| Objective:Myocardial Ischemia refers to various reasons cause reduction of coronary blood flow,result the clinical pathophysiology in myocardial oxygen etc. material supply insufficient and metabolites clear ability reduced.In clinical common in Coronary heart disease(CDH),also known as Ischemic Heart Disease.In recent years,with our Countri’es economic rapid development,people’s living standards improved greatly,dietary changes in the structure and the average life expectancy extended,so that the incidence of ischemic heart disease increased year by year,and it has become one of the main reasons people dead.So to find and explore effective prevention and treatment method of ischemic heart disease has become the priorities and hot spots to the majority medical profession research.Existing studies of acupuncture in myocardial ischemia improved take more on single points,mainly in "Neiguan(PC6)" and "Xinshu(BL15)",most did not consider diagnosis and treatment facilities with acupoints compatibility.The mechanism of multiple acupuncture point combined effect and its effects whether Betterthan a single hole,we need further inquiry.In this study,the rat model of chronic myocardial ischemia as the research object,adopt the Electro "Acupuncture of Combined Biao and Ben Acupoints" Treatment, and take the single "Neiguan(PC6)" treatment as a control,by observing the ECG,cardiac pathological,myocardial infarct size,apoptosis index and the of mitochondrial ultrastructure,analysis the protective effects of Electro "Acupuncture of Combined Biao and Ben Acupoints" Treatment on myocardial ischemia in rats,through the activation and inhibition of PI3K/Akt pathway to observe changes in PI3K/Akt pathway and apoptosis-related genes,proteins and other indicators,systematic collation and analysis the regulatory mechanism of Electro "Acupuncture of Combined Biao and Ben Acupoints" Treatment on chronic myocardial ischemia rat’s PI3K/Akt signaling pathways and related gene and protein expression,discussion the protective effect and mechanism of Electro "Acupuncture of Combined Biao and Ben Acupoints " Treatment on chronic myocardial ischemia,to provide experimental evidence for clinical acupoints compatibility.Materials and Methods:Use 140 healthy SPF Wistar male rats with 3-month-old,weight180-220 g,According to random number tabler divided into 7 groups:the Normal group,the Model group,the LY294002 group,the IGF-1 group,the Neiguan group,the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group, the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 group.each group had 20 rats,feeding with general food in separately cage,after adaptive feeding one week to conduct modeling and deal with.Except the normalgroup,the other six groups were injected in abdominal subcutaneous with isoproterenol(ISO) to copy the model of chronic myocardial ischemia,a total injection 14 days,once in daily.The LY294002 group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 group in addition to modeling, but also gives PI3K/Akt pathway specific inhibitor LY294002 solution injected into subcutaneously,from start modeling to end modeling,a total of14 days, once a day.The IGF-1 group in addition to modeling,to give the activator insulin growth factor-1(IGF-1) of PI3K/Akt pathway injected into abdominal subcutaneous,from start modeling to end modeling,a total of 14 days, once a day.The Neiguan group except modeling to give electrical acupuncture with the single "Neiguan(PC6)"(near right of the "Neiguan(PC6)" next to 0.5cm shallow thorn a needle as an auxiliary electrode);the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment proup and the the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 group specimens and specimen group with points with points + LY294002 group was given electrical acupuncture on "Neiguan(PC6)", "Zusanli(ST36)", "Guanyuan(RN4)"(the ipsilateral "Neiguan(PC6)" pick with the same side "Zusanli(ST36)",near the "Guanyuan" right of to 0.5cm shallow thorn a needle as an auxiliary electrode).Electro-acupuncture treatment contact with HANS-200electro-acupuncture device,continuous wave,frequency 2HZ,intensity 1mA,degree with the rats’ s limbs quivering slightly,since the beginning of modelingto last 21 days,therapy once a day,every time10 mins.After acupuncture treatment end,use the BL-420 S biological and functional experimental system software to test the changes ofthe rat’s heart rate(HR),ST segment.Adopt HE staining to observe the morphological changes of myocardial tissue in rats.TTC staining test infarct size of myocardial in rats.Used Tunel method to detect myocardial apoptosis index.Used the electron microscope to observe structure changes in rat myocardium mitochondrial.Adopt the immunohistochemistry method to detect PI3 K, Akt, Caspase3,Caspase9,Bax,Bcl-2 and other apoptosis-related protein expression levels in myocardial tissue.Used RT-PCR(Real time-PCR) to detect the PI3 KmRNA and AktmRNA expression levels of myocardial tissue in rats.Simultaneously,Using immunoblotting(Western Blot) detection PI3 K and Akt protein expression levels.Result:1.Before intervention the HR and ST segment voltage pairwise comparison, the difference was not statistically significant(P>0.05), between the two groups were comparable.Except the normal group,compared with the rest of the intervention group before intervention HR faster,ST segment elevation voltage,the differences were highly significant statistical significant(P<0.01).the rest groups compared with before interventionthe,The HR faster, the ST segment voltage raised, the differences were statistically significant(P<0.01).After the intervention,compared with the normal group, all group’s HR faster,ST segment voltage elevate significantly(all P<0.01).Compared with the model group,the HR of the IGF-1 group,the Neiguan group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group were slower,the ST segment voltage decreased significantly(All P<0.01);the LY294002 group ’ s HR faster(P <0.05),ST segment voltage increased(P<0.01);the difference of the Electro Acupuncture of Combined Biaoand Ben Acupoints Treatment + LY294002 group’s HR and ST segment voltage was not statistically significant(P>0.05).Compared with the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group,the LY294002 group and the sElectro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 groups’ HR and ST segment voltage chang were higher than specimens Points group(P<0.01);the IGF-1 group compared with the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group,the difference of HR, ST segment voltage change were not statistically significant(P>0.05);the Neiguan group’s HR was faster and ST segment elevation voltage more than that in the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group(P<0.05).2.In the light microscope,the HE staining of myocardial tissue can see:the Normal group ’s myocardial fibers was compact,orderly rows,the nucleus was visible clearly,the muscle fibers was red,the nucleus was blue;the model group showed the myocardial hypertrophy of myocardial damage,Sarcoplasmic cohesion was strongly eosinophilic staining(magenta),partial collapse broken into irregular coarse particles or agglomerates,myocardial fibers was disordered twisted and broken,the gap of muscle fiber was widened,local fibrosis,the nuclear rupture,dissolution and dispersion confusion,the necrotic area were more than 1/2 of the thickness of the heart;the myocardial injury of the LY294002 group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 group were heavier than the model group.After electro-acupuncture treatment,the myocardial injury of the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group and the Neiguan group significantly reduced, and the degree ofimprovement in the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group was better than the Neiguan group,that was consistent with the degree of improvement in the IGF-1 group.3.Compared with the normal group,the other groups IS/LV and ARR/LV were significantly increased,the difference was significant(P<0.05 or P<0.01).Compared with model group,the IGF-1 group the Neiguan group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment groups’ IS/LV and ARR/LV had declined,the difference was significant(P<0.01);the LY294002 group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment+ LY294002 groups’ IS/LV and ARR/LV were significantly increased,the difference had statistically significant(P<0.01 or <0.05).Compared with the specimen Points group,the IGF group’s IS/LV and ARR/LV was decreased,but the difference had not statistically significant(P> 0.05);The Neiguan group,the LY294002 group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment+ LY294002 groups’ IS/LV and ARR/LV ratio was higher than the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group’s,the difference was significant(P<0.01).4.Compared with the normal group, the other groups’ apoptosis index(Apoptotic Index, AI) were significantly increased,the difference was significant(P<0.05 or P<0.01).Compared with model group,the IGF-1 group,the Neiguan group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment groups ’ AI were decreased,the difference was highly significant(P<0.01);the LY294002 group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 groups’ AI were significantly increased,the difference was statistically significant(P<0.01 or<0.05).Compared with the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group,the IGF group’s AI was decreased,but the difference was not statistically significant(P>0.05);the Neiguan group,the LY294002 group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 groups’ AI were higher than that with the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group,the difference there were highly significant(P<0.01).5.The myofibrils of the Normal group arranged in parallel orderly,filaments clear,Mitochondrial rich, consistent shape with oval and structural integrity,ridge full,no mitochondrial swelling and vacuolar degeneration;A larger number of mitochondria withe layered form,cristae were arranged substantially parallel and lamellar;good Shows wieh nucleus and nucleolus.The Model group:myocardial cell damaged severe,cytoplasmic edema obvious,filaments were dissolved,mitochondrial swelling,vacuolization,focal cavitation,cristae rupture,disorder and the number significantly reduced,formed a wide area electron lucent;Matrix electron density and dense granules reduced or even disappeared,with vacuolar formation;sarcomere fracture,myofibrils disorganized,part Z line is not obvious;Nuclear chromosome condensation and margination,forming a crescent shape,could see the early typical apoptotic bodies.The LY294002 group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 group had similar degree of mitochondrial damage, but were heavier than the model group.After elect-acupuncture treatment,the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group and the Neiguan group’s degree of myocardial mitochondrial damage wereimproved.And sthe Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group’s improvement extend was better than the Neiguan group’s,that was consistent with the IGF-1 group’s improvement degree.6.Compared with the normal group,the Caspase3, Caspase9 and Bax protein expression in other groups’ cardiac muscle of rats were increased in varying degrees,the difference was hihgly significant(P<0.01),it means that myocardialhad varying degrees cell apoptosis after modeling,the model was success.Compared with the model group,the Caspase3, Caspase9 and Bax protein expression of the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment+ LY294002 group had no significant difference(P>0.05);the Caspase3,Caspase9 and Bax protein expression of the LY294002 group was more obvious(P<0.05),described the extent of apoptosis was heavier;The Caspase3,Caspase9 and Bax protein expression in the IGF-1 group,the Neiguan acupoints group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group compared with model group had different degree of decline(P<0.01 or 0.05),it described The three groups’ degree of apoptosis in rat cardiomyocytes were lighter than the model group.Compared with the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group,the Neiguan group,the LY294002 group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 groups’ Caspase3,Caspase9 and protein expression were increased in varying degrees,the difference was statistically significant(P<0.05 or0.01),it description myocardial apoptosis extent of the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group was lower than these three groups;the IGF-1 group’s Caspase3, Caspase9 and Bax protein expression compared with the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group had no significant difference(P>0.05),it means the extent of myocardial cell apoptosis in two groups almost unanimously.7.Compared with the normal group,the other groups’ Bcl-2 protein in the rats myocardial tissue expression were increased in different degrees,the difference was significant(P<0.01).Compared with the model group,the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 group’s Bcl-2 protein expression slightly higher than the model group,but the difference was not statistically significant(P>0.05);the LY294002 group ’ s Bcl-2protein expression lower than the model group,the difference was significant(P<0.01);The Bcl-2 protein expression in the IGF-1group,the Neiguan group and the model group,the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group compared with model group had varying degrees of increased(P<0.01 or0.05).Compamred with the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group,the Bcl-2 protein expression in the Neiguan group,the LY294002 group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 group were belower,the difference was statistically significant(P<0.05 or0.01);The IGF-1 group’s Bcl-2 protein expression was no significantly difference with the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group’s(P>0.05).8.Compared with normal group,the PI3 KmRNA expression of each other group increased significantly(P<0.05).Compared with the model group,the PI3 KmRNA expression of the IGF-1 group,the Neiguan group and the Electro Acupuncture of Combined Biao and Ben AcupointsTreatment group increased(P<0.01),PI3 KmRNA expression of the LY294002 group lower than the model group(P<0.01),PI3 KmRNA expression of the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 group had no difference with the model group(P>0.05);Compared with the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group,IGF-1 group’s PI3 K expression had no difference with it(P>0.05),the Neiguan group’s PI3 K expression lower than the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group(P<0.05),PI3 KmRNA expression of the LY294002 group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 group were lower than samples with acupoint group(P<0.01).9.Compared with the normal group,the model group’s AKT1 mRNA and Akt2 mRNA expression increased(P<0.01),AKT1 mRNA and Akt2 mRNA expression of the LY294002 group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 group reduced significantly(P<0.01 or <0.05).the IGF-1 group,the Neiguan group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment groups’ AKT1 mRNA and Akt2 mRNA expression increased significantly(P<0.01).Compared with the model group,AKT1 mRNA and Akt2 mRNA expression of the IGF-1 group,the Neiguan group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group increased(P<0.01 or <0.05),the LY294002 group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002groups’ AKT1 mRNA and Akt2 mRNA expression level were lower than the model group(P<0.01).Compared with the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group,the IGF-1 group’s AKT1 mRNA and Akt2 mRNA expression had no difference withit(P>0.05),the Neiguan group’s AKT1 mRNA and Akt2 mRNA expression was lower than the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group’s(P<0.01 or <0.05).10.Compared with the normal group,the PI3 K protein expression of other group were increased significantly(P<0.05).Compared with the model group,the PI3 K protein expression of the IGF-1 group,the Neiguan group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group were significantly increased(P<0.01),the LY294002 group’s PI3 K protein expression group was lower than the model group(P<0.01),the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 group’s PI3 K protein expression had no difference with the model group(P>0.05).Compared with the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group,the IGF-1 group’s PI3 K protein expression had no difference(P>0.05),the PI3 K protein expression of the LY294002 group,the Neiguan group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 group were lower than the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group(P<0.01).11.For Akt1 and Akt2 protein,the Akt1 and Akt2 protein expression of the LY294002 group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment + LY294002 group were significantly lower than other groups,the difference was significant(P<0.01);The Akt1 and Akt2 protein expression of the model group,the IGF-1group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group were higher than the normal group,the difference was significant(P<0.01),the Akt1 and Akt2 protein expression of the IGF-1 group and the Electro Acupuncture of Combined Biao and BenAcupoints Treatment group increased most significantly.Compared with the IGF-1 group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group, the Neiguan group’s Akt1 and Akt2 protein expression level decreased,the difference was significant(P<0.01),whereas the IGF-1 group ’ s Akt1 and Akt2 protein expression had no significant difference with the IGF-1group and the Electro Acupuncture of Combined Biao and Ben Acupoints Treatment group(P>0.05).Conclusion:1.The Electro “Acupuncture of Combined Biao and Ben Acupoints”Treatment can improve heart rate(HR), ST segment voltage changes,ease myocardial pathological damage of the chronic myocardial ischemia model rat,reduced ischemic myocardial infarct size,recovery of the myocardial mitochondrial structure and function,improve myocardial ischemia state,play a good myocardial protection role in treatment of chronic myocardial ischemia in rats.2.The “Electro Acupuncture of Combined Biao and Ben Acupoints Treatment” can reduce apoptosis index,and adjustable the expression of Caspase3,Caspase9,Bcl-2,Bax and other apoptosis-related proteins,increased expression of anti-apoptotic proteins,inhibiting the expression of pro-apoptotic protein,play a inhibition role in cardiac apoptosis,thereby protecting the cardiomyocytes.3.Compared with single Neiguan points treatment,The “Electro Acupuncture of Combined Biao and Ben Acupoints treatment” had more advantage in the degree of improvement extent on the above indicators,with points ahead of specimens theory acupoints coordinatingrole, providing strong evidence for the effect of acupoint compatibility.Prompt the “Electro Acupuncture of Combined Biao and Ben Acupoints treatment” had acupoints synergistic effect,provide strong evidence for the effect of acupoint compatibility.4.By the addition LY294002 of PI3 K in the modeling/Akt pathway specific inhibitor,resulting in the HR and ST segment voltage of myocardial ischemia model rat were increased significantly,the HE staining display cardiac pathology injury,myocardial mitochondrial structure damage,myocardial infarct area is more serious than the model group,Akt1, Akt2 mRNA and PI3 K, Bcl-2, Akt1, Akt2 protein expression of inhibition apoptosis levels down,Pro-apoptotic protein:Caspase3,Caspase9,Bax protein expression levels regulated activity;Through PI3K/Akt pathway activator after IGF-1 injection,reverse the above phenomenon.Descriptionthe intracellular P13K/Akt signaling pathways involved in the gene and protein regulation of the “Electro Acupuncture of Combined Biao and Ben Acupoints treatment” in chronic myocardial ischemia protective effect,but PI3 K / Akt pathway whether is an advantage protection path of myocardial ischemia,and whether produce associate role with other pathways,need Experimental studies to further verification in the future. |
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