Study On The Mechanisms And Clinical Efficacy Of Extracorporeal Cardiac Shock Wave Therapy

Backgrounds:Coronary heart disease is one of the leading causes of death and disability worldwide,and the main treatment options include drug therapy,percutaneous coronary intervention(PCI),and coronary artery bypass grafting(CABG).However,a portion of the population continues to have recurrent angina after these treatments.As a complementary treatment for coronary artery disease,Cardiac Shock Wave Therapy(CSWT)is an effective,non-invasive and safe treatment developed in recent years and has been put into use in several clinical centers in China.In vitro and in vivo experimental studies had shown that Shock Wave Therapy(SWT)could promote vascular endothelial renewal,inhibit local inflammatory responses,inhibit myocardial fibrosis after myocardial infarction,reduce oxidative stress,and inhibit apoptosis.Ferroptosis is distinguished from apoptosis and is a novel form of programmed cell death that has been discovered in recent years.More and more studies have confirmed that ferroptosis is involved in the development of cardiovascular diseases.Whether CSWT can protects cardiomyocytes against Hypoxia-Reoxygenation(H/R)by inhibiting ferroptosis has not been reported in the literature.Objectives:1.Whether SWT can alleviate H/R injury of rat cardiomyocytes by inhibiting ferroptosis.2.To explore the possible mechanisms of SWT inhibiting ferroptosis in H/R model.Methods:The rat H9c2 cardiomyocyte cell line was selected as the research object.1.Place the synchronized H9c2 cells in a hypoxia incubator containing 1%O2 for 3-9 hours,and reoxygenate for 24 hours after hypoxia.The cell viability was detected by MTT method,the degree of cell injury was detected by LDH level,the ultrastructure of cells was observed by electron microscope,and the expression of ferroptosis core regulators(xCT,GPX4)and apoptosis key effect medium Cleaved caspase-3 were detected by Western Blot(WB).Select the moderate damage degree to establish the H/R model.2.After the model was established,the H9c2 cells were divided into 4 groups,namely the normoxia control group,the NC+SWT(energy:0.07 mJ/mm2)group,the H/R control group,and the H/R+SWT group.The MTT method and LDH method were used to detect the cell viability and the degree of damage.The content of ROS,MDA and GSH were used to detect the level of oxidative stress.The morphology of mitochondria was observed by electron microscope.The ferroptosis signaling pathway-related proteins(xCT,GPX4,ACSL4,NRF2,HO-1),the expression of apoptosis signaling pathway-related proteins(Bcl2,BAX,Cleaved caspase-3,pAkt/Akt)and transfection of siRNA of the core regulator xCT were verified that possible mechanism of SWT in inhibiting ferroptosis in the H/R model.Results:1.MTT and LDH detection showed that with the prolongation of hypoxia time,the cell viability decreased,and the degree of cell damage gradually increased.WB method showed that GPX4 and xCT proteins level were significantly decreased and Cleaved caspase-3 protein level was significantly increased after 5 hours of hypoxia and 24 hours of reoxygenation.The morphology of mitochondria was changed after H/R by electron microscope.Therefore,5 hours of hypoxia and 24 hours of reoxygenation were selected as the H/R model.2.SWT could increase the cell viability of H/R group by about 14.5%on average,and reduce the LDH level by about 28.8%on average.Compared with the H/R group,the ROS level in the H/R+SWT group decreased significantly(P<0.01),and the MDA content decreased by 21%(H/R+SWT group vs H/R group:1.99±0.21 vs 2.52±0.16nmol/mg prot,P<0.05),the expression of ACSL4 protein was significantly decreased(P<0.05),and the content of GSH was increased by about 1.85 times.Electron microscope observation of SWT could improve the mitochondrial morphology after H/R.3.SWT could inhibit the expression level of pro-apoptotic proteins Cleaved-caspase3 and Bax,increased the expression of anti-apoptotic protein Bcl2,and activated the PI3K/Akt pathway.SWT could significantly increase the expression level of GPX4,xCT,NRF2,and HO-1;knockdown of xCT expression level by transfection of siRNA could reverse the protective effect of SWT on cardiomyocytes.Conlusion:1.H/R could cause cardiomyocyte injury and decreased cell viability.Moreover,both apoptosis and ferroptosis could be induced by H/R.2.SWT protected cardiomyocytes by inhibiting lipid peroxidation,promoting NRF2/HO-1 signaling pathway activation and xCT/GPX4 expression level.SWT could attenuate cardiomyocyte injury and ferroptosis caused by H/R.AIM Cardiac shockwave therapy(CSWT)is a non-invasive treatment for patients with refractory angina or ischemia.Several clinical trials have suggested that CSWT can alleviate angina symptoms and improve the quality of life in patients with refractory angina and ischemic cardiomyopathy.Further studies on the effectiveness and long-term follow-up results of CSWT in patients with refractory angina who have undergone CABG are lacking.This study is aimed to demonstrate the efficacy and safety of CSWT in patients with refractory angina who have undergone coronary artery bypass grafting(CABG).In addition,the long-term effects of CSWT on enrolled patients were further explored.Methods CAD patients undergone CABG who were not suitable for coronary revascularization or a second CABG were enrolled.By using myocardial perfusion imaging(MPI),the most severe ischemic segments were identified.All patients received CSWT for 9 sessions.Evaluation was performed before and after CSWT,including the Canadian Cardiovascular Society(CCS)classification,New York Heart Association(NYHA)classification,6 min walk test(6MWT),Seattle Angina Questionnaire(SAQ),nitroglycerin dosage,echocardiography and MPI.All patients were followed up in the 4th month and 12th month after CSWT.Results A total of 30 patients were enrolled.Compared with baseline values,CCS(P=0.20),NYHA(P=0.21),6MWT(P=0.025),5 domains of SAQ score(P<0.05),nitroglycerin dosage(P<0.001),left ventricular ejection fraction(LVEF)(P=0.03)in echocardiography,summed stress score(P<0.001),summed difference score(P=0.004),target stress score(P=0.001),target difference score(P<0.001),and ischemic area in MPI were all improved significantly in the 4th month post-CSWT.18 patients completed the 12-month follow-up.The results showed that 6MWT(P=0.002),nitroglycerin dosage(P=0.001).SAQ score(physical activity limitation,stable state of angina pectoris,treatment satisfaction and disease awareness,P<0.05),summed difference score(P=0.046)and target difference score(P=0.003)were still significantly improved.There were no change in hemodynamic parameters and cardiac biomarkers before and after CSWT.Conclusions Our results suggest that CSWT could improve the symptoms of angina pectoris in patients with refractory angina pectoris after CABG,reduced the dosage of nitroglycerin,improved activity tolerance and quality of life,and improved the cardiac function and myocardial ischemia degree of patients after 4 months of follow-up.Angina pectoris and myocardial ischemia can still be improved to some extent by CSWT at 12 months of follow-up.