The Roles And Regulatory Mechanisms Of Ferroptosis In Vascular Endothelial Cell Inflammation And Senescence

Background Ferroptosis is a new regulatory cell death mode caused by oxidative damage induced by the accumulation of intracellular free iron ions.Studies have found that oxidative stress mediated by ferroptosis is involved in the pathogenesis of many diseases.Inflammation,oxidative stress,and lipid metabolism disorders are the main triggers of atherosclerosis(AS).Cell apoptosis,necrosis,autophagy,and other regulatory cell death modes are involved in the pathogenesis of atherosclerosis.At the same time,dead cells can release harmful mediators,which will induce oxidative stress and inflammation,aggravating cell and tissue damage.Vascular endothelial dysfunction is an important pathological mechanism of atherosclerosis.At present,whether ferroptosis is involved in the inflammatory injury of vascular endothelial cells and its mechanism remains unclear.Fully exploring the roles and regulatory mechanisms of ferroptosis on vascular endothelial cell injury is of great significance for guiding the treatment of atherosclerosis and improving its prognosis.NRF2 is an important antioxidant transcription factor,which plays an oxidative defense function by regulating the expression of various antioxidant factors.Studies have found that NRF2 is also a negative regulator of ferroptosis,which can activate the glutathione transport system,reduce intracellular free iron levels by inhibiting ferritin degradation,and regulate multiple ferroptosis key genes expression by modulating mitochondrial activity and lipid peroxidation.In addition,NRF2 can regulate oxidative homeostasis.However,the regulatory mechanisms of NRF2 in vascular endothelial cell ferroptosis,inflammation,cell senescence,and the roles of NRF2-mediated redox adaptation in vascular endothelial cell ferroptosis induced by oxidative damage still need to be systematically studied.Objective 1.To explore the relationship between ferroptosis and inflammation and cell senescence in a model of vascular endothelial cell inflammatory injury;2.To explore the regulatory effect and mechanisms of NRF2 in vascular endothelial cell ferroptosis,inflammation,and cell senescence;3.To clarify the effect of NRF2-mediated redox adaptation on oxidative stressinduced vascular endothelial cell injury and ferroptosis;4.To explore whether ferroptosis is involved in the pathogenesis of acute myocardial infarction.Methods 1.HUVECs were treated with LPS to construct the inflammatory injury and senescence model of vascular endothelial cells.(1)HUVECs were treated with different concentrations(0 μg/m L,0.5 μg/m L,1 μg/m L,2 μg/m L,4 μg/m L,and 8 μg/m L)of LPS for 24 hours to determine the optimal drug action concentration.After that,HUVECs were treated with 2 μg/m L LPS at different times(0 h,6 h,12 h,24 h,48 h).Cell viability was detected by the CCK8 kit.(2)The inflammatory-related protein expression levels of HMGB1,IL-6,and TNF-α in HUVECs treated with 2 μg/m L LPS at different times(0 h,1 h,3 h,6 h,9 h,12 h,24 h)were detected via western blot assay method.(3)Cell senescence-related β-galactosidase staining was used to detect HUVECs senescence under LPS stimulation at different concentrations(0 μg/m L,1 μg/m L,2μg/m L,4 μg/m L)and 2 μg/m L LPS at different time(0 h,12 h,24 h,48 h).The senescence-related protein expression levels of p53,p21,and p16 in HUVECs treated with 2 μg/m L LPS at different times were detected by western blot.2.To explore the effects of ferroptosis in LPS-induced inflammation and senescence of vascular endothelial cells.(1)HUVECs were treated with 2 μg/m L LPS at different times(0 h,1 h,3 h,6 h,9 h,12 h,24 h).Intracellular iron concentration was detected by the Calcein-AM assay kit and iron ion detection kit.The intracellular glutathione(GSH)and malondialdehyde(MDA)levels were detected by GSH and MDA assay kit,ROS levels were detected by the DCFH-DA assay kit,and Mito Tracker was used to label mitochondria.The ferroptosis-related m RNA and protein expression levels of GPX4,x-CT,PTGS2,NCOA4,and FTH were detected via western blot and q RT-PCR.(2)HUVECs were pretreated with ferroptosis inhibitor Fer-1 for 3 hours,then cocultivated with 2μg/m L LPS for the corresponding time.Intracellular iron ions,MDA,GSH,ROS levels,mitochondrial morphology,and the m RNA and protein expression levels of GPX4,x-CT,PTGS2,NCOA4,and FTH were detected by the corresponding kit.All of these proved that ferroptosis was involved in LPS-induced inflammation in HUVECs.(3)Western blot method was used to detect the inflammatory-related protein expression levels of HMGB1,IL-6,and TNF-α in LPS-treated HUVECs after Fer-1 inhibited ferroptosis.(4)Cell senescence-related β-galactosidase staining was used to detect LPSinduced HUVECs senescence after Fer-1 inhibiting ferroptosis.Senescence-associated protein expression levels of p53,p21,and p16 in LPS-induced HUVECs after Fer-1 inhibiting ferroptosis were detected by western blotting.3.To investigate the regulatory mechanisms of NRF2 on vascular endothelial cell ferroptosis,inflammation,and senescence.(1)The expression levels of NRF2 total protein,nuclear protein,cytoplasmic protein,and NRF2 m RNA were detected in LPS-treated HUVECs before and after inhibiting ferroptosis.To verify the effect of LPS on NRF2 nuclear migration before and after inhibition of ferroptosis,an immunofluorescence assay was used to observe the nuclear entry of NRF2.(2)NRF2 high expression and low expression cell models were established by transfecting NRF2 overexpression plasmid or adding NRF2 inhibitor ML385.By inhibiting or overexpressing NRF2 in LPS-treated HUVECs,the cell proliferation activity,intracellular iron ions,GSH,MDA,and ROS levels,mitochondrial morphology,and ferroptosis-related proteins expression levels of GPX4,x-CT,PTGS2,NCOA4,and FTH were detected.To explore the regulatory role of NRF2 on oxidative damage-mediated ferroptosis in HUVECs.(3)The inflammatory protein expression levels of HMGB1,IL-6,and TNF-α were detected to explore the effect of NRF2 on ferroptosis-related inflammatory.(4)Cell senescence-related β-galactosidase staining was used to detect HUVECs senescence,and senescence-related proteins expression levels of p53,p21,and p16 were detected in HUVECs,to explore the effect of NRF2 on LPS-induced HUVECs senescence.4.To explore whether the NOX4/ROS/NRF2 axis is involved in the process of LPS-induced ferroptosis and inflammation in HUVECs,and the adaptive regulation of NRF2 on oxidative damage and ferroptosis induced by NOX4 overexpression in HUVECs.(1)To explore whether NOX4 is involved in LPS-induced HUVECs inflammation and ferroptosis,the expression level of NOX4 protein was detected via western blot in LPS-treated HUVECs before and after inhibiting ferroptosis.(2)A high expression model of NOX4 was established by NOX4 overexpression plasmid,the ROS level,mitochondrial morphology,and ferroptosis-related protein expression levels of GPX4,x-CT,PTGS2,and FTH were detected in HUVECs,to explore the effect of NOX4 on oxidative damage and ferroptosis.(3)The protein level of NRF2 in HUVECs after NOX4 overexpression was detected using the western blot method.The expression level of NRF2 protein was also detected in NOX4 overexpression and LPS-treated HUVECs after NAC(5 m M)pretreatment.(4)The NOX4-overexpressed HUVECs were treated with ML385,then the cell viability and ferroptosis-related protein expression levels of GPX4,x-CT,PTGS2,and FTH were detected.To verify NRF2-mediated redox adaptation in NOX4-induced oxidative injury and ferroptosis of vascular endothelial cells.5.To investigate the roles of ferroptosis in acute myocardial infarction.(1)The general medical history,clinical biochemical indices,and serum were collected from patients with acute myocardial infarction(AMI)and coronary heart disease(CHD).Serum iron,GSH,and malondialdehyde(MDA)levels were detected by serum iron,GSH,and MDA assay kit.(2)The general medical history,clinical biochemical indices,serum iron ion,GSH,and MDA levels in patients with AMI and CHD were compared.Correlations among serum iron levels,MDA levels,and blood lipid levels were analyzed by spss26.Results 1.The effect of LPS on inflammation and senescence of vascular endothelial cells was clarified,and the inflammatory and senescence model was established.(1)Under the cultured with LPS,the proliferation activity of HUVECs was decreased in a time-and concentration-dependent manner;(2)Under the cultured with LPS,the inflammatory-related proteins expression levels of HMGB1,IL-6,and TNF-α were increased in a time-dependently manner;(3)Under the cultured with LPS,β-galactosidase positive cells ratio of HUVECs was increased in a concentration-and time-dependent manner,and the senescencerelated protein expression levels of p53,p21,and p16 were increased in a timedependent manner.2.Ferroptosis is involved in LPS-induced HUVECs inflammation,inhibition of ferroptosis can improve LPS-induced vascular endothelial cell inflammation and senescence.(1)Ferroptosis was involved in LPS-induced HUVECs oxidative damage.Under the cultured with LPS,the iron ions levels,MDA levels,ROS levels,and mitochondrial damage were aggravated,and the GSH levels were decreased.The m RNA and protein levels of anti-ferroptosis factors GPX4,x-CT,and FTH were decreased,and the proferroptosis factors PTGS2 and NCOA4 were increased.(2)Under the culture with LPS,Fer-1,a ferroptosis inhibitor,was added.It was found that inhibition of ferroptosis can increase HUVECs survival viability,decrease intracellular iron ion,MDA,and ROS levels,ameliorate mitochondrial damage,and increase GSH levels.Inhibition of ferroptosis can also increase m RNA and protein expression levels of anti-ferroptosis factors GPX4,x-CT,and FTH,and decrease the m RNA and protein expression levels of pro-ferroptosis factors PTGS2 and NCOA4.(3)Under the cultured with LPS,it was found that inhibition of ferroptosis can decrease inflammatory-related protein expression levels of HMGB1,IL-6,and TNF-α in HUVECs.(4)Under the cultured with LPS,inhibition of ferroptosis decreased the proportion of β-galactosidase positive HUVECs cells and the senescence-related protein expression levels of p53 and p21.3.NRF2 is activated during oxidative damage in vascular endothelial cells,overexpression of NRF2 improves LPS-induced HUVECs ferroptosis,inflammation,and senescence.(1)Under the cultured with LPS in HUVECs,the expression levels of NRF2 m RNA,total protein,and nuclear protein increased in a time-dependently manner.Immunofluorescence assay also found that the LPS could increase cell nuclear accumulation of NRF2.Under LPS treatment,inhibition of ferroptosis decreased the expression levels of NRF2 total protein,nuclear protein,and the nuclear transfer of NRF2.(2)Under LPS treatment,NRF2 high expression can improve the HUVECs survival viability,decrease intracellular iron ions,MDA,ROS levels,and decrease mitochondrial damage,increase GSH levels,and increase the anti-ferroptosis protein expression levels of GPX4,x-CT,and FTH,decrease the pro-ferroptosis protein levels of PTGS2 and NCOA4.On the contrary,inhibition of NRF2 can increase HUVECs death rate,increase intracellular iron ions,MDA,and ROS levels,aggravate mitochondrial damage,decrease intracellular GSH levels,decrease x-CT,GPX4,and NCOA4 protein expression,and increase PTGS2 and FTH protein expression.(3)Under LPS treatment,NRF2 high expression can decrease inflammatoryrelated protein expression levels of HMGB1,IL-6,and TNF-α in HUVECs.Inhibition of NRF2 increases inflammatory-related protein expression levels of HMGB1,IL-6,and TNF-α in HUVECs.(4)Under LPS treatment,NRF2 high expression can decrease the percentage of β-galactosidase-positive cells and the protein expression levels of p53 and p21 in LPSinduced HUVECs;inhibition of NRF2 can increase the ratio of β-galactosidase-positive cells and the protein expression levels of p53 and p21 in LPS-induced HUVECs.4.The NOX4/ROS/NRF2 axis is involved in LPS-induced HUVECs ferroptosis,NRF2-mediated redox adaptation regulated NOX4 high expressioninduced HUVECs ferroptosis resistance.(1)Under LPS treatment in HUVECs,NOX4 protein expression level was increased in a time-dependently manner.Ferroptosis inhibitor Fer-1 reduced the expression level of NOX4 protein in LPS-induced HUVECs.(2)Under LPS treatment in HUVECs,NOX4 high expression can increase ROS level,aggravate mitochondrial damage,increase anti-ferroptosis protein expression levels of GPX4,x-CT,and FTH,and decrease pro-ferroptosis protein expressions levels of PTGS2.(3)High expression of NOX4 increased the protein expression level of NRF2.Pretreatment with NAC,an inhibitor of ROS production,decreased NRF2 protein expression level in NOX4 overexpression and LPS-treated HUVECs.(4)High expression of NOX4 increased the proliferation activity of HUVECs.Inhibition of NRF2 decreased the proliferation activity in NOX4 overexpressed HUVECs.Inhibition of NRF2 down-regulated the anti-ferroptosis protein expressionlevel of GPX4,x-CT,and FTH,and up-regulated the protein expression level of PTGS2 in NOX4-overexpressed HUVECs.5.Serum iron level is an independent risk factor for acute myocardial infarction.Ferroptosis may be involved in the progress of acute myocardial infarction.(1)The general medical history and clinical biochemical indices of patients with acute myocardial infarction(AMI)(Disease group)and coronary heart disease(CHD)(Control group)were analyzed,and it has found that the two groups were statistically different(P < 0.05)in gender,smoking history,LDL/HDL,folate,and fasting blood glucose levels.(2)Serological analysis found that patients with acute myocardial infarction had higher serum iron and serum MDA levels than patients with coronary heart disease,(P < 0.05).(3)Binary Logistic regression analysis showed that serum iron ion levels were an independent risk factor for acute myocardial infarction(OR=11.042,95%CI=1.769-68.923).(4)The correlation analysis of serum iron,blood lipids,and serum MDA levels in the AMI group and CHD group has shown that with the increase of serum iron level,serum MDA level,LDL,and LDL/HDL also showed an increasing trend.The correlation analysis results between serum MDA levels and lipid levels in the two groups showed that with the increase of serum MDA level in the AMI group,the serum LDL level and LDL/HDL also showed an increasing trend.However,no such trend was observed in patients with CHD.Conclusion 1.LPS-induced inflammatory and cell senescence in HUVECs.2.Ferroptosis is involved in the pathogenesis of inflammatory and LPS-induced senescence of vascular endothelial cells.Inhibiting ferroptosis can improve LPSinduced inflammation and cell senescence of vascular endothelial cells.3.NRF2 reduced LPS-induced ferroptosis by activating the system Xc--glutathione-GPX4 axis and inhibiting the ferritinophagy signaling pathway.NRF2 reduces ferroptosis-dependent inflammation by decreasing the protein levels of HMGB1,IL-6,and TNF-α in LPS-induced vascular endothelial cells.4.P53-mediated ferroptosis is involved in LPS-induced vascular endothelial cell senescence.NRF2 can improve the LPS-induced senescence of vascular endothelial cells by regulating the p53/p21 senescence pathway.5.The NOX4/ROS/NRF2 axis is involved in LPS-induced vascular endothelial cell ferroptosis,NRF2-mediated redox adaptation improves vascular endothelial cell ferroptosis induced by the pro-ROS generation factor NOX4.NRF2 is an important regulator of oxidative homeostasis in vascular endothelial cells.6.Serum iron level is an independent risk factor for acute myocardial infarction.Ferroptosis may be involved in the progression of acute vascular events of coronary atherosclerosis.Innovation and Significance Innovation: 1.This study confirmed that the antioxidant defense factor NRF2 can reduce LPSinduced vascular endothelial cell inflammation and cell senescence by inhibiting ferroptosis.2.We further confirmed that NOX4/ROS/NRF2 pathway was involved in the regulation of ferroptosis in vascular endothelial cells from the mechanism of cellular redox homeostasis.Significance: By establishing a model of LPS-induced inflammatory injury and senescence in HUVECs,it was confirmed that ferroptosis is associated with LPS-induced HUVECs inflammation and cellular senescence.Inhibition of ferroptosis can effectively improve LPS-induced vascular damage.The novel role of NRF2 in anti-oxidative injury was revealed from the perspective of vascular endothelial cell ferroptosis,inflammation,cell senescence,and redox homeostasis.In addition,the analysis of clinical data showed that serum iron levels were an independent risk factor for acute myocardial infarction,and ferroptosis may be a risk factor in the development of acute vascular events of coronary atherosclerosis.Therefore,this study demonstrates from both in vitro and in vivo that the treatment targeting ferroptosis will provide a new idea for improving the prognosis of atherosclerotic-related vascular diseases.