Link between organic nanovescicles from vegetable kingdom and human cell physiology: intracellular calcium signalling

Abstract

Background: Plant-derived nanovesicles (PDNVs) are a novelty in medical and agrifood environments, with several studies exploring their functions and potential applications. Among fruits, apples (sp. Malus domestica) have great potential as PDNVs source, given their widespread consumption, substantial waste production, and recognized health benefits. Notably, apple-derived nanovesicles (ADNVs) can interact with human cell lines, triggering anti-inflammatory and antioxidant responses. This work is dedicated to the comprehensive biochemical characterization of apple-derived nanovesicles (ADNVs) through proteomic and lipidomic analysis, and small RNAs sequencing. This research also aims to shed light on the underlying mechanism of action (MOA) when ADNVs interface with human cells, through observation of intracellular calcium signalling in human fibroblasts, and to tackles differences in ADNVs content when isolated from fruits derived from integrated and organic production methods cultivars.

Results: The ADNVs fraction is mainly composed of exocyst-positive organelles (EXPOs) and MVB-derived exosomes, identified through size and molecular markers (Exo70 and TET-3-like proteins). ADNVs' protein cargo is heterogeneous and exhibits a diverse array of functions, especially in plant's protection (favouring ABA stress-induced signalling, pathogen resistance and Reactive Oxygen Species (ROS) metabolism). Noteworthy plant miRNAs also contribute to phytoprotection. In relation with human cells lines, ADNVs elicit spikes of intracellular Ca²⁺ levels, utilizing the cation as second messenger, and produce an antioxidant effect. Lastly, organic samples yield a substantial increase in ADNV production and are particularly enriched in bioactive lysophospholipids.

Conclusions: We have conclusively demonstrated that ADNVs confer an antioxidant effect upon human cells, through the initiation of a molecular pathway triggered by Ca²⁺ signalling. Within ADNVs, a plethora of bioactive proteins, small RNAs, and lipids have been identified, each possessing well-established functions within the realm of plant biology. While ADNVs predominantly function in plants, to safeguard against pathogenic agents and abiotic stressors, it is noteworthy that proteins with antioxidant power might act as antioxidants within human cells.

Keywords: Apple-derived extracellular vesicles; Calcium signalling; Lipidomic; Plant-derived extracellular vesicles; Proteomic; miRNA.

Fig. 1

Quantification, size distribution and morphological characterization of ADNVs. A TEM Imaging of ADNVs from GD and GDBio samples; B TRPS analysis output. The graph represents the distribution size (diameter) in nm, correlated to the population percentage for each bean

Fig. 2

Networks obtained from Cystoscape visualization software, following enrichment analysis of proteomic outcome with g: Profiler online software. A, B The figure shows Biological Process (BP) network of all proteins (A), each node is labeled with the pathway entry name (in reference to GO database), and (B) their clustering through AutoAnnotate tool; C Cellular Compartment (CC) network of said proteins as for BP; D–E Molecular Function (MF) pathway analysis showing all pathways (D) and pathway clustering with annotation (E) as for BP

Fig. 3

Comparison of proteomic results between GD and GDBio samples. A Principal Component Analysis (PCA) of GD (yellow) and GDBio (red) samples pertaining the protein characterization of samples; B Significant proteins’ expression in GD and GDBio samples replicate

Fig. 4

miRNA expression in GD (A) and GDBio (B) samples. The graph reports READs value on a logarithmic (log₁₀) scale. The dotted line represents READs value of 100

Fig. 5

A–D Lipid classification for each sample belonging to the GD and GDBio clusters. A Overall lipid content in each sample replica; B Lipidic function distribution in membrane (MEM), storage compartment (STO) and lysosome (LYS); C Characterization of lipidic structure in sterol esters (STE), sterols (ST), sphingolipids (SL), glycophospholipids (GPL), glycolipids (GL) and fatty acids (FA); D Lipidic class distribution as percentage

Fig. 6

Pathway analysis on lipidomic profiles, performed with MetaboAnalyst 5.0. for GD and GDBio samples. “Pathway impact” represents a combination of the centrality and pathway enrichment results; higher impact values represent the relative importance of the pathway; circle size indicates the impact of the pathway, the colour (from yellow to red) represents the significance which is also represented by –log₁₀(p-value)

Fig. 7

Intracellular Ca²⁺ levels. A The curves are representations of intracellular calcium variations, depicted as a ratio of the Relative Fluorescence Units (RFU) and frames, in cells standing in a medium with or without EGTA. The arrow indicates the addition of ADNVs to the cell medium; B Area Under the Curve (AUC) values for calcium variation provoked by ADNVs addition, in cells standing in a medium with or without EGTA. There is no statistical significance between the two distributions

Fig. 8

A Total antioxidant capacity. Measure of the ABTS + scavenging ratio of ADNVs, expressed as percentage relative to the control condition. B Mitochondrial superoxide level. The Relative Fluorescence Unit (RFU), normalized on the number of cells in each sample, is compared between cells treated with TNFα, cells treated with TNFα and ADNVs at increasing concentrations, and untreated cells. *p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001