Ameliorating Doxorubicin-induced Cardiotoxicity Through Modulating INOS/NO Signaling By Electroacupuncture At PC6

ObjectiveDoxorubicin(DOX)-induced cardiotoxicity is the most common and serious side effect of anthracycline drugs,which caused cardiac hypertrophy,dilated cardiomyopathy,left ventricular dysfunction ect.The aim of this study is to establish a mouse model of DOX-induced cardiotoxicity and explore the mechanism of electroacupuncture(EA)at Neiguan(PC6)on DOX-induced cardiotoxicity mice.Methods1.DOX(3 mg/kg/day)was injected intraperitoneally for 5 days to prepare the DOX-induced cardiotoxicity mice.Different frequencies(2 Hz,50 Hz,100 Hz)of EA at PC6 were used,20 minutes a day,in total of 7 days.On the seventh day after EA treatment,the motor function of mice was detected by Rotarod test,the cardiac function of mice was observed by echocardiography on the 21st day after EA treatment.2.To further explore the protection of EA at PC6 on DOX-induced cardiotoxicity mice,hematoxylin and eosin staining(HE)and wheat germ agglutinin staining(WGA)were used,and nitric acid reductase method was used to detect the NO level in myocardial tissue and serum;3.To explore the mechanism of EA at PC6 in the treatment of DOX induced-cardiotoxicity mice,the experiment was conducted by following groups:the vehicle control group(9%NaCl,ip),the control+L-arginine group,the DOX group(DOX,ip.),the DOX+EA group,the DOX+EA+L-arginine group,feeding L-arginine(30 mg/ml).Then the cardiac function test and test of NO production were carried out.4.RT-PCR and western blot were used to detect iNOS,eNOS and nNOS mRNA expression and protein level of the cardiac tissue.5.iNOS knockout mice(iNOS-/-)were used to further verify the mechanism of EA at PC6.Littermate wild-type mice were randomly divided into control group and DOX group were.iNOS-/-mice were divided into control group,DOX and DOX+EA group.On the 21st day after EA treatment,the cardiac function of mice was observed,and serum NO was detected.Results1.The value of EF%,FS%,and SV in DOX group decreased(P<0.01).After the 2 Hz EA treatment,the cardiac function of DOX-induced cardiotoxicity mice was improved,the value of EF%,FS%,and SV increased(P<0.01).The total distance and time on the rod decreased significantly(P<0.01)in DOX group compared with the control group,while 2 Hz EA group increased significantly(P<0.01)compared with the DOX group,indicating that the cardiac function and motor ability of mice decreased after DOX treatment,and 2 Hz EA at PC6 could improve the cardiac function and the motor ability of DOX-induced cardiotoxicity mice.In addition,the results of echocardiography and rotarod test showed that there was a statistical difference between 50 Hz and 100 Hz EA at PC6 group compared with 2 Hz EA group(P<0.05)and indicated that the selection of frequency may be important for the EA treatment in DOX-induced cardiotoxicity model.2.In our experiment,the ratio of body weight/heart weight of DOX group was significantly higher(P<0.01).HE staining and WGA staining were performed on the hearts of mice.The results showed that the left ventricular area and myocardial cells in DOX group were significantly larger(P<0.01).But EA at PC6 reduced the ratio of body weight/heart weight,the area of left ventricular chamber and myocardial cells(P<0.01).It is suggested that EA at PC6 prevented DOX-induced cardiac hypertrophy in mice.3.By measuring the NO level in serum and myocardial tissue of mice,we found that DOX increased the NO level in serum and myocardial tissue of mice(P<0.01).The NO level in serum and myocardial tissue of mice decreased significantly in DOX+EA group(P<0.01).L-arginine increased the NO production,it is fed to DOX-induced cardiotoxicity mice treated with EA.The protective effect of EA at PC6 on DOX-induced cardiotoxicity was blocked.It is suggested that EA at PC6 might achieve its therapeutic effect by regulating NO level.4.The results of RT-PCR and western blot showed that DOX increased the level of iNOS mRNA and protein in the myocardium of mice significantly(P<0.01),but there was no change in the level of eNOS and nNOS mRNA and protein in the myocardium.EA at PC6 decreased the level of iNOS mRNA and protein in DOX group(P<0.05).It is suggested that EA at PC6 might be used to treat DOX-induced cardiotoxicity through iNOS/NO pathway.5.iNOS knockout mice(iNOS-/-)were used to verify the mechanism.The iNOS+/+ mice were randomly divided into the control group and DOX group.The iNOS-/-mice were divided into the control group,DOX group and DOX+EA group.On the 21st day after EA treatment,the cardiac function of the mice was observed,and NO level of the serum was detected.Conclusion1.EA at PC6 improves the DOX-induced cardiotoxicity in mice effectively,including the impairment of left heart function and hypertrophy,and improved the impaired motor ability in DOX induced cardiotoxicity in mice;2.EA at PC6 protects the DOX-induced cardiotoxicity mice by regulating iNOS/NO pathway.