ABSTRACT
Objective
This study investigated the molecular mechanism of whether hUC-MSCs-EVs repressed PTEN expression and activated the PI3K/AKT pathway through miR-29b-3p, thus inhibiting LPS-induced neuronal injury.
Methods
hUC-MSCs were cultured and then identified. Cell morphology was observed. Alizarin red, oil red O, and alcian blue staining were used for inducing osteogenesis, adipogenesis, and chondrogenesis. EVs were extracted from hUC-MSCs and identified by transmission electron microscope observation and Western blot. SCI neuron model was established by 24h lipopolysaccharide (LPS) induction. After the cells were cultured with EVs without any treatment, uptake of EVs by SCI neurons, miR-29b-3p expression, cell viability, apoptosis, caspase-3, cleaved caspase-3, caspase 9, Bcl-2, PTEN, PI3K, AKT, and p-Akt protein levels, caspase 3 and caspase 9 activities, and inflammatory factors IL-6 and IL-1β levels were detected by immunofluorescence labeling, RT-qPCR, MTT, flow cytometry, Western blot, caspase 3 and caspase 9 activity detection kits, and ELISA. The binding sites between PTEN and miR-29b-3p were predicted by the database and verified by dual-luciferase assay.
Results
LPS-induced SCI cell model was successfully established, and hUC-MSCs-EVs inhibited LPS-induced apoptosis of injured spinal cord neurons. EVs transferred miR-29b-3p into LPS-induced injured neurons. miR-29b-3p silencing reversed EV effects on reducing LPS-induced neuronal apoptosis. miR-29b-3p reduced LPS-induced neuronal apoptosis by targeting PTEN. After EVs-miR-inhi and si-PTEN treatment, inhibition of the PI3K/AKT pathway reversed hUC-MSCs-EVs effects on reducing LPS-induced neuronal apoptosis.
Conclusion
hUC-MSCs-EVs activated the PI3K/AKT pathway by carrying miR-29b-3p into SCI neurons and silencing PTEN, thus reducing neuronal apoptosis.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Author contributions
XX, WL, ZX contributed to the study concepts, study design, and definition of intellectual content; ZS, HY, YW contributed to the literature research; XX, WL contributed to the manuscript preparation and ZX, ZS, HY, XW contributed to the manuscript editing and review; YZ, LX, DJ, RJ contributed to the experimental studies and data acquisition; XX, WL, ZX contributed to the data analysis and statistical analysis. All authors read and approved the final manuscript.
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/03008207.2022.2060826
Availability of data and materials
All the data generated or analyzed during this study are included in this published article.
Ethics declarations
The experiments were authorized by the academic ethics committee of The Xiangya Hospital of Central-South University. All procedures strictly followed the code of Declaration of Helsinki. All the subjects involved were fully informed of the objective of the study and signed informed consent before sampling.