RESUMO
Background: After peripheral nerve injury, Schwann cells proliferate and migrate to the injured site, thereby promoting peripheral nerve regeneration. The process is regulated by various factors. Endothelial cells participate in the process via angiogenesis. However, the effects of endothelial cells on Schwann cells are not yet known. The present study sought to evaluate whether endothelial cells accelerate Schwann cell proliferation and migration. Methods: We established a co-culture model of rat Schwann cells (RSC96s) and rat aortic endothelial cells (RAOECs), and studied the effects of endothelial cells on Schwann cells by evaluating changes in Schwann cell proliferation and migration and related multiple genes and their protein expressions in the co-culture model. Results: The results showed that increasing the proportion of endothelial cells in the co-culture model enhanced the proliferation. At days 1 and 3 following the co-culturing, the relative growth rates of the co-cultured cells were 122.87% and 127.37%, respectively, which showed a significant increase in the viability compared to that of the RSC96s (P<0.05). In this process, the expression of Ki67 increased. The migration ability of Schwann cells was also enhanced. The migration capacity of Schwann cells was detected by wound-healing and Transwell assays. The results of the group with 15% of endothelial cells was significantly higher than the results of the other groups (P<0.0001 and P<0.05, respectively). Further, neuregulin 1 and glial fibrillary acidic protein increased the process of Schwann cell migration. Conclusions: The results showed that endothelial cells can promote the proliferation and migration of Schwann cells and participate in peripheral nerve regeneration.
RESUMO
Our present study investigated whether exosome secretion of nucleus pulposus cells (NPCs) is regulated by autophagy. Different autophagic states of NPCs were induced by rapamycin (Rap), bafilomycin A1 (Baf) and other agents, and it was found that exosomes were secreted in an autophagy-dependent manner. Activation or inhibition of autophagy increased or decreased, respectively, the amount of exosomes that were released into the extracellular space. In addition, in order to confirm that Rap-promoted release of exosomes was mediated by autophagy rather than other pathways, we used autophagy associated gene 5 (ATG5) small-interfering RNA (siRNA) to silence the expression of ATG5 gene, which is indispensable for autophagy. The results showed that siRNA against ATG5 (siATG5) induced an accumulation of intraluminal vesicles (ILVs) in NPCs and a concomitant decrease in the amount of exosomes isolated from supernatant. Ras homolog gene (Rho) and Rho-associated coiled-coil forming protein kinase (ROCK) family molecules are capable of cytoskeletal remodeling and affecting vesicle transport. Therefore, we carried out targeted interventions and evaluated the effects of the RhoC/ROCK2 pathway on the secretion of exosomes within autophagic environment. Knockdown of RhoC and ROCK2 with corresponding siRNA significantly inhibited the secretion of exosomes originating from ILVs in NPCs, even when NPCs were subsequently treated with Rap. Taken together, our findings suggest that autophagy positively regulates expression levels of RhoC and ROCK2, and that the RhoC/ROCK2 pathway exerts a key function on NPCs-derived exosome secretion.