Gat1 mediated Schwann cell proliferation after nerve decompression in peripheral constriction injury of rat sciatic nerve

Peripheral compressive neuropathy results in significant neuropathic pain, muscle weakness, and prolonged neuroinflammation. Surgical decompression remains the gold standard for treatment; however, the outcomes are often suboptimal, and patients have a high recurrence rate. In this study, we employed a reproducible and controllable chronic constriction injury (CCI) rat model to simulate early nerve decompression. The rat sciatic nerve was constricted for a short duration, followed by release, which induced reversible sensorimotor dysfunction. We further explored the genes involved in nerve regeneration via NanoString analysis. Compared with the control nerves, the differentially expressed genes (DEGs) Mmp12, Il1rn, Fcgr3, Hmox1, Lst1, Ccl2, Nlrp3, Il2rg, and Sell were downregulated in the constricted nerves at days 7 and 14 after release. Other significant DEGs, such as Gsn, Ugt8a, Snca, Slc6a1, Pmp22, Kdm5d, Mal, Plp1, Slc2a1, Gjb1, Cyp27a1, and Plekhb1, were upregulated on days 7 and 14. When the correlations between these upregulated DEGs and nerve regeneration were investigated, we found that Slc6a1, also known as GABA transporter 1 (Gat1), was associated with nerve regeneration. To further understand its role, we overexpressed Gat1 in a rat neuronal Schwann cell line, RT4-D6P2T. This led to increased viability of the Gat1-overexpressing cells. Additionally, neuronal markers including Nefh, S100b, and Ngfr, along with cell proliferation signaling molecules, such as p-Akt and p-Mapk, were elevated in these cells. The increase in viability and neuronal marker expression was inhibited by the Gat1 inhibitor tiagabine in Gat1-overexpressing cells. To simulate neuronal damage and recovery, we treated the cells with H2O2 for 2 hours and then allowed them to recover in growth medium for 1 and 7 days. The data indicated that the mRNA and protein levels of Gat1 were reduced on day 1 but upregulated by day 7. Consistently, the decrease in the expression of neuronal markers observed on day 1 was reversed by day 7. Taken together, our findings demonstrate that Gat1 plays a significant role in neuronal regeneration in both a cell model and an animal model of CCI.周围性压迫性神经病可引发剧烈神经病理性疼痛、肌肉无力，并伴随长期神经炎症。外科减压术仍是该疾病治疗的金标准，但临床疗效往往欠佳，且患者复发率较高。本研究采用可重复、可控的慢性压迫损伤（chronic constriction injury, CCI）大鼠模型，模拟早期神经减压过程，具体为将大鼠坐骨神经短时压迫后解除压迫，由此诱导出可逆的感觉运动功能障碍。本研究通过NanoString分析技术，深入探究了参与神经再生的相关基因。与对照组神经相比，解除压迫后第7天和第14天，坐骨神经组织中的差异表达基因（differentially expressed genes, DEGs）Mmp12、Il1rn、Fcgr3、Hmox1、Lst1、Ccl2、Nlrp3、Il2rg及Sell均呈下调表达；另有一批显著差异表达基因，包括Gsn、Ugt8a、Snca、Slc6a1、Pmp22、Kdm5d、Mal、Plp1、Slc2a1、Gjb1、Cyp27a1及Plekhb1，则在上述两个时间点呈上调表达。在分析上述上调差异表达基因与神经再生的关联时，我们发现Slc6a1（亦称γ-氨基丁酸转运体1（GABA transporter 1, Gat1））与神经再生密切相关。为进一步明确Gat1的生物学功能，我们在大鼠雪旺细胞系RT4-D6P2T中过表达Gat1，结果显示该细胞的活力显著提升；同时，神经标志物（如Nefh、S100b及Ngfr）以及细胞增殖信号分子（如p-Akt及p-Mapk）的表达水平均显著升高。Gat1抑制剂替加宾（tiagabine）可抑制过表达Gat1细胞的活力提升及神经标志物表达上调。为模拟神经元损伤与修复过程，我们用H₂O₂处理细胞2小时，随后将细胞置于生长培养基中分别恢复培养1天和7天。实验数据显示，Gat1的mRNA及蛋白表达水平在恢复培养第1天出现下调，但至第7天则呈现上调；与之相符的是，恢复培养第1天观测到的神经标志物表达下调现象，在第7天得到了逆转。综上，本研究结果证实，Gat1在细胞模型及CCI动物模型的神经元再生过程中均发挥重要调控作用。