Effect And Mechanism Of Paeonol On Doxorubicin-induced Cardiomyopathy

Background:Doxorubicin hydrochloride,an anthracycline drug,is mainly used in the treatment of tumors clinically and has a significant effect.When doxorubicin reaches a certain cumulative amount in the body,it is easy to cause damage to myocardial cells and eventually cause severe cardiotoxicity.The clinical manifestations of cardiotoxicity are irreversible cardiomyopathy and congestive heart failure,which limits the clinical application of doxorubicin.Although the mechanism of doxorubicin-induced cardiotoxicity has not been fully elucidated,the resulting cardiomyocyte damage is an important reason for the development of doxorubicin-induced cardiomyopathy.How to effectively reduce the myocardial damage caused by doxorubicin is an important task in the current research field of tumor cardiomyopathy.Paeonol is an active ingredient isolated from Moutan Cortex,a traditional Chinese medicine,which has the effects of clearing heat,promoting blood circulation and removing blood stasis.It is often used to relieve pain and resist inflammation clinically.However,whether paeonol has a protective effect against doxorubicin-induced cardiomyopathy remains unclear.In this study,the effect and mechanism of paeonol on doxorubicin-induced cardiomyopathy were explored by establishing a model of doxorubicin-induced heart disease.Objectives:1.To explore whether paeonol reduces doxorubicin-induced cardiomyopathy;2.If possible,to elucidate the mechanism of paeonol to reduce doxorubicin-induced cardiomyopathy.Methods:1.The SPF male SD rats were randomly divided into four groups: Control group,Control+Pae group,Dox group,Dox+Pae group.The model was established by intraperitoneal injection of doxorubicin(15 mg/kg),and rats of paeonol treatment were given intragastric gavage throughout the course(75,150,300 mg/kg/d).After the model was successful,the following tests were performed.Echocardiography was used to evaluate cardiac function of rats.Left ventricular pressure of rats was detected by hemodynamics.Myocardial fibrosis was observed by masson staining.Myocardial hypertrophy was observed by WGA staining.Serum of SD rats was collected,and the contents of lactate dehydrogenase(LDH)and creatine kinase isoenzyme(CK-MB)in serum were detected to reflect the degree of myocardial injury.TUNEL staining was used to detect cardiomyocyte apoptosis.The oxidative stress level of cardiomyocyte was detected by dihydroethylamine(DHE)staining.Myocardial mitochondrial morphology was observed by transmission electron microscopy.Western blotting was used to detect the expression level of related proteins such as cell apoptosis,oxidative stress,mitochondrial respiratory chain and mitochondrial dynamics.2.C57BL/6 mice were subcutaneously inoculated with luciferase B16 cells to create a melanoma model.After the model was successful,they were randomly divided into four groups: Control group,Control+Pae group,Dox group,Dox+Pae group.The model was built in the same way as above,then,the tumor size was evaluated by mouse live imaging analysis.3.Primary rat cardiomyocytes were isolated and divided into four groups randomly:Control group,Control+Pae group(50 μM),Dox group(3 μM),Dox+Pae Groups(3μM +50 μM).Each group was given corresponding treatments and cultured for 24 hours,followed by subsequent detection.CCK-8 reagent was used to detect cell viability.LDH content reflects the degree of cardiomyocyte damage.Cardiomyocyte apoptosis was detected by TUNEL staining.Mito-SOX fluorescence intensity reflects the mitochondrial ROS content of cardiomyocytes.Mito-Tracker Red Staining was used to observe the morphological changes of cardiomyocytes mitochondria.Western blotting was used to detect the expression level of mitochondrial dynamics related proteins.4.Primary rat cardiomyocyteswere isolated and transfected with adenovirus to knock down and overexpress Mfn2 before doxorubicin treatment.Then,the following tests were performed.CCK-8 reagent was used to detect cell viability.LDH content reflects the degree of cardiomyocyte damage.Cardiomyocyte apoptosis was detected by TUNEL staining.Mito-SOX fluorescence intensity reflects the mitochondrial ROS content of cardiomyocytes.Morphological changes of cardiomyocytes mitochondria was observed by Mito-Tracker Red Staining.Western blotting was used to detect Mfn2 expression level.Results:1.At the animal level in vivo,doxorubicin induced cardiac dysfunction,increased myocardial fibrosis and myocardial hypertrophy,and increased cardiomyocyte apoptosis,oxidative stress,serum myocardial injury markers LDH and CK-MB.At the same time,myocardial mitochondria fission was increased and down-regulation of Mfn2 expression by doxorubicin,while no significant changes were found in the expression of Mfn1,Opa1,Drp1 and Fis1.The expression of mitochondrial respiratory chain complex protein was partially down-regulated by doxorubicin.However,the above effects were inhibited by paeonol.2.The result of in vivo imaging in mice showed that paeonol reduced the cardiotoxicity of doxorubicin without affecting its anti-tumor activity.3.At the cellular level in vitro,paeonol alleviated the cardiomyocyte injury,apoptosis and oxidative stress induced by doxorubicin,restored the expression of intracellular fusion protein Mfn2,and inhibited the over fission of mitochondria.4.At the cellular level in vitro,Knock down of Mfn2 blocked the protective effect of paeonol on cardiomyocytes.Over expression of Mfn2 inhibited mitochondrial fission induced by doxorubicin and directly alleviated cell injury and apoptosis.Conclusions:1.Paeonol improves cardiac function and structure of rats with doxorubicin-induced cardiomyopathy,reduces myocardial damage,and inhibits mitochondrial fission caused by doxorubicin.2.Paeonol inhibits mitochondrial fission and alleviates myocardial injury by upregulating mitochondrial fusion protein Mfn2.