RESUMO
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-1ß 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.
Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , MicroRNAs , Traumatismos da Medula Espinal , Azul Alciano/metabolismo , Azul Alciano/farmacologia , Apoptose , Caspase 3/metabolismo , Caspase 9/metabolismo , Caspase 9/farmacologia , Vesículas Extracelulares/metabolismo , Humanos , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Neurônios/metabolismo , PTEN Fosfo-Hidrolase , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/terapia , Cordão Umbilical/metabolismoRESUMO
Following recent advances in osteoimmunology, there is growing recognition of the vital role of immune cells in the osteogenesis process. The 3D-printed scaffold, as a substitute for injured and/or diseased bone tissues, has demonstrated satisfactory pro-osteogenetic performance. However, whether immune cells prompt the above pro-osteogenetic performance has not been elucidated in detail. In the present study, highly controllable Ti-6Al-4V scaffolds with different pore geometries were fabricated using a selective laser-melting technique, to reveal their osteoimmunological functions with macrophages. The results showed that macrophages displayed characteristics of M2 phenotype in response to scaffolds. As a result, an anti-inflammatory microenvironment was generated. When the pore geometry was considered, such observations were more apparent with the hexagonal pore scaffold than with the triangular one. In addition, inhibition of the toll-like receptor signaling pathway in macrophages has been proposed to cause the above phenomenon. Upon applying conditioned media derived from macrophages on pre-osteoblasts, the hexagonal pore scaffold group was found to significantly enhance osteoblastic differentiation, via macrophage-to-implant interactions. However, the effect of triangular pore scaffold was not statistically significant compared to that of hexagonal pore scaffolds or nonporous samples. In an attempt to quantify scaffold pore geometries, it was suggested that pores with higher circularity values tended to induce M2 polarization of macrophages, promote an anti-inflammatory milieu, and therefore, achieve better osteogenetic performance via immunomodulation.