探究CCI大鼠海马铁死亡与神经病理性疼痛诱发情绪紊乱的相关性分析

李亚南; 薛纯纯; 王开强

doi:10.13406/j.cnki.cyxb.003826

重庆医科大学学报 ›› 2025, Vol. 50 ›› Issue (05) : 630 -639. DOI: 10.13406/j.cnki.cyxb.003826

基础研究

探究CCI大鼠海马铁死亡与神经病理性疼痛诱发情绪紊乱的相关性分析

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Association between hippocampal ferroptosis and emotional disorders induced by neuropathic pain in rats with chronic constriction injury

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摘要

目的观察坐骨神经慢性压迫损伤（chronic constriction injury，CCI）大鼠模型中海马区铁死亡现象及变化，探讨海马铁死亡与神经病理性疼痛（neuropathic pain，NP）诱发情绪紊乱的相关性。方法 32只SD雄性大鼠随机分为Sham组、CCI组、CCI+F组、CCI+E组，每组8只（n=8）。CCI+F组和CCI+E组于术后7 d分别腹腔注射铁抑素1（ferrostatin-1）（10 mg/kg）和铁死亡激活剂（erastin）（10 mg/kg），连续注射2 d。分别于术前，术后7 d、14 d测定机械足反射阈值（mechanical withdrawal threshold，MWT）、热缩足潜伏期（paw withdrawal threshold，PWL）、开放旷场实验（open field test，OFT）。术后14 d处死大鼠取海马组织，Western blot检测谷胱甘肽过氧化物4（glutathione peroxide 4，GPX4）、溶质载体家族成员11（solute carrier family 7 member 11，xCT）、铁蛋白重肽1（ferritin heavy polypeptide 1，FTH1），试剂盒检测丙二醛（malondialdehyde，MDA）、谷胱甘肽（glutathione，GSH）表达水平，组织HE、尼氏、免疫荧光染色，透射电镜观察线粒体结构变化。结果行为学结果，CCI组大鼠MWT和PWL值较Sham组术后7 d明显下降（P<0.001），术后14 d较7 d进一步下降（P<0.001）；与CCI相比，CCI+F组大鼠MWT和PWL术后14 d明显增加（P<0.001），CCI+E组MWT无明显变化，PWL明显降低（P<0.01）。CCI组大鼠术后14 d开放旷场实验测定（open field test，OFT）的总距离和中央跨格次数较Sham组明显减少（P<0.001），而静止不动时间明显增加（P<0.001）；与CCI组比，CCI+F组总距离和中央跨格次数明显增加，静止不动时间明显减少，CCI+E组OFT差异无统计学意义。生化结果，与Sham组相比，CCI组大鼠GPX4、xCT、FTH1明显下降（P<0.01），MDA明显增加，GSH明显下降。与CCI组相比，CCI+F组大鼠GPX4、xCT、FTH1明显增加（P<0.05），MDA明显下降（P<0.001），GSH明显增加（P<0.01）；CCI+E组xCT、FTH1表达明显减少，MDA、GSH差异无统计学意义。形态学结果，术后14 d CCI组较Sham组海马神经元形态损坏甚至坏死和尼氏小体减少，c-fos基因（FBJ murine osteosarcoma viral oncogene homolog，c-fos）明显减少（P<0.05），线粒体嵴破坏明显，CCI+F组较CCI组海马神经元细胞形态完整，尼氏小体数量增加，c-Fos增加（P<0.05），线粒体相对完整；CCI+E组c-Fos、尼氏小体较CCI组差异无统计学意义。对术后14 d PWL、MWT、GPX4、OFT、c-Fos值做两两线性相关分析，结果显示呈正相关（P<0.01）。结论外周神经损害诱发海马区铁死亡现象，出现情绪焦虑、抑郁，进而加剧铁死亡程度，加速中枢痛敏形成。

Abstract

Objective To investigate the phenomenon of hippocampal ferroptosis and its change in a rat model of chronic constriction injury（CCI） of the sciatic nerve，as well as the association between hippocampal ferroptosis and emotional disorders induced by neuropathic pain. Methods A total of 32 male Sprague-Dawley rats were randomly divided into Sham group，CCI group，CCI+F group，and CCI+E group，with 8 rats in each group. The rats in the CCI+F group and the CCI+E group were given intraperitoneal injection of Ferrostatin-1（10 mg/kg） and Erastin（10 mg/kg），respectively，since day 7 after surgery for two consecutive days. Mechanical withdrawal threshold（MWT），paw withdrawal latency（PWL），and open field test（OFT） were measured before surgery and on days 7 and 14 after surgery. On day 14 after surgery，the rats were sacrificed to collect hippocampal tissue；Western blot was used to measure the levels of GPX4，xCT，and FTH1；related kits were used to measure the expression levels of MDA and GSH；HE staining，Nissl staining，and immunofluorescent staining were performed；transmission electron microscopy was used to observe the structural changes of the mitochondria. Results In terms of behavioristics，compared with the Sham group，the CCI group had significant reductions in MWT and PWL on day 7 after surgery（P<0.001），with further reductions from day 7 to day 14 after surgery（P<0.001）；compared with the CCI group，the CCI+F group had significant increases in MWT and PWL on day 14 after surgery（P<0.001），and the CCI+E group had no significant change in MWT and a significant reduction in PWL（P<0.01）. In terms of the OFT test，compared with the Sham group，the CCI group had significant reductions in total distance and number of central squares crossed （P<0.001） and a significant increase in immobility time（P<0.001）；compared with the CCI group，the CCI+F group had significant increases in total distance and number of central squares crossed and a significant reduction in immobility time，while the CCI+E group had no significant changes in these indices. In terms of biochemical results，compared with the Sham group，the CCI group had significant reductions in GPX4，xCT，and FTH1（P<0.01），a significant increase in MDA，and a significant reduction in GSH；compared with the CCI group，the CCI+F group had significant increases in GPX4，xCT，and FTH1（P<0.05），a significant reduction in MDA（P<0.001），and a significant increase in GSH（P<0.01），and the CCI+E group had significant reductions in xCT and FTH1，with no significant changes in MDA and GSH. In terms of morphology，compared with the Sham group，the CCI group had damage and necrosis of hippocampal neurons，significant reductions in Nissl bodies and c-Fos level（P<0.05），and significantly damaged mitochondrial cristae on day 14 after surgery；compared with the CCI group，the CCI+F group had intact structures of hippocampal neurons，significant increases in Nissl bodies and c-Fos（P<0.05），and relatively intact mitochondria，while there were no significant differences in c-Fos and Nissl bodies between the CCI+E group and the CCI group. The pairwise linear correlation analysis of PWL，MWT，GPX4，OFT，and c-Fos on day 14 after surgery showed a significantly positive correlation between any two indicators（P<0.01）. Conclusion Peripheral nerve injury triggers ferroptosis in the hippocampus and causes anxiety and depression，thereby aggravating ferroptosis and accelerating the development of central hyperalgesia.