| Background Cardiovascular disease is the leading cause of death in the developed countries and even affects up to 80 million people in the United States. Myocardial ischemia/reperfusion (I/R) injury contributes to adverse cardiovascular outcomes after myocardial ischemia, cardiac surgery or circulatory arrest. The molecular mechanisms underlying myocardial I/R injury, however are complex.Some scholars at home and abroad have demonstrated that myocardial ischemia can be effectively treated via electroacupuncture pretreatment. Electroacupuncture therapy is nervous electrical stimulation therapy combined with traditional Chinese medicine acupuncture, which on the basis of needling acupoints of traditional Chinese medicine, using pulse generator on the needle at human bioelectricity to trace current wave, on the basis of the original needle stimulates, combined with different amplitude continuous wave, discontinuous wave to stimulate acupuncture points, through sustained, stable and accurate for the body, controllable stimulation to generate electricity physiological effect, electroacupuncture pretreatment as an effective method of myocardial protection, through activated endogenous protective mechanism to mobilize the body’s potential, improve the body’s ability to adapt to the stress response. The operation of electroacupuncture pretreatment is convenient, and can avoid many adverse reactions of drug pretreatment, and can be widely applied to patients, which clinical application prospect is wide.Although clinical studies have convincingly demonstrated that myocardial ischemia can be effectively treated via acupuncture at the single Neiguan point, the mechanisms in charge of the cardioprotective effect through acupuncture therapy remains undefined.MicroRNA, an endogenous small nucleotide segment, has been detected more than 700 kinds. About 30% of the human genome is regulated by the microRNA. MiRNA-214 plays an important role in the regulation of many types of diseases through a variety of target genes. Accumulating evidence suggests that miRNA-214 has an active role in myocardial injury and immunity. MiRNA-214 attends the procedure of myocardial ischemia/reperfusion by inhibition of cell apoptosis, PYEN and HIF1AN mechanism. It may become a new novel targeted marker in the prevention and treatment of myocardial ischemia/reperfusion injury, and miRNA-214 will provide ideas and methods for myocardial ischemia/reperfusion injury disease. The recent studies have revealed that microRNAs (miRNAs) play important roles in various aspects of cardiac function involved in the regulation of cardiovascular physiological and pathological processes, including cardiac hypertrophy, cardiac fibrosis, cardiac apoptosis and angiogenesis. Using a rat model of myocardial I/R, we observed that electroacupuncture pretreatment could reduce the infarct size, reverse cardiac function injury as well as decrease serum lactate dehydrogenase (LDH) and creatine kinase (CK) activities, which were consistent with the previous in vivo studies, emphasized that electroacupuncture has a cardioprotective effect against myocardial ischema. However, the role of miRNAs in the cardioprotection by electroacupuncture pretreatment on myocardial I/R injury remains unknown. Since miRNA-214 is a sensitive marker of a variety of cardiac stresses, its expression is up-regulated in cardiac hypertrophy, myocardial infarction and heart failure. The overload of intracellular free Ca2+ concentration ([Ca2+]i) is an important cause of cell injury. It is well known that following myocardial I/R injury, intracellular Ca2+ overload occurs in cardiomyocytes, causing activation of sodium/calcium exchanger 1(NCX1), protein and downstream effectors of Ca2+ handling, including BCL2-like 11(BIM), calmodulin-depandent protein kinaseⅡδ(CaMKⅡδ) and Cyclophilin D(CypD), and leads to cell death ultimately. Also, acupuncture-serum could decreases Ca2+ content in cultured rat myocardial cells,and even electroacupuncture pretreatment was involved in maintaining cardiomyocyte Ca2+ homeostasis. Several studies have demonstrated that electroacupuncture pretreatment was involved in maintaining cardiomyocyte Ca2+ homeostasis, and this mechanism was important to the protective effect of electroacupuncture on myocardial ischemia/reperfusion injury.Recently, miRNA-214 has been shown to protect against I/R-induced cardiac injury in mice by attenuating Ca2+ overload induced cell death. In addition, a recent study found that electroacupuncture pretreatment can mediate Ca2+ and relative protein levels of NCX1, BIM, CaMKⅡδ and CypD of normal H9c2 cells of rat.To further explore the mechanisms underlying the cardioprotective effect of electroacupuncture pretreatment, we focused on miRNAs which exert effects on cardioprotection. Since miRNA-214 is a sensitive marker of a variety of cardiac stresses, its expression is up-regulated in cardiac hypertrophy, myocardial infarction and heart failure. Recently, Aurora et al suggested that miRNA-214 is up-regulated in I/R-induced cardiac injury in mice and it plays a protective role against I/R injury in their study. As a result, we assume that the miRNA-214 mediated alleviating myocardial I/R injury in electroacupuncture pretreatment, and System to test whether miRNA-214 was involved in cardioprotection mechanism by the electroacupuncture pretreatment in vivo and in vitro.Chapter 1 cardioprotection of electroacupuncture pretreatment on myocardial ischemia/reperfusion injury in vivo and in vitroObjective To evaluate the cardioprotection of electroacupuncture pretreatment on myocardial ischemia/reperfusion injury in vivo and in vitro.Methods 24 male Sprague-Dawley rats were divided into 4 groups randomly (n=6):control group (Sham group), Sham+electric acupuncture group (Sham+EA) group, ischemia/reperfusion (I/R group), ischemia/reperfusion+electric acupuncture group (I/R+EA group). Rats in group Sham+EA and I/R+EA received electroacupuncture stimulating 30 min per day for 3 days before myocardial ischemia/reperfusion injury. Rats in group I/R and I/R+EA received left anterior descending (LAD) occlusion for 40min and reperfusion for 180 min. And Rats in group Sham received the same procedures without occlusion of the LAD.H9c2 cells were divided into 4 groups randomly (n=3):the Control group, the Control+electric acupuncture group (Control+EA group), oxygen-sugar deprivation (OGD), oxygen-sugar deprived of oxygen+electric acupuncture group (OGD+EA group). Cells in group Control+EA and OGD+EA received received electroacupuncture treatment daily for 30 min and continued for 10 days. Cells in group OGD and OGD+EA received oxygen-glucose deprivation.The rats were anesthetized by intraperitoneal injection of 3.5% chloral hydrate (50 ml/kg). Then the animals were placed in the supine position and intubated, ventilated artificially with a respirator with a tidal volume of 20 ml/kg body weight and 50 breaths per minute. Electrocardiography electrodes were connected to the limbs through small needles inserted subcutaneously. Following the skin incision, the hearts were exposed through a left thoracotomy in the fourth intercostal space. The LAD coronary artery was ligated with 6-0 silk suture using a snare occluder. Ischemia was confirmed by visual observation and continuous ECG monitoring. Evidence for a successful intervention was a cyanotic left anterior ventricular wall and local wall distension, as well as elevated ST segments and peak T waves on the electrocardiogram. After completion of 40 min of occlusion, the coronary artery was reperfused by releasing the knot. Following reperfusion for 180 min, the rat hearts were harvested. Sham surgery animals underwent the same procedures without occlusion of the LAD.The cells were maintained as monolayers in high glucose (25mmol/L) Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum, at 37℃ in a humidified atmosphere with 5% CO2. Medium was changed every three days. Osmotic control cells (to account for medium hyper-osmolarity) were exposed to mannitol (24.5mmol/L) in normal medium containing glucose (5.5mmol/L). For OGD-treated cells, the complete growth medium was replaced by DMEM without glucose, and cells were incubated at 37℃ with 5% CO2 and 95% N2 (v/v) for 10 h. After hypoxia, the culture medium was removed, replaced by fresh high glucose DMEM, supplemented with 10% FBS and cells were maintained in a regular 5% CO2 incubator.Two stainless steel needles were inserted into "Neiguan" acupoints which were located in the interosseal muscles between the radius and the ulna of the distal medial thoracic limb at the level of 3 mm superior to the wrist joint in rats. These two needles were connected to positive and negative poles of an acupuncture apparatus. Application of electroacupuncture was continued for 30 min a day and for three consecutive days before the myocardial I/R experiment. The stimulatory frequency of electroacupuncture was 4/20 Hz,0.5 ms duration and at an intensity of 1 mA, just strong enough to elicit a slight twitch of the foot. Electroacupuncture stimulation was applied to cells. The frequency was 50Hz and the duration was 0.5ms. Cells received electroacupuncture treatment daily for 30 min and continued for 10 days.The indices of the cardiac function, including heart rate(HR), mean arterial pressure(MAP), left ventricular pressure(LVSP), maximal rate for left ventricular pressure rising and declining (±p/dt) max in the groups of sham, sham+EA, I/R and I/R+EA, as well as myocardial infarct size were measured and recorded. LDH and CK activities as well as cell apoptosis rate in vivo and in vitro were measured.Results It has been shown that all the indices of the cardiac function were not significantly different between the sham and the sham+EA group. Compared with the sham group, all the indices of the cardiac function were decreased significantly in I/R group (P<0.05). However, electroacupuncture pretreatment could reverse this cardiac function injury. I/R+EA group showed that all the indices of the cardiac function were increased compared with I/R group (P<0.05). Myocardial infarct size of I/R+EA group was significant decreased than I/R group (P<0.05). Compared with the sham group, the activity of LDH was not altered in the sham+EA group, but significantly increased in the serum of I/R group (P< 0.01), the activity of CK was not altered in the sham+EA group, but significantly increased in the serum of I/R group (P<0.05). The activity of LDH was significantly increased in the serum of OGD group (P<0.01), the activity of CK was not altered in the OGD+EA group, but significantly increased in the serum of OGD group (P< 0.05). The cell apoptosis rate of OGD group(7.2+1.5%) was higher than OGD+EA group(4.6+0.5%) significantly, P<0.05.Conclusions Electroacupuncture pretreatment can protect the heart against myocardial ischemia/reperfusion injury in vivo and in vitro.Chapter 2 miRNA-214 was involved in cardioprotection by the electroacupuncture pretreatment on myocardial ischemia/ reperfusion injury in vivo and in vitroObjective To evaluate whether miRNA-214 and calcium overload mechanisms involved in the cardioprotection by electroacupuncture pretreatment on myocardial ischemia/reperfusion injury in vivo and in vitro.Methods 24 male Sprague-Dawley rats were divided into 4 groups randomly (n=6):control group (Sham group), Sham+electric acupuncture group (Sham+EA) group, ischemia/reperfusion (I/R group), ischemia/reperfusion+electric acupuncture group (I/R+EA group). Rats in group Sham+EA and I/R+EA received electroacupuncture stimulating 30 min per day for 3 days before myocardial ischemia/reperfusion injury. Rats in group I/R and I/R+EA received left anterior descending (LAD) occlusion for 40min and reperfusion for 180 min. And Rats in group Sham received the same procedures without occlusion of the LAD.H9c2 cells were divided into 4 groups randomly (n=3):the Control group, the Control+electric acupuncture group (Control+EA group), oxygen-sugar deprivation (OGD), oxygen-sugar deprived of oxygen+electric acupuncture group (OGD+EA group). Cells in group Control+EA and OGD+EA received received electroacupuncture treatment daily for 30 min and continued for 10 days. Cells in group OGD and OGD+EA received oxygen-glucose deprivation.The rats were anesthetized by intraperitoneal injection of 3.5% chloral hydrate (50 ml/kg). Then the animals were placed in the supine position and intubated, ventilated artificially with a respirator with a tidal volume of 20 ml/kg body weight and 50 breaths per minute. Electrocardiography electrodes were connected to the limbs through small needles inserted subcutaneously. Following the skin incision, the hearts were exposed through a left thoracotomy in the fourth intercostal space. The left anterior descending coronary artery was ligated with 6-0 silk suture using a snare occluder. Ischemia was confirmed by visual observation and continuous ECG monitoring. Evidence for a successful intervention was a cyanotic left anterior ventricular wall and local wall distension, as well as elevated ST segments and peak T waves on the electrocardiogram. After completion of 40 min of occlusion, the coronary artery was reperfused by releasing the knot. Following reperfusion for 180 min, the rat hearts were harvested. Sham surgery animals underwent the same procedures without occlusion of the LAD.The cells were maintained as monolayers in high glucose (25mmol/L) Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum, at 37℃ in a humidified atmosphere with 5% CO2. Medium was changed every three days. Osmotic control cells (to account for medium hyper-osmolarity) were exposed to mannitol (24.5mmol/L) in normal medium containing glucose (5.5mmol/L). For oxygen-glucose deprivation -treated cells, the complete growth medium was replaced by DMEM without glucose, and cells were incubated at 37℃ with 5% CO2 and 95% N2 (v/v) for 10 h. After hypoxia, the culture medium was removed, replaced by fresh high glucose DMEM, supplemented with 10% FBS and cells were maintained in a regular 5% CO2 incubator.Two stainless steel needles were inserted into "Neiguan" acupoints which were located in the interosseal muscles between the radius and the ulna of the distal medial thoracic limb at the level of 3 mm superior to the wrist joint in rats. These two needles were connected to positive and negative poles of an acupuncture apparatus. Application of electroacupuncture was continued for 30 min a day and for three consecutive days before the myocardial I/R experiment. The stimulatory frequency of electroacupuncture was 4/20 Hz,0.5 ms duration and at an intensity of 1 mA, just strong enough to elicit a slight twitch of the foot. Electroacupuncture stimulation was applied to cells. The frequency was 50Hz and the duration was 0.5ms. Cells received electroacupuncture treatment daily for 30 min and continued for 10 days.examined expression of miRNA-214, intracellular free Ca2+ concentration ([Ca2+]i) as well as the relative protein levels of sodium/calcium exchanger 1(NCX1), BCL2-like 11(BIM), calmodulin-depandent protein kinaseⅡδ(CaMKⅡδ) and Cyclophilin D(CypD).Results The expression of miR-214 in the I/R group was higher than sham group (P<0.01). Electroacupuncture pretreatment increased miR-214 expression of sham+EA group and the I/R+EA group. Compared with the normal cardiac muscle cells of control group, expression of miR-214 were raised by electroacupuncture pretreatment or oxygen-glucose deprivation (P<0.01). In addition, the miR-214 expression of OGD+EA group was higher than OGD group(P<0.01). The [Ca2+]i in OGD cells was significantly increased over control (P<0.01). The relative protein levels of NCX1, BIM, CaMKⅡδ and CypD were also increased in the OGD group compared with the control group. And those relative protein levels in OGD+EA group were significantly lower than in OGD group, P<0.05 or P<0.01.Conclusions Electroacupuncture pretreatment can promote the expression of miRNA-214 and rescue the down-regulating Ca2+ and elevated protein levels of NCX1, BIM, CaMKⅡδ and CypD to protect the heart against myocardial ischemia/ reperfusion injury in vivo and in vitro.Chapter 3 miRNA-214/Ca2+ signal pathway mechanisms in electroacupuncture pretreatment on myocardial ischemia/ reperfusion injuryObjective To study the miRNA-214/Ca2+ signal pathway and and molecular mechanisms in electroacupuncture pretreatment on myocardial ischemia/reperfusion injury through miRNA-214 expression and gene silencing model.Methods H9c2 cells were divided into 4 groups randomly (n=3):the Shaml group, oxygen deprivation (OGD) group, OGD+EA+miRNA-214 silence group(miR-NC group), OGD+EA+miRNA-214 expression group(miR-214 group). Cells in group miR-NC received slow virus with the miRNA-214 simulation transfection 10 days before OGD. Cells in group miR-214 received slow virus with the miRNA-214 transfection 10 days before OGD. Cells in group miR-NC and miRNA-214 received e received electroacupuncture treatment daily for 30 min and continued for 10 days. Cells in group OGD, miR-NC and miRNA-214 received oxygen-glucose deprivation.The H9c2 cells were transfected with the miR-NC, or miR-214, using lipofectamine 2000, according to the manufacturer’s instructions. Subsequently, cells were washed and maintained in the medium.The cells were maintained as monolayers in high glucose (25mmol/L) Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum, at 37℃ in a humidified atmosphere with 5% CO2. Medium was changed every three days. Osmotic control cells (to account for medium hyper-osmolarity) were exposed to mannitol (24.5mmol/L) in normal medium containing glucose (5.5mmol/L). For oxygen-glucose deprivation (OGD)-treated cells, the complete growth medium was replaced by DMEM without glucose, and cells were incubated at 37℃ with 5% CO2 and 95% N2 (v/v) for 10 h. After hypoxia, the culture medium was removed, replaced by fresh high glucose DMEM, supplemented with 10% FBS and cells were maintained in a regular 5% CO2 incubator.Electroacupuncture stimulation was applied to cells. The frequency was 50Hz and the duration was 0.5ms. Cells received electroacupuncture treatment daily for 30 min and continued for 10 days.Lactate dehydrogenase (LDH) and creatine kinase (CK) activities as well as cell apoptosis rate were measured and recorded, examined expression of miRNA-214, intracellular free Ca2+ concentration ([Ca2+]i) as well as the relative protein levels of sodium/calcium exchanger 1(NCX1), BCL2-like 11(BIM), calmodulin-depandent protein kinaseⅡδ(CaMKⅡδ) and Cyclophilin D(CypD).Results The apoptosis rates of sham group, OGD group, miR-NC group and miR-214 group were 9.86±3.98%,15.86±2.26%,46.99±4.41%,17.35±1.34% separately. Cell apoptosis is not obvious in the Sham group, the OGD group has certain apoptosis phenomenon, cell apoptosis of miR-214 group was slightly lower than OGD group, and there are obvious cell apoptosis in miR-NC group. The activity of LDH and CK of miR-214 group were significantly lower than miR-NC group(P<0.01). The expression level of miRNA-214 mRNA in miR-214 group was up-regulated about 3-fold over miR-NC group (P<0.01). In addition, the miRNA-214 mimic group showed a significant decrease in [Ca2+]i compared with the miR-NC group (P<0.05). The relative protein levels of NCX1, BIM, CaMKII8 and CypD were also significantly decreased in miRNA-214 group compared with the miR-NC group(P<0.01).Conclusions miRNA-214/Ca2+ signal pathway in electroacupuncture pretreatmen contributes to cardioprotection on myocardial ischemia/reperfusion injury.The main conclusion of the research1. Electroacupuncture pretreatment can protect the heart against myocardial ischemia/reperfusion injury in vivo and in vitro.2. Electroacupuncture pretreatment can promote the expression of miRNA-214 and rescue the down-regulating Ca2+ and elevated protein levels of NCX1, BIM, CaMKⅡδ and CypD to protect the heart against myocardial ischemia/ reperfusion injury in vivo and in vitro.3. miRNA-214/Ca2+ signal pathway in electroacupuncture pretreatmen contributes to cardioprotection on myocardial ischemia/reperfusion injury. |
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