[Effect of "Xingnao Kaiqiao" needling on expression of ferroptosis-related factors in rats with cerebral ischemia-reperfusion injury].

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 (<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 (<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 (<0.01, <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 (<0.01, <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.