Transcranial direct current stimulation promotes post-SCI motor recovery via miR-298-5p/ LC3-dependent autophagy activation

While transcranial direct current stimulation (tDCS) has been shown to contribute to motor recovery after spinal cord injury (SCI), the mechanisms remain unclear. This study investigated whether tDCS exerts neuroprotective effects by regulating apoptosis and autophagy. To achieve this aim, SCI was induced in rats using a modified Allen’s method with tDCS treatment. Otherwise,Furthermore, tDCS-responsive miRNAs were detected by using microRNA (miRNA) sequencing and validated via RT-qPCR. Similarly, tDCS regulatory mechanisms were verified through dual-luciferase assays and miRNA overexpression. Subsequently, Hâ‚‚Oâ‚‚-treated PC-12 cells simulated SCI in vitro to examine miR-298-5p's effects on apoptosis and autophagy. The findings revealed that miR-298-5p was upregulated post-SCI and downregulated after tDCS. In vitro, miR-298-5p silencing promoted autophagy and reduced apoptosis, whereas overexpression exacerbated neuronal injury. LC3 was a direct of miR-298-5p, and tDCS enhanced autophagy, reduced apoptosis, improved nerve regeneration, and minimized motor deficits after SCI. Notably, all tDCS-induced effects were counteracted after the overexpression of miR-298-5p by agomir. In summary, this study showed that while miR-298-5p could be detrimental to SCI, tDCS could increase autophagy flux and inhibit neuronal apoptosis by negatively regulating miR-298-5p, thereby improving the recovery of function in SCI.虽然经颅直流电刺激（transcranial direct current stimulation, tDCS）已被证实可促进脊髓损伤（spinal cord injury, SCI）后的运动功能恢复，但其具体作用机制仍尚不明确。本研究旨在探讨tDCS是否通过调控细胞凋亡与自噬发挥神经保护作用。为达成这一研究目标，本研究采用改良艾伦造模法（Allen’s method）构建大鼠脊髓损伤模型，并予以tDCS干预。此外，本研究通过微小RNA（microRNA, miRNA）测序技术筛选tDCS响应的miRNA，并借助实时定量聚合酶链反应（RT-qPCR）进行验证。同理，本研究通过双荧光素酶报告基因实验以及miRNA过表达技术验证了tDCS的调控机制。随后，本研究采用过氧化氢（H₂O₂）处理PC-12细胞以体外模拟脊髓损伤环境，以此探究miR-298-5p对细胞凋亡与自噬的影响。研究结果显示，脊髓损伤后miR-298-5p表达上调，而tDCS干预可使其表达下调。体外实验结果表明，沉默miR-298-5p可促进细胞自噬并抑制细胞凋亡，而过表达miR-298-5p则会加重神经元损伤。微管相关蛋白1轻链3（LC3）是miR-298-5p的直接作用靶点，tDCS可增强细胞自噬通量、抑制神经元凋亡、促进神经再生，并减轻脊髓损伤后的运动功能缺损。值得注意的是，通过agomir（miRNA激动剂）过表达miR-298-5p后，tDCS所诱导的所有保护效应均被抵消。综上所述，本研究证实，尽管miR-298-5p对脊髓损伤具有不利影响，但tDCS可通过负向调控miR-298-5p的表达，增强细胞自噬通量并抑制神经元凋亡，从而改善脊髓损伤后的功能恢复。