Gardenia-derived extracellular vesicles exert therapeutic effects on dopaminergic neuron apoptosis-mediated Parkinson's disease

Abstract

Plant-derived extracellular vesicles (EVs) show health benefits. Gardenia jasminoides Ellis, known for its neuroprotective properties, lacks therapeutic investigation on gardenia-derived extracellular vesicles (GDEVs). This study investigated the value of GDEVs in Parkinson's disease (PD) using rotenone (Rot)-induced Parkinsonism models in dopaminergic PC12 neuron cells and Caenorhabditis elegans. PD features apoptosis in dopaminergic neurons, while GDEVs alleviate PD by mitigating mitochondrial-mediated apoptosis. Specifically, GDEVs improve Rot-induced mitochondrial dysfunction to reduce cytochrome C release and apoptosis. Consequently, GDEVs reduce the risk of PD by lowering α-synuclein levels and regulating dopamine release. RNA sequencing and subsequent studies showed that GDEVs reduce p38 MAPK and p53 phosphorylation levels, and increase the Bcl-2/Bax ratio to prevent apoptosis in PC12 cells. In Caenorhabditis elegans, we verified that GDEVs reduce PD progression by increasing dopaminergic neurons using BZ555 mutants, and enhance dopamine release and motility. This study highlights the therapeutic potential of GDEVs in preventing neurodegenerative diseases.

Fig. 1. Isolation and characterization of GDEVs.

A The schematic diagram illustrates the extraction of GDEVs from gardenia. The Fig. 1A was created with Biorender.com. B NTA analyzed the particle concentration and size of GDEVs. C TEM observed the morphology of GDEVs. GDEVs were obtained using different crushing speeds and extraction methods, with particle concentrations as follows: a: 1.40 ± 0.03 × 10¹¹ (particles/mL), b: 1.61 ± 0.13 × 10¹⁰ (particles/mL), c: 1.01 ± 0.05 × 10¹¹ (particles/mL), d: 3.62 ± 0.22 × 10¹¹ (particles/mL).

Fig. 2. Characterization and cellular uptake of GDEVs by PC12 cells.

A Protein concentration of GDEVs. B Total RNA concentration of GDEVs. C Zeta potential of GDEVs. D–E Visualization of DiD-labeled GDEVs taken up by PC12 cells with quantification of fluorescence intensity. DiD was incorporated into GDEVs, loaded into 100 kDa ultrafiltration tubes (Millipore, American), and recovered by PBS elution to obtain GDEVs-DiD.

Fig. 3. GDEVs decreased free radical generation and cellular damage in PC12 cells.

A MTT assay revealing the effect of different Rot concentrations on cell viability. B MTT assay displaying the effect of different GDEVs protein concentrations on cell viability. C GDEVs at different concentrations improve the reduction in cell viability induced by 1 μM Rot. D and E Statistical analysis of the images shown in G and H. F The bright-field of PC12 cells. G DCF staining indicates ROS production. H DHE staining shows superoxide ions (O₂⁻) levels. I DAPI staining shows nuclear damage. The scale bar in Fig. 3G–I is 50 μm.

Fig. 4. GDEVs improved PD associated with mitochondria-dependent apoptosis in dopaminergic PC12 neuronal cells.

A and B Mitochondrial membrane potential was assessed by Rh123 probe and fluorescence intensity was quantified. C and D Mitochondrial membrane integrity was evaluated by NAO probe and fluorescence intensity was quantified. E and F Cytochrome C levels and quantification of band intensity. Unprocessed blots for Fig. 4E are provided in Supplementary Fig. 10. G The left peak (percentage) in (a) and (b) is normal cells, and the right is (a) apoptotic cells labeled with Annexin V-FITC and (b) apoptotic or dead cells stained with PI. H Late apoptotic cells were counted by flow cytometry. I TUNEL assay detected DNA breaks and fragmentation. J and K Levels of α-synuclein aggregation and its phosphorylation form. L Dopamine release levels. The scale bar in Fig. 4A, C, and I is 50 μm.

Fig. 5. RNA sequencing screened the differential mRNA enrichment in the MAPK/p53 pathway.

A Venn diagram demonstrating the number of differential genes between groups. B Volcano plot displaying the differentially expressed mRNAs. C Enrichment of mRNAs in the KEGG pathway. a. the Rot vs. Ctrl group, b. the GDEVs vs Rot group. D Differential genes in the MAPK and p53 signaling pathways. E and F GSEA-KEGG analyzed the importance of the MAPK and p53 signaling pathways, with significant enrichment indicated by |NES | > 1, p-value < 0.05, and adjusted p-value < 0.25.

Fig. 6. GDEVs inhibited apoptosis in dopaminergic PC12 neuronal cells by inactivating the p38 MAPK/p53 signaling pathway.

A Expression levels of p-p38 MAPK, p38 MAPK, p-p53, p53, and GAPDH proteins in PC12 cells. B Expression levels of Bcl-2, Bax, cleaved Caspase-3, Caspase-3, and GAPDH proteins in PC12 cells. C and D Quantitative analysis of p-p38 MAPK/p38 MAPK and p-p53/p53 levels. E–F Quantitative analysis of Bcl-2/Bax and cleaved Caspase-3/Caspase-3 levels. Unprocessed blots for Fig. 6A and B are provided in Supplementary Fig. 10. (G) Graphs depicting the interaction between the p38 MAPK/p53 and Caspase-3 signaling pathways in regulating apoptosis. The Fig. 6G was created with Biorender.com.

Fig. 7. GDEVs alleviated apoptosis-related PD in dopaminergic PC12 cells via the p38 MAPK/p53 pathway: validation with SB203580 inhibition.

PC12 cells in the Rot+SB203580 group were treated with the SB203580 (10 μM) for 24 h. A and B Protein expression levels detected by WB, and unprocessed blots for Fig. 7A, B are provided in Supplementary Fig. 11. C and D Quantification of mitochondrial membrane potential and fluorescence intensity. E and F Mitochondrial membrane integrity and quantification of fluorescence intensity. G and H Cytochrome C levels and band intensity quantification. Unprocessed blots for Fig. 7G are provided in Supplementary Fig. 11. I and J Cell membrane integrity was assessed by Annexin V-FITC binding to PS. K DNA breaks were labeled with YSFluor™ 488-12-dUTP. L and M Levels of α-synuclein and its phosphorylation form. N Dopamine release levels. The scale bar in Figs. 7C, E, and K is 50 μm.

Fig. 8. GDEVs improved dopaminergic loss-associated PD in C. elegans.

A A schematic diagram illustrating the mechanisms by which GDEVs improve dopaminergic loss-associated PD in N2 nematodes. The Fig. 8A was created with Biorender.com. B Survival rate of N2 nematodes. C DCF probed ROS generation. D DHE probed O₂⁻ production. E Rh123 probed mitochondrial membrane potential. F NAO probed mitochondrial membrane integrity. G The mRNA levels of pmk-1 and cep-1 in nematodes homologous to p38 MAPK and p53 in PC12 cells. H The mRNA levels of ced-9 homologous to Bcl-2, egl-1 homologous to Bax, and ced-3 homologous to Caspase-3. I Expression of dopaminergic neurons with GFP expression in BZ555 mutants. J and K Levels of α-synuclein and its phosphorylation form in N2 nematodes. L Dopamine release levels in N2 nematodes. (M-O) Motility of N2 nematodes for foraging, head swings, and body bends. The quantitative analysis of fluorescence intensity in Fig. 8C, D and Fig. 8I is presented in Supplementary Fig. 8. The scale bar is 250 μm in Fig. 8C–F and 25 μm in Fig. 8I.