RSL3 Promote Ferroptosis Of Macrophages And Vulnerability Of Atherosclerotic Carotid Plaque By Inhibiting GPX4

BackgroundAlthough the incidence of ischemic stroke has declined globally over the past decade,it has increased significantly in East Asia,particularly in China.Vulnerable atherosclerotic plaque of the carotid artery is the primary cause of ischemic cerebral strokes,which is one of the leading causes of severe morbidity and mortality.Plaque stability rather than size is more closely associated with complications of atherosclerosis.Inflammation is the key of the progression of atherosclerosis,and macrophage is the key of maintaining inflammatory homeostasis in plaque.Recruitment and death of macrophages promoting plaque vulnerability.Ferroptosis is a form of regulated cell death that is iron-dependent and mediated by excessive lipid peroxidation.ROS accumulation,lipid peroxidation,intraplaque hemorrhage,and iron overload in macrophages are key characteristics of advanced plaques and ferroptosis.At present,ferroptosis has been deeply studied in the field of cancer.Many studies have found that inducing ferroptosis can promote tumor cell death and improve cancer progression in various disease models.However,a study has found that the use of ferroptosis inhibitors can inhibit iron accumulation and lipid peroxidation in Apo E(-/-)mice with high fat diet,and inhibit plaque formation.The role of ferroptosis in atherosclerosis may promote plaque progression,which means that systematic use of ferroptosis inducers in cancer treatment may promote atherosclerosis progression and even further increase stroke incidence rate.A single cell sequencing study shows that macrophages are associated with a differential gene associated with ferroptosis in atherosclerotic vessels.This suggests that ferroptosis may occur in macrophages of atherosclerotic plaques,leading to macrophage death and thereby affecting plaque stability.This study aims to provide direct evidence for pig ferroptosis in macrophages in atherosclerotic plaques,confirm the correlation between ferroptosis and the vulnerability of atherosclerotic plaques,and investigate the feasibility of regulating the occurrence of ferroptosis through the intervention of RSL3 inhibiting GPX4 activity,thus interfering with plaque vulnerability.MethodsPart I: In clinical sample study,the correlation between ferroptosis and macrophages in carotid atherosclerotic plaques is found through immunofluorescence detection of clinically carotid atherosclerotic plaques.The clinical samples are grouped according to the symptom grouping commonly used in current international studies,and the plaque vulnerability index is used as an evaluation index of plaque vulnerability to evaluate the correlation between clinical symptoms and plaque vulnerability.The expression of ferroptosis in plaques is evaluated according to the immunohistochemical results,and the data of ferroptosis expression and plaque vulnerability are analyzed to explore the correlation between ferroptosis and plaque vulnerability.Part II: In vitro cell experiment,atherosclerosis modeling is performed by ox-LDL cultured RAW264.7 macrophages,and the conclusion of the correlation between ferroptosis and macrophages found in the first part is verified by immunofluorescence.The expression of ferroptosis in atherosclerotic macrophages after used RSL3 is evaluated by Western Blot technique.The mitochondrial changes and cell death are observed by transmission electron microscopy and flow cytometry after the intervention,so as to obtain the incidence of ferroptosis in atherosclerotic macrophages after the intervention of RSL3.Part III: In vivo animal experiments,Apo E(-/-)mice are fed high fat for atherosclerosis modeling,and intraperitoneal injection of drugs is used to intervene,and experimental animals are grouped according to drugs.Oil red staining of the aorta and aortic valve is used to evaluate the progression of atherosclerosis,and the plaque vulnerability index is used to evaluate the stability of carotid artery plaque,so as to explore the effect of ferroptosis intervention on the progression of atherosclerosis and plaque stability.The data of carotid plaque vulnerability and ferroptosis in each sample are analyzed to verify the conclusion of correlation between ferroptosis and plaque vulnerability found in the first part.ResultsPart I of the clinical sample study: 1)There is colocalization of macrophage and ferroptosis in carotid atherosclerotic plaque,which occurs in inflammatory infiltration around the necrotic core;2)There was no significant difference in plaque stability between symptomatic and asymptomatic groups,but the symptomatic group had more lipid peroxide in plaque and more ferroptosis.3)There was a linear correlation between the occurrence of lipid peroxide and ferroptosis and plaque stability.Part II of vitro cell experiments: 1)Atherosclerotic macrophages colocalize with the ferroptosis regulatory proteins GPX4 and FTH1;2)RSL3 inhibited GPX4 and FTH1 expression in atherosclerotic macrophages;3)RSL3 can increase or even rupture the mitochondrial crista of macrophages in atherosclerosis.4)RSL3 intervention increased the cell death of atherosclerotic macrophages.Part III of vivo animal experiments: 1)RSL3 promotes the progression of atherosclerosis;2)RSL3 promotes ferroptosis by inhibiting GPX4 expression;3)Ferroptosis increases plaque vulnerability.Conclusions1)Ferroptosis happens in macrophages of carotid atherosclerotic plaques;2)RSL3 inhibits GPX4 and promotes ferroptosis in atherosclerotic macrophages;3)Ferroptosis is negatively correlated with carotid plaque stability;4)RSL3 promoting ferroptosis increases the vulnerability of carotid plaque.