Rosiglitazone Regulates Ferroptosis Of HUVEC By Inhibiting The ACSL4-mediated Lipid Peroxidation Pathway

Objectives:With the aging of the population and the improvement of people’s living standard,as well as the poor lifestyle,the incidence of cardiovascular diseases,diabetes,hyperlipidemia and other diseases is gradually increasing,which has become a major threat to human health.Vascular endothelial dysfunction is closely related to atherosclerosis,diabetes mellitus and hyper lipidemia.Ferroptosis is a kind of cell death discovered in recent years.Study found that lipid peroxidation plays an important role in Ferroptosis,acyl coenzyme A synthetase long chain members of the family of 4(ACSL4)is the key enzyme of lipid ingredients,especially by means of phosphatidyl ethanolamine(PE)adjusted,ACSL4 can promote the lipid peroxidation,and promote cell death,ACSL4 catalyst containing arachidonic acyl(AA)and adrenaline acyl(Ad A)PE priority is the main cause of Ferroptosis.TZDs is a kind of insulin sensitization agent,a recent study found that rosiglitazone(RSG)can restrain specificity ACSL4,but the ACSL other subtypes,there is no obvious inhibition,we assume that rosiglitazone by inhibiting ACSL4 reduce lipid peroxidation products and the accumulation of oxygen free radicals(ROS),maintain membrane integrity,thus to reduce Ferroptosis to protect the role of cell damage,RSA selective death compounds(RSLs)include Erastin and RSL3 to induce cell death,This experiment investigated whether rosiglitazone can reduce Ferroptosis of the human umbilical vein endothelial cells(HUVEC)induced by RSL3,RSG inhibit Ferroptosis mechanism,and observation of RSG and Ferroptosis inhibitors Ferrostatin-1 whether there is interaction,it may be to improve the tissue and cell damage caused by Ferroptosis and treatment of chronic diseases,to provide a new strategy.Methods:1.To detect the cytotoxicity of RSL3: HUVEC were treated with RSL3 solution at different concentrations(0μM,0.1μM,0.2μM,0.5μM,1μM,2μM).Cell proliferation was detected by CCK-8 method at 6h,12 h and 24 h to determine the optimal concentration and time of RSL3.2.RSL3 induced Ferroptosis: HUVEC were treated with RSL3 at 1μm concentration for 12 h,at the same time add Ferroptosis inhibitors Ferrostatin-1 interfere,by CCK 8 method influence on HUVEC cell survival rate,and by RT-PCR method and protein immunoblot electrophoresis detecting different intervention group of Ferroptosis GPX4,SLC7A11 m RNA and protein expression level,determine Ferroptosis,build RSL3 Ferroptosis HUVEC cell death model;3.Cytotoxicity of RSG: cells were treated with different concentrations of RSG(0μM,1μM,15μM,25μM 50μM,75μM and cultured for 24 hours.Cell proliferation was detected by CCK-8 method.4.RSG and lipid peroxidation levels: MDA content in each group after treatment with RSG(0μM,1μM,15μM,25μM)was determined by thiobar bituric acid(TBA)method to reflect lipid peroxidation level.DCFH-DA immunofluorescence staining was used to detect the ROS content in each group after different concentrations of RSG and Ferrostatin-1 intervention..5.RSG and Ferroptosis: Western Blotting was used to detect the relative expression levels of ACSL4,GPX4,FTH1,and COX2 proteins,and RT-PCR was used to detect the expression levels of ACSL4 and GPX4 m RNA after the addition of rosiglitazone and other drugs to the iron-death cell model constructed in the earlier stage,and to investigate the possible molecular mechanism of rosiglitazone inhibiting iron death.Results:1.CCK-8 results of RSL3 showed that,compared with the control group without RSL3,HUVEC was treated with RSL3 at different concentrations and at different times,and RSL3 decreased cell viability in a dose-dependent and time-dependent manner.The HUVEC cell damage model was constructed by the intervention of RSL3 with a concentration of 1μM for 12 h.2.Ferroptosis induced by RSL3:compared with control group,RSL3 intervention cell RT-PCR and protein immunoblot electrophoresis results showed that GPX4,SLC7A11 protein expression significantly lower,RSL3 and Ferrostatin-1 training can improve the survival rate of the HUVEC and reversal at the level of gene and protein level in GPX4,SLC7A11 expression,explain successfully established RSL3 induced HUVEC cell death model,Ferrostatin-1 effectively inhibits Ferroptosis;3.RSG CCK-8 results showed that HUVEC treated with low concentration(≤25μM)of rosiglitazone showed no significant difference in cell proliferation compared with the control group without RSG(P>0.05),while higher concentration(≥50μM)of RSG inhibited cell proliferation and was toxic to cells.4.RSG inhibits intracellular lipid peroxidation: After the cells are intervened by RSG,the MDA content of intracellular lipid peroxidation products shows a downward trend.DCFH-DA immunofluorescence staining results show that ROS accumulation decreases.Small doses of RSG have a significant inhibitory effect.5.The effect of RSG on Ferroptosis-related factors: protein immunoblot electrophoresis results showed that compared with RSL3 intervention group,ASCL4,COX2 expression in cells levels significantly decreased(P<0.05)after pretreatment RSG,increase GPX4 protein levels,no obvious difference was found between FTH1 protein expression,compared with group Ferrostatin-1,RSG and Ferrostatin-1 cells after cut ACSL4 expression,GPX4 protein levels rise significantly,RT-PCR and protein immunoblot electrophoresis results are consistent.Conclusions:Research results show that:(1)RSL3 successfully induced HUVEC Ferroptosis,RSL3 with dose and time dependent on the way to reduce the cell survival rate,RSL3 intervention group at the genetic level and protein level GPX4,SLC7A11 expression significantly lower,Ferrostati-1 set of relieve RSL3 cytotoxic effect,to improve the survival rate of HUVEC,and at the genetic level raised GPX4,SLC7A11 expression and protein level,RSL3 induction success HUEVC cell death,Ferrostatin-1 can reverse this phenomenon.2.RSG intervention group to detect the contents of MDA and ROS accumulation is on the decline,RSG groups cut ASCL4 and COX2 protein expression level,raise GPX4 protein expression,RSG inhibit ACSL4 m RNA,increased GPX4 m RNA relative expression,intervention group Ferrostatin-1 ACSL4 on protein and gene level,the GPX4 with RSG similar,RSG + Ferrostatin-1 set of both use intervention effect is more apparent.ACSL4 activation of unsaturated fatty acids(PUFA)generated PUFA-CoA,PUFA-CoA by lysophospholipids single alkali single-base transferase 3(LPCAT3)on lipid reshaping,generate PUFA-PE by lipoxygenase(ALOX)and Fenton reaction generates harmful lipid active oxygen(L-OOH),on the one hand plenty of L-OOH accumulation directly induce death,on the other hand L-OOH was reduced to non-toxic lipid alcohol(L-OH)under the action of GPX4,when GPX4 inactivation or activity is restrained leads to the accumulation of L-OOH and membrane lipid peroxidation damage,which triggers Ferroptosis.RSG may reduce Ferroptosis by targeting the inhibition of ACSL4 or increasing the activity of GPX4 to reduce the accumulation of lipid peroxidation products and ROS in the downstream pathway of Ferroptosis.In addition,Ferrostatin-1 and RSG have been found to have a synergistic effect on the alleviation of Ferroptosis.