Objectives: To observe the effect of eye-acupuncture on the antioxidant function axis：System xc(-)-glutathione-glutathione peroxidase 4 (System xc［-］-GSH-GPX4) in the cortical tissue of ischemic penumbra of acute cerebral ischemia-reperfusion injury (CIRI) rats, so as to explore its underlying mechanism in improvement of CIRI by ameliorating the ferroptosis of neurons via antioxidant function axis.
Methods: Male SD rats were randomly divided into sham operation, model, eye-acupuncture and GPX4-inhibitor groups, with 15 rats in each group. The CIRI model was replicated by occlusion of the middle cerebral artery and reperfusion for 24 h. For rats of the eye acupuncture group and inhibitor group, manual acupuncture stimulation was applied to bilateral eye-acupuncture-points "Gan"(Liver), "Shangjiao"(Upper-energizer), "Shen"(Kidney) and "Xiajiao"(Lower-energizer) of both eyes, 30 min, 12 h and 24 h after modeling, 3 times altogether. For rats of the inhibitor group, intraperitoneal injection of GPX4 inhibitor RSL3 (an activator of ferroptosis) 10 mg/kg (dissolved in 5% DMSO) was conducted 30 min before every acupuncture stimulation. The neurological function was assessed by using Garcia JH scoring method. The brain infarct size was detected after triphenyl tetrazolium chloride (TTC) staining. The ischemic penumbral cortex tissue of the brain was taken for observing morphological changes after H.E. staining, and for observing ultrastructural changes of the mitochondria by using transmission electron microscope. The contents of malondialdehyde (MDA) and GSH were detected by using photocolorimetric method, the content of ferrous ions (Fe2+) detected using spectrophotometry, and the activity of reactive oxygen species (ROS) assayed by ELISA. The expression levels of solute carrier family 7 member 11 (SLC7A11), solute carrier family 3 member 2 (SLC3A2) and GPX4 proteins of the ischemic penumbral cortex were detected by Western blot, and those of SLC7A11 and GPX4 mRNAs were detected using real-time quantitative PCR.
Results: Compared with the sham operation group, the Garcia JH neurological deficit score, GSH content, and expression levels of SLC7A11, SLC3A2 and GPX4 proteins, and GPX4 and SLC7A11 mRNAs were significantly decreased (P<0.01), and the contents of Fe2+ and MDA, and ROS activity considerably increased (P<0.01) in the model group. In contrast to the model group, the decreased and increased levels of the above mentioned indexes were reversed in the eye-acupuncture group (P<0.01, P<0.05) but not in the inhibitor group. The therapeutic effects of eye-acupuncture were reduced by GPX4 inhibitor in increasing the levels of Garcia JH neurological deficit score, GSH content, and expression levels of SLC7A11, SLC3A2, and GPX4 proteins, and GPX4 and SLC7A11 mRNAs, as well as in lowering the contents of Fe2+ and MDA and ROS activity(P<0.05, P<0.01). Results of TTC staining displayed that the brain tissue on the right side showed obvious gray infarct loci in the model group, which was evidently smaller in the eye-acupuncture group, but not in the inhibitor group. H.E. staining displayed disordered arrangement of cells, with shriveled or broken nucleus, and interstitial edema and vacuolation, and a number of large area typical cerebral infarction, and net-like necrotic loci with blurred necrotic lesion boundaries in the model group, which was apparently milder in the eye-acupuncture group. In the inhibitor group, an increased number of cerebral infarction foci, and disordered arrangement and severe injury of cells were found.
Conclusions: Eye-acupuncture can ameliorate ferroptosis in neurons of CIRI rats by modulating System xc(-)-GSH-GPX4 antioxidant function axis.
目的: 观察眼针对急性脑缺血再灌注损伤（CIRI）大鼠脑缺血半暗带皮质组织中抗氧化功能轴系统XC［System xc（-）］-谷胱甘肽（GSH）-谷胱甘肽过氧化物酶4（GPX4）的影响，探讨眼针改善CIRI大鼠神经元铁死亡的作用机制。方法: SD大鼠随机分为假手术组、模型组、眼针组和抑制剂组，每组15只。改良线栓法复制CIRI大鼠模型；眼针组和抑制剂组针刺双侧“上焦区”“肝区”“下焦区”“肾区”，留针30 min，于脑缺血再灌注后30 min、12 h、24 h各施针１次。抑制剂组每次针刺前30 min腹腔注射GPX4抑制剂RSL3（10 mg/kg），共3次。改良Garcia JH评分法评估大鼠神经功能损伤程度；TTC染色观察脑组织梗死灶面积；HE染色观察缺血区脑组织形态；透射电子显微镜观察缺血区脑组织神经元线粒体结构；比色法检测缺血区脑组织丙二醛（MDA）、GSH、亚铁离子（Fe2+）含量；ELISA法检测活性氧（ROS）的活性；Western blot法检测缺血区脑组织溶质载体家族7成员11（SLC7A11）、溶质载体家族3成员2（SLC3A2）和GPX4的蛋白表达；实时荧光定量PCR法检测缺血区脑组织SLC7A11、GPX4的mRNA表达。结果: 与假手术组比较，模型组大鼠改良Garcia JH神经功能损伤评分降低（P<0.01），缺血侧脑组织可见明显的灰白色梗死灶，HE染色示出现多个较大面积的典型脑梗死改变，线粒体超微结构呈典型铁死亡改变，缺血区脑组织Fe2+、MDA含量及ROS活性显著升高（P<0.01）；GSH含量，SLC7A11、SLC3A2、GPX4蛋白及GPX4、SLC7A11 mRNA表达显著降低（P<0.01）。与模型组比较，眼针组治疗后改良Garcia JH神经功能损伤评分升高（P<0.01），脑组织的梗死灶面积缩小，脑组织细胞损伤程度明显减轻，线粒体损伤减轻，缺血区脑组织Fe2+、MDA含量及ROS活性显著降低（P<0.01）；GSH含量，SLC7A11、SLC3A2、GPX4蛋白及GPX4、SLC7A11 mRNA表达升高（P<0.01，P<0.05）。与眼针组比较，抑制剂组神经功能损伤评分降低（P<0.01），梗死灶面积增大，细胞损伤程度加重，线粒体损伤加重，缺血区脑组织Fe2+、MDA含量及ROS活性显著升高（P<0.01，P<0.05）；GSH含量，SLC7A11、SLC3A2、GPX4蛋白及GPX4、SLC7A11 mRNA表达降低（P<0.01，P<0.05）。结论: 眼针可通过调控System xc（-）-GSH-GPX4抗氧化功能轴改善CIRI大鼠神经元铁死亡。.
Keywords: Antioxidant function axis; Cerebral ischemia-reperfusion injury (CIRI); Eye-acupuncture; Ferroptosis; Ischemic penumbra; System xc(-)-GSH-GPX4.