Clinical Effect Of Acupuncture And Moxibustion On Patients With Atherosclerotic Plaque And Its Molecular Mechanism: Novel Links With Apolipoprotein A-? And Platelet Function

Objective1.To explore the interactions between apolipoprotein A-IV and platelet integrin GPIIb IIIa,and to identify the roles of apolipoprotein A-IV(apo A-IV)in inhibiting platelet function and atherothrombosis.2.To evaluate the clinical effects of acupuncture and moxibustion in patients with carotid atherosclerosis based on Fu's “Heart-Gallbladder Theory”.Through a randomized,blinded and controlled trial,this study will compare the effects of acupuncture and moxibustion on the thickness and area of carotid plaques,the intima-media thickness of carotid artery,blood lipid profiles,platelet aggregation,coagulation function and apolipoprotein A-IV levels.We will also explore molecular mechanisms in mediating the anti-atherothrombotic effects of acupuncture and moxibustion.Methods1.Fundamental study to investigate the novel mechanisms in the prevention and treatment for atherothrombosis(1)Generation of recombinant human and mouse apo A-IV,and biotinylated apo A-IV(2)Identification of the interactions between apoA-IV and integrin GPIIb IIIa,as well as their potential binding sites: Using flow cytometry to evaluate the platelet activation markers;Using ELISA,flow cytometry and immunofluorescent to study the platelet activation,and platelet related inflammation.Recombinant apo A-IV mutants(e.g.double mutations of aspartic acids(D5 and D13)to glutamate)were be generated.The effects of these mutants were determined via platelet functional studies in vitro,ex vivo and in vivo.Size-exclusion chromatography,circular dichroism spectroscopy and thermal denaturation assays were also performed to measure the protein secondary structure and stability.(3)Determination the roles of apo A-IV in platelet activity and thrombus formation in vitro: Recombinant apo A-IV were used for platelet aggregation assays in vitro and thrombosis models ex vivo(perfusion chamber)(4)Determination the roles of apoA-IV in platelet activity and thrombus formation in vivo: The intravital microscopy thrombosis models were employed to evaluate the roles of apo A-IV in thrombosis in vivo.(5)Evaluation of the in vivo role of apo A-IV following high fat diet:Platelet function assays were performed in platelets from C57BL/6J wild-type,transgenic mice overexpressing apo A-IV(apo A-IV-Tg),apo A-IV wild-type(apo A-IV+/+),heterozygous(apo A-IV+/-),and apo A-IV knockout(apo A-IV-/-)mice under fasting conditions,and after a high fat diet.(6)Clinical study of the relationship between levels of apo A-IV and platelet aggregation: Platelet aggregation assays were performed at 6-hour intervals for 24 hours in 20 healthy volunteers.Plasma samples to be assayed for the concentration of apo A-IV were stored at-80?C until analyzed by ELISA.(7)Investigation of the diurnal changes in apo A-IV levels in humans: ELISA was developed and performed to detect the apo A-IV levels in humans at different times of the day,i.e.,06:00,12:00,18:00 and 24:00.2.Clinical effect of acupuncture and moxibustion on the treatment of atherothrombosis and its molecular mechanismThe clinical research of this PhD project is the pilot study of the large randomized controlled trial of “Acupuncture and Moxibustion for the Treatment of Carotid Atherosclerosis based on the “Heart – Gallbladder Theory(AMTCA Study)”.Between May 2016 and October 2017,25 patients from Guangdong Provincial Hospital of Chinese Medicine were recruited in this study,including Acupuncture Group 23 cases,and pharmaceutical control Group 9 cases.This study also recruited 6 healthy volunteers to investigate the molecular mechanisms in mediating the anti-atherothrombotic effects of acupuncture and moxibustion.The acupoints used in this study were: Neiguan,Yang Ling Quan,Renying,Xinshu,Geshu,Danshu,Baihui,Yintang,Zhongwan,Xiawan,Qihai and Guanyuan.The patients in the control group were orally taken Aspirin 75mg/day and Atorvastatin 10mg/day.Evaluation indicators include: the thickness and area of carotid plaques,the intima-media thickness of carotid artery,blood lipid profiles(Total cholesterol,Triglyceride,High-density lipoprotein,Low-density lipoprotein,and lipoprotein(a)),platelet aggregation function,coagulation function(Prothrombin time,Activated partial clotting enzyme time,Thrombin time,Fibrinogen)and apolipoprotein A-IV levels and its ciradian rhythm.Results1.Fundamental study to investigate the novel mechanisms in the prevention and treatment for atherothrombosis(1)Succefully generation of recombinant human and mouse apo A-IV,and biotinylated apo A-IV;(2)Apo A-IV is a novel ligand of platelet integrin GPIIb IIIa;(3)Apo A-IV inhibits platelet aggregation in vitro;(4)Apo A-IV inhibits thrombus growth under flow conditions;(5)N-terminal aspartic acids(D5 and D13)of apoA-IV are required for its inhibitory effect on platelets;(6)Increasing shear rates enhances the anti-thrombotic effects of apoA-IV under flow conditions;(7)Double mutations at apo A-IV N-terminal aspartic acids(D5 and D13)do not affect the structure and stability of apo A-IV;(8)ApoA-IV inhibits thrombus growth in vivo;(9)In vivo dynamic visualization of apo A-IV localization and deposition(10)Apo A-IV attenuates postprandial platelet hyperactivity;(11)Human apoA-IV has a circadian rhythm that is inversely correlated with platelet aggregation.2.Clinical effect of acupuncture and moxibustion on the treatment of atherothrombosis and its molecular mechanism(1)Acupuncture and Moxibustion decreased the thickness and area of atherosclerotic plaques,and the intima-media thickness of carotid artery in patients with carotid atherosclerosis;(2)Acupuncture and Moxibustion did not significantly alter levels of total cholesterol,triglyceride,low-density lipoprotein,and lipoprotein(a)in patients with carotid atherosclerosis;(3)Acupuncture and Moxibustion markedly dereased platelet aggregation levels of patients with carotid atherosclerosis;(4)Acupuncture and Moxibustion did not significantly alter the Fibrinogen levels,coagualtion function and platelet counts in patients with carotid atherosclerosis;(5)Acupuncture and Moxibustion significantly enhanced levels of plasma apo A-IV and its circadian rhythm in healthy donors(6)Acupuncture and Moxibustion increased the plasma apo A-IV levels in patients with carotid atherosclerosis.Conclusion1.We demonstrate that apo A-IV,by antagonizing platelet integrin GPIIb IIIa activity,attenuates platelet aggregation and thrombosis,postprandial platelet hyperactivity,and may also slow the progression of atherosclerosis(i.e.both early and late stages of atherothrombosis).Furthermore,we identified a circadian rhythm of apo A-IV in humans,which negatively correlates with platelet aggregation and the risk of cardiovascular events.Thus apo A-IV is an important endogenous inhibitor of atherothrombosis.Control of food consumption(e.g.increase of unsaturated fat),which raises apo A-IV synthesis and secretion,may have a significant impact on the prevention of the early developmental stages of atherosclerosis.Infusion of recombinant apo A-IV may be able to directly intervene in thrombosis and control cardiovascular diseases and stroke.2.Acupuncture and Moxibustion markedly increase plasma levels of apo A-IV in both healthy donors and patients with carotid atherosclerosis,thus inhibiting platelet aggregation and attenuating atherothrombosis.We thus identified that apo A-IV may mediate the anti-atherothrombotic effects of acupuncture and moxibustion.