Protective Effects Of Ginsenoside Rb3on Myocardial Ischemia Reperfusion Injury And Effects Of MMPs At Different Stages Of Myocardial Infarction In Rats

Ischemic heart disease is one of the major diseases of clinical cause of death. Coronary atherosclerosis caused by coronary artery stenosis or occlusion induce decreasing cardiac blood perfusion, leading to reduced oxygen supply of the heart and abnormal myocardial energy metabolism. In recent years, arterial bypass surgery, thrombolytic therapy, percutaneous coronary vessels intervention, cardiopulmonary bypass cardiac surgery and cardiopulmonary cerebral resuscitation (CPR) and other means to establish and promote the application makes the ischemic heart in a short time to regain blood perfusion and restore oxygen supply. However, coronary reperfusion while improving myocardial infarction prognosis, also increased myocardial injury due to reperfusion, arising arrhythmia, and expansion of the infarct size, persistent ventricular systolic dysfunction and other conditions. On the other hand, on the basis of coronary artery disease, prone to coronary blood supply drastically reduced or interrupted, so that the corresponding myocardium will be undering serious and sustained acute ischemia and myocardial infarction. Compensatory changes of the myocardium caused by severe left ventricular remodeling resulting into heart failure.In this study, through the establishment of myocardial ischemia-reperfusion injury (MIRI) model, we will study the effect and mechanism of ginsenoside Rb3(G-Rb3) on myocardial ischemia and reperfusion injury in rats. Meanwhile through the establishment of myocardial infarction model to detect the variations of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase with or without G-Rb3interference at different stages of myocardial infarction and to identify the effect of G-Rb3in anti-ventricular remodeling.1. Protective effect of ginsenoside Rb3on myocardial ischemia reperfusion injury(1) Methods120rats were divided into sham operation group, myocardial ischemia and reperfusion model group, and the G-Rb35,10,20mg·kg-1group randomly. Each group of rats were24and were administered medicines continously for3days. After the last administration, the left anterior coronary descending arteries of rats were ligated to induce myocardial ischem After30min ligation, released the ligature for24h reperfusion. Then10rats from ea group were anesthesied by intraperitoneal injection of300mg'kg-1chloral hydrate, and intubated the left common carotid artery to measure systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate (HR) changes through the RM-6000multi-channel polygraph. Catheters were inserted into the left ventricle from right common carotid artery, through the multi-channel polygraph determination of left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP) and the change of ventricular pressure rate (±dp/dt max).were measured. Then1ml of blood was taken from left common artery, and3.8%sodium citrate was added at1:9ratio as anticoagulant. Under an optical microscope to count the number of platelet, the remaining blood was used rotated in LBY-F5platelet adhesion instrument ball for15min, and then counted the number of platelet again. The platelet adhesion rate (PAR) was caculated. Another3ml of blood was taken and treated with3.8%sodium citrate as mentioned above. The mixture were prepared into the platelet-rich plasma (PRP) and platelet-poor plasma (PPP) respectively. The function of platelet aggregation was measured by LB Y-NJ2platelet aggregation instrument, then platelet aggregation rates (PAG) at1,3and5min and maximum platelet aggregation rate (MPAG) were calculated. At last, rat heart was dissected and washed away residual intracardiac blood with chloride sodium solution, removed blood vessels, fatty tissue and other non-myocardial tissues, then cut the atrium along the coronary sulcus, to weigh the wet left ventricle. Parallel to the atrioventricular groove, left ventricle was cross-cut into4-5pieces, and immersed in NBT phosphate buffer at37℃water bath. After dyeing, ischemic myocardial tissue was cut down and weighed. The myocardial infarct size (MIS) was calculated as the percentage of ischemic myocardium weight by left ventricular wet weight. Another10rats from each group were taken abdominal aorta blood for analyis of serum creatine kinase isoenzyme (CK-MB), lactate dehydrogenase (LDH) using the COBAS-FARA automatic biochemical analyzer; the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were testsed following the kit manufacture's instruction. More blood was taken and treated with anticoagulant, then the plasma was gained by centrifuge for endothelin (ET), angiotensin Ⅱ (AngⅡ), prostacyclin (PGI2) and thromboxane A2contents by radioimmunoassay. At last, rat hearts were dissected.6hearts from each group were used for HE staining and observed under the microscope, and4were used for further electron microscopy measurment. The rest4animals from each group were decapitated, and open the chest, quickly removed the heart, clipping two pieces myocardial infarction ventricle for semi-quantitative reverse transcription-polymerase cha reaction (RT-PCR), to determine the myocardial expressions of oncogene c-fos, c-myc, and c-jun mRNA.(2) Results(1) compared with the MIRI model group, the G-Rb310,20mg· kg-1treatments could significantly increase the levels of HR SBP, DBP, MAP, LVSP, and dp/dtmax, and significantly reduced the value of LVEDP (P<0.05or P<0.01);(2) compared with the MIRI model group, the G-Rb310,20mg· kg-1treatments could significantly reduce the PAR, and ADP-induced PAG at1-,3-, and5min, and MPAG (P<0.05or P<0.01);(3) compared with the MIRI model group, the G-Rb35,10,20mg kg-1treatments could significantly narrow the MIS, and significantly reduce the activities of serum CK-MB and LDH (P<0.05or P <0.01);(4) compared with the MIRI model group, the G-Rb35,10,20mg· kg-1treatments could significantly reduce the serum MDA content, and significantly increased SOD activity (P<0.05or P<0.01);(5) compared with the MIRI model group, the plasma Ang Ⅱ and ET contents could be significantly reduced after G-Rb35,10,20mg· kg-1treatments (P<0.05or P<0.01);(6) compared with the MIRI model group, the G-Rb310,20mg· kg-1could significantly increase the PGI2level in plasma and PGI2/TXA2ratios (p<0.05or P<0.01);(7) compared with the MIRI model group, the G-Rb35,10,20mg· kg-1treatments could relieve the pathological changes of myocardial tissue induced by MIRI under optical microscope and electron microscope obversation;(8) compared with the MIRI model group, the G-Rb320mg kg-1could significantly inhibit the proto-oncogene c-myc, c-fos and c-jun expression in myocardial tissue (P<0.05or P<0.01).2. Effects of Ginsenoside Rb3on MMPs at Different Stages of Myocardial Infarction in Rats(1) Methods400rats were divided into sham operation group, model of myocardial infarction group, positive control group the G-Rb320mg· kg-1group randomly. Each group of rats were100. The Myocardial infarction model was induced by left anterior descending coronary occulusion. Rats were administrated different medicines until the end of the experiment. At the1st,7th,14th,21st day after ligation,10rats from each group were anesthesied by intraperitoneal injection of300mg·kg-1chloral hydrate, and abdominal aorta blood was taken for serium analyis of aspartate aminotransferase (AST), serum creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase (LDH) using the COBAS-FARA automa biochemical analyzer; the levels of transforming growth factor-beta1(TGF-β1), mat metalloproteinase-2(MMP-2), matrix metalloproteinase-9(MMP-9), tissue inhibitor of metalloproteinase factor-1(of TIMP-1) and metalloproteinase inhibitor-3(TIMP-3) in serum were measured by ELISA method; the changes of tumor necrosis factor-a(TNF-a)and interleukin-1β (IL-1β) were measured by radioimmunoassay. At the1st and21st day after ligation,10rats from each group were taken hearts and parallel to the atrioventricular groove, left ventricle was cross-cut into4~5pieces, and immersed in NBT phosphate buffer at37℃water bath as mentioned above to calculate the myocardial infarct size (MIS). At the7th,14th and28th day after ligation,20rats from each group were taken hearts.4hearts from each group were used for HE staining and observed the pathological changes under the microscope; and4were used for further electron microscopy measurment with uranyl acetate and lead citrate double staining;4rats from each group were sacrificed for immunohistochemical analysis of the change of type I and III collagens;4hearts from each group were taken for total protein extraction to measure the protein levels of MMP-2, MMP-9, TIMP-1and TIMP-3; the rest4animals from each group were decapitated, and the heart tissue was used for RNA extraction to determine the myocardial expressions of MMP-2, MMP-9, TIMP-1and TIMP-3.(2) ResultsCompared with the model of myocardial infarction group, after G-Rb3administration:(1) the serum CPK, LDH and AST activities were significantly decreased at the1st,7th,14th,21st and28th day after ligation (P<0.05or P<O.01);(2) the contents of TNF-a, IL-1β and TGF-β1in serum were significantly lower than the myocardial infarction model rats at the1st,7th,14th,21st and28th day after ligation (P<0.05or P<O.01);(3) MMP-2and MMP-9concentrations in serum were significantly lower than the model of myocardial infarction group rats, but TIMP-1and TIMP-3contents appeared obviously restoration compared with the model group at the1st,7th,14th,21st and28th day after ligation (P<0.05or P<O.01);(4) rat myocardial infarct size was significantly reduced at1st and21st day after ligation(P<0.05or P<O.01);(5) G-Rb320mg kg-1could significantly reduce the abnormal form of pathology under the light microscope observation and ultrastructure changes in electron microscopy of myocardial tissue at the7th,14th and28th day after ligation;(6) G-Rb320mg.kg-1could reduce the type I and III collagen fibers proliferateion at the7th,14th and28th day after ligation;(7) MMP-2and MMP-9protein expression was significantly increased, TIMP and TIMP-3protein expression was significantly reduced of the model of myocard infarction group at the7th,1th and28th day after ligation, but there was no significant changes after G-Rb320mg.kg-1treatment;(8) the RNA levels of MMP-2and MMP-9after ligation were significantly increased, and TIMP-1and TIMP-3mRNA levels were significantly decreased; G-Rb320mg.kg-1treatment reversed these tendency and had siginificant differences compared with model group (P<0.05or P<0.01).Conclusions:1. G-Rb3can significantly improve hemodynamic parameters of myocardial ischemia and reperfusion injury in rats, decrease MIS, lower the activities of CK-MB and LDH in serum, and reduce the pathological changes of myocardial tissue. This proven that G-Rb3may have protective effects on myocardial ischemia reperfusion injury.2. The mechanism of protective effects of G-Rb3on myocardial ischemia and reperfusion injury may be related to its antioxidant effects, reducing Ang II and ET levels, improve PGI2/TXA2ratio, inhibition of platelet adhesion and aggregation, and inhibition of myocardial tissue proto-oncogene c-myc and c-fos and c-jun expression.3. CPK and AST activities were significantly increased at14th day after ligation, and it maintained a steady level at21or28days; LDH activity was significantly increased at7th day after ligation, and it maintained a steady level at14,21or28days. G-Rb3treatment could significantly reduce myocardial infarction size at different stages of MIS, also reduced serum CPK, LDH and AST activities, and reduced the pathological changes of myocardial tissue.4. TNF-a and TGF-β1levels in serum were significantly increased at7th day after ligation, and it maintained a steady level at14,21or28days; IL-1β level was significantly increased at14th day after ligation, and it maintained a steady level at21or28days. G-Rb3treatment could significantly reduce the levels of TNF-a, TGF-β1and IL-1β at different stages of MIS5. The1st day after ligation, the activity of MMP-2and MMP-9in serum showed the increasing tendency, and it arrived the highest level at7-14days; meanwhile, the TIMP-1level decresed at14days, and TIMP-3decreased at7days after ligation. G-Rb3treatment could significantly reduce the levels of TNF-a, TGF-β1and IL-1β at different stages of MIS. G-Rb3could significantly reduce MMP-2and MMP-9levels and increase TIMP-1and TIMP-3contents in serum at different stages of MIS.6. At the7th,14th and28th day after ligation, type I and III collagen fibers proliferatei was significantly increased; both the protein and mRNA levels were significantly increase TIMP-1and TIMP-3protein and mRNA expressions were significantly decrease. G-Rb320mg kg-1treatment could significantly reverse the parameters changes after myocardiol infarction.In summary, G-Rb3on one hand, by reducing TNF-alpha, IL-1β, TGF-β1in content, on the one hand, by regulating TIMP-1and TIMP-3levels, thus affect matrix metalloproteinase expression at different stages of myocardial infarction, thereby inhibiting myocardial type Ⅰ and Ⅲ collagen fibers, and prevention of ventricular remodeling after myocardial infarction occurred.