Influence Of Electroacupuncture Pretreatment At “Neiguan” On The P38MAPK Signaling Pathway In Rats With Myocardial Ischemia-reperfusion Injury

With the constant progress of the society,the improvement of the people living standard,infectious disease is no longer the main cause of death..Non-communicable chronic disease has gradually become a danger to human health,among which,the cardiovascular morbidity and mortality has accounted for 29% of all-cause death,and been more than malignant tumor,leaps to the first.In China,the amount of the death caused by acute myocardial infarction has been more than 1million,meanwhile the morbidity and mortality rate of the acute myocardial infarction is still risin g.Myocardial/ischemia has become the adult's primary causes of heart disease in hospital in China.The basic therapeutic prin ciples of the acute myocardial infarction is the early recovery of coronary artery unimpeded,which can make the obstruction site flow again.The method of making the myocardial cell oxygen restore power as soon as possible can reduce the damage to cardiac function.There fore,spasmolysis thrombolysis,surgery and angioplasty have become common treatments widely.With the development of interventional medical technology,the coronary artery bypass grafting,the stent implantation,and the percutaneous coronary angio plasty cavity have gradually become the routine treatments of acute myocardial infarction.However as the coronary arteries flow again,the patho logical process of coronary heart disease(CHD)is going on,and a series of changes in ultrastructure and even irreversible damage appeares.All of these is seriously affecting the effects of the treatment of coronary heart disease.The aggravating tissue damage and the irreversible damage,based on myocardial ischemia after restore blood flowing,are called myocardial ischemia/reperfusion injury(I/R).The mechanism of myocardial ischemia/reperfusion in jury involved in oxidative stress,inflammation,calcium over load,mitochondrial damage,obstacle of energy use,cell apoptosis and so on.These factors influence each other.Oxidative stress is the common pathway of the cell necrosis and apoptosis Some studies have found that because of the I/R,the cell apoptosis,the formation of NOS and NF-?B activation will accure.At the same time,the NF-?B activation and NOS in turn can promote cell apoptosis,elevating the content of CK and LDH,reducing the content of the NO in seru m.Yi-min bao and her colleagues have found I/R lead to abnorma l myocardial mechanics and hemodynamic information:arrhythmia(dif ferent extent and duration),myocardial suppression and reduced myocardial diastolic function etc.Some other tudies have confirmed that,a large number of ROS production is the main fac-tor,which will lead to acute myocardial injury.The generation of ROS which eliminates the effect of various endogenous scavenger will cause cell membrane damage and calcium overload by the lipid peroxida tion,shrink sarcoplasmic reticulum injury and dysfunction by cal cium balance,stimulate the increase of PMN adhesion molecule by activating endothelial adhesion molecules and cause the damage toDNA by causing proinflammatory enhanced coagulation.The impo-rtant measure of the treatment of I/R and the protection of the heart function is to Control the concentration of reactive oxygen species and the oxidative stress response.Nowadays the common medical pr-evention measures of I/R is still at the experimental stage,it can not be widely used.Based on the etiology and pathogenesis of my-ocardial ischemia reperfusion in TCM,on the platform of the im-portant acupuncture disciplines and collaborative innovation ce-nter in hubei province,we have chosen classic common acupoints achieved gratifying results by teamwork.We have found that Acu-puncture in PC6 have a protective effect on electrical activity of ischemic myocardial cells On the one hand,there are central nervous nuclei(including the medulla oblongata,solitary nucleus nuclear paraventricular nucleus,etc.),regulating cell cAMP and c GMP;On the other hand,there are the aid of angiotensin,endothelin,ca-techolamine neurotransmitters,through MAPK signal pathway,for the regulation of the myocardial cell integrity and stability.This experiment around the two centers,"acupuncture prevention" and "protection of cardiac function in myocardial cell ",is for the study of the protection and the mechanism of preconditioning of myocardial ischemia-reperfusion injury by acupuncture in PC6,and to provide experimental basis of the characteristic and advantage of acupuncture in PC6 for the I/R.ObjectiveUsing the method of ejector sleeve,the myocardial ischemia reperfusion model is established successfully,and improved a lot;the role is made sure of the Oxidative stress and mitochondrial activity in reducing I/R.By the use of acupuncture,s advantage of low trauma,easy operation and the strong controllabili-ty,combined with theclassical theory of acupuncture and mox-ibustion,with the research results of zang-fu organs-meridian correlation,we will observe the ventricular remodeling,myocardial infarction,cardiac function parameters,factors in serum and myocardial cell,the influence of the mitochondrial respiratory function during the early preconditioning on myocardial ischemia reperfusion in rats by acupuncture in PC6,and by the use of p38 MAPK pathway blockers SB203580,analyze the mechanism from the pers-pective of p38 MAPK signaling pathways,and show the foundation of the clinical prevention and treatment of the various I/R.MethodsIn this project,200 male Wistar rats,3-month-old,weighting about220±28g,were randomly divided into five group: sham operation group(S group),myocardial ischemia-reperfusion injury model group(M group),electroacupuncture preconditioning group(EA group),SB203580group(SB group)and electroacupuncture precondit-ioning+SB203580group(EA+SB group).Ischemia-reperfusion injury model was achieved by the ligation and deligation of left anterior descending artery with ischemia for 20 min and reperfusion for 40 min.Before establishing model,the acupuncture group with electric acupuncture for 20 min,1mA,continuous wave.Then the cardiac function after I/R(left ventricular endiastolic pressure LVED P,left ventricular systolic mean presure LVSP,fractional shortening FS and ejection fraction EF)were investigate d,myocardial tissue were taken and gauging the Content of ROS?SOD?MDA?ATP,and the ultrastructures were observed by the use of a laser confocal microscope,HE stain and transmission electron microscope in the rat myocardial cells especially.Serum creatine(CK),lactate dehydrogenase(LDH)and cardiac troponin I(cTnI) were detected among the 5 groups.Results(1)Compared with S group,the ratio of infarct area and degree of infarction in the M group was obviously higher(P<0.05),and the ratio of infarct area and infarct degree of SB group,EA group and EA+SB group were lower than this in M group,and the difference was significant(P<0.05).But there was no significant difference among the two indicators in EA group?EA+SB group and in SB group(P>0.05).(2)Compared with S group,the index of LVEDP in M group,SB group,EA group and EA+SB group significantly increased and the index of LVSP,FS and EF in these four groups decreased(P<0.05).The four indicators of EA group,SB group and EA+SB group compared with M group,the difference was statistically significant(P<0.05).(3)Compared with S group,the three serum indicators included LDH,CK and c Tnl in M group,SB group,EA group and EA+SB group significantly increased(P<0.05),the three indexes such as LDH,CK and cTnl in SB group,EA group and EA+SB group were lower than those in the M group,and they had significant difference(P<0.05).The two indexes cTnl in the EA group and the EA+SB group were lower than this in SB group.(P<0.05)(4)Compared with S group,the content of TNF-??IL-1 and IL-6 in M group,SB group,EA group and EA+SB group increased(P<0.05),and the difference was significant.Compared with M group,the content of TNF-??IL-1?and IL-6 in SB group,EA group and EA+SB group could reducesignificantly(P<0.05).Especially in the EA group,the content of TNF-??IL-1?? IL-6 were significantly better than those in EA+SB group and SB group(P<0.05).Compared with S group,the content of IL-4 and IL-10 in M group were not significant difference.The content of IL-4 andIL-10 in SB group,EA group and EA+SB group were significantly higher than those in M group and S group(P<0.05),but there was not significantly difference in SB group,EA group and EA+SB group(P>0.05).The content of IL-4 and IL-10 in EA group was a litter higher than the others.(5)Compared with S group,the protein expression of ICAM-1 and VCAM-1 were increased in M group,SB group,EA group and EA+SB gr-oup,the difference was obvious(P<0.05).Compared with M group,the protein expression of ICAM-1 and VCAM-1 in SB group,EA group and EA+SB group could apparently bring down(P<0.05).Especially the protein expression of ICAM-1 and VCAM-1 in EA group was lower than those in EA+SB group and SB group(P<0.05).(6)Compared with S group,and the content of ATP had a significant decline,whereas the content of ROS and MDA had an obvious rise in M group,EA group and EA+SB group(P<0.05).Compared with M group,the content of ROS?MDA?ATP and the activity of SOD were significantly different in EA group,SB group,and EA+SB group(P<0.05).(whether lack of ATP enzyme).(7)Compared with S group,The protein expression of Na+-K+-ATPase and Ca2+-ATPase in M group,EA group,SB group and EA+SB group were sign-ificantly higher(P < 0.05),and compared with M group,the protein expression of EA group,SB group and EA+SB group had significant difference(P<0.05).The protein expression of Na+-K+-ATPase and Ca2+-ATPase in EA group was higher than those in EA+SB group and SB group(P<0.05).(8)Compared with S group,The protein expression of MKK3/6,p38 and p-p38 in M group,EA group,SB group and EA+SB group weresign-ificantly higher(P < 0.05),and compared with M group,the protein expression of EA group,SB group and EA+SB group had significant difference(P < 0.05).(9)In S group,myocardial fiber arranged regular,cell structure was well,the morphology and amount of cell nuclear and mitochondia were regular,mitochondria cristae was clear,there was no abn-ormality among myocardia.In M group,myocardial fiber became dissolved even nenecrosis,interstice increased,swelling obvio-us,some parts of mitochondria pyknosis and smaller,some parts swelled and membrane damaged.Mitochondria cristae became irregular fractured into floc and deletion.In EA group,SB group and EA+SB group,the gap between myocardial fiber widened slightly,necrosis reduced,myocardial cell swelled slightly,cell nuclear and mitoch-ondia structure kept basic integrity,whereas a minority of mito-chondia vacuole still existed.Conclusion1.Electroacupuncture pretreatment at“neiguan”decreases the infarct area and infarct degree of I/R model rat,obviously reduces mitochondrial swelling and necrosis,reduce myocardial cell infiltration of inflammatory cytokines,and improve the impaired myocardial pathomorphology obviously.2.Electroacupuncture pretreatment at “neiguan”decreases the index of LVEDP of I/R model rat,increases the index of LVSP,FS and EF,decrease the content of LDH,CK and cTnI in serum,and protects the damaged heart function.3.Electroacupuncture pretreatment at “ neiguan ” inhibits the expression of inflammatory cytokines of TNF-a?IL-1??IL-6,increases the expression of anti inflammatory factors of IL-4 and IL-10,and decreases the expression of adhesion molecule such as ICAM-1 andVCAM-1 protein,meanwhile,it could protect damaged car-diac tissue through the regulation of inflammation related factors.4.Electroacupuncture pretreatment at“neiguan”reduces the content of ROS and MDA in myocardial cells of I/R model rat-s,increases the content of ATP,improves the activity of SOD,Na+-K+-ATPase and Ca2+-ATPase,and make the RCR better.It reduces I/R myocardial injury by protecting the integrity of the structure and function of mitochondria.5.MKK3/6/p38 MAPK signal transduction pathway was involved in the pathological process of I/R model rats,the expression of MKK3/6and p38 MAPK in I/R model rat was up-regulated,electroacupuncture pretreatment at“Neiguan” could inhibit the protein expression of MKK3/6?p38MAPK in I/R and the phosphorylation of p38 MAPK.On the regulation of myocardial cell p38 signal transduction pathway might be one of the mechanisms of electroacupuncture pretreatment to protect I/R.Compared with EA+SB group and SB group,the index of EA group showed obviously differences,but between the EA+SB group and SB group there was not significantly difference,illustrating that the mechanism of electroacupuncture preconditioning to protect I/R might be a multi-level and comprehensive effect in many ways.