Sacubitril/Valsartan Ameliorates Doxorubicin Cardiotoxicity By Mediating Ferroptosis

Objective:To investigate the role of sacubitril/valsartan in doxorubicin cardiotoxicity by constructing doxorubicin-induced cell injury and cardiotoxicity models.The role of ferroptosis in this process and possible mechanism were initially investigated to provide theoretical basis and strategies for clinical application of sacubitril/valsartan in the prevention of doxorubicin cardiotoxicity.Methods:1.Rat cardiomyocytes H9c2 was pretreated with different concentration of sacubitril/valsartan,CCK-8 method was used to detect the influence of sacubitril/valsartan on the cell viability of H9c2 treated with doxorubicin.2.Sacubitril/valsartan or valsartan pretreated rat H9c2 cells and primary cardiomyocytes induced by doxorubicin,western blot and qPCR were used to detect the protein expression of atrial natriuretic peptide（ANP）,B-type natriuretic peptide（BNP）.The level of lactate dehydrogenase（LDH）in cell supernatant was detected by automatic biochemical detection machine.The effect of sacubitril/valsartan or valsartan on the surface area of doxorubicin-induced H9c2 cells and primary cardiomyocytes were detected by immunofluorescence.3.6-8-week-old male C57BL/6 mice were randomly divided into four groups:control group（n=12,saline group）,doxorubicin treated group（n=12,Dox+vehicle group）,doxorubicin plus valsartan treated group（n=12,Dox+VAL group）,and doxorubicin plus sacubitril/valsartan treated group（n=12,Dox+SAC/VAL group）.Echocardiography was performed after 19 days,and on day 20,protein of cardiac tissue was extracted and paraffin-embedded sections of cardiac tissue were prepared.The protein expression of ANP,BNP were detected by Western blot.The levels of plasma LDH and creatine kinase MB（CK-MB）in mice were detected by automatic biochemical detection machine.HE staining,Masson staining and DHE staining were used to observe the structure,the degree and the reactive oxygen species of cardiac tissue.4.The ferroptosis inhibitor ferrostatin-1 treated with H9c2 cells induced by doxorubicin,and then the protein expression of long-chain acyl-coenzyme A（Co A）synthetase 4（FACL4）,the enzyme glutathione peroxidase 4（GPX4）,ANP and BNP was detected by Western blot.Detected the level of LDH,malondialdehyde（MDA）,glutathione（GSH）and Fe²⁺.C11-BODIPY 581/591 staining was used to detect lipid peroxidation levels of cells.5.Sacubitril/valsartan pretreated H9c2 cells induced by doxorubicin,the expression of FACL4 and GPX4 protein were detected by Western blot.Cytoplasmic reactive oxygen species（ROS）level was detected by immunofluorescence and flow cytometry.Detected the level of MDA,GSH,Fe²⁺.C11-BODIPY 581/591 staining was used to detect lipid peroxidation levels of cells.Then,transmission electron microscope was used to observe the mitochondrial,the mitochondrial membrane potential was detected by JC-1 fluorescence probe,and the mitochondrial ROS level was detected by Mito-SOX.The ferroptosis activator Erastin treated with H9c2 cells,the protein expression of ANP,BNP,FACL4,GPX4,and the level of LDH were detected.The protein expression of FACL4,GPX4 in cardiac tissue were detected by Western blot.Immunohistochemistry staining was used to detect the expression of GPX4 in cardiac tissue.In addition,we also examined the levels of Fe²⁺,GSH in the cardiac tissue.Mitochondria in cardiac tissue of mice in different treatment groups were observed by transmission electron microscope.6.Sacubitril/valsartan pretreated H9c2 cells and mice induced by doxorubicin,the protein expression levels of silent information regulator 2homolog 1（Sirt1）and nuclear factor erythroid 2-related factor 2（Nrf2）were detected by Western blot.Immunohistochemistry staining was used to detect the protein expression levels of Sirt1 and Nrf2 in cardiac tissue.7.H9c2 cells were transfected with Sirt1 or Nrf2 si RNA for 24 h,and then pretreated with sacubitril/valsartan for 6 h and doxorubicin for another24 h in the presence of sacubitril/valsartan.The protein expression levels of Sirt1,Nrf2,FACL4,GPX4,ANP,BNP were detected by western blot.Detected the level of LDH,MDA,GSH,Fe²⁺.C11-BODIPY 581/591staining was used to detect lipid peroxidation levels of cells.Then,transmission electron microscope was used to observe the mitochondrial,the mitochondrial membrane potential was detected by JC-1 fluorescence probe,and the mitochondrial ROS level was detected by Mito-SOX.Results:1.Cell viability had no significant differences at 5,10,20μmol/L sacubitril/valsartan treatment,but significantly reduced at 30 mmol/L（p<0.05）.Sacubitril/valsartan significantly and concentration-dependently up-regulated the H9c2 cell viability induced by doxorubicin.2.Compared with the valsartan,sacubitril/valsartan down-regulated the increased protein and m RNA expression of ANP and BNP induced by doxorubicin in H9c2 cells,decreased LDH levels and reduced the increased of cardiomyocyte surface area induced by doxorubicin,and the effect is more obviously than that of valsartan.3.Compared with the valsartan,sacubitril/valsartan significantly increased the decreased left ventricular ejection fraction（LVEF）and left ventricular fractional shortening（LVFS）induced by doxorubicin in mice,decreased the increased LDH,CK-MB levels,ANP,and BNP expression by doxorubicin treatment.HE staining showed that sacubitril/valsartan significantly reduced doxorubicin-induced cardiomyocyte vacuolization,cardiac tissue inflammation,and myocardial fiber arrangement disorder,and the effect is more obviously than that of valsartan.4.The ferroptosis inhibitor ferrostatin-1 reduced the increased expression levels of FACL4,ANP,BNP,and LDH,Fe²⁺,MDA levels induced by doxorubicin,and increased GSH levels and GPX4 expression levels.C11-BODIPY 581/591 fluorescence result showed that ferrostatin-1 reduced lipid peroxidation level.5.Sacubitril/valsartan decreased the increased FACL4 protein expression levels,Fe²⁺,MDA levels and lipid peroxidation levels induced by doxorubicin,and increased GSH levels and GPX4 protein expression level.Sacubitril/valsartan improved mitochondrial morphological changes induced by doxorubicin,increased mitochondrial membrane potential,and decreased production of cellular ROS and mitochondrial ROS.The ferroptosis activator Erastin increased the protein expression levels of FACL4,ANP,BNP and cell supernatant LDH levels,and decreased the expression level of GPX4.Secondly,sacubitril/valsartan decreased the expression level of FACL4 protein and increased GPX4 expression in cardiac tissue.DHE staining showed that sacubitril/valsartan reduced the production of reactive oxygen species induced by doxorubicin.In addition,we found that sacubitril/valsartan increased the GSH levels and decreased Fe²⁺levels of cardiac tissue in doxorubicin-induced mice.Transmission electron microscope showed that sacubitril/valsartan could improve doxorubicin-induced mitochondrial outer membrane breakage and mitochondrial cristae reduction or disappearance.6.Sacubitril/valsartan pretreatment increased the decreased protein expression levels of Sirt1 and Nrf2 in doxorubicin-induced H9c2 cells and mice cardiac tissue.7.Silencing Sirt1 or Nrf2 increased the expression levels of ANP,BNP,FACL4,and the levels of Fe²⁺,MDA,LDH,lipid peroxidation,decreased GSH level and GPX4 expression level.Silencing Sirt1 induced break of mitochondrial outer membrane,mitochondrial cristae reduced or absent,and decreased mitochondrial membrane potential,and increased production of cellular ROS and mitochondrial ROS.Conclusion:Sacubitril/valsartan ameliorates doxorubicin cardiotoxicity by inhibiting ferroptosis through the Sirt1/Nrf2 signaling pathway.