| Objective: Myocardial ischemia-reperfusion injury is an important risk factor which can cause the aggravation of myocardial injury during the treatment of coronary heart disease.In this study,the myocardial ischemia-reperfusion model rats were taken as the research object.Under the idea of "treatment aiming at its pathologenesis" of guidance from traditional Chinese medicine,"Neiguan(PC6)","Zusanli(ST36)" and "Guanyuan(CV4)",three acupoints were selected for electroacupuncture pretreatment by using the method of "Combined Biao and Ben Acupoints Treatment".The myocardial cell-associated cytoskeletal proteins were taken as the breakthrough point by observing the effects of the electroacupuncture preconditioning with "Biao-Ben Acupoints" on the ultrastructure of sarcomere and the expression of cytoskeletal proteins(desmin and ?-actinin)in cardiomyocytes to explore the possible mechanism of electroacupuncture preconditioning with "Biao-Ben Acupoints" in the prevention and treatment of ischemic heart disease,and to provide experimental basis for acupuncture prevention and treatment of myocardial ischemia-reperfusion injury.Methods: 1.Fifty SPF male SD rats,weighing about 220-250 g,were randomly divided into 5 groups with 10 rats in each group after adaptive feeding for 1 week.These 5 groups are model group(myocardial ischemia/ reperfusion injury group,MI/RI group),sham operation group(sham operation group,SO group),Neiguan pretreatment group(MI/RI+PC6 group),Zusanli pretreatment group(MI/RI+ST36 group),and electroacupuncture precondi-tioning with Biao-Ben Acupoints group(myocardial ischemia reperfusion group with acupuncture pretreatment on Neiguan,Zusanli and Guanyuan,MI/RI+AP group).2.MI/RI group and SO group were bound once a day for 20 min for 7 days.On the 8th day,myocardial ischemia-reperfusion model was established after thoracotomy,in which ischemia was implemented for 20 min and then reperfusion for 40 min.However,no ligation of the left anterior descending coronary artery was implemented in SO group when thoracotomy modeling on the 8th day.MI/RI+PC6 group,MI/RI+ST36 group and MI/RI+AP group were all preconditioned 7 days before modeling in which the rats were pretreated with electroacupuncture at the same time every day and myocardial ischemia-reperfusion was performed on the 8th day.The rats in MI/RI+PC6 group were treated with acupuncture at bilateral "Neiguan(PC6)" point every day 7 days before modeling,and at the same time,another needle was inserted subcutaneously at 0.3-0.5cm outside the "Neiguan(PC6)" point as auxiliary electrode which would form a pair of electrodes were formed with "Neiguan(PC6)" point.And the MI/RI+ST36 group was treated with acupuncture at bilateral "Zusanli(ST36)" point every day for 7 days before modeling,meanwhile,another subcutaneous shallow needle at 0.3-0.5cm was opened outside the "Zusanli(ST36)" point as auxiliary electrode therefore a pair of electrodes were formed with the acupoints of "Zusanli(ST36)".And in the MI/RI+AP group,the bilateral "Neiguan(PC6)","Zusanli(ST36)" and "Guanyuan(CV4)" were acupunctured 7 days before the establishment of the model,and a pair of electrodes were formed by the "Neiguan(PC6)" and "Zusanli(ST36)" acupoints at the same side.Another needle was inserted subcutaneously outside the "Guanyuan(CV4)" point to 0.3-0.5cm as auxiliary electrode,and a pair of electrodes were formed with "Guanyuan(CV4)" point.Han's electroacupuncture therapeutic apparatus was set as continuous wave,frequency 2Hz,intensity 1m A.The suitable intensity was determined by the trembling sensation of rat limbs,and the treatment should last for 20 min.The rats were treated once a day for 20 minutes for 7 days.And myocardial ischemia-reperfusion was performed on the 8th day.3.At the end of acupuncture preconditioning treatment(the 8th day),10% chloral hydrate rats were injected intraperitoneally with anesthesia(3ml/kg)and tracheal intubation was implemented in each group,connecting with the small animal ventilator for mechanical ventilation.After thoracotomy,the anterior descending branch of left coronary artery was blocked by suture at 2mm below the root of left atrial appendage and pulmonary artery,resulting in myocardial ischemia of left ventricular anterior wall for 20 min,and then the left anterior descending branch of left coronary artery was opened to restore blood supply and reperfusion for 40 min,resulting in MI/RI model.The electrocardiogram(ECG)changes of 40 min were recorded respectively before ligation and 20 min after ligation.The elevation of ST segment higher than 0.2mv would mark myocardial ischemia,and the decrease of ST segment would mark reperfusion.4.On the 8th day,blood samples were collected from abdominal aorta and heart after the establishment of MI/RI.The contents of myocardial ischemia markers which are ischemic modified albumin(IMA)and brain natriuretic peptide(BNP)would be measured by ELISA.The degree of myocardial injury in HE stained sections was observed by optical microscope and the ultrastructure of sarcomere observed by high power transmission electron microscope.The expression of myocardial cytoskeleton desmin and ?-actinin were detected by Western blot.Statistical analysis was performed on the data of each group.Results: 1.Comparison of ST segment of ECG in rats:(1)compared the data of 20 min ligation with before ligation: the value of ST segment in all groups except SO group significantly increased(P<0.01),which indicated that the model of myocardial ischemia in each group was successful;(2)compared the data of 40 min reperfusion with 20 min ligation: except for SO group,the ST segments of the other four groups significantly decreased(P<0.05),and the ST segment significantly decreased after reperfusion,which indicated that the reperfusion was successful.(3)after reperfusion for 40min: compared with MI/RI group,the ST segment values of the other four groups significantly decreased(P<0.01);compared with MI/RI+PC6 group,the value of ST segment in MI/RI+ST36 group significantly increased after reperfusion(P<0.05);and the value of ST segment in MI/RI+AP group significantly decreased after reperfusion(P<0.05).Compared with MI/RI+ST36 group,the value of ST segment in MI/RI+AP group decreased significantly after reperfusion(P<0.01).Results showed that electroacupuncture preconditioning could reduce the degree of myocardial ischemia reperfusion injury and had a certain protective effect on the heart.The ST segment value drops most obviously in the MI/RI+AP group.The results showed that the protective effect of MI/RI+AP group on heart was better than that of MI/RI+PC6 group and MI/RI+ST36 group.2.Morphological comparison of myocardial tissue in rats: in MI/RI group,myocardial injury was the most obvious in which fiber arrangement was disordered,edematous occurred,and gap widened.And most of the myocardial fibers were twisted,squeezed,dissolved and broken as well as myocardial fibers coagulate ischemic necrosis,strong eosinophilic cytoplasm,inflammatory cell infiltration,loss of some nuclear pyknosis and even fragmentation and dissolution.There was no significant difference in myocardial tissue structure in SO group.In MI/RI+AP group,the structure of myocardial tissue was intact,and the arrangement of myocardial fibers was neat and clear,and cardiomyocytes were accompanied by a small number of inflammatory cell infiltration and mild interstitial edema.The pathological changes of myocardial histomorphology in MI/RI+AP group were lighter than those in MI/RI+PC6 group and MI/RI+ST36 group.3.Serum markers of myocardial ischemia in rats:(1)compared with MI/RI group: the serum concentration of IMA and BNP in SO group,MI/RI+PC6 group,MI/RI+ST36 group and MI/RI+AP group significantly decreased(P<0.01);SO group < MI/RI+AP group < MI/RI+PC6 group < MI/RI+ST36 group < MI/RI group.(2)compared with MI/RI+PC6 group,the serum concentration of IMA in MI/RI+ST36 group significantly increased(P<0.01)and the serum concentration of IMA in MI/RI+AP group significantly decreased(P<0.01);the serum concentration of BNP in MI/RI+ST36 group increased(P<0.05);the serum concentration of BNP in MI/RI+AP group did not decrease significantly,and the difference was not statistically significant(P >0.05).(3)compared with MI/RI+ST36 group: the serum concentration of IMA and BNP in MI/RI+AP group significantly decreased(P < 0.01).4.Ultrastructural comparison of myocardial segments in rat cardiomyocytes: in MI/RI group,myocardial injury was extremly serious,in which myofibrils atrophied,ruptured,dissolved,the gap increased,and the bright and dark zone structural areas of myocytes were seriously destroyed in the form of fragments.The arrangement was blurry and disordered.M-line bent or disappeared,and part of the cytoskeleton was even completely broken,and the end actin filament breakage of sarcomere protein chain was reduced,separated and dissolved,with interstitial edema and vacuolar degeneration.MI/RI+PC6 group and MI/RI+ST36 group were slightly improved compared with MI/RI group.The ultrastructural damage of myocardial cells in MI/RI+AP group was significantly less than that in MI/RI group.The myocardial myofibrils were arranged neatly,and the myofilament was clear with little rupture,and the structure of bright band and dark zone in myometrium was clearly observed.Z line and M line were uniformly distributed,clear and complete.5 Expression of skeletal protein desmin and ?-actin 5.1The ratio content of desmin gray value of skeletal protein:(1)Compared with SO group,the expression of desmin in MI/RI group,MI/RI+PC6 group,MI/RI+ST36 group and MI/RI+AP group was significantly lower than that in SO group(P<0.01).The ratio of gray value of skeleton protein desmin was significantly lower than that in MI/RI+PC6 group(P<0.01).(2)Compared with MI/RI group,the expression of desmin in MI/RI+PC6 group,MI/RI+ST36 group and MI/RI+AP group were significantly higher than that in MI/RI+PC6 group.(3)Compared with MI/RI+PC6 group,the expression of desmin in MI/RI+ST36 group decreased,but there was no significant difference(P>0.05),and the expression of desmin in MI/RI+AP group increased,but there was no significant difference(P>0.05).(4)Compared with MI/RI+ST36 group,the expression of desmin in MI/RI+AP group was significantly increased(P<0.01).5.2The ratio of gray value of skeletal protein ?-actinin protein:(1)Compared with SO group,the expression of ?-actinin in MI/RI group,MI/RI+PC6 group,MI/RI+ST36 group and MI/RI+AP group was significantly decreased(P<0.01).(2)Compared with MI/RI group,the expression of ?-actinin in MI/RI+PC6 group,MI/RI+ST36 group and MI/RI+AP group was significantly increased(P<0.01).(3)Compared with MI/RI+PC6 group,the expression of ?-actinin in MI/RI+ST36 group was decreased,but the difference was not statistically significant(P>0.05).The expression of ?-actinin was significantly increased in MI/RI+AP group(P<0.05).(4)The expression of ?-actinin protein in MI/RI+AP group was higher than that in MI/RI+ST36 group(P<0.01).Conclusions 1.Electroacupuncture preconditioning with "Biao-Ben Acupoints" can effectively protect MI/RI model rats,and promote the decrease of ST segment of ECG,also improve the degree of myocardial tissue damage and reduce the content of IMA and BNP in serum,so as to attenuate the extent of myocardial ischemia-reperfusion injury,and improve cardiac function.2.Electroacupuncture pretreatment with "Biao-Ben Acupoints" can obviously stabilize the myocardial sarcomere structure in MI/RI model rats,reduce the injury of myocardial fibers,improve the contractile function of the heart and stabilize the cytoskeleton structure of myocardial cells.3.Electroacupuncture pretreatment could up-regulate the expression of myocardial cytoskeleton proteins(desmin and ?-actinin),but the up-regulation was more significant in the "Biao-Ben Acupoints" pretreatment group(MI/RI+AP group > MI/RI+PC6 group > MI/RI+ST36 group),indicating that MI/RI+AP group has better protective effect on myocardium. |
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