| Objective:Cardiovascular diseases and tumors are two important causes of human death.Doxorubicin(DOX)is currently one of the most commonly used broad-spectrum anthracycline anti-tumor drugs and is widely used in the treatment of patients with leukemia,breast cancer,ovarian cancer,liver cancer and lung cancer.However,some cancer patients may experience obvious cardiotoxicity after long-term use of DOX,leading to changes in cardiac structure and function,and then leading to cardiomyopathy or congestive heart failure.Therefore,the myocardial damage caused by DOX limits its use in cancer patients.Currently,there is a lack of effective prevention and treatment methods for DOX induced cardiotoxicity.Therefore,clarifying the mechanism of DOX induced cardiotoxicity and finding intervention targets will lay the foundation for the clinical prevention and treatment of DOX induced cardiotoxicity.Ferroptosis is a non-apoptotic form of cell death,characterized by iron-dependent intracellular lipid peroxidation products and the accumulation of reactive oxygen species.When ferroptosis occurs,mitochondria become smaller,mitochondrial membrane density becomes concentrated,mitochondrial cristae reduces or disappears,and mitochondrial outer membrane ruptures.Research suggests that ferroptosis is involved in a variety of pathological processes,including cancer,neurodegenerative diseases,acute liver and renal failure.Recent studies have found that ferroptosis plays an important role in cardiovascular diseases such as DOX induced myocardial injury and myocardial ischemia-reperfusion injury.CREG(cellular repressor of E1A-stimulated genes)gene is discovered in Hela cells by Professor Gill of Harvard Medical School in 1998.CREG is a secreted glycoprotein with 220 amino acids and 3 M6P glycosylation sites.CREG is mainly located in the lysosome and perinuclear endoplasmic reticulum,and is widely expressed in adult tissues and cells.CREG plays an important role in the regulation of the differentiation of tissues and cells.CREG is an important cardioprotective factor,which plays an important role in maintaining cardiomyocyte differentiation and homeostasis regulation.Studies have found that in a model of transverse aortic constriction,CREG expression is significantly decreased,and overexpression of CREG reduces myocardial fibrosis,however,the roles and mechanisms of CREG in DOX induced cardiotoxicity have not been reported.Based on the above results,we hypothesize that CREG,as an endogenous key regulatory gene regulating ferroptosis,plays a protective role in the development of DOX induced cardiotoxicity.Therefore,in this study,CREG cardiac-specific knockout mice and CREG transgenic mice were used to establish DOX induced cardiotoxicity models.At the same time,to establish DOX related cell model,DOX was used to stimulate cardiomyocytes,and CREG expression was determined in vivo and in vitro levels.The effect and its molecular mechanism of CREG on ferroptosis after DOX induced cardiotoxicity were preliminarily explained.Methods:1.The relationship between CREG expression and doxorubicin induced cardiotoxicity.(1)DOX was injected by intraperitonea to establish DOX induced cardiotoxicity model,the control group was injected intraperitoneally with the same dose of PBS.By detecting cardiac systolic and diastolic functions,body weight,heart weight,hematoxylin-eosin(HE)staining,Sirius staining and wheat germ agglutinin(WGA)staining of cardiac tissue to evaluate whether the model of DOX induced cardiotoxicity was successful.(2)The serum of each group of mice was taken,and the expression of serum myocardial injury markers Creatine Kinase Isoenzyme(CKMB),Lactate dehydrogenase(LDH),and glutamic oxalacetic transaminase(AST)were detected by Elisa.Quantitative Polymerase Chain Reaction(PCR)was used to detect the expression of brain natriuretic peptide(BNP),a marker of heart failure in each group of mice.(3)Quantitative PCR,Western blot and immunohistochemical staining were used to detect the expression of CREG gene m RNA and protein in the heart tissues of the DOX group and the control group.(4)Establish DOX induced cardiotoxicity cell model,extract neonatal mouse cardiomyocytes(NMCMs),stimulate DOX(10?M)for 24 hours to establish cell model,Quantitative PCR,western blot and immunofluorescence staining methods were used to detect the expression of CREG m RNA and protein.2.The role of CREG in doxorubicin induced cardiotoxicity(1)Using CREG transgenic mice(TG-CREG)and its littermate control mice(ctrl)to establish DOX induced cardiotoxicity model.Compare the heart size of mice in each group by measuring the body weight,heart weight,and calculating the mouse heart weight/body weight ratio.Cardiac ultrasound,HE staining,Sirius staining and WGA staining of cardiac tissue were used to evaluate the effect of CREG overexpression on cardiac function and cardiac morphology after DOX induced cardiotoxicity.The serum of each group of mice was taken,and the expression of serum myocardial injury markers CKMB,LDH,and AST were detected by Elisa.Quantitative PCR was used to detect the expression of BNP,a marker of heart failure in each group of mice.Finally,the heart tissues were taken,and the transcription level and protein expression of CREG in the heart tissue of each group were detected by quantitative PCR and Western blot.(2)Using cardiac-specific CREG knockout mice(CREG-CKO)and its littermate control mice CREG fl/fl(ctrl)to establish DOX induced cardiotoxicity model.Compare the heart size of mice in each group by measuring the mouse body weight,heart weight,and calculating the mouse heart weight/body weight ratio.Cardiac ultrasound,HE staining,Sirius staining and WGA staining of cardiac tissue were used to evaluate the effects of low expression of CREG on cardiac function and cardiac morphology after DOX induced cardiotoxicity.Afterwards,the serum of each group of mice was taken,and the expression of serum myocardial injury markers CKMB,LDH,and AST were detected by Elisa.Quantitative PCR was used to detect the expression of BNP,a marker of heart failure in each group of mice.Finally,heart tissues were taken,and quantitative PCR and Western blot methods were used to detect the transcription level and protein expression of CREG in the heart tissue of each group of mice.3.Study on the mechanism of CREG in doxorubicin induced cardiotoxicity.(1)The Elisa method was used to detect the serum level of iron ion and malondialdehyde(MDA)level in the TG-CREG and ctrl groups of mice after modeling.Fresh ventricular tissues from each group of mice were taken,and abnormal mitochondria were detected by electron microscope.Quantitative PCR was used to detect the expression of CREG and ferroptosis-related factors,including NRF2,HO-1,ptgs2,and COX4 in the heart tissues of each group.Immunohistochemical staining was used to detect the expression of CREG and ptgs2 in the myocardial tissue of mice in both groups.Western blot was used to detect the expression of CREG and ferroptosis-related proteins NRF2,HO-1,4-HNE,ptgs2,and COX4 in the heart tissues of mice in both groups.(2)The Elisa method was used to detect serum iron ion and MDA levels in CREG-CKO and ctrl groups of mice after modeling.Fresh ventricular tissues from each group of mice were taken,and abnormal mitochondria were detected by electron microscope.Quantitative PCR was used to detect the expression of CREG and ferroptosis-related factors,including NRF2,HO-1,ptgs2,and COX4 in the heart tissues of each group.Immunohistochemical staining was used to detect the expression of CREG and ptgs2 in the myocardial tissue of mice in the DOX group.Western blot was used to detect the expression of CREG and ferroptosis-related proteins NRF2,HO-1,4-HNE,ptgs2,and COX4 in the heart tissues of mice in the CREG-CKO group and ctrl groups of mice after modeling.(3)To establish the cell model of DOX,CREG overexpression(Ad-CREG)NRCMs model was established by infection with adenovirus,and then stimulated with DOX.Western blot was used to detect the protein level changes of CREG and ferroptosis indicators such as NRF2,HO-1,4-HNE,ptgs2,and COX4.PCR was used to detect the m RNA level changes of CREG and ferroptosis indicators such as NRF2,HO-1,ptgs2,and COX4.DHE staining was used to detect the changes of oxidative stress in each group of NMCMs,and JC-1 staining was used to detect the changes of mitochondrial function in each group of NMCMs.(4)In NMCMs,CREG low expression(Si-CREG)NRCMs model was established by transfection with small interfering RNA and then stimulated by DOX.Western blot was used to detect the protein level changes of CREG and ferroptosis indicators such as NRF2,HO-1,4-HNE,ptgs2,and COX4.PCR was used to detect the m RNA level changes of CREG and ferroptosis indicators such as NRF2,HO-1,ptgs2,and COX4.DHE staining was used to detect the changes of oxidative stress in each group of NMCMs,and JC-1 staining was used to detect the changes of mitochondrial function in each group of NMCMs.(5)Determine the upstream and downstream relationship between CREG and NRF2.Established NRF2 over expression(Ad-NRF2)and NRF2 low expression(Si-NRF2)models by infection with adenovirus or transfection with RNA interference,and stimulated by DOX,and the expressions of CREG,NRF2,HO-1,4-HNE,ptgs2,COX4were detected by western blot.(6)To determine whether overexpression of NRF2 could relieve the ferroptosis of cardiomyocytes caused by CREG knockdown,NRF2 was overexpressed when CREG was knockdown,and DOX was given to stimulate at the same time.Western blot was used to detect the changes of CREG,NRF2,HO-1,4-HNE,ptgs2.The oxidative stress was detected by DHE staining,and cell mitochondrial function was evaluated by JC-1staining.Results:1.The transcription and protein levels of CREG decreased significantly in doxorubicin induced cardiotoxicity.(1)Compared with the control group,the contractive and diastolic functions of the heart were significantly reduced;the body weight in the DOX group was significantly reduced,the hearts were shrunk and the myocardial cells were smaller.(2)Compared with the control group,the myocardial injury markers CKMB,LDH,AST and BNP in the DOX group were significantly increased.(3)The results of quantitative PCR showed that compared with the control group,the CREG gene transcription level in the heart tissue of the DOX group was significantly reduced;Western blot and immunohistochemistry showed that,compared with the control group,the CREG protein expression was significantly reduced in the hearts of the DOX group.(4)In the cell model of DOX induced cardiotoxicity,the transcription and protein levels of CREG in the DOX group were significantly reduced compared with the control group.In summary,the animal model and cell model of DOX cardiotoxicity have been successfully established.After DOX induced cardiotoxicity,CREG transcription level and protein level were significantly reduced.2.CREG plays a protective role in doxorubicin induced cardiotoxicity.(1)Compared with TG-CREG group and ctrl group,the contractive and diastolic functions of the heart,the body weight,heart weight in the TG-CREG+DOX group and DOX group were significantly reduced;compared with ctrl group,in the DOX group,the hearts were shrunk and the myocardial cells were smaller;compared with DOX group,the contractive and diastolic functions of the heart,the body weight,the hearts weight in the TG-CREG+DOX group were elevated and the myocardial cells were increased.Compared with TG-CREG group and ctrl group,the myocardial injury markers CKMB,LDH,AST,and BNP in the TG-CREG+DOX group and DOX group were significantly elevated;compared with DOX group,the myocardial injury markers CKMB,LDH,AST,and BNP in the TG-CREG+DOX group were reduced.The results of quantitative PCR and Western blot showed that,the expression of CREG in the heart tissue of TG-CREG mice was significantly higher than that in the ctrl group of mice;compared with TG-CREG group and ctrl group,the m RNA level and protein level of CREG in TG-CREG+DOX group and DOX group were reduced;compared with DOX group,the m RNA level and protein level of CREG in the TG-CREG+DOX group were elavated.(2)Compared with CREG-CKO group and ctrl group,the contractive and diastolic functions of the heart,the body weight,heart weight in the CREG-CKO+DOX group and DOX group were significantly reduced,myocardial cells were smaller;compared with DOX group,the contractive and diastolic functions of the heart,the body weight,the hearts weight were further reduced and the myocardial cells were smaller in the CREG-CKO+DOX group.the myocardial injury markers CKMB,LDH,AST,and BNP in CREG-CKO+DOX group and DOX group were increased;compared with DOX group,myocardial injury markers CKMB,LDH,AST,BNP in the CREG-CKO+DOX group were significantly increased and myocardial damage was significantly worse.Compared with mice in the ctrl group,the expression of CREG in the heart tissue of CREG-CKO group was significantly reduced;compared with CREG-CKO group and ctrl group,the m RNA level and protein level of CREG in CREG-CKO+DOX group and DOX group were reduced;compared with DOX group,the m RNA level and protein level of CREG in the CREG-CKO+DOX group were further reduced.3.CREG could affect the ferroptosis of cardiomyocytes after doxorubicin induced cardiotoxicity by regulating NRF2 protein expression(1)Compared with TG-CREG group and ctrl group,the heart tissues of the TG-CREG+DOX group and the DOX group showed significant ferroptosis,manifested as the serum iron ion level and MDA level were significantly increased.The serum iron ion level?MDA level of the TG-CREG+DOX group were lower than that of the DOX group.Compared with TG-CREG group and ctrl group,mitochondria were severely damaged in the TG-CREG+DOX group and the DOX group;mitochondrial damage in the TG-CREG+DOX group was significantly relieved compared with DOX group.Compared with TG-CREG group and ctrl group,the expression of ferroptosis predictor ptgs2 and oxidative stress indicator 4-HNE was increased,and the expression of ferroptosis pathway proteins NRF2,HO-1 and COX4 was decreased in TG-CREG+DOX group and the DOX group;compared with the DOX group,the expression of ptgs2 and 4-HNE was decreased and the expression of NRF2,HO-1 and COX4 in the TG-CREG+DOX group was increased.(2)Compared with CREG-CKO group and ctrl group,the heart tissues of the CREG-CKO+DOX group and the DOX group showed significant ferroptosis,manifested as the serum iron ion level and MDA level were significantly increased.The serum iron ion level?MDA level of the CREG-CKO+DOX group were significantly higher than that of the DOX group.Compared with CREG-CKO group and ctrl group,mitochondria were damaged in the CREG-CKO+DOX group and the DOX group;compared with DOX group,the mitochondrial damage in the CREG-CKO+DOX group was significantly aggravated.Compared with CREG-CKO group and ctrl group,the expression of ferroptosis predictor ptgs2 and oxidative stress indicator 4-HNE was increased,and the expression of ferroptosis pathway proteins NRF2,HO-1 and COX4was decreased in CREG-CKO+DOX group and the DOX group;compared with the DOX group,the expression of ptgs2 and 4-HNE was significantly increased and the expression of NRF2,HO-1 and COX4 was significantly decreased in the CREG-CKO+DOX group.(3)Ferroptosis was increased both in the Ad-CREG group and the Ad-ctrl group after DOX stimulated,the main manifestations were increased expression of ferroptosis predictor ptgs2 and oxidative stress indicator 4-HNE;compared with Ad-ctrl group,ferroptosis pathway proteins NRF2,HO-1 and COX4 were decreased in Ad-ctrl+DOX group;compared with the Ad-ctrl+DOX group,the expression of ptgs2 and 4-HNE was decreased,and the expression of NRF2,HO-1 and COX4 was increased in Ad-CREG+DOX group.Compared with the control group,oxidative stress marker ROS staining was significantly enhanced in the Ad-CREG group and the Ad-ctrl group were stimulated by DOX;compared with Ad-ctrl+DOX group,the oxidative stress marker ROS staining was significantly weakened in Ad-CREG+DOX group.Compared with the control group,the Ad-CREG group and the Ad-ctrl group showed abnormal staining of the mitochondrial membrane potential detection index JC-1 after DOX stimulated,the JC-1monomeric significantly increased,and the JC-1 aggregate significantly decreased;compared with the Ad-ctrl+DOX group,the mitochondrial membrane potential detection index JC-1 monomeric was significantly reduced and JC-1 aggregate was significantly increased in the Ad-CREG+DOX group.(4)Compared with the control group,the ferroptosis was increased in both the Si-CREG group and the Si-ctrl group after stimulated by DOX,the main manifestations were increased expression of ferroptosis prediction marker ptgs2 and oxidative stress indicator 4-HNE,and decreased expression of ferroptosis pathway proteins NRF2,HO-1and COX4;compared with the Si-ctrl+DOX group,the expression of ptgs2 and 4-HNE was increased and the expression of NRF2,HO-1 and COX4 was decreased in Si-CREG+DOX group.Compared with the control group,after DOX stimulated,the oxidation marker ROS staining was significantly enhanced in the Si-CREG group and the Si-ctrl group;compared with the Si-ctrl+DOX group,the oxidative stress marker ROS staining in the Si-CREG+DOX group was significantly enhanced.Compared with the control group,the Si-CREG group and the Si-ctrl group showed abnormal staining of the mitochondrial membrane potential detection index JC-1 after DOX stimulated,the JC-1monomeric significantly increased,and the JC-1 aggregate significantly decreased;compared with the Si-ctrl+DOX group,the mitochondrial membrane potential detection index JC-1 monomeric was significantly increased and JC-1 aggregate was significantly reduced in the Si-CREG+DOX group.(5)Compared with the Ad-ctrl group,the protein level of the CREG gene in the Ad-NRF2 group was not changed;compared with the Si-NRF2 group,the protein level of CREG gene in the Si-NRF2 group was not changed.(6)Compared with the Si-CREG group,the expression of NRF2 protein in the Si-CREG+Ad-NRF2 group was increased,the CREG protein expression did not change;after DOX stimulation,compared with the Si-CREG+DOX group,there was no change in the expression of CREG protein,the protein expression of NRF2 and HO-1 was significantly increased,the protein expression of 4-HNE and ptgs2 was significantly decreased in the Si-CREG+Ad-NRF2+DOX group.Compared with the Si-CREG group,the fluorescence intensity of ROS in the Si-CREG+Ad-NRF2 group was significantly weakened;compared with the Si-CREG+DOX group,the fluorescence intensity of ROS in the Si-CREG+Ad-NRF2+DOX group was obvious weaken;Compared with the Si-CREG group,the number of JC-1 monemeric decreased and JC-1 aggregate increased significantly in the Si-CREG+Ad-NRF2 group,indicating that mitochondrial function was recovered;compared with the Si-CREG+DOX group,the number of JC-1monemeric decreased and JC-1 aggregate increased significantly in the Si-CREG+Ad-NRF2+DOX group.Conclusion:1.After doxorubicin induced cardiotoxicity,the transcription level and protein level of CREG was significantly decreased.2.CREG has a protective effect on doxorubicin induced cardiotoxicity.When Doxorubicin induced cardiotoxicity occurred,CREG transgenic mice could significantly relieve myocardial damage,while myocardial-specific CREG knockout mice could significantly aggravate myocardial damage.3.As the upstream molecule of NRF2,CREG could regulate the ferroptosis of cardiomyocytes by regulating NRF2 protein expression,and then participate in the occurrence and development of doxorubicin induced cardiotoxicity. |
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