Prevention Of Noninvasive Vagal Nerve Stimulation Against Doxorubicin-induced Cardiotoxicity And The Underlying Mechanism

Background: Doxorubicin(DOX),one of the most potent antitumor anthracycline antibiotics,is widely used to effectively treat various cancers such as leukemias,lymphomas and solid tumors.However,the clinical use of DOX is limited by its serious cumulative dosedependent cardiotoxicity.This may severely impair the prognosis and long-term survivor of cancer patients.The molecular mechanisms of DOX-induced cardiotoxicity are known to involve DNA damage,transcriptome alterations,mitochondrial dysfunction,inflammation,oxidative stress and cardiomyocytes apoptosis,resulting in causing irreversible cardiomyopathy and even congestive heart failure.Therefore,highly efficient and safe adjuvant intervention for preventing DOX-induced cardiotoxicity is urgently needed to be developed for cancer survivors undergoing DOX chemotherapy.Autonomic nervous system(ANS),consist of sympathetic nervous system(SNS)and parasympathetic nervous system(PNS),plays an important role in maintaining body homeostasis.Autonomic dysfunction has been showed closely associated with cancer patients and further chemotherapy potentially activates sympathetic nervous system and exacerbates cardiotoxicity in long-term therapy.Moreover,ANS dysfunction,especially hyperactivity of SNS,and impaired vagal tone,could increase the risk of many cardiovascular events under pathological conditions such as myocardial ischemia,heart failure,hypertension and arrhythmia.Vagal nerve stimulation(VNS),identified as a highly effective autonomic neuromodulation strategy,has revealed huge benefits in treating cardiac disorders in animal researches and clinical trials via its multiple mechanical actions especially classic anti-inflammatory action,oxidative stress inhibition and mitochondrial function improvement.The auricular branch of the vagus nerve is the only nerve located on body surface.Transcutaneous VNS(tVNS)is a non-invasive approach to regulate the autonomic nervous system via activating vagal effect.Our previous study has found that tVNS prevented cardiac injury caused by myocardial infarction or myocardial ischemia-reperfusion and even malignant cardiac arrhythmias.However,the effects of tVNS on DOX-induced cardiotoxicity are unknown.Objective:1.To explore whether tVNS plays a cardio-protective role on DOX-induced cardiotoxicity.2.To explore the effects of DOX on cardiac ANS and the modulation of tVNS on cardiac ANS.3.To investigate the underlying mechanisms of tVNS of preventing cardiac injury induced by DOX.Materials and Methods: Healthy male Sprague Dawley rats(weighing 160-200 g,SPF)were included in this study.Before the experiment,all rats were fed in a controlled environment(12-h day/night)with free food and water for one week.All rats were marked and randomly divided into four groups:(1)control group: rats were received the sham procedure without DOX injection or tVNS;(2)DOX group: rats were injected with DOX via 3 weekly injection(5 mg/kg i.p.at 0,7 and 14 days);(3)tVNS group: equivalent volume of vehicle control was administrated(i.p.at 0,7 and 14 days)and tVNS(0.5 h per day)was performed;(4)DOX + tVNS group: rats were received the same DOX injection and tVNS lasted for 6 weeks.A minimum of 6 animals were studied in per group.1.To generate a DOX-induced cardiopathy model in DOX group and DOX + tVNS group,a cumulate dose of 15 mg/kg DOX was administrated via intraperitoneal(i.p.)injection during three weeks(5 mg/kg on days 0,7 and 14).Chronic intermittent tVNS was delivered daily through a voltage stimulator for 30 minutes over 6-week period on DOX + tVNS group and tVNS group.At the end of the experiment,firstly we measured the cardiac function and left ventricular structure represented by left ventricular ejection fraction(LVEF%),fraction shortening(FS%),left ventricular internal dimension in systole and diastole(LVIDs and LVIDd,respectively).After euthanasia,the blood sample and cardiac tissues of rats were collected to further detection.Biochemical analysis,HE staining,Masson's staining and TUNEL analysis were used to detect serum creatine kinase(CK)level,cardiac histopathological changes,respectively.2.At the end of the experiment,the electrocardiogram of rats was recorded for heart rate variability(HRV)analysis,and the serum was collected to detect serum acetylcholine(Ach)and norepinephrine(NE)levels.Moreover,cardiac tissues were quickly excised for detection of tyrosine hydroxylase-positive(TH+)nerve fibers.3.After euthanasia,cardiac tissues were quickly excised for further analysis.ELISA analysis was used to detect superoxide dismutase(SOD),malonaldehyde(MDA)and catalase(CAT)levels in cardiac tissue.Immunofluorescence was used to measure the expression of F4/80,interleukin-1?(IL-1?)expression and myocardial apoptosis,respectively.Additionally,the transcriptome of cardiac tissue was detected via RNAseq and the relative mRNA expression of CXCL9,CXCL10,CCR1 and CCR2 were measured by QPCR.Results: 1.Compared to the control group,DOX significantly decreased LVEF% and FS%(P<0.0001),and increased LVIDs(P<0.0001),LVIDd(P<0.01).Moreover,there were increased serum CK(P<0.05),inflammatory cells infiltration and fibrosis in cardiac tissue in DOX group.However,in the DOX+tVNS group,increase in LVEF%(P<0.001),FS%(P<0.01)and decrease in the LVIDs(P<0.01),LVIDd(P<0.05)were observed compared to the DOX group.And tVNS also reduced CK level(P<0.05),cardiac injury and fibrosis in the cardiac tissue compared to the DOX group.From TUNEL analysis,DOX significantly increased myocardial apoptosis(P < 0.0001)compared to the control group.However,tVNS significantly prevented the apoptosis in cardiac tissue induced by DOX(P<0.001).2.Compared to the control group,DOX significantly increased the LF(P < 0.0001)and LF/HF(P < 0.05),decreased HF of rats(P < 0.01).And increase in serum NE level and decrease in Ach were also observed in DOX group(all P < 0.05).Additionally,the expression of TH+ nerve fibers in cardiac tissue was also significantly increased(P < 0.01).However,tVNS could significantly decrease LF(P < 0.0001)and LF/HF(P < 0.05),increase HF(P < 0.001)in rats and revers the changes of serum NE(P < 0.05)and Ach levels(P < 0.01),and also decreased the expression of TH+ nerve(P < 0.01)in cardiac tissue.3.Compared to control,increases in MDA(P<0.05),IL-1? and a decrease in SOD(P<0.0001)were observed in the DOX group.However,tVNS significantly reversed the changes compared to the DOX group.From transcriptome analysis of cardiac tissue,197 genes were significantly up-regulated,while 48 genes were down-regulated in the DOX group compared to the control group.However,39 differentially expressed genes(DEGs)altered by DOX was down-regulated by tVNS and most of these were involved in immune system via KEGG analysis.Moreover,tVNS significantly downregulated the mRNA expressions of CXCL9,CXCL10,CCR1 and CCR2 in heart compared to the DOX group(all P < 0.05).Conclusions: 1.Chronic tVNS significantly alleviated dox-induced myocardial injury,pathological remodeling and improved cardiac insufficiency.This evidence suggests that tVNS may be an adjuvant treatment to prevent dox-induced cardiotoxicity.2.Chronic DOX exposure significantly increased the activity of SNS,while decreased the vagal nerve activity.While long-term tVNS could reverse these changes to rebalance cardiac autonomic nervous system.3.Chronic tVNS prevented dox-induced cardiotoxicity by inhibiting oxidative stress status,decreasing myocardial apoptosis,down-regulating chemokine-related gene expressions and recruitment of inflammatory monocytes-macrophages in the myocardium.