Objectives: To investigate the mechanism of Yiyuan moxibustion in improving lipid peroxidation and urine retention in spinal cord injury (SCI) rats by regulating ferroptosis via the nuclear factor erythroid 2-related factor 2 (NRF2)/glutathione peroxidase 4 (GPX4) pathway.
Methods: Forty-eight female Wistar rats were randomly divided into sham operation, model, moxibustion and moxibustion + inhibitor (combination) groups, with 12 rats in each group. SCI induced urinary retention model was established by using Allen's method. Moxibustion was applied to "Shenque" (CV8), "Guanyuan"(CV4) and "Zhongji" (CV3) for 15 min, once daily for 14 d. Rats in the combination group received moxibustion combined with intraperitoneal injection of the NRF2 inhibitor ML385 (30 mg/kg) once daily for 14 consecutive days. Urodynamic analysis was used to record bladder leak point pressure, maximum bladder volume, and bladder compliance to evaluate bladder function. H.E. staining was used to observe morphological changes in spinal cord tissue. Prussian blue staining was used to observe iron deposition in spinal cord tissue. Transmission electron microscopy (TEM) was used to observe mitochondrial morphological changes in spinal cord tissue. Colorimetric assays were used to measure serum Fe2+, malondialdehyde (MDA), and reduced glutathione (GSH) levels. Western blot was used to detect the protein expressions of NRF2, GPX4, and solute carrier family 7 member 11 (SLC7A11) in spinal cord tissue.
Results: Rats in the model group had disrupted spinal architecture, vacuolated neurons, swollen mitochondria with lost cristae, and brownish iron deposits, which were relatively milder in the moxibustion group. Compared with the sham operation group, the bladder leak point pressure, serum GSH content, and expression levels of NRF2, SLC7A11, and GPX4 proteins in spinal cord tissue were all significantly reduced (P<0.01), while the maximum bladder volume and bladder compliance, as well as the serum contents of Fe2+ and MDA, were significantly increased (P<0.01) in the model group. When compared to the model group and the combination group, the moxibustion group exhibited a decrease in maximum bladder volume, bladder compliance, serum Fe2+ and MDA content (P<0.01), but an increase in bladder leak point pressure, GSH content, and the expression of NRF2, SLC7A11, and GPX4 proteins (P<0.01, P<0.05).
Conclusions: Yiyuan moxibustion may alleviate lipid peroxidation caused by ferroptosis in spinal neurons by up-regulating the NRF2/GPX4 pathway, thereby promoting the recovery of neural regulation of bladder function and improving urine retention in SCI rats.
目的: 基于核因子类红细胞2相关因子2（NRF2）/谷胱甘肽过氧化物酶4（GPX4）通路观察益元灸改善脊髓损伤（SCI）大鼠尿潴留的作用机制。方法: Wistar大鼠按随机数字表法选取12只作为假手术组，其余大鼠按照改良Allen’s垂直打击法制备SCI模型，脊髓休克期过后，筛选出符合标准的SCI后尿潴留模型，每组12只。造模成功的大鼠按照随机数字表法分为模型组、益元灸组、益元灸+抑制剂组，每组12只。对益元灸组大鼠“神阙”“关元”“中极”进行益元灸治疗，每次15 min;益元灸+抑制剂组大鼠予益元灸联合腹腔注射NRF2抑制剂ML385（30 mg/kg）。两组均1次/d，连续治疗14 d。采用尿动力学分析仪记录膀胱漏尿点压力、膀胱最大容量，并分析膀胱顺应性，评价大鼠膀胱功能;HE染色观察脊髓组织的形态变化;普鲁士蓝染色观察脊髓组织铁沉积情况;透射电镜观察脊髓神经元线粒体形态学变化;比色法测血清Fe2+、丙二醛（MDA）、还原型谷胱甘肽（GSH）含量;Western blot法检测脊髓组织中NRF2、GPX4和溶质载体家族7成员11（SLC7A11）蛋白表达。结果: 假手术组大鼠脊髓组织结构完整;模型组脊髓神经元呈空泡化，神经元减少，线粒体边缘模糊、嵴消失且呈现空泡化，脊髓组织出现黄褐色铁沉积。益元灸组较模型组和益元灸+抑制剂组大鼠脊髓组织结构排列更有规律，空泡减少，神经元完整，线粒体嵴清晰，结构较为完整，无黄褐色铁沉积。与假手术组比较，模型组大鼠的膀胱漏尿点压力降低（P<0.01），膀胱最大容量和膀胱顺应性升高（P<0.01）;血清中的Fe2+、MDA含量升高（P<0.01），GSH含量降低（P<0.01）;脊髓组织NRF2、SLC7A11、GPX4蛋白表达水平均降低（P<0.01）。与模型组和益元灸+抑制剂组比较，益元灸组大鼠膀胱最大容量、膀胱顺应性降低，膀胱漏尿点压力升高（P<0.01）;血清Fe2+、MDA含量降低（P<0.01），GSH含量升高（P<0.01）;脊髓组织NRF2、SLC7A11、GPX4蛋白表达升高（P<0.01，P<0.05）。结论: 益元灸可能通过上调NRF2/GPX4通路，减轻脊髓神经元铁死亡引起的脂质过氧化，有助于SCI后脊髓对膀胱的神经调控功能恢复，改善SCI大鼠的尿潴留。.
Keywords: Ferroptosis; Lipid peroxidation; Spinal cord injury; Yiyuan moxibustion.