| BackgroundIschemic heart disease is one of the major diseases that harm human health,with the high morbidity and mortality.Myocardial ischemia is a pathophysiological state of myocardial ischemia and hypoxia that result from coronary artery atherosclerosis,spasm,and embolism.Myocardial ischemia-reperfusion injury is a partial or complete acute obstruction of the coronary artery followed by recanali-zation within a certain period.Although the ischemic myocardium can restore perfusion,the reperfusion will instead aggravate the myocardial injury.For myocardial ischemia-reperfusion injury,the current treatment strategies mainly include pharmacological intervention,ischemic pre-adaptation,and post-ischemic adaptation,but the effect is not ideal.Therefore,it is extremely important to further investigate its mechanism.Ferroptosis is an iron-dependent,newly discovered mode of regulated cell death distinct from apoptosis,necrosis,and autophagy.When ferrous iron or ester oxygenase is abnormally increased,unsaturated fatty acids undergo lipid peroxida-tion and induce cell ferroptosis.Studies have shown that ferroptosis can induce diseases such as tumors,stroke,liver insufficiency,renal insufficiency,and cardiomyopathy.However,inhibition of ferroptosis can effectively intervene in related diseases.At present,the effect of ferroptosis on acute myocardial ischemia/reperfusion injury has been reported,but its specific mechanism remains to be elucidated.AMPK is an energy receptor that could respond rapidly to changes in cellular energy.AMPK is involved in regulating pathophysiological processes such as metabolism,oxidative stress,inflammation,apoptosis,and autophagy in cardiomyocytes.Nrf2 is a transcription factor with strong redox sensitivity that ameliorates oxidative stress and reduces cellular damage by regulating the expression of phase II detoxification enzymes and antioxidant enzymes.Studies have shown that Nrf2 activation by upstream regulatory factors such as AMPK induces the expression of downstream antioxidant factors such as HO-1.Heme was catabolized by HO-1 into ferrous iron,chlorophyll and carbon monoxide,inhibiting their pro-oxidant effects.Moreover,chlorophyllin and its metabolites have ROS scavenging effects.In addition,HO-1 has a protective effect against acute myocardial ischemia/reperfusion injury.Targeting the activation of the AMPK/Nrf2/HO-1 pathway has been reported to be protective against embryonic fibroblast,liver,neurological,and respiratory diseases.Apigenin,a flavonoid,is widely distributed in vegetables and fruits,especially in celery,which has a high content.Apigenin has significant antioxidant,anti-inflam-matory,antiviral and anti-cancer effects.Studies have shown that apigenin can reduce endotoxin-induced cardiotoxicity and streptozotocin-induced diabetic cardio-myopathy,and inhibit TGF-β1-stimulated cardiac fibroblast differentiation and extracellular matrix production.Apigenin attenuated renal fibroblasts in vitro by activating AMPK and ERK pathways,and exhibited cytotoxicity in multiple myeloma cell lines.However,whether apigenin can ameliorate acute myocardial ischemia/reperfusion injury by activating the AMPK/Nrf2/HO-1 signaling pathway remains unclear.Therefore,an in-depth study of the mechanism of apigenin on acute myocardial ischemia/reperfusion injury could provide an experimental basis and theoretical foundation for the potential clinical application of apigenin.In conclusion,we aim to explore the role of the AMPK/Nrf2/HO-1 signaling pathway in mediating apigenin at the cellular and animal levels by constructing an acute hypoxia/reoxygenation model for H9c2 cardiomyocytes and an acute ischemia/reperfusion model for SD rats,respectively.We propose that apigenin may exert its anti-myocardial ischemia/reperfusion injury effect via activating the AMPK/Nrf2/HO-1 signaling pathway,with apigenin involved in the injury process.Part 1 The protective effect and mechanism of apigenin in H9c2 cardiomyocyte anoxia/reoxygenation injury by regulating AMPK/Nrf2/HO-1 signaling pathwayPurposes1.Exploring whether apigenin has a protective effect on acute anoxia/reoxyg-enation injury of H9c2 cardiomyocyte-like cells.2.Exploring whether ferroptosis is involved in the process of acute anoxia/reoxygenation injury of H9c2 cardiomyocytes.3.Exploring whether apigenin ameliorates ferroptosis involved in acute anoxia/reoxygenation injury of H9c2 cardiomyocyte-like cells by activating the AMPK/Nrf2/HO-1 signaling pathway.Methods1.Culture H9c2 cardiomyocyte-like cells were used to construct a hypoxia/r-eoxygenation model.2.According to different treatment methods,H9c2 cardiomyocyte-like cells were divided into 6 groups:（1）normal control group,（2）Anoxia/reoxygenation group,（3）Anoxia/reoxygenation+apigenin group,（4）Anoxia/reoxygenation+Fer-1group,（5）Anoxia/reoxygenation+apigenin+ML385 group,（6）Anoxia/reoxyge-nation+apigenin+Compound C group.Hypoxia for 3h,reoxygenation for 2h.Among them,Fer-1 is a ferroptosis inhibitor;ML385 is an inhibitor of Nrf2,and Compound C is an AMPK inhibitor.3.To observe the effect of different concentrations of apigenin on the anoxia/reoxygenation of H9c2 cardiomyocyte-like cells,the cells were divided into 7groups:（1）normal control,（2）Anoxia/reoxygenation,（3）Anoxia/reoxygena-tion+apigenin（5μM）,（4）Anoxia/reoxygenation+apigenin（10μM）,（5）Anoxia/reoxygenation+apigenin（20μM）,（6）Anoxia/reoxygenation+apigenin（40μM）,（7）Anoxia/reoxygenation+apigenin（80μM）.H9c2 cardiomyocyte-like cells were treated with different concentrations of apigenin 24 hours before Anoxia/reoxygen-ation.The survival rate of cells in each group and LDH activity in the culture medium was observed,with the optimal concentration of apigenin was selected.4.The morphology of H9c2 cardiomyocytes was observed by transmission electron microscope.5.Flow cytometry was used to detect the apoptosis and ROS level of H9c2cardiomyocyte-like cells.6.CCK-8 kit to was adopted to detect the viability of H9c2 cardiomyocyte-like cells.7.Microplate reader was used to detect the activities of lactate dehydrogenase,superoxide dismutase,glutathione peroxidase,catalase and Caspase 3,as well as the malondialdehyde,Fe2+,and reduced glutathione the content of glutathione and oxidized glutathione.8.Detect the AMP/ATP ratio by enzyme-linked immunosorbent assay（Elisa method）.9.The opening degree of mitochondrial permeability transition pore was measured by the Ca2+-induced enzyme labeling method.10.The PTGS2 m RNA level of H9c2 cardiomyocytes was detected by fluorescence quantitative reverse transcription-polymerase chain reaction（RT-q PCR）.11.Western blot was used to detect the protein expression levels of NOX4,GPX4,NDUFB8,UQCRC2,m TOR,Nrf2,GSK3β,HO-1,and AMPKα2 in H9c2cardiomyocyte-like cells in each group.Results1.20μM apigenin significantly increased cell viability and decreased LDH activity without cytotoxicity.Therefore,20μM was chosen as the optimal concen-tration.2.Cell damage indicators:In the H9c2 cardiomyocyte-like cell acute anoxia/reoxygenation model,apigenin and Fer-1 pretreatment increased H9c2 cardiomy-ocyte-like cell viability and decreased LDH content.Compound C and ML385attenuate the protective effect of apigenin.3.Expression of pathway proteins:In the H9c2 cardiomyocyte-like cell acute anoxia/reoxygenation model,apigenin pretreatment significantly up-regulated the expressions of AMPKα2,GSK3β,Nrf2 and HO-1,and down-regulated the expression of m TOR.However,both of the above effects of apigenin were reversed by Compound C and ML385.In addition,Fer-1 pretreatment exhibited an apigenin-like effect.4.Oxidative stress indicators:In the H9c2 cardiomyocyte-like cell acute anoxia/reoxygenation model,apigenin and Fer-1 pretreatment significantly reduced ROS generation and MDA content,and increased SOD,CAT,GSH-Px activity and GSH/GSSG ratio,which promoted the nuclear translocation of Nrf2,decreased cytoplasmic Nrf2 levels and increased nuclear Nrf2 levels.The above-mentioned effects of apigenin were inhibited by adding Compound C and ML385.5.Mitochondrial function indicators:In the H9c2 cardiomyocyte-like cell acute anoxia/reoxygenation model,after adding apigenin and Fer-1 pretreatment,the opening of mitochondrial permeability transition pore was reduced,the mitochondrial membrane potential was increased,and the expressions of NDUFB8and UQCRC2 were increased,ATP production increases and the AMP/ATP ratio decreases.However,the effect of apigenin was attenuated by the addition of Compound C and ML385.6.Apoptosis indicators:In the H9c2 cardiomyocyte-like cell acute anoxia/reoxygenation model,apigenin and Fer-1 pretreatment significantly reduced the apoptotic rate and caspase 3 activity,while Compound C and ML385considerably attenuated apigenin-induced apoptosis.7.Results of transmission electron microscopy:The size of mitochondria in the control group was normal,and the cristae structure was complete.The mitochondria of the anoxia/reoxygenation group were shrunken,and the mitochondrial cristae were arranged disorderly,showing distinct characteristics of ferroptosis.Apigenin and Fer-1 pretreatment significantly improved mitochondrial morphological changes induced by acute anoxia/reoxygenation injury.With the addition of Compound C and ML385,the damage to mitochondria and their cristae was further aggravated.8.Ferroptosis index:In H9c2 cardiomyocyte-like cell acute anoxia/reoxyg-enation model,Fe2+content and PTGS2 m RNA level were sharply decreased,NOX4expression reduced,and GPX4 expression increased after apigenin and Fer-1pretreatment.The above effects of apigenin were reversed after treatment with Compound C and ML385.Conclusions1.The acute anoxia/reoxygenation injury of H9c2 cardiomyocyte-like cells is the outcome of the combined effect of multiple regulatory cell death modes.Apigenin has multiple mechanisms and multiple targets and can ameliorate acute anoxia/reoxygenation injury of cells by inhibiting ferroptosis and apoptosis.2.The acute anoxia/reoxygenation injury of H9c2 cardiomyocyte-like cells is involved in ferroptosis,and Fer-1 can significantly reduce this injury.3.In acute anoxia/reoxygenation injury of H9c2 cardiomyocyte-like cells,apigenin can play a significant anti-ferroptosis effect by activating the AMPK/Nrf2/HO-1 signaling pathway.Part 2 The protective effect and mechanism of apigenin in myocardial ischemia-reperfusion injury by regulating AMPK/Nrf2/HO-1 signaling pathwayThrough the first part of the study on acute anoxia/reoxygenation injury of H9c2 cardiomyocyte-like cells,we clarified that apigenin ameliorated ferroptosisinduced acute anoxia/reoxygenation of H9c2 cardiomyocyte-like cells by activating the AMPK/Nrf2/HO-1 signaling pathway Reoxygenation injury at the cellular level.However,whether it is consistent at the animal level needs further verification.Therefore,in this part,we use SD rats as the study object to construct an ischemia/reperfusion injury model at the animal level and verify whether it is different from the cellular level by detecting relevant indicators.Purposes1.Verifying whether apigenin has a protective effect on myocardial ischemia/reperfusion injury at the animal level.2.Verifying whether ferroptosis is involved in the process of myocardial ischemia/reperfusion injury at the animal level.3.Verifying whether apigenin can improve myocardial ischemia/reperfusion injury involving ferroptosis by activating the AMPK/Nrf2/HO-1 signaling pathway at the animal level.Methods1.To establish an ischemia/reperfusion injury model,SD rats were randomly divided into 6 groups:（1）normal control group,（2）ischemia/reperfusion group,（3）ischemia/reperfusion+apigenin group,（4）ischemia/reperfusion+Fer-1 group,（5）ischemia/reperfusion+apigenin+ML385 group,（6）ischemia/reperfusion+apigenin+Compound C group.30 min of ischemia and 3h of reperfusion.2.TTC method staining was used to detect myocardial infarct size.3.The ultrastructure of the myocardium was observed by transmission electron microscope.4.TUNEL kit was used to detect the apoptosis of cardiomyocytes.5.Using a DHE probe to detect the amount of ROS generated in cardiomyocytes.6.The microplate reader was used to detect the activities of LDH,SOD,GSH-Px,CAT,and Caspase 3,and to detect the content of MDA,Fe2+ and the ratio of GSH/GSSG.7.The AMP/ATP ratio was detected by enzyme-linked immunosorbent assay（Elisa method）.8.The opening degree of mitochondrial permeability transition pore was measured by the Ca2+-induced enzyme labeling method.9.Detect the level of PTGS2 m RNA in cardiomyocytes by fluorescence quantitative reverse transcription-polymerase chain reaction（RT-q PCR）.10.Western blot was used to detect the protein expression levels of NOX4,GPX4,NDUFB8,UQCRC2,m TOR,Nrf2,GSK3β,HO-1,and AMPKα2 in cardiomyocytes.Results1.After acute myocardial ischemia/reperfusion,the myocardial infarction area increased significantly,serum IL-6,TNF-α,CK-MB,CTNI content,and MPO,LDH activities increased,indicating that the acute myocardial ischemia/reperfusion injury model was successfully constructed.apigenin and Fer-1 pretreatment significantly reduced myocardial infarction size,decreased IL-6,TNF-α,CK-MB,CTNI content and MPO,LDH activities.However,the cardioprotective effect of Api was attenuated by Compound C and ML385.2.Expression of pathway proteins: In the acute myocardial ischemia/reperfusion model,apigenin and Fer-1 preconditioning significantly up-regulated the expression of AMPKα2,while increasing the expression of GSK3β,Nrf2,HO-1,and reducing the expression of m TOR,suggesting that AMPK/Nrf2/ HO-1 pathway proteins can be activated by apigenin.And Fer-1 and apigenin showed a similar regulatory effect.However,the above-mentioned effects of apigenin were significantly attenuated by Compound C and ML385.3.Oxidative stress indicators: In the acute myocardial ischemia/reperfusion model,apigenin and Fer-1 pretreatment significantly reduced ROS generation,increased SOD,CAT and GSH-Px activities,improved GSH/GSSG value,and decreased MDA content.Reduces cytoplasmic Nrf2 expression and promotes nuclear Nrf2 accumulation.Compound C and ML385 reversed the above-mentioned effects of apigenin.4.Mitochondrial function indicators: In the acute myocardial ischemia/reperfusion model,pretreatment with apigenin and Fer-1 significantly increased the ATP content,decreased the AMP/ATP ratio,and up-regulated the expression of NDUFB8 and UQCRC2,which was remarkably reversed by Compound C and ML385.The above effects of apigenin.5.Apoptosis indicators: After acute myocardial I/R injury,the number of apoptotic cells and the activity of Caspase 3 was significantly increased.ML385 remarkably weakened the effect of apigenin.6.Results of transmission electron ischemia/reperfusion microscopy: The mitochondrial papillary muscles of the normal control group were normal in size and arranged neatly,the mitochondrial cristae were intact,and the intercalated discs of the myocardium were neatly arranged.In the group,the structure of the intercalated disc was incomplete,with rupture,mitochondria shrinking,and mitochondrial cristae decreasing or disappearing,which were distinct morphological features of ferroptosis.Apigenin and Fer-1 pretreatment significantly ameliorated the above myocardial ischemia/reperfusion injury.However,Compound C and ML385 attenuated the protective effect of apigenin.7.Ferroptosis index: Acute myocardial ischemia/reperfusion treatment significantly increased Fe2+ content and PTGS2 m RNA level,up-regulated NOX4 expression,and down-regulated GPX4 expression.After apigenin and Fer-1pretreatment,Fe2+ content and PTGS2 m RNA level was significantly decreased,NOX4 expression decreased,and GPX4 expression increased.Treating with Compound C and ML385 reversed the above effects of apigenin.Conclusions1.Acute myocardial ischemia/reperfusion injury is the combined outcome of multiple regulatory cell death models.Apigenin has multiple mechanisms and multiple targets,which can improve acute myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis.2.Acute myocardial ischemia/reperfusion injury is involved in ferroptosis,and Fer-1 can significantly reduce this injury.3.Apigenin ameliorated acute myocardial ischemia/reperfusion injury involving ferroptosis by activating the AMPK/Nrf2/HO-1 signaling pathway. |
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