Study On The Mechanism Of Propofol Alleviating Myocardial Ischemia/Reperfusion Injury By Inhibiting Ferroptosis Via Regulating AKT/P53 Pathway

IntroductionIschemic heart disease often occurs in myocardial ischemia and aggravates the injury after blood supply,which is called myocardial ischemia/reperfusion(I/R)injury.Myocardial I/R injury is often seen on percutaneous coronary intervention,heart valve surgery,and open-heart surgery,resulting in myocardial infarction,arrhythmia,heart failure,and cardiomyocyte death,which seriously affects the patient’s rehabilitation.In recent years,perioperative myocardial protection has attracted much attention.Therefore,exploring the mechanism of preventing and treating myocardial I/R injury is of great significance in clinical anesthesia.Ferroptosis is an important form of cell death in myocardial I/R injury.Inhibiting myocardial ferroptosis may reduce myocardial I/R injury.Under the action of lipoxygenase or Fe2+,reactive oxygen species(ROS)increase and lipid peroxidation,resulting in cell ferroptosis.Ferroptosis is characterized by mitochondrial atrophy,cristae reduction,and outer membrane rupture.When cardiomyocytes are stimulated by ischemia and hypoxia,p53 activity increases and enters the nucleus to regulate the expression of downstream proteins to promote ferroptosis.Therefore,how to inhibit p53 activity and then inhibit cardiomyocyte ferroptosis has become the key to reducing myocardial I/R injury.Propofol is similar to vitamin E in structure and has a strong antioxidant effect.By promoting antioxidant enzymes expression,propofol inhibits ROS production and lipid peroxidation,reducing myocardial I/R injury.Excessive producing cellular ROS and lipid peroxidation are important factors in ferroptosis occurrence.Therefore,propofol may play a myocardial protective role by inhibiting cardiomyocytes’ferroptosis.Propofol inhibits p53 activity in myocardial I/R injury,but its mechanism is unclear.P53 activity is negatively regulated by protein kinase B(PKB/AKT).Phosphorylated AKT promotes the binding of mouse double minute 2(MDM2)to p53,induces p53 decomposition,and inhibits cell ferroptosis.Propofol has been proved to inhibit rat liver I/R injury through AKT/p53 signaling pathway.Does propofol reduce myocardial I/R injury by inhibiting cardiomyocyte ferroptosis?Does AKT/p53 signaling pathway play a role in propofol inhibiting ferroptosis in cardiomyocytes?So far,it has not been studied.Our research group put forward the hypothesis that propofol reduces myocardial I/R injury by inhibiting cardiomyocytes’ ferroptosis.The above mechanism is related to AKT/p53 signaling pathway.To test this hypothesis,our research group designed relevant experimentsPart Ⅰ:Study on propofol inhibiting myocardial ischemia/reperfusion injury by inhibiting cardiomyocyte ferroptosisObjective1.To observe the role of cell ferroptosis in propofol reducing H9C2 cells injury.2.To detect the role of cell ferroptosis in propofol reducing rats myocardial I/R injury.Methods1.Routine culturing and passaging of H9C2 cells.2.Cell grouping,modeled with hydrogen peroxide(H2O2)and erastin(ferroptosis inducer)(Figure 1-1 A):(1)Group C:routine culturing for 25h.(2)Group H:routine culturing for 60min and adding 200μM H2O2 for 24h.(3)Group H+P:adding 50μM propofol for 60min and 200μM H2O2 stimulating for 24h.(4)Group E:routine culturing for 60min and adding 2.5 μM erastin for 24h.(5)Group E+P:adding 50μM propofol for 60min and 2.5μM erastin for 24h.3.The cell viability was detected by phase-contrast microscope,trypan blue staining,and CCK-8 experiment(Figure 1-2).4.The contents of reactive oxygen species and superoxide anion were detected by flow cytometry and fluorescent staining.5.Cellular immunofluorescence was used to evaluate the nuclear translocation level of nuclear factor E2 related factor 2(NRF2).6.Malondialdehyde(MDA),superoxide dismutase(SOD)activity and ferrous concentration were detected by kits.7.Establishing Langendorff model of isolated rat heart and grouping(Figure 1-1B):(1)Group C:infusing KH solution for 120min.(2)I/R group:infusing KH solution for 30min,stopping perfusion for 30min,and reperfusion for 60min.(3)I/R+P group:perfusing with KH solution for 10min and 50μM propofol for 20min,stopping perfusion and reperfusion.8.The activity of lactate dehydrogenase(LDH),SOD,the content of creatine kinase isoenzymes(CK-MB),the ratio of reduced glutathione/oxidized glutathione(GSH/GSSG),MDA,and ferrous content were measured by kits.9.The area of myocardial infarction,pathology,and mitochondria were evaluated by triphenyl tetrazolium chloride(TTC),HE staining,and fluoroscopy electron microscope.10.The nuclear translocation of myocardial NRF2 was measured by immunohistochemistry.11.The expressions of NRF2,heme oxygenase-1(HO-1),ferritin heavy chain 1(FTH1),glutathione peroxidase 4(GX4),and cystine/glutamate reverse transport body(XCT)in H9C2 cells and myocardium were evaluated by Western blot.Results1.Compared with the control group,H2O2 decreased cell viability and increased reactive oxygen species.After propofol was used,cell viability and reactive oxygen species fell back compared with group H.2.H2O2 induced NRF2 nuclear translocation and HO-1 expression were slightly higher than that in the control group.After propofol application,NRF2 nuclear translocation and HO-1 increased significantly compared with group H.3.Compared group control,erastin caused a decrease in cell viability and an increase in superoxide anion.After propofol pretreatment,cell viability and superoxide anion were relieved compared with group E.4.The contents of MDA and ferrous in erastin group were higher than those in the control group,and SOD activity and anti-ferroptosis enzyme expression were lower.After propofol application,MDA,ferrous content,SOD activity,and anti-ferroptosis enzyme decreased.5.Compared with group control,I/R caused the increase in LDH,CK-MB,MDA,and ferrous content,and the decrease in SOD activity and GSH/GSSG ratio.Compared with group I/R,the contents of LDH,CK-MB,MDA,ferrous decreased,and the activity of SOD and the ratio of GSH/GSSG increased after propofol treatment.6.Compared with normal myocardium,I/R induced increased myocardial infarction area,fibrous edema,mitochondrial ridge distortion,and decreased GSH/GSSG ratio;Compared with group I/R,propofol decreased myocardial infarction area,fiber edema,mitochondrial ridge regularity,and GSH/GSSG ratio.7.After I/R treatment,NRF2 nuclear translocation and HO-1 increased slightly compared with the control group.After propofol pretreatment,NRF2 nuclear translocation and HO-1 increased remarkably compared with group I/R.8.Compared with the control group,the expression of the anti-ferroptosis enzyme induced by I/R decreased.The expression of the anti-ferroptosis enzyme in group propofol was remarkably more than that in the I/R group.Conclusion1.Propofol inhibits H9C2 cells ferroptosis.2.Propofol inhibits cardiomyocytes’ ferroptosis to reduce myocardial I/R injury.Part Ⅱ:Propofol alleviated myocardial ischemia/reperfusion injury by inhibiting ferroptosis via regulating AKT/P53 pathwayObjective1.To investigate the effect of propofol on AKT knockout H9C2 cells ferroptosis.2.To explore whether propofol alleviates myocardial ischemia/reperfusion injury by inhibiting ferroptosis via regulating AKT/P53 pathway.Methods1.Routine culturing and passaging of H9C2 cells.2.Cell grouping(Figure 2-1A):(1)Group C:routine culturing for 25h.(2)Group E:routine culturing for 60min and adding 2.5μM erastin for 24h.(3)Group E+P:adding 50 μM propofol for 60min and 2.5μM erastin for 24h.3.The cell viability was examined by the phase-contrast microscope,trypan blue staining,and CCK-8 experiment.4.The effects of propofol on AKT,p-AKT,anti-ferroptosis enzymes(FTH1,GPX4,XCT),and cell viability of H9C2 cells treated with AKT siRNA and erastin were evaluated by Western blot and CCK-8 experiments.5.Establishing Langendorff model of isolated rat heart,and using AKT inhibitor MK2206.Grouping(Figure 2-1B):(1)Group C:infusing KH solution for 120min.(2)I/R group:infusing KH solution for 30min and stopping perfusion for 30min and reperfusion for 60min.(3)I/R+P group:perfusing with KH solution for 10 min and 50μM propofol for 20min,stopping perfusion and reperfusion.(4)I/R+P+MK:injecting 15nm mk2206 for 10min and propofol,stopping perfusion and reperfusion.(5)I/R+MK:injecting 15nm mk2206 for 10min and KH solution for 20min,and stopping perfusion and reperfusion.6.The GSH/GSSG ratio and ferrous content of the myocardium were detected by kits.7.Myocardial pathology and mitochondria were measured by HE staining and fluoroscopy electron microscope.8.The expression of myocardial anti-ferroptosis enzyme and p53 were measured by Western blot.9.The expression of AKT and p-AKT in the myocardium was evaluated by Western blot and immunofluorescence.Results1.Compared with groupscramble siRNA,AKT,p-AKT,and anti-ferroptosis enzyme expression and cell viability decreased in AKT siRNA group.Compared with group scramble siRNA E,the expression of anti-ferroptosis enzyme and cell viability in AKT siRNA E group decreased slightly.Compared with group scramble siRNA E+P,the expression of anti-ferroptosis enzyme and cell viability in AKT siRNA E+P group decreased slightly.2.After I/R treatment,GSH/GSSG ratio decreased and ferrous content increased compared with group control.Compared with group I/R and I/R+MK group,propofol pretreatment increased GSH/GSSG ratio and decreased ferrous content respectively.3.Compared with group control,the I/R group caused myocardial fiber edema,wavy changes,and mitochondrial ridge distortion.Compared with group I/R and I/R+MK group,propofol treatment reduced myocardial fiber edema and wavy changes and promoted mitochondrial ridge regularity,respectively.4.Compared with group control,the anti-ferroptosis enzyme expression increased and p53 expression decreased in I/R treatment.Compared with group I/R and I/R+MK group,propofol treatment increased the expression of the anti-ferroptosis enzyme and decreased the expression of p53,respectively.5.Compared with group control,the p-Akt expression in group I/R decreased.Compared with group I/R and group I/R+MK,propofol treatment promoted the p-AKT expression.There was no difference in AKT expression among groups.Conclusion1.Propofol inhibits H9C2 cell’s ferroptosis through AKT signaling pathway.2.Propofol regulates AKT/p53 signaling pathway,inhibits cardiomyocytes ferroptosis,and reduces myocardial I/R injury.Full-text conclusionPropofol alleviated myocardial ischemia/reperfusion injury by inhibiting ferroptosis via regulating AKT/P53 pathway.Innovation and significance1.Propofol was found to inhibit cardiomyocytes’ ferroptosis for the first time.2.It was found for the first time that Propofol regulates AKT/p53 signaling pathway,inhibits cardiomyocytes ferroptosis,and reduces myocardial I/R injuryLimitations1.The concentration-dependent changes of myocardial protective effect on propofol were not discussed.2.The expression of other anti-ferroptosis enzymes was not detected.