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Title: [Effect of "Xingnao Kaiqiao" needling on expression of ferroptosis-related factors in rats with cerebral ischemia-reperfusion injury].
Author: Wang Q, Liu YY, Hou ZW.
Journal: Zhen Ci Yan Jiu; 2024 Oct 25; 49(10):1019-1029. PubMed ID: 39433362.
Abstract:
OBJECTIVES: To observe the effect of "Xingnao Kaiqiao" needling on the expression of ferroptosis-related proteins in neurons of rats with cerebral ischemia-reperfusion injury (CIRI), so as to explore its mechanism underlying improvement of CIRI. METHODS: Male Wistar rats were randomly divided into sham operation, model, acupuncture and deferoxamine (DFO) groups, with 18 rats in each group. The CIRI model was established by occlusion of the middle cerebral artery. In the acupuncture group, "Xingnao Kaiqiao" needling was applied to "Shuigou" (GV26), "Neiguan" (PC6) and "Sanyinjiao"(SP6) for 20 min with electroacupuncture (2 Hz/15 Hz, 1 mA) at PC6 and SP6, twice daily for continuous 3 days. Rats of the DFO group received intraperitoneal injection of iron chelator DFO (0.1 g/kg, once daily). The severity of neurological impairment (neurological deficit score, 0-5 points, the lower the score, the severer is the neurological impairment) was evaluated by using Zausinger 6-poins scaling method. The cerebral infarct volume was measured after 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, and the histopathological changes of the ischemic brain tissue were observed after H.E. staining. The mitochondrial structure of the hippocampal neurons on the ischemic side of the brain was observed by using transmission electron microscope. The levels of iron deposition rate (%) in the ischemic penumbra of the brain tissue and hippocampus were observed after Prussian blue staining, and the reactive oxygen species (ROS) content of the cerebral ischemic penumbra was assayed using flow cytometry, and the content of glutathione (GSH) content in the ischemic penumbra was detected by using microplate method. The real-time quantitative PCR was used to detect the expression of glutathione peroxidase 4 (GPX4), divalent metal transporter 1 (DMT1), transferrin (TF), transferrin receptor 1 (TFR1), and ferroportin 1 (FPN1) mRNAs in the ischemic penumbra, and the Western blot was used to detect the expression of GPX4, DMT1, TF, TFR1, FPN1, and ferritin (FER) proteins in the ischemic penumbra. RESULTS: Compared with the sham operation group, the neurological deficit score, GSH content, expression of GPX4 and FPN1 mRNAs and proteins were significantly decreased (P<0.01), while the percentage of cerebral infarct volume, iron deposit rates of the cerebral ischemic penumbra and hippocampus, ROS content, and the expression levels of DMT1, TF, and TFR1 mRNAs and proteins and FER protein were considerably increased (P<0.01) in the model group. In comparison with the model group, the decrease of neurological deficit score, GSH content, expression of GPX4 and FPN1 mRNAs and proteins, and the increase of the percentage of cerebral infarct volume, iron deposit rates of the cerebral ischemic penumbra and hippocampus, ROS content, and the expression levels of DMT1, TF, and TFR1 mRNAs and proteins and FER protein were all reversed in both DFO and acupuncture groups (P<0.01, P<0.05). The effects of acupuncture were significantly superior to those of DFO in lowering the levels of cerebral cortical and hippocampal iron deposit rates, ROS content and in elevating the expression of GPX4 mRNA and protein (P<0.01, P<0.05). H.E. staining showed large necrotic cells, disordered arrangement of cells in the cerebral cortex and hippocampus, with hyperchromic nuclei, vacuole-like changes, widening of cellular space, and cell swelling in the model group, which was relatively milder in the cell damage in both acupuncture and DFO groups. In addition, the ultrastructure of cells in the hippocampus showed irregular cellular nuclear morphology, atrophy of some mitochondria in the cytoplasm, partial mitochondrial membrane rupture and edema, and loosening of the ridge structure in the model group, which was milder in the mitochondrial impairment (including reduced number of mitochondria, broken mitochondrial membrane and reduced ridge structure in fewer cells) in the acupuncture group. CONCLUSIONS: The "Xingnao Kaiqiao" needling intervention has a neuroprotective effect in CIRI rats, which may be related to its functions in regulating ferroptosis-related targets and iron metabolism in cerebral ischemic penumbra, reducing oxidative stress injury, and suppressing neuronal ferroptosis. 目的: 观察“醒脑开窍”针刺法对脑缺血再灌注损伤（CIRI）大鼠神经元铁死亡相关蛋白表达的影响，探讨针刺抑制CIRI大鼠神经元铁死亡的作用机制。方法: 将雄性Wistar大鼠随机分为假手术组、模型组、针刺组和去铁胺（DFO）组，每组18只。采用线栓法制备CIRI大鼠模型，针刺组予针刺“水沟”“内关”“三阴交”，每日针刺2次，每次20 min，连续3 d。DFO组腹腔注射DFO注射液（0.1 g/kg），每日1次，连续3 d。采用Zausinger 6分法评估大鼠神经功能缺损程度；TTC法测定脑梗死体积百分比；HE染色观察脑组织形态结构；透射电子显微镜观察缺血侧海马组织神经元线粒体结构；普鲁士蓝染色观察脑组织铁沉积水平；流式细胞术检测缺血半暗带活性氧（ROS）含量，微板法检测缺血半暗带谷胱甘肽（GSH）含量，实时荧光定量PCR法检测缺血半暗带谷胱甘肽过氧化物酶4（GPX4）、二价金属转运体1 （DMT1）、转铁蛋白（TF）、转铁蛋白受体1（TFR1）、膜铁转运蛋白1 （FPN1）mRNA表达水平，Western blot法检测缺血半暗带GPX4、DMT1、TF、TFR1、FPN1及铁蛋白（FER）的蛋白表达水平。结果: 与假手术组比较，模型组大鼠神经功能缺损评分降低、脑梗死体积增大（P<0.01），脑组织病理损伤严重、铁沉积增多（P<0.01），线粒体呈典型铁死亡改变；GSH含量降低、ROS含量升高（P<0.01），GPX4、FPN1 mRNA和蛋白表达水平降低（P<0.01），TF、TFR1、DMT1 mRNA和蛋白表达水平升高（P<0.01），FER蛋白表达水平升高（P<0.01）。与模型组比较，DFO组和针刺组大鼠神经功能缺损评分升高（P<0.05），脑梗死体积减小（P<0.01），脑组织病理学和线粒体铁死亡改变减轻，铁沉积减少（P<0.01）；ROS含量降低、GSH含量升高（P<0.05，P<0.01），DMT1、TF、TFR1 mRNA和蛋白表达水平降低（P<0.05，P<0.01），FER蛋白表达水平降低（P<0.05），GPX4、FPN1 mRNA和蛋白表达水平提高（P<0.01）。与DFO组比较，针刺组铁离子沉积面积和ROS含量更低、GPX4 mRNA和蛋白表达水平更高（P<0.01，<0.05）。结论: “醒脑开窍”针刺法对CIRI大鼠具有神经保护作用，其机制可能与调节缺血半暗带铁死亡相关靶点，调控铁代谢，抑制氧化应激损伤，抑制神经元铁死亡有关。.

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