Bone marrow derived stem cells facilitate axonal regeneration in a rat model of 2, 5-hexanedione-induced neuropathy via miRNA-dependent and independent routes

Abstract Background Peripheral polyneuropathies are clinically difficult situations and can be caused by exposure to occupational hazardous materials, such as N-hexane, which is still being used in developing countries. Autologous transplantation with bone marrow-mesenchymal stem cells (BMSCs) could be a promising therapeutical approach but its effect on toxic chemical-induced neuropathy remains undetermined. The present study sought to understand whether BMSCs could improve axon regeneration in Hexane-caused polyneuropathies and attempted to understand the associated molecular mechanisms. Methods A rat model of 2,5-hexanedine (HD)-induced polyneuropathy was established by intraperitoneally injection of 400 mg/kg/day HD, 5 times/week for 5 weeks. After the development of characteristic phenotypes, rats were given a single bolus tail-vein injection of BMSCs (5×107 cells/kg) and followed-up for 5 weeks. Sciatic nerves of HD/HD + BMSCs treated rats, as a representative tissue for peripheral nerve, were analyzed using electron microscopy, immunohistochemistry, western blotting and RNA sequencing. To further dissect the underlying mechanism, cultured DRG neurons +/- Schwann cells were challenged with HD/BMSC-derived conditional medium (BMSC-CM), in the presence or absence of NGF/Akt/mTOR inhibitors, recombinant NGF, miRNA mimics or miRNA inhibitors and further tested for changes in cellular and molecular levels. Results BMSCs engrafting significantly improved motor function recovery. Accelerated axon regeneration in sciatic nerves was evidenced by transmission electron microscopy, GAP43 western blotting and MBP/SMI312 immunostaining. Increased NGF expression and signaling activation was evidenced in response to BMSC/BMSC-CM treatment. In cultured cells, BMSC-CM’s effect was abolished by anti-NGF antibody or TrkA inhibitor K252a but reinforced by recombinant NGF. The activation of PI3K-Akt-mTOR-CREB signaling was observed and Akt and mTOR inhibitors were shown to attenuate BMSCS-promoted axon regeneration. While NGF was shown to be secreted by BMSCs in culture medium, miRNA array suggested certain miRNAs might also participate to regulate NGF expression. Three let-7 family miRNAs, including let-7a-5p, let-7d-5p and let-7e-5p, and 69 other miRNAs were found to be differentially expressed in HD and HD/BMSC groups. The regulatory effect of let-7e-5p on NGF expression was examined using miRNA mimics and inhibitors. Conclusions Together, our study demonstrated that BMSC transplantation significantly improve axon regeneration in hexane-induced polyneuropathy by activating NGF-PI3K-Akt-mTOR-CREB signaling via two independent mechanisms.