Hepcidin-Mediated Brain Iron Dysbolism In The Pathogenesis Of Cognitive Impairment In Postmenopausal Women

Objective:Studies have demonstrated a significantly higher incidence of cognitive dysfunction in postmenopausal women compared to men within the same age group,characterized by accelerated disease progression and extensive cognitive impairment.The underlying cause for this gender disparity in cognitive decline remains unclear,although it is hypothesized to be associated with lower estrogen levels;however,the specific mechanism has yet to be elucidated.Recent reports suggest that cessation of iron loss after menopause may lead to abnormal iron metabolism in women.Our research team has identified a close association between white matter lesions(WMLs)and cognitive impairment in postmenopausal women.White matter primarily consists of myelin-sheathed nerve fibers formed by oligodendrocytes(OLs),which are the main source of myelin production within the central nervous system(CNS)and are particularly vulnerable during WMLs pathogenesis caused by various factors due to their high iron content and lack of antioxidant enzymes.Hepcidin,predominantly secreted by the liver,plays a crucial role in both peripheral and central iron metabolism regulation.Estrogen exerts an inhibitory effect on hepcidin expression.Therefore,we propose that hepcidin may contribute to the development and progression of cognitive impairment among postmenopausal women.This study aims to investigate the relationship between hepcidin-mediated brain iron metabolism regulation and cognitive decline through clinical investigations as well as animal models,providing novel experimental evidence for understanding the pathogenesis of cognitive impairment in postmenopausal women while identifying potential targets for intervention.Methods:Part Ⅰ:(1)A total of 181 postmenopausal female patients consecutively admitted to our hospital between September 2021 and September 2023 were included in this study and categorized into two groups based on their cognitive function,namely the Cognitive Impairment(CI)group and the Normal Cognitive Function(NC)group,as determined by the Beijing version of Montreal Cognitive Assessment Scale(Mo CA).Propensity score matching successfully paired forty-nine individuals according to age.General clinical data and peripheral blood samples were collected.(2)Serum levels of hepcidin,non-heme-bound iron,malondialdehyde(MDA),and lipid hydroperoxide(LPO)were quantified using enzyme-linked immunosorbent assay(ELISA).(3)The correlation between Mo CA scores and hepcidin levels was assessed through correlation analysis,while logistic regression was employed to investigate risk factors associated with cognitive impairment in postmenopausal women.Part Ⅱ:(1)The brain MRI scan sequences utilized in this study included 3D-BRAVO,DTI,ESWAN and multi-echo SWI.Participants were divided into high hepcidin(H-hepcidin)and low hepcidin(L-hepcidin)groups based on their respective levels of hepcidin.(2)Hippocampal volume was calculated through image segmentation,with correlation analysis conducted between hippocampal volume and hepcidin level.(3)Changes in white matter microstructure and iron deposition were evaluated using Tract Based Spatial Statistics(TBSS)and quantitative magnetic sensitivity maps respectively,with further analysis conducted to determine the correlation between hepcidin level and both iron deposition as well as damage to white matter microstructure.(4)Graph theory analysis was employed to explore changes in structural brain network attributes among participants from both groups.Part Ⅲ:(1)Female SD rats were randomly allocated into the Sham group and OVX group.(2)The cognitive function of rats in the two groups was evaluated at 8 weeks postsurgery using Morris Water Maze(MWM)and Y-maze tests.(3)Hepcidin levels in peripheral blood and brain tissue of rats were measured by ELISA.(4)White matter damage in the corpus callosum and hippocampus of rats was assessed through Immunofluorescence(IF),Luxol Fast Blue(LFB)staining,and Western Blotting(WB).(5)Iron metabolismrelated proteins as well as iron death-related proteins expression in the hippocampus of rats were examined by WB.Part Ⅳ:(1)SD rats were initially randomly divided into the Sham group and OVX group,and the blood-brain barrier(BBB)permeability of rats was assessed 3 weeks postsurgery.In case of BBB disruption in the OVX group,these rats were further randomized into two groups receiving hypodermic injections of Glycol split heparin(GSHP)or Normal saline(NS)for a duration of 4 weeks.Simultaneously,the Sham group received subcutaneous injections of NS for 4 weeks.(2)Cognitive function evaluation was performed using MWM and Y maze tests on the rats.(3)The levels of hepcidin in peripheral blood and brain tissue,as well as the expression of iron death-related proteins,were measured through ELISA;additionally,Prussian blue staining was conducted to detect iron deposition in hippocampus and corpus callosum brain regions.(4)LFB staining,IF staining,and WB methods were used to assess corpus callosum and hippocampus regions’ white matter damage along with determining levels of iron metabolism-related proteins and expression patterns of iron death-related proteins.Results:Part Ⅰ:(1)There were no significant differences in age,history of hypertension,history of diabetes,tobacco and alcohol use,and previous stroke history between post-matching p CI and p NC groups;(2)ELISA results revealed a significant increase in the levels of hepcidin,MDA,and LPO in peripheral blood samples from postmenopausal women in the p CI group(all P<0.05).The level of non-heme iron showed a decreasing trend but did not reach statistical significance(P=0.084).(3)Correlation analysis demonstrated a significant negative correlation between serum levels of hepcidin and MDA with cognitive function;(4)Logistic regression analysis adjusted for age indicated that high levels of hepcidin and low education level were independent risk factors for cognitive impairment in postmenopausal women.Part Ⅱ:(1)The serum Hepcidin level in postmenopausal women exhibited a negative correlation with bilateral hippocampal volume,and both the left hippocampal volume(P=0.014)and the right hippocampal volume(P=0.016)were significantly reduced in the Hhepcidin group;(2)QSM sequence magnetic resonance examination revealed a significant increase in magnetic susceptibility of both the left hippocampus(P=0.003)and the right hippocampus(P=0.001)in the H-hepcidin group,indicating elevated iron deposition within the hippocampus.Additionally,there was a significant increase in mean magnetic susceptibility(MMS)observed in various brain regions including the left prethalamic radiation,right fronto-occipital tract,corpus callosum radiation,and left cingulate gyrus within the H-hepcidin group(all P<0.001);(3)Compared to the L-hepcidin group,fractional anisotropy(FA)demonstrated reduced anisotropy within several brain regions including the left cingulate gyrus,right upper longitudinal tract,right inferior occipitofrontal tract,right prethalamic radiation and right corticospinal tract within the H-hepcidin group(P<0.05,cluster value ≥50).Furthermore,the radial diffusion rate(RD),indicative of white matter microstructure damage at multiple sites,were significantly increased for both the right upper longitudinal tract and left upper longitudinal tracts within the H-hepcidin group(P<0.05,cluster value≥50);(4)In the H-hepcidin group,global efficiency(Eg)and local efficiency(Eloc)of small-world attributes were significantly decreased compared to those of the Lhepcidin group,while the shortest path length(Lp)became longer(all P<0.01),indicating damage to the brain network structure within the H-hepcidin group.Part Ⅲ:(1)After 8 weeks of modeling,during the Morris water maze(MWM)learning period,the escape latency of ovariectomized(OVX)rats significantly increased on the 5th day,indicating impaired cognitive function.Additionally,there was a significant decrease in both the target quadrant residence time and the number of platform crossings.(2)ELISA results revealed a significant increase in hepcidin levels in both peripheral blood and brain homogenate samples from OVX rats.(3)Prussian blue staining demonstrated a notable increase in iron deposition within the hippocampus and corpus callosum of OVX rats.(4)LFB staining of brain sections showed that myelin fibers within the corpus callosum area exhibited looseness and obvious white matter injury in the OVX group.Furthermore,there was a significant decrease in mean fluorescence intensity of MBP within the hippocampus,indicating white matter injury present in this region.Immunofluorescence analysis also revealed a significant reduction in OLs positive cells within the corpus callosum of OVX rats.(5)Western blot analysis indicated a significant upregulation of ACSL4 expression and downregulation of GPX4 expression within the hippocampus of OVX rats.Moreover,mitochondrial morphology observed within OLs cells from this region appeared significantly smaller,suggesting iron-induced cell death occurring specifically within these cells.Part Ⅳ:(1)After administering GSHP and NS interventions to OVX rats,the cognitive function of OVX+GSHP rats was significantly superior to that of NS rats,as evidenced by improved performance in the MWM and Y maze tests.(2)ELISA results revealed a significant reduction in hepcidin,MDA,and ROS levels in both peripheral blood and hippocampal homogenate of the OVX+GSHP group compared to the NS group.Additionally,there was a notable increase in GSH levels and GPX4 activity,indicating a lower oxidative stress level in the OVX+GSHP group.(3)Prussian blue staining demonstrated a substantial decrease in positive iron particles within the hippocampus and corpus callosum of the OVX+GSHP group.(4)IF staining of brain sections exhibited increased MBP fluorescence intensity within the hippocampus of rats from the OVX+GSHP group.LFB staining further indicated an enhanced average absorbance within the corpus callosum area for these rats,suggesting an improvement in white matter damage within both regions.(5)WB results displayed a significant decrease in ACSL4 expression along with an increase in GPX4 expression within the hippocampus of rats from the OVX+GSHP group.Transmission electron microscopy revealed improved demyelination status within white matter areas of their hippocampus compared to those from the NS group.Furthermore,OPCs cell mitochondria morphology appeared mostly normal,indicating amelioration of iron-induced death within this region.Conclusions:The levels of hepcidin,as well as the lipid metabolites MDA and LPO,were significantly elevated in peripheral blood samples from postmenopausal women with cognitive impairment during iron death.Furthermore,there was a significant negative correlation between the levels of hepcidin and MDA and cognitive function.These findings also revealed that hepcidin levels were negatively associated with hippocampal size but positively correlated with iron deposition in the hippocampus.In the H-hepcidin group,extensive microstructural changes occurred in brain white matter,leading to disruption of the brain network structure.Additionally,an OVX model demonstrated cognitive and behavioral impairments along with increased hepcidin levels in both peripheral blood and hippocampus.Moreover,there was an increase in iron deposition within the corpus callosum area and hippocampus accompanied by abnormal expression of white matter demyelination markers,OLs injury indicators,and iron death-related factors.However,treatment with GSHP(a hepcidin inhibitor)resulted in improved cognitive behavior among rats while reducing iron deposition within both corpus callosum and hippocampus regions as well as repairing white matter damage.This study suggests that inhibiting hepcidin expression may effectively reduce iron deposition while promoting repair of white matter damage within the brain thereby improving cognitive function among postmenopausal women.