| BackgroundHypercholesterolemia and the corresponding artherosclerosis are the pathological foundation of coronary atherosclerotic heart disease (CAHD) and heart muscle ischemia, and are critical risk factors of ischemic heart disease. Hypercholesterolemia plays an important role in both the beginning of ischemia and the development of the injury. We and other researchers have found the myocardium has increased susceptibility to ischemia/reperfusion injury in hypercholesterolemia, but the mechanisms are not clear. We know that lipid can accumulate and later form artherosclerosis plaques. When such plaque covers the coronary, it will cause spasm. So do the increase of active oxygen production and reactive nitrogen production, which induce dysfunction of endothelium. However, these initial factors can not explain the phenomenon of increased susceptibility. There are still lots of questions.Membrane fluidity is the dynamical character of lipid bilayer, which is important in signal transduction, chemical transportation, ion exchange, energy transmission and activity of enzymes. Stable membrane fluidity is so significant to the maintenance of internal environment and normal function of cells that it affects the development of many diseases. The change of membrane fluidity comes earlier than the morphological and biological changes of cells in a disease. The changes of membrane fluidity can also reflect the early cell injury and the development of disease. Some studies found that membrane fluidity of red blood cell decreases in hypercholesterolemia. This indicates that the disorder of lipid metabolism may change the lipid proportion of the membrane and further affect its fluidity. Will hypercholesterolemia change the fluidity of myocardium and affect its signal transduction? If so, is there any relationship between the changes of myocardium membrane fluidity and heart muscle increased that susceptibility caused by hypercholesterolemia? Study considering these questions will help us to investigate the mechanism of increased susceptibility of myocardium to ischemia/reperfusion injury caused by hypercholesterolemia.Myeloperoxidase (MPO), which expresses in neutrophil, is an important cytokine and an independent risk factor of CAHD. MPO stimulates the apoptosis of endothelium, degrades the relaxing factor NO, lowers its biological activity, causes endothelium dysfunction and increases the vulnerability of atherosclerotic plaque, which enhances the development of heart muscle ischemia. Moreover, MPO released by activated and effused neutrophils could catalyze H2O2 produced by neutrophils and injured tissue forms hypochloric acid and other oxidants, which are toxic and will cause cardiovascular injury. However, inflammation reaction plays an important role in myocardium ischemic/reperfusion injury. So, whether MPO plays a direct function of injury to myocardium? Does it take part in the increased susceptibility of ischemia/reperfusion injury heart muscle in hypercholesterolemia? And how does it work?From 1990, researchers detected many G protein-coupled receptor autoantibodies from serum of patients with cardiovascular diseases, includingα1-AA,β1-AA,β3-AA,M2-AA and AT1-AA etc. Some study indicatesα1-AA existence would cause increased peripheral resistance and myocardium remodel.β1-AA induces heart rate and later exacerbates heart burden and then results in injury. M2-AA destroys mitochondria, which would lead to myofibrilla dissolve and heart injury. These results suggest that G protein-coupled Receptors autoantibody may play an important role in the development of cardiovascular diseases. Evidence proved that hypercholesterolemia causes ultrastructure changes of myocardium and inflammation response in cardiovascular diseases. These indicate that hypercholesterolemia probably induce the production of G protein-coupled receptors autoantibody by the dysfunction of immune system. If this hypothesis is true, these autoantibodies have high probability to increase the susceptibility of myocardium in hypercholesterolemia and aggravate heart injury. As a result, investigation of the production of G protein-coupled receptors autoantibody in hypercholesterolemia is critical in cardiovascular diseases.Overall, basing on a dietetic hypercholesterolemia rat model, current research aim to discuss the functions and some related mechanisms of cardiac muscle cell fluidity, myeloperoxidase and certain portion of G protein-coupled receptors autoantibody in hypercholesterolemia increased susceptibility of heart muscle.PART 1 Hypercholesterolemia-induced myocardial cell membrane fluidity decline in ratsObjective:To determine whether decreased membrane fluidity of myocardial cells may contribute to enhanced myocardial injury in hypercholesterolemia.Materials and Methods:1.1 Materials:Male Wistar rats (4-5 weeks) weighing110±10g, provided by Shanxi Medical University Laboratory Animal Center.1.2 Experimental groups1.2.1 The groups of diet-derived HC rats(1) Control group: Rats were given a normal diet for 10 weeks;(2) Vehicle + high cholesterol group: Rats were given a HC diet for 10 weeks and receive vehicle for 4 week in the 6th week;(3) PIO + high cholesterol group: Rats were given a HC diet for 10 weeks and receive PIO (10 mg/kg/day, oral gavage) for 4 week in the 6th week.1.2.2 The groups of rats'regional myocardial ischemia/ reperfusion (MI/R) in vivo(1) Sham group: Left Anterior Descending (LAD) without occlusion, total time course is 24 hours;(2) MI/R+ normal diet group: LAD with reversible occlusion, 30min ischemia followed by 24 hours reperfusion;(3) MI/R+ Vehicle + HC diet group: LAD with reversible occlusion, 30min ischemia followed by 24 hours reperfusion;(4) MI/R+ PIO + HC diet group: LAD with reversible occlusion, 30min ischemia followed by 24 hours reperfusion.1.3 Methods1.3.1 Establishment of experimental model and sample collection: (1) The establishment of diet-derived HC rat model and tissue collection;(2) The establishment of rat via a polyethylene catheter inserted into the left ventricular cavity through the right carotid artery in vivo and tissue collection;(3) Isolation of cardiac myocyte and sample collection;(4) The establishment of rat regional MI/R model in vivo and tissue collection1.3.2 Index determination(1) Measurement of Total Cholesterol (TC), Triglyceride (TG) and Low Density Lipoprotein Cholesterol (LDL-cho) in serum of rat on an empty stomach;(2) Determination of myocardial function;(3) Detection of membrane Na+-K+-ATPase activity;(4) Detection of cholesterol and phospholipid content in myocardial cell membrane;(5) Membrane fluidity measurements(6) Determination of cAMP with radioimmunoassay(7) Determination of myocardial infarct size(8) Correlation analysis between membrane fluidity and myocardial contractile function or cardiac infarct sizeResults:2.1 PIO had effects on plasma lipid profiles after high cholesterol dietTen weeks of HC diet resulted in a dramatic increase in plasma cholesterol (TC) (3.44±0.86 mmol/l vs. 1.24±0.26 mmol/l, P<0.01), triglyerde (TG) (1.13±0.34 mmol/l vs. 0.26±0.05 mmol/l, P<0.01) and low-density lipoprotein (LDL-cho) (1.66±0.55 mmol/l vs. 0.45±0.14 mmol/l, P<0.01). There were no significant differences between groups in plasma lipid profiles before the onset of the study (time 0) or after 6 weeks of a HC diet (before PIO treatment). After an additional 4 weeks of high cholesterol diet and treated with vehicle, plasma TC, TG and LDL levels further increased. At all these post-cholesterol times, PIO-treated rats on the HC diet exhibited significantly lower plasma TC (2.16±0.33 mmol/l, P<0.01), TG (0.59±0.18 mmol/l, P<0.05) and LDL-cho (1.24±0.28 mmol/l, P<0.05) than the vehicle group. These results indicate that PIO improved plasma lipid profiles in this diet-induced HC model, and therefore, any cardiovascular effects observed could be attributed to this mechanism.2.2 Hypercholesterolemia worsens cardiac functional and increases post-ischemic myocardial injuryHC diet markedly aggravated myocardial contractile function. The values of LVSP, +dp/dtmax and–dp/dtmax were significantly decreased in the HC diet group compared with the normal diet group (LVSP: 13.64±1.37 Kpa vs. 20.79±2.87 Kpa, P<0.01; +dp/dtmax: 537.43±102.24 Kpa/s vs. 953.38±171.12 Kpa/s, P<0.01; -dp/dtmax: 452.19±88.34 Kpa/s vs. 793.10±112.82 Kpa/s, P<0.01). Additionally, ischemia/reperfusion-induced myocardial infarct size in the HC rats (i.e. vehicle) was significantly larger than the normal rats (56.81±6.45% vs 35.25±2.52%, P<0.01). There was no difference in ischemic area (AAR) expressed as percent of left ventricle, indicating a comparable degree of ischemic insult between sham-, normal diet-, vehicle- and PIO-treated groups after left anterior descending artery occlusion.2.3 Hypercholesterolemia decreased membrane Na+-K+-ATPase activity, increased C/P and reduced the membrane fluidity of cardiac myocytesNa+-K+-ATPase reside in cell membrane nullity of the organelle membrane, which activity is usually to identify the cell membrane. A clear decrease in Na+-K+-ATPase activity was observed in HC rats compared with that in normal diet rats (5.99±1.69μmol Pi/mg protein/hour vs. 7.88±1.25μmol Pi/mg protein/hour, P<0.01). The C/P of cardiac myocytes membrane was largely higher in rats with HC diet than normal diet (0.44±0.04 vs. 0.33±0.03, P<0.01). Most importantly, in myocardial cells from HC rats, the DPH fluorescence polarization (P) and microviscosity (η) were markedly higher than that seen in the normal diet group (0.34±0.07 vs 0.24±0.05, P<0.01; 7.99±1.37 vs 2.41±0.59, P<0.01, respectively), indicating that hypercholesterolemia decreased cardiac myocytes membrane fluidity.2.4 Hypercholesterolemia decreased myocardial cAMP contentTo further elucidate the effect by which hypercholesterolemia may disturb myocardial function, myocardial cAMP content, a downstream product in signal transduction pathway of membrane receptor, was determined. Hypercholesterolemia markedly decreased myocardial cAMP content (53.77±22.17 pmol/mg protein vs. 127.50±19.72 pmol/mg protein, P<0.01). The result demonstrated that hypercholesterolemia may cause myocardial function injury and thus contribute to increased myocardial vulnerability after ischemia and reperfusion.2.5 PIO treatment improved myocardial contractile function, attenuated myocardial infarct size, increased Na+-K+-ATPase activity and cAMP content, reduced C/P, and restored membrane fluidityPrevious studies have demonstrated that PPARγagonists, such as rosiglitazone (RSG), exert beneficial cardiovascular effects in diabetic patients. In recent studies, researchers have demonstrated that treatment with RSG significantly improved endothelial and myocardial function in hypercholesterolemic rabbits. To determine whether the PPARγsignaling pathway may improve cardiac function by restoring membrane fluidity in a non-diabetic model, 18 rats fed with a high cholesterol diet were treated with PIO for 4 weeks. Treatment with PIO in hypercholesterolemic rats exerted significant protective effects as evidenced by markedly improveded cardiac contractile dysfunction (LVSP: 20.40±2.83 Kpa, P<0.01; +dp/dtmax : 827.56±172.49 Kpa/s, P<0.01;–dp/dtmax: 684.01±113.43 Kpa/s, P<0.01) and attenuated myocardial infarct size (39.82±2.74, P<0.01). PIO treatment tended to significantly increase Na+-K+-ATPase activity (7.43±1.42μmol Pi/mg protein/hour, P<0.01) and decrease C/P (0.40±0.04, P<0.05) in cardiac myocytes membrane. In addition, treatment with PIO markedly decreased both DPH fluorescence polarization (0.26±0.06, P<0.01) and microviscosity (3.29±0.63, P<0.01), a parameter that primarily reflects membrane fluidity. To further determine the effect by which PIO may exert its cardiac protective properties, the cAMP content in myocardial cell of PIO treatment was determined. Treatment with PIO in hypercholesterolemia significantly increased myocardial cAMP content (102.05±25.51, P<0.05).2.6 Negative correlation between myocardial contractile function and DPH fluorescence polarization, positive correlation between cardiac infarct size and DPH fluorescence polarizationTo determine the direct relation between membrane fluidity and myocardial function or cardiac injury, correlation between DPH fluorescence polarization and LVSP, +dp/dtmax, -dp/dtmax and infarct size were analyzed. LVSP, +dp/dtmax and -dp/dtmax negatively correlated to DPH fluorescence polarization respectively (r=-0.191, P<0.05; r=-0.323, P<0.01; r=- 0.322, P<0.01, respectively.). Cardiac infarct size positively correlated to DPH fluorescence polarization (r=0.599, P<0.01). In other words, membrane fluidity positively correlated to myocardial contractile function and negatively correlated to cardiac infarct size. These results suggest that hypercholesterolemia-induced cardiac impairment may be associated with membrane fluidity decrease, and PIO treatment protects hypercholesterolemia-induced myocardial dysfunction by preserving the integrity of membrane fluidity.Summary1. Hypercholesterolemia impairs cardiac function, and increases myocardial infarct size after ischemic/reperfusion, suggesting that hypercholesterolemia may enhance cardiac vulnerability.2. Hypercholesterolemia increases C/P in the myocardial cell membrane, decreases membrane fluidity and myocardial cAMP content, as well as membrane fluidity positively correlates to myocardial contractile function and negatively correlates to cardiac infarct size, suggesting that hypercholesterolemia may exacerbate cardiac injury by decreased membrane fluidity-induced myocardial dysfunction.3. PIO treatment markedly inhibited hypercholesterolemia-induced C/P increase, membrane fluidity decrease of cardiac myocyte membrane, and myocardial cAMP content decrease, thus recovered cardiac function and decreased infarct size after myocardial ischemia/reperfusion.PART 2 The role of MPO in the increased susceptibility of ischemic-reperfusion injury in hypercholesterolemia ratsObjective:To confirm the role of MPO in the increased susceptibility of ischemic-reperfusion injury in hypercholesterolemia rats and to explore the mechanism (such as the reduction in the effective utilization of NO etc.) by observing the changes of MPO activity and distribution in myocardial tissue of ischemia/reperfusion HC rats.Materials and Methods:1.1 Materials:(1) Male Wistar rats (4-5 weeks) weighing110±10g, provided by Shanxi Medical University Laboratory Animal Center.(2) H9c2 (2-1) cell line 1.2 Experimental groups1.2.1 The groups of diet-derived HC rats(1) Control group: Rats were given a normal diet for 10 weeks;(2) Vehicle + high cholesterol group: Rats were given a HC diet for 10 weeks and intraperitoneal injection of 10% DMSO for 1 week in the 9th week;(3) ABAH (MPO inhibitor) + high cholesterol group: Rats were given a HC diet for 10 weeks and intraperitoneal injection of ABAH (25.00mg/kg?d) for 1 week in the 9th week.1.2.2 The groups of rats'regional myocardial ischemia/ reperfusion (MI/R) in vivo(1) Sham group: Left Anterior Descending (LAD) without occlusion, total time course is 3 or 24 hours;(2) MI/R+ normal diet group: LAD with reversible occlusion, 30min ischemia followed by 3 or 24 hours reperfusion;(3) MI/R+ Vehicle + HC diet group: LAD with reversible occlusion, 30min ischemia followed by 3 or 24 hours reperfusion;(4) MI/R+ ABAH + HC diet group: LAD with reversible occlusion, 30min ischemia followed by 3 or 24 hours reperfusion.1.2.3 The groups of H9c2 (2-1) cell lines(1) Normoxia group: cultured in atmosphere of 5%CO2, 95% air for 5h;(2) ABAH+ Normoxia group: added 100μM ABAH into the cultured medium then cultured in atmosphere of 5%CO2, 95% air for 5h;(3) H/R group: hypoxia with 95% N2 and 5% CO2 for 3h then followed by 2h reoxygenation in 5% CO2 and 95% air;(4) MPO+H/R group: added 0.1 u/ml MPO into the cultured medium then operated as H/R group; (5) ABAH+H/R group: added 100μM ABAH into the cultured medium then operated as H/R group;(6) ABAH+MPO+ H/R group: added 100 mΜABAH into the cultured medium for 30 min before adding 0.1 u/ml MPO then operated as H/R group.1.3 Methods1.3.1 Establishment of experimental model and sample collection(1) The establishment of diet-derived HC rat model and tissue collection;(2) The establishment of rat regional MI/R model in vivo and tissue collection;(3) The establishment of H9c2 (2-1) H/R model and cell collection.1.3.2 Index determination(1) Measurement of Total Cholesterol (TC), Triglyceride (TG) and Low Density Lipoprotein Cholesterol (LDL-cho) in serum of rat on an empty stomach;(2) Measurement of Creatine Kinase (CK) content in serum and culture medium supernatant;(3) Measurement of Lactate Dehydrogenase (LDH) content in serum and culture medium supernatant;(4) Myocardial apoptosis is detected by TdT-mediated dUTP nick end labeling (TUNEL);(5) Caspase-3 relative activity assay;(6) Determination of myocardial infarct size;(7) Measurement of cardiac function;(8) Determination of myocardial tissue MPO distribution by immunohistochemistry;(9) Myocardial tissue MPO activity assay; (10) Correlation analysis of myocardial tissue MPO activity , serum CK concentration, LDH leakage, apoptotic index, caspase-3 activity , myocardial infarction size and cardiac function;(11) Measurement of nitric oxide (NO) content in myocardial tissue;(12) Determination of myocardial cGMP content by Radioimmunoassay kit;(13) Detection of myocardial nitric oxide synthase (NOS) protein expression by Western-blot;(14) Detection of the mRNA expression of NOS in myocardial tissue by real time PCR;(15) Detected H9c2 (2-1) cell proliferation by Cell counting Kit-8.Results:2.1 The establishment of Diet-derived HC rat modelThe lipid levels (TC, TG and LDL-cho) in rats of each group had no significant difference before given HC diet feeding (0 week). The lipid levels of vehicle + high cholesterol group was significantly higher than control group after feeding with high-cholesterol diet for 10 weeks (TC: 3.01±0.75 mmol/L vs. 1.44±0.14 mmol/L, P<0.01; TG: 0.88±0.35 mmol/L vs. 0.26±0.05 mmol/L, P<0.01; LDL-cho: 1.53±0.65 mmol/L vs. 0.45±0.14 mmol/L, P<0.01, respectively.). All these demonstrated that diet-derived HC rat model was set up successfully.2.2 HC rats had increased vulnerability of MI/R2.2.1 A significantly exacerbated myocardial MI/R injury was found of HC rats2.2.1.1 CK content in serum of HC rats were increasedCompared with sham group, the CK content in MI/R group increased significantly (0.17±0.01 U/ml vs. 0.05±0.02 U/ml, P<0.01); Compared with normal diet MI/R group, the CK content in HC MI/R group increased significantly (0.70±0.08 U/ml, P<0.01).2.2.1.2 LDH content in serum of HC rats were increased Compared with sham group, the LDH content in MI/R group increased significantly (3091.59±33.69 U/L vs. 1854.45±422.97 U/L, P<0.01); Compared with normal diet MI/R group, the LDH content in HC MI/R group increased significantly (3596.18±99.81 U/L, P<0.01).2.2.2 Increased myocardial apoptosis in HC rats with ischemia/reperfusion2.2.2.1 TUNEL detectionThe apoptotic index in MI/R group was significantly higher than that in sham group (16.07±1.02% vs. 1.44±0.14%,P<0.01); the apoptotic index in HC MI/R group was significantly higher than that in normal diet MI/R group (22.63±1.02%, P<0.01).2.2.2.2 Caspase3 activity assay:Caspase3 activity in MI/R group was also significantly increased compared with sham group (6.66±0.61 vs. 1.00±0.08,P<0.01); caspase3 activity in HC MI/R group was also significantly increased compared with normal diet MI/R group (12.29±0.92,P<0.01).2.2.3 Myocardial infarct size in HC group enhanced after MI/RThe ratio of area at risk to left ventricular area (AAR/LV): no significant differences were observed among these groups (P>0.05), which indicated that the ischemic area induced by coronary ligation is roughly identical among each experimental group, so that these groups are comparable. The myocardial infarct size of HC MI/R group was significantly enhanced as compared with normal diet MI/R group (56.05±3.91% vs. 34.90±2.52%, P<0.01).2.2.4 The cardiac function in HC MI/R group were aggravatedAt the end of 3 h reperfusion, compared with the sham group, the LVSP and±dP/dtmax in MI/R group decreased obviously (LVSP: 15.12±1.38 Kpa vs. 18.28±3.02 Kpa, P<0.05; +dp/dtmax: 610.43±110.26 Kpa/s vs. 785.22±162.22 Kpa/s, P<0.01; -dp/dtmax: 500.57±116.20 Kpa/s vs. 667.00±234.83 Kpa/s, P<0.05, respectively.);compared with the normal diet MI/R group,the LVSP and±dP/dtmax in HC MI/R group decreased obviously (LVSP: 9.95±1.85 Kpa, P<0.01; +dp/dtmax: 386.43±100.76 Kpa/s, P<0.01; -dp/dtmax: 264.53±98.76 Kpa/s, P<0.01, respectively.).These results confirmed that, compared with the normal diet group, the MI/R injury in HC group increased, which indicated the increased susceptibility of ischemic-reperfusion injury in hypercholesterolemia rats.2.3 Myocardial tissue MPO changes in HC rats with ischemia/reperfusion2.3.1 Myocardial tissue MPO activity assay in HC rats with ischemia/reperfusionCompared with the sham group, the myocardial tissue MPO activity in MI/R group increased obviously (13.48±4.58 U/g protein vs. 8.29±2.80 U/g protein, P<0.05), and further increased in HC MI/R group (19.44±2.35 U/g protein, P<0.05).2.3.2 Myocardial tissue MPO distribution of HC rats with ischemia/reperfusionThe result of immunohistochemistry indicated that rare MPO was detected in the area not at risk (ANAR) of myocardial tissue from normal diet rats, but clear staining was observed in the vascular tissue and cardiac myocytes from ischemia/reperfusion area. The results suggested that infiltrating neutrophils in ischemia/reperfusion heart release a large number of MPO, these MPO possibly invasive ischemia/reperfusion myocardial cells through active or passive manner then cause myocardial cell injury through a variety of mechanisms. Moreover, the MPO immunostaining further enhanced in HC MI/R group than the normal diet MI/R group.This is consistent with the above-mentioned results that MPO activity increased in HC MI/R group.Prompted MPO may be involved in the increased susceptibility of ischemia/reperfusion myocardial injury in HC rats.2.4 Correlation analysis of myocardial tissue MPO activity with MI/R injury.2.4.1 Positive correlation between MPO activity in ischemia/reperfusion myocardial tissue and serum CK and LDH levels (CK: r = 0.618, P<0.01; LDH: r = 0.64, P<0.01, respectively.).2.4.2 Positive correlation between MPO activity and apoptotic index and caspase-3 activity in ischemia/reperfusion myocardial tissue (apoptotic index: r = 0.651, P<0.01; caspase-3: r = 0.619, P<0.01, respectively.).2.4.3 Positive correlation between MPO activity and myocardial infarct size in ischemia/reperfusion myocardial tissue (r = 0.663, P< 0.01). 2.4.4 Negative correlation between MPO activity in ischemia/reperfusion myocardial tissue and myocardial contractile function (LVSP and±dP/dtmax) of ischemia/reperfusion heart (LVSP: r = -0.555, P<0.01; +dP/dtmax: r = -0.794, P< 0.01; -dP/dtmax: r =-0.748, P<0.01, respectively.).These results confirmed the closely correlation between MPO activity in ischemia/reperfusion myocardial tissue and increased susceptibility of ischemia/reperfusion injury in hypercholesterolemic rats. Changes of MPO in myocardial tissue of HC rats with ischemia/reperfusion were directly related to ischemia/reperfusion myocardial vulnerability of HC rats.2.5 MI/R HC rats'myocardial tissue MPO and cardiac injury changed after the intervention with MPO inhibitorsIn order to further explore whether or not MPO participate in and directly affect the HC rat MI / R myocardial vulnerability increased, ABAH was applicated to HC rats in vivo to observe the corresponding changes.2.5.1 Effect of ABAH on the H9c2 (2-1) cellThere was no statistical difference of cell survival rate between ABAH + normoxia group and normoxia group (0.98±0.01 vs. 1.01±0.05); there was no statistical difference of cell survival rate between ABAH+H/R group and H/R group too (0.63±0.06 vs. 0.60±0.09). The results above showed that ABAH itself had no effect on myocardial cells and removed the experimental error which forms by the medicine intervention.2.5.2 The change of MPO activity and distribution in myocardial tissue, after ABAH interventionCompared with the MI/R+ Vehicle + HC diet group, the MPO immunostaining and MPO activity (13.66±2.63 U/g protein, P<0.05) in MI/R+ ABAH + HC diet group significantly decreased.2.5.3 After ABAH intervention, the myocardial ischemia/reperfusion injury of HC rats mitigated.Compared with the MI/R+ Vehicle + HC diet group CK (0.31±0.06 U/ml, P<0.01) and LDH (3286.65±40.04 U/L, P<0.01) content in MI/R+ ABAH + HC diet group significantly decreased.2.5.4 After ABAH intervention, cardiomyocyte apoptosis of HC rats with ischemia/reperfusion mitigated.Compared with the MI/R + Vehicle + HC diet group, apoptotic index (18.55±1.29%, P<0.01) and Caspase3 activity (7.97±0.29, P<0.01) in MI/R+ ABAH + HC diet group significantly decreased.2.5.5 After ABAH intervention, the myocardial infarct size of HC rats decreasedThe ratio of AAR/LV: no significant differences were observed among these groups (P>0.05). The myocardial infarct size of MI/R+ ABAH + HC diet group was significantly decreased as compared with MI/R+ Vehicle + HC diet group (38.89±2.47%, P<0.01).2.5.6 After ABAH intervention, HC rats with ischemia/reperfusion cardiac dysfunction mitigated Compared with the MI/R + Vehicle + HC diet group, the cardiac dysfunction in MI/R+ ABAH + HC diet group had some degrees recovery (LVSP: 13.73±2.20 Kpa, P<0.05; +dP/dtmax: 564.00±128.56 Kpa/s, P<0.05; -dP/dtmax: 444.00±96.08 Kpa/s, P<0.05, respectively.).These results indicated: First, ABAH itself had no effect on myocardial cells. Second, after ABAH intervention, MPO activity and distribution in myocardial tissue of HC rats with ischemia/reperfusion decreased and ischemia/reperfusion injury mitigated. Third, MPO changes in myocardial tissue of HC rats with ischemia/reperfusion had close related to ischemia/reperfusion myocardial vulnerability of HC rats. Howerve, it needs to be further confirmed that whether there is direct relation between them.2.6 Effect of MPO on the H9c2 (2-1) cellIn order to remove various influence of neural and humoral regulation to experimental result in vivo, the experimental use of H9c2 (2-1) cells to observe the MPO on the direct effects of myocardial cells.2.6.1 H9c2 (2-1) H/R model was established successfullyCompared with the normoxia group (control group), the CK and LDH content in culture medium supernatant in H/R group significantly increased (CK: 0.08±0.03 U/ml vs. 0.02±0.01 U/ml, P<0.01; LDH: 856.22±28.91 U/L vs. 33.11±21.18 U/L, P<0.01, respectively.), showing that H9c2 (2-1) H/R model had been established successfully. 2.6.2 MPO aggravated hypoxia/reoxygenation injury in H9c2 (2-1) cellsCompared with H/R group, the CK and LDH content in culture medium supernatant in 0.1u/ml MPO+H/R group significantly increased (0.42±0.11 U/ml, P<0.01; 1362.78±106.89 U/L, P<0.01, respectively.), showing that MPO could not only cause myocardial cells' damage but also further aggravate the myocardial cell hypoxia/reoxygenation injury, which prompted that the MPO distribution and activity possibly directly participated in myocardial cell's damage, and further aggravated the myocardial cell hypoxia/reoxygenation injury.2.6.3 After ABAH intervention, H9c2 (2-1) cell H/R damage changeCompared with 0.1 u/ml MPO+H/R group, the CK and LDH content in ABAH+0.1 u/ml MPO+H/R group significantly decreased (0.13±0.07 U/ml, P<0.01; 880.81±80.86 U/L, P<0.01, respectively.), further prompted that MPO distribution and activity possibly directly participated in myocardial cells'damage, and had directly relationship with ischemia/reperfusion myocardial vulnerability of HC rat.2.7 HC rats with ischemia/reperfusion myocardial tissue NO-cGMP signaling pathway impaired2.7.1 NOx content increased in myocardial tissue of HC rats with ischemia/reperfusionCompared with MI/R + normal diet group, the NOx content in MI/R + Vehicle + HC diet group significantly increased (972.78±77.45μmol/g protein vs. 421.44±58.96μmol/g protein, P<0.01). ABAH could effectively reduce the NOx content in HC rat ischemia/reperfusion myocardial tissue (615.76±50.54μmol/g protein, P<0.01), but had not restored completely to the normal physiological condition.2.7.2 The cGMP content in myocardial tissue of HC rats with ischemia/reperfusion decreasedCompared to sham group the cGMP content in MI/R+ normal diet group decreased (31.53±11.37 pmol/mg protein vs. 74.22±22.23 pmol/mg protein, P<0.01); compared to MI/R+ normal diet group, the cGMP content in MI/R+ Vehicle + HC diet group decreased (7.35±2.47 pmol/mg protein, P<0.01); compared to MI/R+ Vehicle + HC diet group, the cGMP content in MI/R+ ABAH + HC diet group restored partly (28.84±9.08 pmol/mg protein, P<0.01).2.7.3 The changes of mRNA and protein expression of NOS in myocardial tissue of HC rats with ischemia/reperfusion 2.7.3.1 The change of mRNA expression of NOS in myocardial tissue of HC rats with ischemia/reperfusionCompared with sham group, the mRNA expression of iNOS in MI/R+ normal diet group increased (4.80±1.73 vs. 1±0.70, P<0.01); compared with MI/R+ normal diet group, the mRNA expression of iNOS in MI/R+ Vehicle + HC diet group increased (10.09±2.15, P<0.01); compared with MI/R+ Vehicle + HC diet group the mRNA expression of iNOS in I/R+ ABAH + HC diet group decreased (3.41±1.50, P<0.01).Compared with control group the mRNA expression of eNOS in vehicle + high cholesterol group decreased significantly (0.87±0.02 vs. 1.00±0.04, P<0.01). No significant differences of nNOS mRNA expression were observed among these groups.2.7.3.2 The change of protein expression of NOS in myocardial tissue of HC rats with ischemia/reperfusionCompared with sham group, the protein expression of iNOS in MI/R+ normal diet group increased (2.22±0.44 vs.1.00±0.26, P<0.05); compared with MI/R+ normal diet group, the protein expression of iNOS in MI/R+ Vehicle + HC diet group increased (3.17±0.93, P<0.05); compared with I/R+ Vehicle + HC diet group, the protein expression of iNOS in MI/R+ ABAH + HC diet group decreased (2.28±0.46, P<0.05).Compared with control group, the protein expression of eNOS in vehicle + high cholesterol group decreased significantly (0.36±0.14 vs. 1.00±0.37, P<0.05). No significant differences of nNOS protein expression were observed among these groups.These results indicated that NOx content increased in myocardial tissue of HC rats and it further increased in AAR of HC rats with ischemia/reperfusion; cGMP content decreased in myocardial tissue of HC rats and it further decreased in AAR of HC rats with ischemia/reperfusion; both mRNA and protein expression of iNOS increased in myocardial tissue of HC rats and it further increased in AAR of HC rats with ischemia/reperfusion; both mRNA and protein expression of eNOS decreased significantly among control group and vehicle + high cholesterol group; both mRNA and protein expression of nNOS had no significant differences among these groups. These data demonstrated that although NOx content in AAR of HC rats with ischemia/reperfusion increased significantly, nitric oxide bioavailability decreased and NO-cGMP signaling pathway maybe impaired; MPO may impaired NO-cGMP signaling pathway in myocardial tissue by reduced nitric oxide bioavailability, and so to participate in the rat ischemia/reperfusion myocardial injury and played a role in the increased susceptibility of ischemic-reperfusion injury in hypercholesterolemic rats. The increased NOx content i...
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