[Eye-acupuncture ameliorates ferroptosis of cerebral neuronal cells in CIRI rats by up-regulating System xc(-)-GSH-GPX4 antioxidant functional axis activities].

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 (Fe) 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 (<0.01), and the contents of Fe and MDA, and ROS activity considerably increased (<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 (<0.01, <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 Fe and MDA and ROS activity(<0.05, <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.