Journal: International Journal of Molecular Sciences

Article Title: Ellagic Acid Prevents α-Synuclein Aggregation and Protects SH-SY5Y Cells from Aggregated α-Synuclein-Induced Toxicity via Suppression of Apoptosis and Activation of Autophagy

doi: 10.3390/ijms222413398

Figure Lengend Snippet: Preformed α-syn fibrils’ disaggregation by EA in a concentration-dependent manner, prevention of seeding aggregation and cell death. ( A ) Samples of preformed α-syn fibrils were incubated for 48 h at 37 °C with continuous shaking with or without EA at different molar ratios (aged α-syn:EA, 1:1, 1:4, 1:6). The fibril content was then estimated by the Th-S binding assay. The assays were performed in triplicate and all values are mean ± standard deviation. ( B ) α-syn monomers sample (100 μM) containing α-syn seeds (2 μM) incubated alone or in the presence of 10 or 50 μM EA for 5 h with continuous shaking at 37 °C. The amounts of preformed fibrils were estimated by the Th-S binding assay. The assays were performed in triplicate and all values are mean ± standard deviation. ( C ) The cytotoxicity of aged α-syn in SH-SY5Y human neuroblastoma cells was assessed by the MTT assay. The cells were treated for 48 h prior to MTT addition with samples of α-syn incubated alone or in the presence of EA (α-syn:EA, 1:1, 1:2, 1:4) for 11 days at 37 °C with continuous shaking. The final concentration of α-syn used for the MTT assay was 5 µM and EA at a concentration of 5, 10, or 20 µM (α-syn:EA, 1:1, 1:2, 1:4). The results are expressed as percentages of the control. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni Multiple Comparison Test, ***, p < 0.001; **, p < 0.01 compared with aged α-syn alone-treated group for A and C, and with α-syn + seeds for B.

Article Snippet: Wild-type SH-SY5Y human neuroblastoma cells were procured from Addexbio (San Diego, CA, USA).

Techniques: Concentration Assay, Incubation, Binding Assay, Standard Deviation, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Ellagic Acid Prevents α-Synuclein Aggregation and Protects SH-SY5Y Cells from Aggregated α-Synuclein-Induced Toxicity via Suppression of Apoptosis and Activation of Autophagy

doi: 10.3390/ijms222413398

Figure Lengend Snippet: Expression of apoptotic markers, Bax, BCL-2, and tumor suppressor protein p53 in SH-SY5Y wt cells treated with α-syn alone or pre-incubated with different molar ratios of EA. SH-SY5Y wt neuronal cells were seeded in 24-well plates at a density of 5 × 10 4 cells/well, and the cells were maintained for 24 h before treating for 24 h with a 5 µM final concentration of α-syn pre-incubated for 11 days at 37 °C with continuous shaking, in the presence or absence of EA at molar ratios of α-syn:EA of 1:1, 1:2, and 1:4. ( A ) Western blot analysis to examine the effect α-syn:EA on the expression of Bax in SH-SY5Y cells ( left panel), and the results were quantified as percentage relative to GAPDH expression ( right panel). ( B ) Western blot analysis to examine the effect α-syn:EA on the expression of BCL-2 in SH-SY5Y cells ( left panel), and the results were quantified as percentage relative to GAPDH expression ( right panel). ( C ) Ratio of the expression of Bax/BCL-2. ( D ) Western blot analysis to examine the effect α-syn:EA on the expression of the tumor suppressor protein p53 in SH-SY5Y cells ( left panel), and the results were quantified as percentage relative to GAPDH expression ( right panel). Data represent the mean value of three independent experiments ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni Multiple Comparison Test, ***, p < 0.001; **, p < 0.01; *, p < 0.05.

Article Snippet: Wild-type SH-SY5Y human neuroblastoma cells were procured from Addexbio (San Diego, CA, USA).

Techniques: Expressing, Incubation, Concentration Assay, Western Blot, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Ellagic Acid Prevents α-Synuclein Aggregation and Protects SH-SY5Y Cells from Aggregated α-Synuclein-Induced Toxicity via Suppression of Apoptosis and Activation of Autophagy

doi: 10.3390/ijms222413398

Figure Lengend Snippet: Expression of cell proliferation markers AKT/pAKT in SH-SY5Y wt cells treated with α-syn alone or α-syn pre-incubated with different molar ratios of EA. SH-SY5Y wt neuronal cells were seeded in 24-well plates at a density of 5 × 10 4 cells/well, and the cells were maintained for 24 h before treating for 24 h with a 5 µM final concentration of α-syn pre-incubated for 11 days at 37 °C with continuous shaking, in the presence or absence of EA at molar ratios of α-syn:EA of 1:1, 1:2, and 1:4. ( A ) Western blot analysis to examine the effect α-syn:EA on the expression of AKT protein in SH-SY5Y cells ( left panel), and the results were quantified as percentage relative to GAPDH expression ( right panel). ( B ) Western blot analysis to examine the effect α-syn:EA on the expression of pAKT in SH-SY5Y cells ( left panel), and the results were quantified as percentage relative to GAPDH expression ( right panel). ( C ) Ratio of the expression of pAKT/AKT. Data represent the mean value of three independent experiments ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni Multiple Comparison Test, ***, p < 0.001; **, p < 0.01; *, p < 0.05.

Article Snippet: Wild-type SH-SY5Y human neuroblastoma cells were procured from Addexbio (San Diego, CA, USA).

Techniques: Expressing, Incubation, Concentration Assay, Western Blot, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Ellagic Acid Prevents α-Synuclein Aggregation and Protects SH-SY5Y Cells from Aggregated α-Synuclein-Induced Toxicity via Suppression of Apoptosis and Activation of Autophagy

doi: 10.3390/ijms222413398

Figure Lengend Snippet: Aged α-syn causes nuclear condensation and DNA fragmentation in SH-SY5Y cells. SH-SY5Y cultured cells were treated with vehicle, vehicle + Ellagic acid, aged α-syn, and Ellagic acid + aged α-syn for 24 h, as indicated in the figure. ( A ) Cells were fixed, and stained with Hoechst 33342. Aged α-syn exposure significantly increased the apoptotic cells, as shown by the arrow. ( B ) Number of live and apoptotic cells are counted per field and presented as a ratio. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni Multiple Comparison Test, ***, p < 0.001; **, p < 0.01. Scale bar 50 µm.

Article Snippet: Wild-type SH-SY5Y human neuroblastoma cells were procured from Addexbio (San Diego, CA, USA).

Techniques: Cell Culture, Staining

Journal: International Journal of Molecular Sciences

Article Title: Ellagic Acid Prevents α-Synuclein Aggregation and Protects SH-SY5Y Cells from Aggregated α-Synuclein-Induced Toxicity via Suppression of Apoptosis and Activation of Autophagy

doi: 10.3390/ijms222413398

Figure Lengend Snippet: Expression of autophagy markers, LC3, and p62 proteins in SH-SY5Y wt cells treated with α-syn alone or α-syn pre-incubated with different molar ratios of Ellagic acid. SH-SY5Y wt neuronal cells were seeded in 24-well plates at a density of 5 × 10 4 cells/well, and the cells were maintained for 24 h before treating for 24 h with a 5 µM final concentration of α-syn pre-incubated for 11 days at 37 °C with continuous shaking, in the presence or absence of EA at molar ratios of α-syn:EA of 1:1, 1:2, and 1:4. ( A ) Western blot analysis to examine the effect α-syn:EA on the expression of LC3 protein in SH-SY5Y cells ( left panel), and the results were quantified as the ratio percentage of LC3-II/LC3-I relative to GAPDH expression ( right panel). ( B ) Western blot analysis to examine the effect α-syn:EA on the expression of p62 protein in SH-SY5Y cells ( left panel), and the results were quantified as percentage relative to GAPDH expression ( right panel). Data represent the mean value of three independent experiments ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni Multiple Comparison Test, ***, p < 0.001; **, p < 0.01; *, p < 0.05.

Article Snippet: Wild-type SH-SY5Y human neuroblastoma cells were procured from Addexbio (San Diego, CA, USA).

Techniques: Expressing, Incubation, Concentration Assay, Western Blot, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Ellagic Acid Prevents α-Synuclein Aggregation and Protects SH-SY5Y Cells from Aggregated α-Synuclein-Induced Toxicity via Suppression of Apoptosis and Activation of Autophagy

doi: 10.3390/ijms222413398

Figure Lengend Snippet: Improvement of autophagic flux by EA in α-syn-treated cells. Human neuroblastoma SH-SY5Y wt cells were seeded into 24-well plates and incubated for 24 h. The cells were treated with 20 µM of EA 3 h prior to 5 µM preformed α-syn fibrils exposure. To examine the autophagic flux, cells were treated with the autophagy inhibitor, chloroquine (10 µM), 1 h before α-syn fibrils exposure, as mentioned in the Methods Section. ( A ) Western blot analysis to examine the effect on the expression of LC3 protein in SH-SY5Y cells. ( B ) The results were quantified as percentage relative to GAPDH expression. Note that adding CQ increased the accumulation of LC3-II, indicating the activation of autophagic flux by EA. Data represent the mean value of three independent experiments ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni Multiple Comparison Test, ***, p < 0.001; **, p < 0.01, ns: non-significant.

Article Snippet: Wild-type SH-SY5Y human neuroblastoma cells were procured from Addexbio (San Diego, CA, USA).

Techniques: Incubation, Western Blot, Expressing, Activation Assay, Standard Deviation

Journal: Drug Delivery

Article Title: Multifunctional icariin and tanshinone IIA co-delivery liposomes with potential application for Alzheimer’s disease

doi: 10.1080/10717544.2022.2072543

Figure Lengend Snippet: The capability of liposomal formulations to across BBB in vitro . (A) Fluorescent signals within N2a cells are observed through a microscope, scale bar = 50 μm; (B) semi-quantitative analysis of relative fluorescence intensity. *, vs. Cou-6 liposomes. p < .05; (C) fluorescence intensity was analyzed by flow cytometry; (D) quantitative analysis of fluorescence intensity. *, vs. Cou-6 liposomes. p < .05. All data are presented as mean ± SD ( n = 3).

Article Snippet: Mouse brain microvascular endothelial cell line (bEnd.3) and mouse brain neuroma cell line (N2a) were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: In Vitro, Microscopy, Fluorescence, Liposomes, Flow Cytometry