Study On The Mechanism Of GPNMB/Hepcidin/Ferroportin Regulating Neuronal Ferroptosis In Early Brain Injury After Subarachnoid Hemorrhage

Objective(s):To determine the early iron death of neurons after SAH and the relationship between ferroptosis and prognosis,to reveal the role of ferroptosis in EBI,and to clarify the mechanism of GPNMB/Hepcidin/Ferroportin regulating ferroptosis after SAH.Methods:Adult male CD-1 mice were randomly divided into sham operation(Sham)group,subarachnoid hemorrhage(SAH)group,subarachnoid hemorrhage +solvent(SAH+vehicle)group,subarachnoid hemorrhage + GPNMB low dose group(SAH+GPNMB 1μg),subarachnoid hemorrhage + GPNMB medium dose group(SAH + GPNMB3.3 μg)and subarachnoid hemorrhage + GPNMB high dose group(SAH + GPNMB10 μg).The SAH model was established by internal carotid artery puncture.The severity of SAH was graded according to Sugawara method.GPNMB was administered by intraventricular injection.The modified Neurological Severity Scores and Garcia test were used to evaluate the short-term neurological changes in mice.The Rotor-Rod test and Moritz water maze test were used to explore the medium-and long-term neurological changes in mice.The brain water content was measured by drywet method,and the permeability of blood-brain barrier in mice was evaluated by Evansblue staining.Western blotting was used to detect the existence and expression of proteins in the samples,immunofluorescence staining was used to detect brain sections,enzyme-linked immunosorbent assay(Elisa)was used to further detect the expression of ferroptosis related proteins before and after SAH,and transmission electron microscopy was used to observe ferroptosis in cortical neurons of mice in each group.Results:GPNMB increased significantly at 6 hours after SAH,and reached the peak at 24 hours(p < 0.05).GPNMB was widely expressed in microglia,astrocytes and neurons,and increased after SAH.After SAH,the neurological function,brain water content and blood-brain barrier were seriously damaged.The mice interfered with GPNMB could effectively inhibit the ferroptosis of neurons and improve the neural function.After SAH,the expression of Hepcidin increased and the expression of Ferroportin decreased.In the group treated with GPNMB,the increase of Hepcidin and the decrease of Ferroportin were reversed,and the protective effect was blocked by Hepcidin while GPNMB was given.Conclusion(s):The expression of GPNMB increases after SAH.Exogenous administration of GPNMB can improve neurological function score,reduce cerebral edema and prevent further damage of blood-brain barrier.These effects may be achieved through Hepcidin/Ferroportin,a signal pathway regulating iron death.