Effects Of IPQDS On Experimental Myocardial Ischemia And Prevention Of AM Gene On Myocardial Hypertrophic Rats

Part 1 Effects of IPQDS on experimentaL myocardial ischemia Background:Myocardial ischemia, which is caused by coronary atherosclerosis in most casses, is a clinical state with coronary blood flow decrease, myocardial oxygen supply deficiency and less removal of metabolic products.Myocardial ischemia is a higher incidence cardiovascular disease, its mortality is high when acute attack. Present therapeutic measure, such as thrombolylic drug treatment, interventional therapy and surgical treatment, have get good clinical efficacy in usual cases. But after applying them, the myocardial ischemia reperfusion injury which easily cause arrhythmia or heart failure have become new disadvantages. Therefore, looking for safe and effective drug for the prevention and treatment of ischemic heart disease is one of the important issues.Panax quinquefolius L. is one of the special local products of the east region of Changbai Mountain. Its main root and fibrous root are recorded in China Pharmacopeia, while its stem and leaves are not sufficiently used. Therefore, exploitation the overground part of Panax quinquefolius L. is meaningful to systematically utilize the medical resource of Panax quinquefolius L. We have been studying the overground part of Panax quinquefolius L. and confirmed with systemic reseach in chemistry, pharmacology and toxicology that panaxsaponins derived from stem and leaves of Panax quinquefolius L. (PQS) are main effective parts. Panaxsaponin-Rb2,-Rb3,-Rd,-Re,-Rg1,-F2 and para-panaxsaponin-F11,-RT5 were separated from PQS. Panax quinquefolium 20-sprotopanaxdiolsaponins (PQDS) were separated by us from PQS and confirmed that PQDS mainly contain panaxsaponin-Rb2,-Rb3, -Rd and -Rc and they have not haemolysis. Therefore, by means of raw material of PQDS derived from PQS, we reseached and exploited IPQDS which a new drug of traditional Chinese drug about treatment of acute myocardial ischemia diseases with patent technique and got the supporting of country 863 plan.In this paper, after the establishment of rat and dog myocardial ischima model, we analyse protective effects of PQDS on experimental myocardial ischemia and its mechanism on morphology, enzymes, blood biochemistry, epicar dial ECG, cardiac function, hemodynamics, myocardial oxygen metabolism and molecular levels to provide pharmaco dynamic basis for the development of five new Chinese medicine to the treatment of acute myocardial ischima.Methods:1. Establish a rat model of myocardial ischemia reperfusion injury and measure the following indicators:(1)Determinate serum the activities of aspartate aminotransferase enzyme (AST), creatine phosphate kinase (CK) and lactate dehydro genase (LDH) By COBAS-FARA automatic analyzer ;(2)Draw blood from abdominal aorta, detect serum the content of nitric oxide (NO) and malondial dehyde (MDA), activities of superoxide dismutase (SOD) and glutath ione peroxidase (GSH-Px) by kit;(3)Draw 1ml Blood from the abdominal aorta, anticoagulate with 3.8% sodium citrate by ratio of 1:9, add the blood into platelet adhesion LBY-F5 Miriam ball, count the number of platelets after adhesion in the light microscope, calculate platelet adhesion rate (PAR) ;(4)Draw 3mL blood from the abdominal aorta, anticoagulate with 3.8% sodium citrate by ratio of 1:9, utilize LBY-NJ2 to measure platelet aggregation rate(PAG)and maximum platelet aggregation rate (MPAG) ;(5)Stain organizations with chlorination NBT (NB-T) to calculate myocardial infarct size (MIS) with the ratio of myocardial ischemia and ventricular wet weight and myocardial histopathology was observed under light and electron microscope;(6)The levels of plasma endothelin (ET), angiotensinⅡ(AngⅡ) and PGI2 and TXA2 were detected by Radioimmunoassay;(7)mRNA expression of cardiac proto-oncogene c-fos, c-myc and c-jun were detected by RT-PCR.2. Establish canine myocardial infarction model, determinate the following indicators:(1)Separate the right common carotid artery, connecte pressure transducer after intubation, measure systolic blood pressure (SBP) and diastolic blood pressure (DBP) by carrier amplifiers (AP-601G); (2)Separate the left femoral artery, insert the catheter to the left ventricle, connecte carrier amplifier (AP-601G) to detect left ventricular pressure (LVP), left ventricular end diastolic pressure (LVEDP) and the maximum change rate of left ventricular pressure (±dp/dtmax);(3)Separate the aortic root, place the suitable electromagne tic flowmeter probe, measure cardiac output (CO) and compute cardiac index(CI) ;(4)Separate LCX, place the suitable diameter electromagnetic flowmeter probe to measure coronary flow (CBF) and compute myocardial blood flow (MBF) and coronary vascular resistance (CVR) ;(5)Connect ECG electrodes, recordⅡ-lead ECG and heart rate (HR) by AB-601 bioel- ectrical amplifier ;(6)Map the epicardial electrocardiogram in normal, the edge of infarction zone and the central area of infarction by wet-type multi-point adsorption ;(7)Draw blood from the left femoral artery at the different time, measure the content of serum free fatty acid (FFA) with one abstraction colorimetry ;(8)Draw blood from the coronary sinus and the left femoral artery at the same time, measure arterial and venous blood oxygen by the CORNIHG 178 blood gas analyzer, calculate myocardial oxygen consumption (COC), myocardial oxygen utilization (MOUR) and myocardial oxygen consumption index (MOCI) ;(9)Draw Blood from the femoral artery, determinate activities of serum aspartate aminotransferase enzyme (AST), creatine phosphor kinase (CK) and lactate dehydrogenase (LDH) by COBAS-FAARA automatic biochemical analyzer ;(10)Blood from the femoral artery, anticoagulate with heparin, utilize LBY-N6A to measure viscosity of blood and plasma ;(11)Stain tissues with chlorination NBT (NB-T) to calculate myocardial infarct size (MIS) with the ratio of myocardial ischemia and ventricular wet weight.Results(1)IPQDS have significant protective effects on myocardial ischemia reperfusion injury in rats and acute myocardial infarction in dogs, which relate to l as the extent of myocardial ischemia in dogs and ischemic area,and can alleviate the level of the acute myocardial ischemic electrocardiogram and decrease activities of serum CK, LDH and AST ; (2)IPQDS can significantly reduce the content of serum MDA, increase activities of serum SOD and GSH-Px in myocardial ischemia reperfusion injury rats and acute myocardial infarction dogs ;(3)IPQDS can significantly reduce PAR, PAG and MPAG in myocardial ischemia reperfusion injury rats,as well as decrease the viscosity of blood and plasma in acute myocardial infarction dogs ;(4)IPQDS can significantly reduce the level of FFA in myocardial infarction dogs ;(5)IPQDS can significantly reduce levels of serum NO, plasma ET, AngⅡand TXA2, increase plasma levels of PGI2 and PGI2/TXA2 ratio in rats with myocardial ischemia reperfusion injury ;(6)IPQDS could inhibit the expression of proto-nocogene c-myc,c-fos and c-jun after myocardial ischemia reperfusion injury ;(7)IPQDS can significantly reduce oxygen utilization, reduce oxygen consumption in dogs with acute myocardial infarction ;(8)IPQDS can significantly prevent blood pressure (SBP, DBP and MAP) from decreasing, markly increase MBF, LVP,±dp/dtmax, CO and CI, and decrease LVEDP ;ConclusionIPQDS have protective effects on myocardial ischemia reperfusion injury in rats and acute myocardial infaction in dogs, which maybe relate to elevating the activities of endogenous antioxidase,decreasing function of platelet adhesion and aggregation,improving metabolic disorder of myocardial FFA, keeping physiologic equilibrium of vasoactive substance (NO/ET and PGI2/TXA2), reducing cardiac work,decreasing myocardial oxygen consumption and improving heart diastolic and systolic funtion, as well as apposing pump failure under myocardial ischemia,et al, which indicate protective effects of IPQDS on myocardial ischemia should be the result from many synthetic path action. In addition, IPQDS could inhibit the expression of proto-nocogene c-myc,c-fos and c-jun after myocardial ischemia reperfusion injury and significantly decrease levels of plasma ET and AngⅡ,which indicate it maybe prevent and treat ventricular remodelling after acute myocardial ischima. Part 2 Prevention of Ad.CMV-AM on myocardial hypertrophic ratsBackground:AS one major type of cardiac hypertrophy, Left ventricular hypertrophy (LVH) is a compen-satory response to increased cardiac workload and is an independent risk factor for heart failure, myocardial infarction and sudden cardiac death. Prevention of underlying LVH is a major goal in the management of patients with chronic hypertension and heart failure and understanding its mechanistic basis is one of the most important tasks facing medicine and science.Chronic administration of the pharmacological inhibitor of NOS, (L-NAME) into normotensive rats, results in hypertension accompanied by myocardial remodelling, cardiac ischemia and necrosis, and mechanical dysfunction. Reports of prevention or regression of increased LV mass by ACE inhibitors and Ca2+ channel antagonists but not by blood pressure reduction per se indicate that NOS inhibition may have direct consequences for the myocardium.Many evidences support a direct role of Adrenomedullin (AM) produced locally within the heart, in the attenuation of cardiac growth/remodelling and protection against ischemia-reperfusion injury. However, the necessity for intravenous infusion may limit the therapeutic potential of this peptide. Such limitations could conceivably be overcome by delivery of the AM gene. Adenovirus Ad.CMV-AM, in which human AM cDNA is under the control of cytomegalovirus (CMV) promoter, will be administered to Sprague-Dawley rats treated with the inhibitor of nitric oxide synthase, L-NAME (35mg/kg/day in drinking water), which normally exhibit increased oxidative stress, hypertension, left ventricular hypertrophy and myocardial ischemia. Ad.Null injected rats age-matched SD rats given L-NAME (35mg/kg/day in drinking water) and untreated SD rats will serve as controls for treatment, disease and normal ageing, respectively. We measured SBP,parameters of hypertrophy,indicators of oxidative stress in cardiomyocytes and mRNA expression of endogenous AM and the foreign gene. These findings should provide a basis for ethical approval for small pilot studies to be undertaken to assess the feasibility of administration of such a treatment to patients with clinically significant abnormal thickening of heart muscle, in order to reverse this structural change and thereby reduce these patients'cardiovascular risk. Hope to attain the objectives: obtain a supply of Ad.CMV-AM and knowledge of its use for in vivo studies; ascertain the feasibility of delivery of the human AM gene into a novel experimental model system; investigate if increased levels of AM attenuate development or regress established ventricular of AM attenuate development or regress established ventricular remodeling, oxidative stress and ischemia; provide the rationale for pilot clinical studies.1. Large scale production,purification and amplication, as well as titer detection for recombinant Adenovirus.(1)Culture, passage HKE293T cells, furthermore culture the cells in large scale.(2)Amplication of recombinant Adenovirus in cultured HEK293T via infecting the cells with virus at a MOI of 10.(3)Generate cesium chloride ( CsCl ) gradient, spin the virus solution by Optima L-90K ultra low temperature high speed centrifugation to purify and recombinant adenovirus at two steps. Determinate the titer of purified virus by VP and TCID50.2. Ad.CMV-GFP transfect cardiomyocyte in vivo.(1)Ad.CMV-GFP was administered to SD rats by tail intravenous injection.(2) Isolation of ventricular cardiomyocytes was taken by Perfusion of excised hearts with a solution of collagenase in calcium-free Krebs Ringer solution using a Langendorff perfusion apparatus.(3)Observe the infected cardiomyocyte using Axiovert IM35 phase-contrast invert microscope and take pictures using WV BL600 camera.3. Establish cardiomyocyte hypertrophy Model, determinate the following parameters:(1)SD rats at 8 weeks will receive a single intravenous injection of Ad.CMV-AM at a dose of 2x1010 plaque-forming units/rat via the tail vain at 4-weekly intervals concurrently with L-NAME (35mg/kg/day in drinking water) for 8 weeks prior to isolation of left, and for comparison right, ventricular cardiomyocytes at 16 weeks.(2)Isolate ventricular cardiomyocytes by the same way as the second experiment.(3)Measure the systolic blood pressure (SBP) at weekly intervals in each animal during its lifetime by tail cuff sphygmomanometry .(4)Weigh the heart mass before perfusion and calculate the HW/BW ratio.(5)Cell was visualised using an inverted phase contrast microscope and width(μm) was detected.(6)Oxidative status of membrane proteins was assess via OxyblotProtein Oxidation Detection Kit and ECL Advance Western Blotting Detection Kit, and quantified by densitometry, compared to the signal intensity of the molecular weight protein standards provided.(7)Total RNA will be extracted from cardiomyocytes by standard acid guanidinium thiocyanate-phenol-chloroform extraction. Genes expression of c-fos ANP, BNP, sk-α-actin, NADPH oxidase, SOD3, GPx, human-AM and rat-AM were all performed by real-time RT-PCR: reported sequences for each gene will be used to design on Primer Express software, rat and human specific primers adapted to RT-PCR conditions, synthesised by Invitrogen. RT-PCR will be performed in duplicate using Roche FastStart Universal SYBR Green Master (Rox) in a 2:1 reaction, and an ABI Prism Sequence Detector (PE Applied Biosystems). Analysis will be performed using ABI 7000 Prism software and normalised to GAPDH.(8)Detect the expression of exogenous AM by imunofluorescence technique.Results(1)Adequate level of titer of purified recombinant Adenovirus were obtained.(2)Greenfluorescent was visible in infected cardiomyocyte using Ad.CMV-GFP weekly after treatment.(3)SBP was Elevated, Ad.CMV-AM can not prevent this effect.(4)Ad.CMV-AM did not prevent the increase of heart weight and HW/BW ratio under current condition, indicate not to prevent cardiac hypertrophy at organ level.(5)Width of cardiomyocyte was not widened too much with the effect of Ad.CMV-AM, indicate not to prevent cardiac hypertrophy at cell level.(6)Ad.CMV-AM did not appear preventative effect mRNA expression of hypertrophic genes, sk-α-actin and promote increase of ANP level.(7)Ad.CMV-AM did not affect elevated mRNA expression of NADPH oxidase and increased expression of SOD3 and GPx mRNA.(8)Ad.CMV-AM seems not to affect increased oxidation status of cardiomyocyte membrane protein.(9)Exogenous AM seems no expressed and Ad.CMV-AM seem not to promote mRNA expression of endogenous AM.(10)Peptide of exogenous AM was not expressed in cardiomyocyte cells.ConclusionUnder the current experiment conditions, AM gene delivery did not obviously attenuate manifestation of parameters of ventricular cell hypertrophy and oxidative stress in these parameters besides the width of cardiomyocyte in a model of pressure overload and cardiac ischemia induced by chronic nitric oxide deficiency.Expected result that an exogenous supply of AM, acting with elevated levels of the endogenous peptide could potentially prevent or reverse the phenotypic changes occurring in hypertrophying cardiomyocytes seems not to be attained. The close reasons are considered perhaps relevant to titer and activity stability of and necessary targeting confirmation and stability of exogenous genes delivered by Adenovirus.