxidative stress is a well-established event in the pathology of many diseases, including PD.To examine whether the protective effect of Ssd against MPP+-induced cytotoxicity was attributed to its antioxidant activity, the intracellular ROS levels were measured in SH-SY5Y cells.We found that the MPP+ treatment significantly increased the ROS generation, whereas Ssd attenuated the intracellular ROS levels in a concentration-dependent manner. These data suggested that Ssd could reduce oxidative stress in SH-SY5Y cells.3. Ssd reduced MPP+-induced cell apoptosis in SH-SY5Y cells.To examine the effects of Ssd on cell apoptotic deaths, SH-SY5Y cells were incubated with MPP+ alone or in combination with Ssd in different doses. Flow cytometry analysis showed that Ssd treatment significantly inhibited MPP+-induced cell apoptosis in a dose-dependent fashion. Moreover, the activity of caspase-3 was examined in order to evaluate the apoptosis process following Ssd treatment. Following 24 h treatment of SH-SY5Y cells with MPP+, the caspase-3 activity was dramatically increased nearly three folds. However, Ssd administration attenuated the MPP+-induced caspase-3 activation in a dose-dependent manner. These results indicated that Ssd could alleviate cell apoptosis induced by MPP+ in SH-SY5Y cells.4. Up-regulation of SIRT3 by Ssd administration in SH-SY5Y cells.We further investigated the molecular mechanism underlying the protective effects of Ssd on SH-SY5Y cells. Since SIRT3 plays important roles in cell survival and apoptosis, we hypothesized that SIRT3 may be involved in the neuroprotective of Ssd in SH-SY5Y cells. Data from qRT-PCR indicated that SH-SY5Y cells exposed to MPP+ alone significantly decreased SIRT3 mRNA level. However, a dramatic increase of SIRT3 mRNA expression was detected in Ssdtreated SH-SY5Y cells. Similarly, the protein level of SIRT3 was reduced after MPP+ treatment, but was enhanced in Ssd-treated cells. These data suggested that Ssd promoted the expression of SIRT3 in SH-SY5Y cells.5. Ssd protected SH-SY5Y cells against MPP+-induced cytotoxicity via SIRT3In order to determine whether SIRT3 is involved in the protective effects of Ssd, we down regulated SIRT3 with a specific small interfering RNA (siRNA) in SH-SY5Y cells. The expression of SIRT3 was obviously reduced both at mRNA and protein levels.Consequently, our data showed that Ssd failed to promote SH-SY5Y cells viability after treatment with MPP+. In addition, SIRT3 knockdown in SH-SY5Y cells exhibited no significant differences in ROS production and cell apoptosis with or without Ssd treatment. These results demonstrated that SIRT3 knockdown reversed the protective effects of Ssd on SH-S Y5Y cells.ConclusionSsd has neuroprotective effects against MPP+-induced cytotoxicity possibly through regulation of SIRT3 expression. |
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