The Effect And Mechanism Of CSE/Transsulfuration Pathway In Macrophage Phenotypic Transformation Induced By Iron Overload And The Relationship With Atherosclerosis

Background:Atherosclerosis(AS)is one of the main causes of cardiovascular disease(CVD).It is of great significance to explore its key pathological mechanisms for the prevention and treatment of atherosclerotic cardiovascular disease(ASCVD).Macrophages play multiple roles in AS.The number and phenotype of macrophages in atheromatous plaques are closely related to the pathological process of AS.The activation of macrophages has two classical phenotypes:M1 macrophages secrete pro-inflammatory cytokines(IL-1β,TNF-α,IL-6)and chemokines(CXCL9,CXCL10);M2 macrophages play a role in anti-inflammatory(IL-10,IL-4,PPARy)and immunomodulation.Studies have shown that iron homeostasis affects phenotypic transformation of macrophages and plays a key regulatory role in AS,but the mechanism is unclear.Iron overload can provoke cellular oxidative stress,lipid peroxidation and DNA damage,which can lead to genomic instability and defects in DNA repair,ultimately impair cell viability and cause ferroptosis.The cysteine-GSH-GpX4 axis is one of the major systems that can inhibit ferroptosis in mammals by regulating can regulate lipid peroxidation.There are two pathways for the synthesis of cysteine:the exogenous pathway is the absorption of extracellular cysteine by System Xc-in the form of cystine,while the endogenous pathway is the de novo synthesis of cysteine from methionine through methionine cycle and transsulfuration pathway.Cystathionine-gamma-lyase(CSE)is a key metabolic enzyme in the transsulfuration pathway encoded by Cth.The transsulfuration is the main pathway of endogenous cysteine production and its role in the effect of iron overload on macrophage phenotypes remains unclear.Further investigation of its mechanism will provide new clues to the exploration of therapeutic protocols for targeting macrophages in AS.Aim:1.To prove the role of CSE/transsulfuration pathway deletion in the occurrence and development of atherosclerosis;2.Clarifying that the CSE/transsulfuration pathway is the key factor in regulating the phenotype transformation of macrophages caused by iron overload.Methods:1.Aortas of Cth-/-and ApoE-/-double knockout mice fed with high fat diet were photographed after isolated and stained with oil red O.2.RAW264.7 macrophages were treated with Ammonium ferric citrate(FAC).The survival rate of macrophages was detected by CCK8.The levels of marker molecules of M1(IL1-β,TNF-α,INOS)and M2(IL-4,IL-10,PPARγ)macrophages were detected by qPCR.3.RAW264.7 macrophages were treated with FAC.The levels of iron metabolism(Transferrin,Ferritin),mitochondrial iron metabolism(FTMT)and iron autophagy metabolism(NCOA4)were detected by Western blot and the expression levels of iron transport protein(FPN,TRFC)genes were detected by qPCR.4.RAW264.7 macrophages were treated with FAC.The ROS levels and MDA levels of macrophages were detected by Cl1-BODIPY cell fluorescence probe.The MDA levels of macrophages were detected by MDA kit.5,RAW264.7 macrophages were treated with FAC.The cysteine content of macrophages was detected by cysteine kit.The level of glutathione metabolism(GPX4)and the expression levels of glutathione ligase(GCLC,GCLM)genes were detected by Western blot.The levels of exogenous cysteine metabolism(SLC7A11)and endogenous cysteine metabolism(CBS,CSE)were detected by Western blot.6.RAW264.7 macrophages were treated with FAC and Erastin.The survival rate of RAW264.7 macrophages was detected by CCK8 assay.The cysteine content of macrophages was detected by cysteine kit.The GSH content of macrophages was detected by GSH kit.The MDA level of macrophages was detected by MDA kit.The levels of iron metabolism(Transferrin,Ferritin),mitochondrial iron metabolism(FTMT),iron autophagy metabolism(NCOA4),glutathione metabolism(GPX4),exogenous cysteine metabolism(SLC7A11)and endogenous cysteine metabolism(CBS,CSE)were detected by Western blot.7.Marrow macrophages were extracted and induced from wild-type mice and Cth-/type mice injected with Iron-dextran.Then the levels of marker molecules of M1(IL-1β,INOS,TNF-α)and M2(IL-4,IL-10,PPARy)were detected by qPCR.8.Marrow macrophages were extracted and induced from wild-type mice and Cth-/type mice injected with Iron-dextran.Then the expression levels of glutathione ligase(GCLC,GCLM)genes in macrophages were detected by qPCR.The levels of iron metabolism(NCOA4,FTMT,Ferritin)and glutathione(GPX4)metabolism were detected by Western blot.9.RAW264.7 macrophages treated with FAC was further processed with ferroptosis compound library,and the 72h survival rates were detected by CCK8 assay.Result:1.Cth-/-and ApoE-/-double knockout mice have increased aortic atherosclerotic area compared with ApoE-/-mice.2.The marker molecules were increased in M1(ILl-β、TNF-α、INOS)and M2(IL-4、IL-10、PPARy)macrophages with iron overload.3.Iron overload increased the expression of FTMT,Ferritin,Transferrin and iron transport(FPN)and decreased the expression of NCOA4 and TFRC in macrophages.4.Iron overload increased the level of lipid peroxidation and the expression of CSE and decreased the expression of GPX4 and SLC7A11 in macrophages.5.Iron overload lacking exogenous cysteine increased cysteine,GSH,MDA level and CSE expression and decreased iron metabolism in macrophages compared with simple iron overload.6.The BMDM phenotypes of WT mice with iron overload and RAW264.7 cells were in the dynamic balance between M1(IL1-β、TNF-α、INOS)and M2(IL-4、IL-10、PPARγ)while the Cth-/-mice with iron overload were more inclined to M1(Pro-inflammatory).7.The BMDM phenotypes of Cth-/-mice with iron overload showed increased expression of iron autophagy and decreased GPX4 activity compared with RAW264.7 cells.Conclusion:1.The loss of CSE can exacerbate the development of atherosclerosis.2.Iron overload can promote the phenotype transformation of M1 and the alternating activation of M2,affect the expression of genes involved in iron metabolism and iron transport and raise the level of lipid peroxidation and affect exogenous and engenous cysteine metabolism in macrophages.3.Iron overload can further affect iron metabolism and endogenous cysteine metabolism in macrophages in lack of exogenous cysteine.4.The loss of CSE can exacerbates the inflammatory progression,affect cysteine metabolism and iron autophagy in iron overload macrophages.