High-Frequency Repetitive Transcranial Magnetic Stimulation Mediates Autophagy Flux in Human Bone Mesenchymal Stromal Cells via NMDA Receptor-Ca2+-Extracellular Signal-Regulated Kinase-Mammalian Target of Rapamycin Signaling

Aim: Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive and safe technique for treatment of central and peripheral nerve injury. In recent years, this technique has been widely used in clinic, and an increasing number of studies have reported its mechanisms. In this study, we investigated the mechanisms of rTMS-mediated Autophagy flux in human bone mesenchymal stromal cells (BMSCs).

Methods: A frequency of 50 Hz was employed. Cells were divided into five groups: (1) normal, (2) sham, (3) 0.5 T, (4) 1.0 T, and (5) 1.5 T. Cells were stimulated for 20 min/day. The levels of p62, LC3-II/I, phosphorylated extracellular signal-regulated kinase (p-ERK), ERK, phosphorylated-AKT (p-AKT), Akt, phosphorylated mammalian target of rapamycin (p-mTOR), mTOR, phosphorylated protein kinase A (p-PKA), PKA, phosphorylated epidermal growth factor receptor (p-EGFR), EGFR, Nanog, Oct4, Sox2, and NMDA Receptor (NMDAR1) were investigated by western blotting. Intracellular calcium (CA²⁺) levels were quantified by flow cytometry. p62 and LC3 expression was also assessed by immunofluorescence analysis.

Results: In the 0.5 T group, rTMS increased the expression of LC3-II/I, p-ERK/ERK, and NMDAR1 and decreased the levels of p62 and p-mTOR/mTOR than in the normal group. The ratio of p-AKT/Akt, p-PKA/PKA, and p-EGFR/EGFR and the expression of Nanog, Oct4, and Sox2 remained unchanged. Immunofluorescence analysis revealed colocalization of p62 with LC3 puncta, and flow cytometry analysis displayed that CA²⁺ levels were elevated. However, in the 1.0 and 1.5 T groups, no changes in the expression of these Autophagy markers were observed.

Conclusion: In the 0.5 T group, high-frequency rTMS can induce Autophagy through NMDAR-Ca²⁺-ERK-mTOR signaling in BMSCs. In the 1.0 and 1.5 T groups, Autophagy is not activated.