FOXG1 interaction with SATB2 promotes autophagy to alleviate neuroinflammation and mechanical abnormal pain in rats with lumbar disc herniation

Most patients with lumbar disc herniation can be relieved or cured by surgical or non-surgical treatment; however, postoperative persistent radiculopathy is common. This study demonstrates the regulation of autophagy by the FOXG1/SATB2 axis in lumbar disc herniation (LDH). Rat dorsal root neurons were induced with TNF-α <i>in vitro</i>. Sprague Dawley (SD) rats were used to construct the LDH rat model, which was treated with <i>L. paracasei S16</i> or oe-FOXG1. Paw withdrawal threshold or latency assay (PWT/L) was performed. Peripheral blood samples were collected and analysed using ELISA and miRNAseq. RT-qPCR was used to analyse the expression of FOXG1, LC3B, Beclin1, p62, and SATB2. TUNEL staining and flow cytometry were used to analyse apoptosis. The expression of Cyclin D1, PCNA, Ki67, FOXG1, SATB2, and autophagy proteins was measured using western blotting. TNF-α induced low expression of FOXG1 and SATB2 in dorsal root ganglion (DRG) neurons of rats. TNF-α induced an increase in p62 protein and a decrease in LC3II/I and Beclin-1 proteins in neurons, which were blocked by oe-FOXG1. oe-FOXG1 suppressed inflammation and apoptosis in TNF-α-induced DRG neurons and LDH rats and promoted the expression of Cyclin D1, PCNA, and Ki67. Many miRNAs were increased in the peripheral blood of LDH rats, but decreased after <i>L. paracasei S16</i> intervention. <i>L. paracasei S16</i> affects miR-31a-5p and SATB2 expression. Dual luciferase reporter gene assay confirmed that miR-31a-5p bound to SATB2. Co-IP analysis confirmed the interaction between FOXG1 and SATB2. Silencing of SATB2 inhibited the beneficial effects of oe-FOXG1 in TNF-α-induced dorsal root ganglion neurons. Animal experiments further demonstrated that oe-FOXG1 improved LDH disease characteristics by downregulating PWT, PWL, inflammation, and apoptosis levels and upregulating SATB2-autophagy levels. MiR-31a-5p/SATB2 is involved in the treatment of <i>L. paracasei S16</i> in LDH rats. Overexpression of FOXG1 promotes autophagy through SATB2 to improve LDH levels This provides a new approach for the treatment of LDH.

多数腰椎间盘突出症（lumbar disc herniation, LDH）患者可通过手术或非手术治疗实现缓解或治愈，但术后持续性神经根病仍是常见并发症。本研究阐明了FOXG1/SATB2轴对腰椎间盘突出症（LDH）的自噬调控作用。研究通过体外实验采用肿瘤坏死因子-α（TNF-α）诱导大鼠背根神经元，并以斯普拉格·道利（Sprague Dawley, SD）大鼠构建LDH动物模型，分别给予副干酪乳杆菌S16（L. paracasei S16）或过表达FOXG1（oe-FOXG1）干预。采用爪回缩阈值/潜伏期试验（PWT/L）开展行为学检测；采集外周血样本，通过酶联免疫吸附试验（ELISA）与小RNA测序（miRNAseq）进行组学分析；采用实时定量聚合酶链反应（RT-qPCR）检测FOXG1、LC3B、Beclin1、p62及SATB2的表达水平；采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）染色与流式细胞术分析细胞凋亡情况；采用蛋白质印迹法（western blotting）检测细胞周期蛋白D1（Cyclin D1）、增殖细胞核抗原（PCNA）、Ki67、FOXG1、SATB2及自噬相关蛋白的表达水平。TNF-α可降低大鼠背根神经节（dorsal root ganglion, DRG）神经元中FOXG1与SATB2的基础表达水平；同时可升高神经元内p62蛋白含量，降低LC3II/I与Beclin-1蛋白的表达比例，上述效应可被oe-FOXG1所阻断。oe-FOXG1可抑制TNF-α诱导的DRG神经元及LDH模型大鼠体内的炎症反应与细胞凋亡，并促进Cyclin D1、PCNA与Ki67的表达。LDH模型大鼠外周血中多种miRNA表达上调，但经副干酪乳杆菌S16干预后，其表达水平显著下调。副干酪乳杆菌S16可调控miR-31a-5p与SATB2的表达；双荧光素酶报告基因实验证实miR-31a-5p可直接结合SATB2；免疫共沉淀（Co-IP）分析证实FOXG1与SATB2存在蛋白相互作用。沉默SATB2可抵消oe-FOXG1对TNF-α诱导的DRG神经元的保护作用。动物实验进一步验证，oe-FOXG1可通过下调爪回缩阈值与爪回缩潜伏期（PWT/PWL）、炎症反应与细胞凋亡水平，上调SATB2介导的自噬通路活性，从而改善LDH的疾病表型。miR-31a-5p/SATB2轴参与了副干酪乳杆菌S16对LDH模型大鼠的治疗过程。过表达FOXG1可通过SATB2介导的自噬通路改善LDH病情，本研究为腰椎间盘突出症的临床治疗提供了全新的潜在策略。