The Mechanism Of Acupuncture Attenuated Hyperlipidemia-related Cardiovascular Remodeling In ApoE^-/- Mice

ObjectiveHyperlipidemia is a crucial risk factor for cardiovascular disease,leading to cardiovascular remodeling and promoting atherosclerosis and arrhythmia.This study will:（1）Clarify the effect of acupuncture at Neiguan（PC6）on improving cardiovascular remodeling in hyperlipidemia model,that is,reducing the incidence of spontaneous supraventricular arrhythmia and the process of atherosclerosis,and comprehensively evaluate the role of inflammation,fibrosis and ion channels;（2）Further explore the mechanisms of acupuncture in regulating the inflammation,fibrosis and ion channels via the Hippo-YAP pathway.Methods8-week-old male Apolipoprotein E knockout(Apo E^-/-)mice were fed a Western Diet for 12-week to construct a hyperlipidemia model and randomly allocated to the model group（KO,n=10）,acupuncture group（KO+Acu,n=8）,and Mn Cl₂ group（KO+Mn Cl₂,n=9）.C57B6/J mice served as the control group（WT,n=11）feeding with SPF normal diet.Acupuncture was applied to PC6 on both upper limbs for 30 min,once daily for continuous 5 days;all the interventions lasted for 12-week.Using biochemical analysis to test plasma lipid levels of total cholesterol（T-CHO）,triglyceride（TG）,low-density lipoprotein cholesterol（LDL-C）and high-density lipoprotein cholesterol（HDL-C）.Oil red O staining was used to observe the plaques of the aortas.The electrocardiogram and echocardiogram were applied.Using ELISA to examine inflammatory cytokines of interleukin 6（IL-6）,tumor Necrosis Factor Alpha（TNF-α）,vascular cell adhesion molecule-1（VCAM-1）,and Chemokine（C-X-C motif）ligand1（CXCL1）.Heart histology alterations were achieved using hematoxylin and eosin staining and Masson’s trichrome staining.Immunohistochemical were used to detect the expression of myocardial VCAM-1,CXCL1,ion channels（Cav1.2,Kv1.5,Nav1.5）,and connexin43（Cx43）.Using Yes-associated protein（YAP）blocker and calcium channel blocker manganese II chloride（Mn Cl₂）,through western blot to detect the YAP,YAP phosphorylation（p YAP）,p YAP/total YAP ratio and Ras homolog family member A（Rho A）expressions in aortas.Immunofluorescence was used to detect the YAP expression in heart tissues.Results1.The effect of acupuncture on lipid metabolism of Apo E^-/-mice fed a high-fat dietAfter 12 weeks of high-fat diet feeding,compared with the WT group,plasma T-CHO,TG and LDL-C were significantly increased in the KO group（P<0.001）,HDL-C were significantly decreased（P<0.01）,and atherosclerotic plaque were increased（P<0.001）.It suggests that the hyperlipidemia model was successfully constructed and atherosclerosis occurred.Compared with the KO group,acupuncture significantly reduced the T-CHO,TG levels（P<0.05）,and the atherosclerotic plaques（P<0.01）.2.The effect of acupuncture on the electrophysiology and morphological structure of hyperlipidemia modelThe incidence of spontaneous supraventricular arrhythmia in the KO group was 60%（6/10）.Compared with the WT group,in the KO group,the heart rate,P wave duration were significantly increased（P<0.05）,QTc was significantly increased（P<0.01）;left ventricle enlarged,left ventricle ejection fraction（LVEF）was significantly decreased（P<0.01）.The fractional shortening（FS）,end-systolic volume（ESV）,and left ventricular internal diameter at end-systole（LVIDs）were significantly decreased（P<0.05）.Acupuncture reduced the susceptibility of spontaneous supraventricular arrhythmia,only 25%（2/8）.Acupuncture hardly altered the ECG parameters and cardiac systolic function（P>0.05）.3.The effect of acupuncture on the inflammatory response in hyperlipidemia modelCompared with the WT group,the plasma IL-6,TNF-α,CXCL1 levels were significantly increased in the KO group,and the VCAM-1 and CXCL1 protein expression were significantly increased in the heart tissues（P<0.05）.HE staining showed that the cardiomyocytes structure of the model group was arranged disorderly and with inflammatory cell infiltration.Compared with the KO group,acupuncture significantly reduced plasma CXCL1 levels（P<0.05）,but did not significantly reduce myocardial VCAM-1 and CXCL1（P>0.05）.HE staining showed acupuncture reduced inflammatory cell infiltration.4.The effect of acupuncture on myocardial fibrosis in hyperlipidemia model Compared with the WT group,only the right atrium fibrosis in the KO group increased significantly（P<0.05）;while the fibrosis does not significantly alter in the right ventricle,left atrium,and left ventricle（P>0.05）.Compared with the KO group,acupuncture significantly reduced the right atrial fibrosis（P<0.01）.5.The effect of acupuncture on ion channels in hyperlipidemia modelCompared with the WT group,the Cav1.2 expression in the right atrium,right ventricle,and left ventricle of the KO group increased significantly（P<0.01）,while the Kv1.5 expression was significantly reduced（P<0.05 or P<0.01）;right ventricle Cx43 expression was significantly reduced（P<0.05）.Compared with the model group,acupuncture reduced the left ventricle Cav1.2 expression（P<0.05）,while increased the Kv1.5 expression（P<0.05）.6.The effect of acupuncture on the Hippo-YAP signaling pathway in hyperlipidemia model Compared with the WT group,the p YAP,p YAP/total YAP ratio were significantly reduced（P<0.001）in the inner side of the aorta arch of KO group,while the YAP level significantly increased（P<0.01）;The YAP and Rho A expressions were significantly increased on the outer side of the aorta arch（P<0.05）;The expression of proteins in the thorax and abdomen aorta did not change significantly.In myocardial tissues,YAP is expressed highly in both the cytoplasm and nucleus in the KO group.Compared with the KO group,acupuncture only significantly increased the p YAP level of the aorta arch inner side（P<0.01）.Acupuncture decreased the nucleus YAP expression in the myocardial.ConclusionAcupuncture effectively reduces the occurrence of cardiovascular remodeling（arrhythmia and atherosclerosis）in Apo E^-/-mice with hyperlipidemia.The underlying mechanism may via YAP-mediated inflammatory response-induced fibrosis and ion channel remodeling.