Dihydromyricetin and Salvianolic acid B inhibit alpha-synuclein aggregation and enhance chaperone-mediated autophagy
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Progressive accumulation of α-synuclein is a key step in the pathological development of Parkinson’s disease. Impaired protein degradation and increased levels of α-synuclein may trigger a pathological aggregation in vitro and in vivo. The chaperone-mediated autophagy (CMA) pathway is involved in the intracellular degradation processes of α-synuclein. Dysfunction of the CMA pathway impairs α-synuclein degradation and causes cytotoxicity.
In the present study, we investigated the effects on the CMA pathway and α-synuclein aggregation using bioactive ingredients (Dihydromyricetin (DHM) and Salvianolic acid B (Sal B)) extracted from natural medicinal plants. In both cell-free and cellular models of α-synuclein aggregation, after administration of DHM and Sal B, we observed significant inhibition of α-synuclein accumulation and aggregation. Cells were co-transfected with a C-terminal modified α-synuclein (SynT) and synphilin-1, and then treated with DHM (10 μM) and Sal B (50 μM) 16 hours after transfection; levels of α-synuclein aggregation decreased significantly (68% for DHM and 75% for Sal B). Concomitantly, we detected increased levels of LAMP-1 (a marker of lysosomal homeostasis) and LAMP-2A (a key marker of CMA). Immunofluorescence analyses showed increased colocalization between LAMP-1 and LAMP-2A with α-synuclein inclusions after treatment with DHM and Sal B. We also found increased levels of LAMP-1 and LAMP-2A both in vitro and in vivo, along with decreased levels of α-synuclein. Moreover, DHM and Sal B treatments exhibited anti-inflammatory activities, preventing astroglia- and microglia-mediated neuroinflammation in BAC-α-syn-GFP transgenic mice.
Our data indicate that DHM and Sal B are effective in modulating α-synuclein accumulation and aggregate formation and augmenting activation of CMA, holding potential for the treatment of Parkinson’s disease.
Keywordschaperone-mediated autophagy macroautophagy alpha-synuclein protein aggregation Parkinson disease lysosomal-associated membrane protein
Bacterial Artificial Chromosome α-synuclein-green fluorescent protein
Glial fibrillary acidic protein
Heat shock (70kDa) protein
Allograft inflammatory factor 1
Lysosomal-associated membrane protein 1
Lysosomal-associated membrane protein 2, isoform A
Mammalian target of rapamycin
- Sal B
Salvianolic acid B
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Substantia nigra pars compacta
C-terminal modified α-synuclein
- WT α-syn
Aberrant degradation of alpha-synuclein (α-syn) has been implicated in the pathogenesis of Parkinson’s disease (PD) which leads to accumulation of α-syn in Lewy bodies [1, 2]. α-Syn can be selectively translocated into lysosomes for degradation via chaperone-mediated autophagy (CMA), a highly regulated cellular process that mediates the degradation of cytosolic proteins in lysosomes [3, 4, 5, 6]. The protein contains a CMA target motif and is degraded by CMA in neural cells [7, 8]. CMA is controlled by two key CMA regulators: the chaperone HSC70 and the receptor lysosomal-associated membrane protein 2A (LAMP 2A). LAMP-1 is highly structurally homologous to LAMP-2A suggesting that there may be an overlapping function of these two proteins [9, 10]. HSC70 binds to protein substrates containing a KFERQ peptide motif [8, 11]. The substrate–HSC complex interacts with LAMP-1/2A for targeting of identified protein translocation to the lysosome [12, 13, 14, 15]. In addition, the ubiquitin-proteasome system (UPS) and macroautophagy are also involved in α-syn degradation [16, 17, 18].
Several studies have been conducted on the development of small molecular inhibitors of α-syn aggregation for the prevention and treatment of PD [19, 20, 21, 22, 23]. Several important compounds in the daily diet and medicinal plants have been found to be protective against α-syn fibrillation [21, 24]. Dihydromyricetin (DHM), a major active ingredient of flavonoid compounds extracted from the stems and leaves of Ampelopsis grossedentata, has anti-tumor, oxidation resistance and free radical scavenging capabilities [25, 26, 27, 28]. Evidence indicates that DHM has neuroprotective effects by enhancing the formation of autophagosomes and inducing autophagy [29, 30, 31]. Salvianolic acid B (Sal B) is one of the bioactive compounds of Salvia miltiorrhiza Bunge extracted from the root of Salvia miltiorrhiza and has been shown to exert various anti-oxidative and anti-inflammatory effects in both in vitro and in vivo studies [23, 32, 33]. Sal B has recently been associated with preventing fibril aggregation of amyloid proteins and inhibiting neuroinflammation, thereby improving neurological function in animal models of neurodegenerative diseases [23, 24]. However, it is not clear whether DHM and Sal B have any effects on α-syn accumulation and aggregation in synucleinopathies, such as PD.
To further explore the role of CMA mediated degradation of aggregated α-syn and the potential function of autophagy regulated by DHM and Sal B, in the present study, we have investigated the effects of DHM and Sal B on α-syn accumulation and aggregation using both in vitro and in vivo models. We observed that DHM and Sal B upregulated the CMA associated protein LAMP-2A and its homologous protein, LAMP-1, decreased levels of α-syn, reduced cytotoxicity and inhibited inflammatory responses when administered in cell and animal models. Our findings indicate that DHM and Sal B are potential therapeutic compounds that can intervene and halt pathological developments in synucleinopathies.
α-Syn monomers were ordered from Proteos (RP-003) and prepared following the Michael J Fox Foundations guidelines for fibril formation. Briefly, monomeric protein was thawed and spun at 15.000xg for 10 min at 4 °C, to pellet any aggregated materials. The supernatant was then assessed by BCA to determine the α-syn concentration. The monomer sample was diluted to 5 mg/ml in PBS without calcium and magnesium, and transferred to a 1.5 ml Eppendorf tube, then incubated for 7 days in a shaking incubator at 1000 rpm and 37 °C. Final fibril solution was stored at -80 °C in single use aliquots until use.
Inhibitor modulation of α-synuclein aggregation kinetics
Aggregation kinetics were assessed in Corning NBS half-area micro plates(#3881) plates using 70 μM α-syn monomers, 20 μM thioflavin T (Sigma, T3516) and Sal B (Sigma, SML0048, ≥94% (HPLC) ) or DHM (Sigma, SML0295, ≥98% (HPLC) ) at either 15 or 30 μM. Vehicle control wells were set up using a volume equal to that of the highest inhibitor concentration (0,015 μl per well). To initiate the experiment, sonicated α-syn seeds were added to each well at 0.1% of the monomer concentration (70 nM). Kinetics were observed using a BMG FLUOstar Omega plate reader, allowing continual measurements for 7 days at 37 °C. Baseline acquisition was performed for 3 hours before addition of the α-syn seeds, and recordings were continued for 12 hours.
Cell culture and transfection
H4 neuroglioma cells from human (origin) were cultured in Opti-MEM + GlutaMAX (Invitrogen, 51985-034) supplemented with 10% fetal bovine serum (FBS; Gibco, 10100-147) at 37°C, passaged, and plated on chamber slides (Labted-II, Nalgen-Nunc, 154526) or glass cover slips. For intracellular α-syn aggregation experiments, H4 cells were seeded in 24-well plate (5 × 104 cells/well) 24 h prior to transient transfection with SynT (C-terminal tagged form of WT α-syn) and synphilin-1 (Fig. S1). Equi-molar ratios of plasmids were mixed with FuGENE® 6 (Promega, E2691) at a 1:2 mass volume ratio, and incubated for 15 min before the complex of transfection reagent and plasmids was transfected into cells according to the manufacturer’s protocol (2 h transfection and 6 h recovery time). ALP modifiers were incubated during the last 24 h before fixation and processing for immunocytochemistry and toxicity assessments. Co-transfection with an empty backbone-vector [pPAGFP-C1, Addgene, 11910] and mock transfection was used as control. Rapamycin (200 ng/ml, Sigma Aldrich, R0395) was prepared in DMSO, chloroquine diphosphate salt (50 mM, CQ, Sigma Aldrich, C6628) and 3-methyladenine (10 mM, 3-MA, Sigma Aldrich, M9281) in water.
For drug treatment, 24 h post first transfection with SynT and synphilin-1, cells were further incubated with DHM, Sal B, Rapamycin or Chloroquine. Twenty-four hours later, cells were fixed with 4% PFA for 10 min at room temperature (RT), washed two times with PBS and subjected to immunocytochemistry analysis. Briefly, cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min at RT, blocked for 1 h at RT with 5% normal donkey serum in 0.1% Triton X-100 in PBS, incubated with primary antibody (mouse anti-α-syn 1:1000; BD Biosciences) at 4°C overnight followed by secondary antibody incubation (1:1000, donkey anti-mouse IgG-Alexa568, Jackson ImmunoResearch) for 2 h at RT, then incubated for 10 min with DAPI 1:1000 in PBS (SIGMA-ALDRICH). Specimen analyses were performed with a conventional epifluorescence microscope (Nikon Ni-E). Cells were subjected to microscopy analysis for LAMP-1/-2A and α-syn colocalization using laser-confocal microscope (Leica TCS SP8), followed by analysis using ImageJ software. Sequential multi-track frames were acquired to avoid any potential crosstalk from adjacent fluorophore. For ThS labeling, transfected H4 cells were fixed and incubated prior to IF labeling for 10 min in 0.5 mg/ml thioflavin-S (Sigma, T1892), and washed in 85% absolute ethanol. At least 300 cells from three independent wells were assessed for each experiment and the number of cells containing α-syn positive aggregates were quantified in the transfection conditions by a random sampling survey. The percentage of the transfected cells containing α-syn positive aggregates compared with the total number was then recorded.
Immunohistochemistry of transgenic mice
Six- and nine-month old mice were housed (3-4 animals/cage) with food and water available ad libitum under a 12-h light/dark cycle. All animal experiments followed the Institutional Animal Care and all procedures were performed under the specifications set by the Ethical Committee for Use of laboratory animals at Lund University, Sweden and at Northeastern University, China. Homozygous transgenic mice expressing WT human α-syn fused to green fluorescent protein (GFP), under control of the mouse α-syn promoter show an overexpression of α-syn-GFP in the CS and the dopaminergic neurons of the SNpc. The formation of α-syn aggregates in the brain of transgenic mice has been shown to rise with increasing age . DHM and Sal B (10mg/kg/day for two weeks) were utilized for intra-peritoneal administration of nine-month old mice (n=8 mice per group). Mice were then euthanized 6 weeks later. Brains were removed, post-fixed in 4% PFA and a gradient sucrose sedimentation (10% - 30%) was performed. For mouse brains, 30 micrometer-thick free-floating coronal sections were cut on a freezing microtome (Leica, SM2010R), blocked in solution comprising PBS + 5% horse serum + 0.25% Triton-X 100, and incubated with primary antibodies (mouse-anti-α-syn antibody 1:1000, Santa Cruz Biotechnology, sc-12767; mouse-anti-GFAP antibody 1:1000, MERCK MILLIPORE, MAB360; mouse-anti-Iba1/AIF1 antibody 1:1000, MERCK MILLIPORE, MABN92) overnight in a humid chamber at 4°C. Sections (incubated with anti-α-syn, anti-GFAP and anti-Iba1/AIF1) were then subsequently incubated with secondary biotinylated anti-mouse antibody (Vector Biolabs) followed by DAB staining using the ABC kit (Vector Biolabs) and DAB peroxidase substrate (Vector Biolabs) according to the manufacturer’s protocol. For each animal, 3 sections were analyzed and all sections were processed under the same standardized conditions. α-Syn+, GFAP+ and Iba1+ cells in the CS and SNpc were counted on a Nikon microscope using the NIS-Elements BR imaging system.
Western blot analysis
Cytosolic fractions were obtained by manual homogenization and incubation in ice-cold lysis buffer containing 25 mM TRIS-HCl pH 7.4, EDTA 1 mM, protease inhibitor + 0.1% SDS for 2 h, followed by centrifugation at 12000 rpm for 10 min. In the supernatant, equivalent amounts of LAMP-1/-2A protein sample were loaded and separated by 8% SDS-PAGE gels, and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) for 2 h at 4°C or overnight at 4°C. Membranes were blocked with 2.5% nonfat milk solution in Tris-buffered saline with 0.1% Triton X-100 (TBST) for 1 h, and then incubated overnight at 4°C with mouse anti-LAMP-1 (Abcam, ab25630, 1:1000), rabbit anti-LAMP-2A (Abcam, ab18528, 1:1000) or mouse anti-β-actin (Sigma, A1978, 1:5000,), followed by HRP-linked secondary antibodies (CST, 7076S for anti-mouse IgG and 7074S for anti-rabbit IgG, 1:10000,) for 2 h at RT. Bands were detected using an ECL detection kit (Cell Signaling Technology) and exposed to X-ray films. Bands were analyzed and normalized to the corresponding β-actin signal for comparison.
Cell viability and α-syn cytotoxicity was evaluated by the MTT assay and LDH assay. H4 cells were plated on 96-well plates in complete medium, transfected with SynT and synphilin-1, then co-transfected with a plasmid encoding for WT α-syn, or with empty plasmid. For the MTT assay, 10 μl MTT reagent and 100 μl detergent reagent were added into each well in sequence after incubation for twenty-four hours. The resulting intracellular purple formazan can be solubilized and quantified by spectrophotometric means. For the LDH assay, after twenty-four hours transfection, culture media were collected and used to determine the levels of released Lactate dehydrogenase (LDH). After treatment, assays were performed following the manufacturer’s instructions (Promega, Madison, WI, USA). Results were expressed as the percentage of cell death.
Open Field Assay
Exploratory/locomotor activity of the animals (six-month-old and nine-month-old, n=8 mice per group) was assessed in an open-field paradigm equipped with a video trajectory analysis system (42 × 42 × 36 cm [d × w × h] plexiglass boxes) and analyzed using Smart 3.0 software (Panlab, Span). The mice were first allowed to explore the confined arena for 15 min during each session, then their performance was recorded and ambulatory locomotor activity was measured by offline analysis. Track paths were subsequently analyzed by an automatic system to assess the following parameters: distance traveled (cm), time spent in the various sections of the arena and number of rears.
Data analysis and statistics
Statistical analyses were performed using Prism 6 (GraphPad Software). All data shown are representative from a minimum of three independent experiments unless otherwise stated. Statistical analysis for comparison of groups in the in vitro experiments was performed using the Student’s t-test. For both in vitro and in vivo experiments, statistical significance of difference between groups was determined by the 2-tailed unpaired Student t test of the means. Where values have been compared with the normalized control, a one-sample t test was used. In cases of multiple-group comparisons, a one-way ANOVA was used, with Scheffe’s post hoc test where values have been compared with the control.
Aggregation kinetics of α-synuclein in the presence of Sal B and DHM
Alterations of CMA markers in an in vitro model of α-synuclein aggregation.
In order to study alterations in α-syn aggregation in vitro, we performed a co-transfection of SynT and synphilin-1 in human neuroglioma cells (H4 cells) for at least 48 h, leading to α-syn aggregation  (Additional file 1). As we reported previously, in this aggregation model we detected many large α-syn aggregates in contrast to wild-type (WT) α-syn transfected cells, where diffuse α-syn was present in the cytoplasm (Additional file 1Bi, Ci). In addition, the cells transfected with SynT exhibited large ThS positive aggregates, implying that α-syn aggregates consist of a typical β-sheet conformation in the cells (Additional file 1Bii). Small and diffused α-syn positive structures were also found in each model but were not ThS positive (Additional file 1Cii), in agreement with our previous findings .
CMA modulates α-synuclein aggregation and toxicity in vitro
DHM and Sal B treatments activate CMA pathways and degrade α-syn aggregates in vivo.
DHM and Sal B treatment lead to decreased astrogliosis and microgliosis in vivo
In this study, we have shown that DHM and Sal B induce the degradation of α-syn aggregates and we attribute this to the observed activation of the CMA pathway both in vitro and in vivo. Firstly, low doses of DHM and Sal B could reduce the expression level of α-syn aggregates by up-regulating the CMA pathway in the SynT-aggregation cell model. We then confirmed that DHM and Sal B up-regulate LAMP-1, an important marker for the structure and function of lysosomal membranes and LAMP-2A, a key marker of CMA, in α-syn transgenic mice and decrease astrogliosis and microgliosis. This data indicates that treatment with DHM or Sal B upregulates the CMA pathway, which is known to play a key role in degrading abnormal a-syn aggregates.
Autophagy has been considered an essential mechanism in neurodegenerative diseases such as PD and Alzheimer’s disease [39, 40, 41]. Increasing evidence suggests that aggregated and misfolded α-syn drives the pathology of PD. Although it has been reported that Sal B can inhibit Aβ aggregation in cultured cells [24, 42], no evidence exists to indicate whether DHM or SalB have a regulatory effect on α-syn aggregation. Here, we observed decreased α-syn expression in cell models after DHM and Sal B treatments, as well as decreased levels of the α-syn protein in α-syn transgenic mice. Therefore, it is possible that the degradation pathway of aggregated α-syn may be directly targeted by DHM and Sal B. From a structural chemistry point of view, several studies have provided evidence that compounds which have three adjacent dihydroxy groups (e.g. DHM) or vicinal dihydroxy groups (e.g. Sal B) are effective inhibitors of α-syn oligomerization and fibrilization [43, 44, 45]. Thus, the special structures of DHM and Sal B may have a direct inhibitory effect on the aggregation of α-syn.
Lysosomes are the primary compartment for the degradation of intracellular proteins via autophagy . The existence of abnormal intracellular α-syn-positive aggregates in PD indicates that the degradation capability of lysosomes may be impaired [3, 47]. CMA exerts a protective function by selectively targeting damaged or misfolded proteins for lysosomal degradation. Dysfunction of CMA in PD is characterized by reduced expression of the membrane receptor of CMA, lysosomal-associated membrane protein (LAMP) [4, 48, 49]. Several studies using different cell culture models of synucleinopathies have shown that the CMA pathway participates in α-syn degradation and its alteration may support α-syn mediated neurodegeneration [3, 7, 50]. Most of the previous studies report increased accumulation of α-syn by inhibiting CMA pathway, or reduced α-syn levels by activating CMA pathway [39, 51]. LAMP-2A plays an important role in the CMA pathway of α-syn degradation and an increased expression of LAMP-2A can activate the CMA pathway [4, 13, 52]. Here, we observed a reduction of α-syn aggregation by DHM and Sal B in vitro. The aggregation cell model is characterized by ThS-positive α-syn aggregates, because the dye can specifically bind to amyloid-like structures to indicate the formation of large inclusions [36, 53]. Smaller α-syn positive aggregates generated by untagged α-syn are also found in cell models, but are not positive for ThS. Thus, our data suggest that DHM and Sal B not only enhance α-syn degradation by the CMA pathway, but also modulate α-syn aggregation. Rapamycin has been widely used to inhibit the mTOR pathway and thereby induce autophagy . Chloroquine blocks lysosomal function by raising lysosomal pH, thereby inhibiting lysosomal function . Here, blocking autophagy with CQ resulted in SynT aggregation and increased toxicity. However, ALP modulation by rapamycin did not increase the toxicity of SynT and α-syn aggregation was reduced. We can see a clear effect of DHM and Sal B treatment on LAMP-1 and LAMP-2A levels, and a similar effect on aggregation induced by rapamycin. Notably, we observed that both LAMP-1 and LAMP-2A clearly co-localized with α-syn in transgenic mice after administration of DHM and Sal B, which is in agreement with previous findings [48, 56]. Recent studies showed that DHM and Sal B can enhance the level of autophagy by regulating the mTOR pathway [31, 57]. Sal B can stabilize the lysosome membrane by increasing the LAMP-1 protein level by reducing lysosomal enzyme translocation to the cytosol . Levels of LAMP-1 can be increased through the regulation of the nuclear localization of Transcription factor EB (TFEB) via the mTOR signaling pathway . LAMP-2A and the mTOR complex were highly relevant in vivo . Thus, we reasoned that DHM and Sal B may enhance the degradation of α-syn by up-regulating the level of CMA and enhancing the expression of LAMP-1 and LAMP-2A via the mTOR signaling pathway.
As previously reported, DHM exerts a more rapid effect in association with enhancement of brain-derived neurotrophic factor expression and inhibition of neuroinflammation . Administration of Sal B significantly decreased microglial activation in the central nervous system , promoted autophagy and induced the clearance of inflammasome, resulting in neuroprotective actions . In our study, we demonstrated that both DHM and Sal B treatments effectively inhibited astroglia- and microglia-mediated neuroinflammation. It appears that DHM and Sal B can penetrate the blood-brain barrier and display multiple pharmacological activities, including oxidation resistance, anti-tumor properties and neuroprotection [23, 64], indicating potential for clinical application . Both DHM and Sal B displayed a protective role towards dopaminergic neurons by exerting neuroprotective effects [66, 67].
Through small molecule screening, we have identified two small molecules, DHM and Sal B that can inhibit α-synuclein aggregation in cell-free conditions. In α-synuclein overexpressing cell and animal models, we have demonstrated that both DHM and Sal B can inhibit α-synuclein accumulation and aggregation in cells and mouse brains. Decreasing α-synuclein aggregates concomitantly activates CMA pathways by increasing expression of LAMP-2A and macroautophagy by increasing LC3-II and LAMP-1, and is accompanied by the inhibition of microglial activation and neuroinflammation. Our results show that DHM and Sal B are effective in modulating α-synuclein accumulation and aggregate formation and augment CMA and macroautophagy. Furthermore, many chemotherapeutic agents have been reported to induce CMA activation, suggesting that autophagic protein degradation could be a potential approach to prevent and treat synucleinopathies. Our study strongly suggests that these two compounds may represent a detoxification and anti-inflammatory mechanism which could be targeted for clinical interventions of PD caused by abnormal accumulation and aggregation of α-syn.
We thank Dr. Andrew McCourt for linguistic editing.
JYL conceived and coordinated the study. WJZ and JYL designed the experiments and wrote the paper. All authors reviewed and contributed to the writing. WJZ performed in vitro and in vivo (with old animals) experiments, analyzed and interpreted the data. MA, NNV, AS and OMWA performed the cell-free screening of the compounds. CH, MD and WL performed in vivo experiments (with young animals). ZYW provided intellectual input. TFO provided reagents for in vitro experiments. All authors read and approved the final manuscript.
We would like to acknowledge financial supports by the National Natural Science Foundation (81430025, 81701265, 31800898, U801681). Acknowledgements are also to the supports of the Swedish Research Council (K2015-61X-22297-03-4), EU-JPND (aSynProtec) and EU-JPND (REfrAME), EU H2020-MSCA-ITN-2016 (Syndegen), BAGADILICO-Excellence in Parkinson and Huntington Research, the Strong Research Environment MultiPark (Multidisciplinary research on Parkinson’s disease), the Swedish Parkinson Foundation (Parkinsonfonden), Torsten Söderbergs Foundation, Olle Engkvist Byggmästere Foundation. W.L. is supported by a scholarship from the China Scholarship Council. TFO is supported by the DFG Center for Nanoscaly Microscopy and Molecular Physiology of the Brain (CNMPB).
Ethics approval and consent to participate
All animal experiments followed the Institutional Animal Care and all procedures were performed under the specifications set by the Ethical Committee for Use of laboratory animals at Lund University, Sweden and at Northeastern University, China.
Consent for publication
All the authors have approved the manuscript.
The authors declare that they have no competing interests.
- 6.Yang RX, Gao GD, Mao ZX, Yang Q. Chaperone-Mediated Autophagy and Mitochondrial Homeostasis in Parkinson's Disease. Parkinsons Dis-Us; 2016.Google Scholar
- 17.Ebrahimi-Fakhari D, Unni VK, Rockenstein E, Masliah E, Hyman BT, McLean PJ. Distinct roles in vivo for the ubiquitin-proteasome system and the autophagy-lysosomal pathway in the degradation of alpha-synuclein. Movement Disorders. 2011;26:S15–S6.Google Scholar
- 59.Bai LJ, Mei XF, Wang YF, Yuan YJ, Bi YL, Li G, et al. The Role of Netrin-1 in Improving Functional Recovery through Autophagy Stimulation Following Spinal Cord Injury in Rats. Front Cell Neurosci. 2017;11.Google Scholar
- 61.Ren Z, Yan P, Zhu L, Yang H, Zhao Y, Kirby BP, et al. Dihydromyricetin exerts a rapid antidepressant-like effect in association with enhancement of BDNF expression and inhibition of neuroinflammation. Psychopharmacology (Berl). 2017.Google Scholar
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