(C-99) Sea urchin gonad-derived Extracellular vesicles for improving the inflammatory environment and enhancing Schwann cell function in early peripheral nerve defects

Introduction:: The peripheral nervous system (PNS) extends throughout the body and plays an important role in transmitting sensory and motor stimuli to the central nervous system. PNS has limited regenerative capacity when the defect is large, such as from trauma or cancer. In the early stage of PNS regeneration, the role of inflammatory response and Schwann cells is important. Recently, research on the application of extracellular vesicles (EVs) to such inflammation and tissue regeneration has been actively conducted. EVs are nano-sized lipid bilayer vesicles released by all cell types. EVs act as messengers for cell-to-cell signaling. Inside, it contains various biological substances, such as proteins, lipids, nucleic acids, and metabolites. In particular, it contains miRNAs that play a key role in regulating other genes within cells. Besides, EVs reflect the biological characteristics of their parents. Studies on EVs have been actively conducted by extracting them from terrestrial animals or stem cells. However, marine organisms-derived EVs are limited.

In this study, we have extracted EVs from sea-urchins belonging to the Echinoderm group with their excellent regenerative ability. Previous genetic analysis studies, it was confirmed that sea-urchins have complex immune systems than vertebrates, such as Toll-like receptors and NOD-like receptors.

Therefore, we hypothesized sea urchin gonad-derived EVs (UGEVs) contain physiologically active substances with the above characteristics. We aimed to characterize UGEVs and confirm the control of the inflammatory response and the enhancement of Schwann cell function.


Materials and Methods:: We extracted extracellular matrix from sea urchin gonads treated with buffer, followed by collagenase treatment, filtration, and precipitation to collect ECM-bounded EVs. Particle size distribution and Morphology were conducted using dynamic light scattering (DLS) and SEM. Protein concentration and size distribution were performed by Bradford assay and SDS-PAGE. Uptake, cell viability, proliferation, and gene expression analysis were all conducted with macrophage cells and Schwann cells. To confirm endocytosis, PKH-26 red fluorescent was treated. Cell viability was checked after EVs treatment using calcein-AM and ethidium-homodimer. Proliferation was confirmed after EVs treatment using a water-soluble tetrazolium salt assay. Cell migration assay was performed by treating Schwann cells with EVs. Gene expression analysis compared the LPS-induced inflammatory group and EVs-treated group in macrophages and the normal group and EVs-treated group in Schwann cells. mRNA was extracted using an isolation kit and transcribed using a cDNA synthesis kit. For RT-qPCR, SYBR Green Reaction Mix was used. Relative quantification was normalized to GAPDH as an internal control.

Results, Conclusions, and Discussions:: The size of UGEVs was about 30-100 nm and round in shape. The protein content of destroyed UGEVs was higher than normal UGEVs. The protein analysis has the evidence that proteins are contained inside. In addition, EVs are protecting proteins of various sizes. UGEVs were labeled with PKH-26, which means that UGEVs have a lipid membrane. Cells were successfully uptake by PKH-labeled UGEVs.

Macrophages and Schwann cells did not show UGEVs-induced necrosis. The effect of UGEVs on cell proliferation was evaluated through mitochondrial activity. Cell proliferation increased as the concentration of UGEVs increased in both macrophages and Schwann cells.

UGEVs were treated in an LPS-induced inflammatory macrophage model. As a result of RT-qPCR, the UGEVs treatment group effectively inhibited pro-inflammatory cytokines compared to the Inflammation group.

To confirm the improvement of Schwann cell function, migration assay and RT-qPCR for neurotrophic factors were performed. Migration of Schwann cells was promoted by UGEVs. The expression of neurotrophic factors was also high in the UGEVs-treated group.

We demonstrated that the UGEVs have the same characteristics as traditional EVs. UGEVs were not cytotoxic to macrophages and Schwann cells, and had a positive effect on cell proliferation by confirming mitochondrial activity. In an inflammatory environment, UGEVs have been demonstrated to effectively inhibit pro-inflammatory cytokines and increased the expression of neurotrophic factors in Schwann cells. UGEVs can rapidly improve the initial inflammatory environment in peripheral nerve defects. In addition, it can positively affect the regeneration of peripheral nerves by improving the recruitment and function of Schwann cells responsible for the early stage of regeneration.

In future studies, UGEVs will be applied to neuronal cell regeneration and various inflammation-related disease models and their effects will be verified. In addition, this study shows that various values can be created if it is expanded to marine life or marine waste that adversely affects the ecosystem.

Keywords : Extracellular vesicles, Sea urchin gonad, Anti-Inflammation, Peripheral nervous system


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