To Explore The Effect And Mechanism Of CFLIP On Myocardial Ischemia/Reperfusion Injury Based On The Pathway Of Programmed Cell Death

Background Myocardial ischemia/reperfusion injury(MI/RI)is an important factor affecting the efficacy of reperfusion therapy for coronary heart disease.Apoptosis,autophagy,and necroptosis mediated cardiomyocyte programmed death are the key factors of MI/RI.Objective This study was performed to infect primary neonatal rat cardiomyocytes and rat myocardial tissues with cFLIP overexpression recombinant adenovirus vector to explore the effects of cFLIP on MI/RI-induced cardiomyocyte apoptosis,autophagy and necroptosis,in order to provide new therapeutic targets and theoretical basis for preventing MI /RI.Methods(1)In vitro: Primary neonatal rat cardiomyocytes were extracted and randomly divided into six groups: Control group,hypoxia/reoxygenation(H/R)group,adenovirus negative control(Ad-NC)group,Ad-cFLIPL group,Ad-cFLIPS group,and Ad-cFLIPL+S group.After transfecting cardiomyocytes with recombinant adenoviruses of different multiplicity of infection(MOI)for 3 d,fluorescence microscopy and flow cytometry were used to detect the infection efficiency,and CCK-8 was used to detect cytotoxicity to determine the best MOI value of recombinant adenovirus.Establish H/R model(hypoxia for 4h and reoxygenation for 12 h),the CCK-8 was used to detect the effect of H/R on cell viability.Flow cytometry was used to detect changes in the apoptotic rate of each group.Transmission electron microscope was used to observe changes in the number of autophagosomes in each group.The DAPI/PI double staining was used to detect changes in the necrosis rate in each group.Western blot was used to detect the expression levels of proteins which associated with apoptosis,autophagy and necroptosis.(2)In vivo: sixty SD rats were divided into the Sham+Ad-NC group,I/R+Ad-NC group,I/R+Ad-cFLIPL group,I/R+Ad-cFLIPS group,I/R+Ad-cFLIPL+S group.3 d after multiple injections of recombinant adenovirus at the apex of heart.The left anterior descending coronary artery ligation was performed to induce myocardial ischemia for 30 min,and the ligature was loosened for reperfusion 3 h to construct MI/RI model.The level of serum LDH and CK-MB were measured to evaluate the degree of myocardial damage,TTC staining was used to detect the ratio of myocardial infarction area in each group,H&E staining was used to detect the myocardial injury index,TUNEL staining was used to exam the positive rate of apoptotic cells in myocardial tissue,and Western blot was used to detect the expression changes of key proteins of apoptosis,autophagy and necroptosis in each group.Results(1)In vitro:(1)100 MOI was used as the best MOI value,the infection efficiency of recombinant adenovirus reaches more than 90%,and does not affect cell viability.(2)H/R significantly reduced the viability of cardiomyocytes and down-regulated the expression of cFLIP protein,while infection of Ad-cFLIPL,Ad-cFLIPS and Ad-cFLIPL+S up-regulated the protein expression of cFLIPL and cFLIPS respectively.Compared with Ad-NC group,the cell viability was increased significantly in cFLIP overexpression group(all P<0.05).(3)Compared with the Control group,the apoptotic rate of the H/R group and the Ad-NC group increased significantly,while the infection of Ad-cFLIPL,Ad-cFLIPS and Ad-cFLIPL+S significantly reduced the rate of apoptosis(all P<0.05).Western blot was used to detect apoptosis-related proteins and found that H/R significantly induced the cleavage of Caspase-3,Caspase-8 and Caspase-9,and overexpression of cFLIPL and(or)cFLIPS could significantly inhibit activation of these proteins.(4)Transmission electron microscopy was used to observe autophagosomes in each group,the results showed that H/R significantly induced the formation and aggregation of autophagosomes in the myocardial cytoplasm,while overexpression of cFLIP could significantly reduce the number of autophagosomes,while down-regulating the protein expression levels of Beclin-1 and LC3-II(all P<0.05).(5)Immunofluorescence was used to detect the positive rate of PI staining,it was found that compared with the Control group,H/R significantly increased the ratio of PI-positive cells,and overexpression of cFLIP could significantly reduce the ratio of PI-positive cells.Western blot was used to detect the expression of necroptosis-related proteins,the results showed that compared with the Control group,H/R injury significantly increased the expression of p-RIPK1,p-RIPK3 and p-MLKL protein.Infection of Ad-cFLIPL,Ad-cFLIPS and Ad-cFLIPL+S found that overexpression of cFLIPL,cFLIPS and cFLIPL+S can significantly down-regulate p-RIPK1,p-RIPK3 and p-MLKL proteins expression(all P<0.05)compared with Ad-NC group.(2)In vivo:(1)Compared with the Sham+Ad-NC group,I/R significantly down-regulated the protein expression of cFLIPL and cFLIPS,while infection of Ad-cFLIP could up-regulate the protein expression of cFLIPL and cFLIPS(all P<0.05),respectively.(2)Compared with the Sham+Ad-NC group,the serum LDH and CK-MB levels and the degree of myocardial tissue damage in the I/R+Ad-NC group were significantly increased,while overexpression of cFLIP can significantly reduce the serum LDH and CK-MB levels,and significantly improved myocardial damage caused by I/R(all P<0.05).(3)TTC staining was used to detect the myocardial infarction area of rats in each group,I/R caused about 28.83%±2.66% of myocardial infarction area,and overexpression of cFLIP can significantly reduce the myocardial infarction area caused by I/R(all P<0.05).(4)TUNEL staining was used to detect the rate apoptosis in myocardial tissue,this result showed that compared with the Sham+Ad-NC group,I/R significantly increased the ratio of TUNEL-positive cells in myocardial tissue,while overexpression of cFLIP could significantly reduce the ratio of TUNEL-positive cells in myocardial tissue(all P<0.05).Western blot was used to detect the expression of apoptosis-related proteins in myocardial tissue and found that I/R significantly promoted the cleaved activation of Caspase-3,Caspase-8 and Caspase-9,and overexpression of cFLIP can significantly inhibit the cleaved activation of these apoptotic proteins(all P<0.05).(5)Western blot was used to detect the expression of autophagy-related proteins in myocardial tissue and found that I/R can significantly up-regulate the protein expression of Beclin-1 and LC3-II,while overexpression of cFLIP can significantly inhibit the expression of these autophagy-related proteins(all P<0.05).(6)Western blot was used to detect the expression of proteins related to necroptosis in myocardial tissue and found that I/R can significantly promote the phosphorylation of RIPK1,RIPK3 and MLKL,while overexpression of cFLIP can inhibit the activation level of these proteins(all P<0.05).Conclusion The protein expressions of cFLIPL and cFLIPS in cardiomyocytes were significantly down-regulated by H/R and I/R treatment,while large numbers of apoptosis,autophagy and necroptosis were induced.Up-regulating the expression of cFLIP protein can effectively reduce MI/RI via inhibiting apoptosis,autophagy and necroptosis simultaneously.Therefore,cFLIP may be an effective and multi-effect therapeutic target to reduce MI/RI in clinical scenario.