The Mechanism Of FGF21 Inhibiting Vascular Endothelial Cell Ferroptosis And Improving Atherosclerosis Through AMPK/NRF2 Signaling Pathway

BackgroundAtherosclerosis is the main cause of cardiovascular diseases such as coronary heart disease and heart failure.It is currently believed that abnormal lipid metabolism-induced vascular endothelial injury is the key factor leading to the formation of atherosclerosis,and the vascular endothelium is the first to be damaged during the development of atherosclerosis.Therefore,the research on the mechanism of targeting the endothelial cells to reduce atherosclerosis has attracted people’s attention.In recent years,it has been found that growth factors such as fibroblast growth factor 2（FGF2）and FGF5 can alleviate endothelial cell injury by inhibiting cell apoptosis and inflammation.Growth factors are considered to be the main regulators of endothelial cell homeostasis.It is shown that the application of growth factors provides a new strategy to further elucidate the pathogenesis and development of atherosclerosis.FGF21 is considered to be a growth factor associated with cellular damage,and it has been confirmed that cold,fasting/starvation,and certain pathological conditions can cause its levels to increase.However,the role and mechanism of FGF21 level increase remain controversial.One study found a correlation between elevated serum levels of FGF21 and the risk of future adverse cardiovascular events in patients with coronary artery disease.Overexpression of FGF21 promotes muscle atrophy and bone loss.On the contrary,experiments have found that exogenous FGF21 can alleviate endothelial damage by inhibiting endothelial cell apoptosis.Other studies have confirmed that exogenous FGF21 can improve lipid metabolism disorder by promoting adiponectin secretion,inhibiting cholesterol synthesis and promoting cholesterol excretion,and can also inhibit oxidative stress by increasing the levels of superoxide dismutase（SOD）and glutathione（GSH）,and reducing the level of malondialdehyde（MDA）,thereby delaying the progress of atherosclerosis.However,the relationship between FGF21 and vascular endothelial cell injury is not well understood.Therefore,the effect of FGF21 on atherosclerosis mediated by endothelial cell injury induced by abnormal lipid metabolism needs to be further investigated.Ferroptosis is an iron-dependent,lipid peroxidation-driven mode of death that occurs due to an imbalance in the production and degradation of reactive oxygen species（ROS）.Atherosclerosis is mainly characterized by disorders of lipid metabolism,in which increased LDL cholesterol in the blood enters the vascular endothelium and is oxidized to form oxidized low-density lipoprotein cholesterol（ox LDL-C）,which in turn exacerbates endothelial cell damage,suggesting that ferroptosis may be involved in the damage of vascular endothelial cells during the formation of atherosclerosis.Recent studies have also found that inhibiting ferroptosis can alleviate endothelial cell damage caused by abnormal lipid metabolism.In addition,during the process of ferroptosis,the reaction between Fe²⁺and H₂O₂generates hydroxyl radicals with the strong oxidizing ability and Fe³⁺,which is further reduced to Fe²⁺by peroxides(O^2-)and re-reacts with H₂O₂,and a large number of hydroxyl radicals are generated in this cycle,which is one of the stronger reactions for the generation of ROS.In diabetic cardiomyopathy,ferroptosis inhibitors were found to better attenuate myocardial injury than apoptosis and necrosis inhibitors,suggesting that the role of ferroptosis may be more critical in ox-LDL-induced endothelial cell injury.Further studies have found that overexpression of FGF21 or recombinant FGF21 protein can inhibit ferroptosis and alleviate liver cell damage and fibrosis.It is suggested that investigating the effect of FGF21 on ferroptosis in vascular endothelial cells can further elucidate the mechanism of FGF21 on atherosclerosis caused by vascular endothelial cell injury.It is believed that FGF21 binds to its receptor FGFR1 and activates liver kinase B1,which leads to the phosphorylation of the AMPKα-subunit at site 172.Previous studies have shown that AMPK activation inhibits macrophage ferroptosis caused by lipid metabolism disorders by increasing the expression of glutathione peroxidase 4（GPX4）.In addition,AMPK can also activate nuclear factor E2 related factor 2（NRF2）,promoting its nuclear translocation.NRF2 is a transcription activator that plays an important role in antioxidant responses.The activation of NRF2 can increase the expression of GPX4 and ferritin heavy chain（FTH）,which in turn attenuates myocardial injury by inhibiting the production and accumulation of lipid peroxides.It has been demonstrated that related proteins regulated by the AMPK/NRF2 pathway are considered to be key molecules in ferroptosis.These findings suggest that FGF21may inhibit ferroptosis induced by abnormal lipid metabolism in vascular endothelial cells through the AMPK/NRF2 signaling pathway.In this study,we propose to use the fibroblast growth factor 21 as an entry point,this experiment combined in vivo and in vitro experiments to investigate whether FGF21 inhibits abnormal lipid metabolism-induced endothelial cell ferroptosis via the AMPK/NRF2 signaling pathway,which provides a theoretical basis for the therapeutic application of FGF21 in atherosclerosis formation.Methods1.Expression of FGF21 in atherosclerosis and ox-LDL-induced endothelial cell injury.（1）Collect serum from patients with coronary heart disease,and use the Elisa kit to detect the content of FGF21 in the serum of patients with coronary heart disease.（2）Apo E^-/-mice were fed with a high-fat diet for 12 weeks to establish an atherosclerosis model.The content of FGF21 in atherosclerosis tissue and serum of Apo E^-/-mice fed with a high-fat diet was detected by western blot and Elisa kit.（3）Application of 150μg/m L of ox-LDL for 24 hours to construct an endothelial cell injury model,and detect the expression of FGF21 in the endothelial cell injury induced by ox-LDL using western blot.2.The effect of FGF21 on the ox-LDL-induced endothelial cell damage and the high-fat diet-induced atherosclerosis.（1）Treating FGF21 overexpression and FGF21 knockdown endothelial cells with ox-LDL,using CCK-8 kit to detect cell viability,LDH kit to detect LDH release,and tube formation assay to detect endothelial cell angiogenesis capacity,to determine the effect of FGF21 on ox-LDL-induced endothelial cell injury.（2）Exploring whether FGF21 alleviates ox-LDL-induced endothelial injury through intracellular or extracellular secretion to exert its protective effect.We first utilized the protein transport inhibitor Monensin（5μM）pretreatment of endothelial cells overexpressing FGF21 for 6 hours,followed by co-treatment with ox-LDL for24 hours,assessed the intracellular effects of FGF21 by detecting changes in cell viability,LDH release,and angiogenesis ability.Secondly,we pretreated endothelial cells with recombinant FGF21 protein of 200 ng/m L for 6 hours,and then co-treated with ox-LDL for 24 hours.We repeated the above experiments to assess the exogenous role of FGF21.（3）Apo E^-/-mice fed with a high-fat diet were intraperitoneally injected with recombinant FGF21 protein（0.1 mg/kg/day）,and the aortic plaque area was detected using oil red O staining and HE staining,and lipid changes were detected by a biochemical kit,to analyze whether exogenous FGF21 could attenuate atherosclerotic lesions.3.To explore whether FGF21 can alleviate ox LDL-induced endothelial cell injury and improve atherosclerosis by regulating ferroptosis.（1）In the mouse atherosclerosis model,an iron detection kit was applied to detect the level of ferrous ions in aortic tissues,the level of lipid peroxidation was detected by MDA and GSH kits,the expression of ferroptosis-related proteins ACSL4,GPX4,FTH and HO-1 was detected by western blot,and the expression of GPX4 in vascular endothelial cells was detected by immunofluorescence staining to analyze whether high-fat diet could cause ferroptosis of vascular endothelial cells.（2）In the endothelial cell injury model,the expression changes of ROS,mitochondrial membrane potential,ferrous ions,lipid peroxidation levels,and ferroptosis-related proteins were detected to investigate whether ox-LDL induced ferroptosis in endothelial cells.（3）Treating FGF21 overexpression and FGF21 knockdown endothelial cells with ox-LDL,and the regulatory effect of FGF21 on ox-LDL-induced ferroptosis in endothelial cells was comprehensively assessed by detecting the expression changes of ferroptosis-related indicators.（4）To investigate whether FGF21 regulates ferroptosis within cells or through secretion outside cells,we first pretreated overexpressing FGF21 endothelial cells using the protein transport inhibitor Monensin,and analyzed whether FGF21 could inhibit ox-LDL-induced endothelial cell ferroptosis through endogenous pathways by detecting the expression changes of ferroptosis-related indicators.Secondly,recombinant FGF21 protein pretreated endothelial cells and repeated the above experiments to investigate whether exogenous FGF21 regulates ox-LDL-induced endothelial cell ferroptosis.（5）Apo E^-/-mice on a high-fat diet were intraperitoneally injected with recombinant FGF21 protein to investigate the effect of exogenous FGF21 on ferroptosis in mouse vascular endothelial cells by detecting the expression changes of ferroptosis-related indicators.4.Based on the AMPK/NRF2 signaling pathway,elucidate the mechanism of FGF21 regulating ox-LDL-induced endothelial cell ferroptosis.（1）Western blot was used to detect the expression of AMPK/NRF2 in ox-LDL-induced endothelial cells and mouse atherosclerotic tissue.（2）Western blot was used to detect the expression of AMPK/NRF2 in endothelial cells treated with recombinant FGF21 protein in combination with ox-LDL,and in mouse aortic tissues injected intraperitoneally with recombinant FGF21 protein,and clarified the effect of exogenous FGF21 on AMPK/NRF2 expression.（3）In endothelial cells pretreated with recombinant FGF21 protein,the AMPK inhibitor（Compound C,10μM）was applied to pretreat the endothelium for 6 hours,and then co-treated with ox-LDL for 24 hours.By detecting the changes in the expression of the cell viability and ferroptosis-related indexes,we explored the role of the AMPK/NRF2 signaling pathway in the inhibition by exogenous FGF21 of ox-LDL-induced ferroptosis in endothelial cells.Results1.FGF21 expression is elevated in serum from patients with coronary artery disease,in atherosclerotic tissue and serum from mice,and in ox-LDL-induced endothelial cell injury.2.FGF21 attenuates ox-LDL-induced endothelial cell injury and inhibits high-fat diet-induced atherogenesis.（1）Compared with the ox-LDL-treated group,overexpression of FGF21increased cell viability,decreased LDH release,and increased angiogenic capacity after ox-LDL treatment,and the effects knockdown of FGF21 on cell viability,LDH release,and angiogenic capacity were contrary to those described previously,indicating that FGF21 attenuated ox-LDL-induced endothelial cell injury.（2）Compared with the overexpression of the FGF21+ox-LDL group,inhibition of overexpression of FGF21 endothelial cell-secreted proteins resulted in decreased cell viability,increased LDH release,and decreased angiogenic capacity;whereas,compared with the ox-LDL group,the recombinant FGF21 protein intervention group showed increased cell viability,decreased LDH release,and increased angiogenesis ability,indicating that FGF21 mainly alleviates ox-LDL induced endothelial cell damage through secretion to extracellular（3）The results of oil red O staining and HE staining showed that the area of aortic plaques in the intervention group of recombinant FGF21 protein was smaller than that in the high-fat diet group,and the results of the lipid assay showed that compared to the high-fat diet group,the recombinant FGF21 protein intervention group mice had lower levels of triglycerides,total cholesterol,and low-density lipoprotein cholesterol in their serum,while the high-density lipoprotein cholesterol content increased,indicating that the exogenous FGF21 reduced the area of aortic plaques and improved blood lipid abnormalities.3.FGF21 attenuates ox-LDL-induced endothelial cell injury and alleviates atherosclerotic lesions by inhibiting ferroptosis.（1）Compared with the control group,a high-fat diet-induced elevated ferrous ions,increased lipid peroxidation levels,increased expression of ferroptosis-related proteins ACSL4 and HO-1,decreased expression of GPX4 and FTH,and decreased expression of GPX4,which was co-transfected with the vascular endothelial cell marker protein CD31 in the aortic tissues of mice,indicating that high-fat diet-induced ferroptosis in vascular endothelial cells.（2）Compared with the control group,after ox-LDL treatment of endothelial cells,intracellular ROS levels increased,mitochondrial membrane potential decreased,ferrous ion levels increased,MDA levels increased,GSH levels decreased,ACSL4 and HO-1 expression increased,and GPX4 and FTH expression decreased,indicating that ox-LDL can induce ferroptosis in endothelial cells.（3）Compared with the ox-LDL-treated group,overexpression of FGF21improved the mitochondrial structure of endothelial cells after ox-LDL treatment,decreased intracellular ROS,ferrous ions,and MDA levels,increased GSH levels,decreased the expression of ACSL4 and HO-1,and increased the expression of FTH and GPX4.The effect of knocking down FGF21 is opposite to what was previously described,indicating that FGF21 inhibits ox-LDL-induced endothelial cell ferroptosis.（4）Compared with overexpression of FGF21+ox-LDL,inhibition of overexpression of FGF21 endothelial cell-secreted protein resulted in reduced mitochondrial cristae in endothelial cells,increased intracellular levels of ROS,ferrous ions,and MDA,and decreased levels of GSH,and increased expression of ACSL4 and HO-1,and decreased expression of FTH and GPX4,indicating that inhibition of overexpression of FGF21 secreted protein the inhibitory effect of FGF21 on ferroptosis was significantly weakened.However,compared with the ox-LDL-treated group,the recombinant FGF21 protein intervention group improved the mitochondrial structure of ox-LDL-treated endothelial cells,decreased intracellular ROS,ferrous ions,and MDA levels,elevated GSH levels,decreased the expression of ACSL4 and HO-1,and increased the expression of FTH and GPX4,indicating that FGF21 alleviates ox-LDL induced endothelial cell damage by inhibiting ferroptosis through secretion into the extracellular.（5）Compared with the high-fat diet group,the recombinant FGF21 protein intervention group had lower levels of ferrous ions and MDA in the aortic tissue,higher levels of GSH,lower expression of ACSL4 and HO-1,higher expression of FTH and GPX4,and higher expression of GPX4 co-stained with the vascular endothelial cell marker protein CD31,indicating that the exogenous FGF21 can improve the formation of atherosclerosis in mice by inhibiting vascular endothelial ferroptosis.4.Recombinant FGF21 protein inhibits ox-LDL-induced ferroptosis in endothelial cells by activating the AMPK/NRF2 signaling pathway.（1）Compared with the control group,the expression of phosphorylated AMPK in endothelial cell injury group and high-fat diet group was reduced,and the expression of nuclear protein NRF2 was increased.（2）Compared with the endothelial cell injury group and high-fat diet group,exogenous FGF21 increased the phosphorylation level of AMPK in endothelial cell injury and atherosclerosis tissues,and further increased the expression of nuclear protein NRF2.（3）Compared with the r FGF21+ox-LDL group,inhibition of AMPK resulted in decreased cell viability,decreased mitochondrial membrane potential,increased MDA levels,decreased GSH levels,increased expression of ACSL4 and HO-1,and decreased expression of FTH and GPX4.The above results indicate that the exogenous FGF21 inhibits endothelial cell ferroptosis by activating the AMPK/NRF2 signaling pathway.Conclusions1.The increase of endogenous FGF21 levels in the serum of clinical patients,serum and aortic tissue of mice and cells is related to atherosclerosis mediated by ox LDL-induced endothelial cell injury.2.By detecting the cell viability,LDH release,and angiogenesis ability of endothelial cells treated with ox LDL,combined with the changes of aortic plaque area and blood lipids in Apo E^-/-mice fed with a high-fat diet,it was concluded that and it was found that overexpression of FGF21 can alleviate ox-LDL-induced endothelial cell injury,while knowdown of FGF21 aggravated the damage to the endothelial cell;the effect of overexpression of After the inhibition of protein secretion by Monensin,the effect of overexpression of FGF21 in reducing endothelial cell injury was weakened;whereas,recombinant FGF21 protein could significantly inhibit ox-LDL-induced endothelial cell injury,suggesting that FGF21 mainly alleviates ox-LDL-induced endothelial cell injury and atherosclerosis by autocrine.3.Based on the ferroptosis-related indexes such as ROS,ferrous ion,MDA,and GSH levels,and the expression of ACSL4,GPX4,FTH,and HO-1,it was suggested that the inhibitory effect of exogenous FGF21 on ox-LDL-induced endothelial cell injury and atherosclerosis formation might be related to ferroptosis.4.Activation of the AMPK/NRF2 signaling pathway by exogenous FGF21inhibits ferroptosis,which is one of the main mechanisms to attenuate atherosclerosis caused by endothelial cell injury induced by abnormal lipid metabolism.