Cardioprotective Effect Of Anisodamine Against Myocardial Ischemia/Reperfusion Injury And Its Mechanism On Cardiomyocytes Apoptosis

Acute myocardial infarction (AMI) has become one of the leading causes of morbidity and mortality in China, with the increasing incidence trends year by year. Nowadays, the main treatments for AMI are primary percutaneous coronary intervention (PCI) and thrombolysis. The key strategy for treating AMI is to open the infarct-related artery (IRA) and restore the myocardial tissue perfusion as soon as possible in order to salvage the ischemic myocardium, narrow infarction area, inhibit ventricular remodeling, protect cardiac function and decrease the incidence of major adverse cardiovascular events (MACEs). However, still 10% to 30% of patients fail to restore effective myocardial perfusion despite the opening of the epicardial infracted coronary, which is so-called no-reflow phenomenon. The key factor for myocardial microcirculation obstruction (MVO) is ischemia/reperfusion injury (1R1), which may further injure ultrastructure and metabolic functions of the cardiac myocytes in turn. Therefore, how to prevent no-reflow phenomenon and IRI in the setting of AMI is an important subject to deal with MVO following myocardial infarction.Myocardial ischemia-reperfusion injury is closely associated with myocardial apoptosis, which is the main presenting of cell death in the course of AMI. Therefore, inhibition of myocardial apoptosis at early stage may significant decrease the incidence of IRI. Anisodamine, an alkaloid isolated from the Solanaceae tanguticum Belladonna in China, with multiple pharmacological effects, is widely used to treat septic shock and multiple organ failure. Previous studies have demostrated that anisodamine has abilities in the expansion of micro-vessels, regulation of systolic and diastolic function of the capillary sphincters and improvement of microcirculatory perfusion.Our previous studies showed that intracoronary administration of anisodamine could effectively reverse the no-reflow phenomenon in patients with ST-segment elevation myocardial infarction (STEMI) treated with primary PCI. The potential mechanisms were related to the ability of anisodamine in improvement of coronary microcirculation, confirmed by a series of studies in Yoke pig model. Besides, our previous studies also found anisodamine could significantly decrease the incidence of contrast--induced nephropathy (CIN) after PCI for the heart-kidney protective effect. At present, the main strategy for the treatment of IRI is "downstream salvage intervention", which is the pharmaceutical or mechanical intervention during and after PCI. Therefore, "upstream prevention intervention", referring to pharmaceutical intervention administrated before the opening of the IRA and the restoration of the coronary blood supply, could achieve the accommodation of myocardial microcirculation in the infarct-related region to the change of reperfusion, relieve and prevent myocardial IRI after the opening of the IRA.This study focused on the preventive and reverse effects of preventively intracoronary administration of anisodamine on IRI in AMI. The study consists of five parts:Part Ⅰ, regarding STEMI patients as study population, explore the protective effects of preventively intracoronary administration of anisodamine on myocardial reperfusion and cardiac function after primary PCI; Part Ⅱ, to observe the effect of anisodamine on myocardial protection of Spragur-Dawley rat induced by ischemia/reperfusion injury and pretreated with anisodamine. Part Ⅲ, to observe the effect of anisodamine on apoptosis of rat H9c2 cardiomyocytes induced by hypoxic/reoxygenation injury and pretreated with anisodamine. Part IV, to investigate the effect of rhBNP on renal function and contrast-induced nephropathy incidence in STEMI-HF patients undergoing pPCI.Part Ⅰ Effects of preventively intracoronary administration of anisodamine on myocardial reperfusion in patients with acute ST-segment elevation myocardial infarction undergoing primary percutaneous coronary interventionObjectives:To investigate whether preventively intracoronary administration of anisodamine before the opening of the infarct-related artery (IRA) can improve myocardial reperfusion and heart function in acute ST-segment elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (PCI).Methods:Patients suffering from STEMI within 12 hours undergoing primary PCI from October 2012 to February 2014 enrolled in this study. Eligible patients were randomly divided into two groups:anisodamine group, 58 cases, preventively intracoronary administration of anisodamine 2000ug/10ml before the opening of the IRA; control group,60 cases, intracoronary administration of saline 10ml. All patients underwent coronary angiography (CAG) and/or PCI by tranforem artery approach.All quantitative coronary angiography perfusion indexes [including the initial thrombolysis in myocardial infarction (TIMI), corrected TIMI frame count (CTFC) and TIMI myocardial perfusion grade (TMPG)] were judged by two interventional cardiologists. Myocardial infarct size was estimated by areas under curve of creatine kinase-MB (CK-MB) which was tested before and every 6 hours after the procedure. Electrocardiography was recorded both on admission and at 90 min after PCI. A decrease in the sum of ST-segment elevation by≥70% was categorized as complete ST-segment resolution (STR). All calculations were computed with the aid of SPSS statistical software (version 16.0). P values (2-sided) of less than 0.05 were considered statistically significant.Result:No significant differences were found between the two groups about thrombus scores and inatial TIMI grade at baseline. After PCI, the proportion of TIMI 3 in anisodamine group was higher than that in control group (91% vs.75%, P= 0.039), so was the TMPG 3 (84% vs.65%, P= 0.044). No significant differences were observed about the level of CK-MB at baseline on admission between the two groups. The area under curve of CK-MB in anisodamine group was significantly lower than that in control group. No significant differences were observed about the LVEF at baseline on admission between the two groups. Echocardiography at 1 week after pPCI showed that, compared to control group, LVEF was higher in anisodamine group (56.22±8.19% vs.53.17±6.18%, P=0.003). Echocardiography at 30 days after pPCI showed that, compared to control group, LVEF was higher in anisodamine group (65.52±8.27% vs.59.12.17±9.31% P=0.038). At the end of 3 months follow up,2 cases of MACEs and 5 MACEs occurred in anisodamine group and control group respectively (P=0.433).Conclusion:Preventively intracoronary administration of anisodamine could improve myocardial perfusion and cardiac function in STEMI-PCI, while anisodamine is safe.Part Ⅱ Cardioprotective effect of anisodamine against myocardial ischemia/reperfusion injury and cardiomyocytes apoptosis in SD ratsObjectives:To observe the effect of anisodamine on myocardial protection of Sprague-Dawley rat induced by ischemia/reperfusion injury and pretreated with anisodamine.Methods:A total of 54 Male Sprague-Dawley (SD) rats were randomly divided into three groups:Sham Operation group (SO), Anisodamine group (ANI) and ischemia/reperfusion (I/R) group. After the pericardium was opened, a silk suture was placed around the anterior descendant branch of the left anterior descending (LAD) coronary artery about 2 mm from its origin. The LAD was only exposed but not ligated in SO group, the other groups were applied with 40 min of coronary artery occlusion and 3 hours reperfusion. Anisodamine was administered 5 min before reperfusion. Myocardial infarct size was measured using TTC method. Blood samples were collected for measurement CK-MB and LDH. We observated myocardial ultrastructure changes with electron microscopy. Using Western-blot to analysis the apoptosis related protein Caspase-3,8,9 express changes.Result:The myocardial tissue slices exhibited no evident infarction in SO group. In I/R group were observed with noticeable infarct area which were reduced in the ANI pretreatment (P=0.004). In ultrastructure observation, the myocardial in SO group were preserved well, but the I/R injury caused an apparent severe damage to the myocardial tissues in I/R group. Anisodamine remarkably reduced the ultrastrcture injury and the release of myocardial damage biomarkers, increased the ATP release. Anisodamine affected cardiomyocytes apoptosis through regulation of caspase-3, caspase-8 expression.Conclusion:Preventively administration of anisodamine could decrease myocardial infarct area, release ischemia/reperfusion injury and myocardial ultrastructure injury through regulation of caspase-3, caspase-8 expression.Part Ⅲ Effect of anisodamine on apoptosis of H9c2 cardiomyocytes induced by hypoxic/reoxygenation (H/R) injury and its mechanismsObjectives:To observe the effect of anisodamine on apoptosis of rat H9c2 cardiomyocytes induced by hypoxic/reoxygenation (H/R) injury and pretreated with anisodamine.Methods:H9c2 cardiomyocytes were inoculated in a 25cm2 culture bottle, and randomly divided into three groups when cell confluence achieved 90%. Control (CON) group:cells were normally cultured; H/R group:cells were cultured under hypoxic conditions for 3 hours, and then reoxygenated for 12 hours in normal media; Anisodamine (ANI) group:cells were also cultured under hypoxic conditions for 3 hours, and then reoxygenated for 12 hours in media containing anisodamine. Sulfonyl rhodamine B colorimetric method (SRB) was used to detect cell activity. TdT-mediated dUTP nick-end labeling (TUNEL) was performed to observe apoptosis. Agarose gel electrophoresis (AGE) was applied to detect DNA fragment fracture of apoptotic cells. Apoptosis change of H9c2 cardiomyocytes induced by H/R in each group was analyzed by Annexin V-propidium iodide (PI) double-staining and flow cytometry. The total protein was extracted, and protein concentration was determined by BCA method. The apoptotic protein expression of caspase-3, caspase-8, Bcl-2 and Bax was measured using western-blot method, whose mRNA levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR).Result:H9c2 cardiomyocytes activity was significantly decreased in H/R group, while cell activity increased obviously after being pretreated by anisodamine. A few brown stained TUNEL-positive apoptotic cells were observed in CON group. More apoptotic cells were present in H/R group. Compared with the H/R group, the number of apoptotic cells was significantly reduced after anisodamine pretreatment. The caspase-3 protein and mRNA expression of H9c2 cardiomyocytes in H/R group was significantly higher than those in CON group (P<0.05), which was obviously decreased in ANI group compared with the H/R group (P<0.05). The expression change of caspase-8 between groups was similar to caspase-3. The protein and mRNA levels of Bcl-2 in H/R group were significantly higher than those in CON group, which were significantly improved by anisodamine pretreatment. Compared with the CON group, the Bax protein and mRNA levels in H/R group augmented, but the levels obviously declined in ANI group.Conclusion:Anisodamine pretreatment could obviously reduce apoptosis and improve cell viability, which was probably by down-regulating the protein and mRNA levels of caspase-3, caspase-8 and Bax, as well as up-regulating the Bcl-2 protein and mRNA levels.Part Ⅳ Anisodamine combined with rhBNP in patients undergoing primary PCI for acute myocardial infarctionObjectives:To investigate whether preventively intracoronary administration of anisodamine combined with rhBNP before the opening of the infarct-related artery (IRA) can improve myocardial reperfusion and heart function in acute ST-segment elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (pPCI).Methods:96 Patients suffering from STEMI within 12 hours undergoing primary PCI from February 2014 to February 2015 enrolled in this study. Eligible patients were randomly divided into four groups:primary PCI group (CON), anisodamine infusion group (ANI), rhBNP infusion group (rhBNP), PCI with both treatments group (ANI+rhBNP). Anisodamine was preventively intracoronary administration of 2000μg/10ml before the opening of the IRA; rhBNP was intracoronary administration of 1.5 loading followed by 0.0075 μg/kg/min for 72 h. μg/kg All patients underwent coronary angiography (CAG) and/or PCI by tranforem artery approach.All quantitative coronary angiography perfusion indexes [including the initial thrombolysis in myocardial infarction (TIMI), corrected TIMI frame count (CTFC) and TIMI myocardial perfusion grade (TMPG)] were judged by two interventional cardiologists. Myocardial infarct size was estimated by peak levels of creatine kinase-MB (CK-MB) which was tested before and every 6 hours after the procedure. Electrocardiography was recorded both on admission and at 90 min after PCI. A decrease in the sum of ST-segment elevation by≥ 70% was categorized as complete ST-segment resolution (STR). All calculations were computed with the aid of SPSS statistical software (version 16.0).P values (2-sided) of less than 0.05 were considered statistically significant.Result:No significant differences were found between the four groups about thrombus scores and inatial TIMI grade at baseline. After PCI, the proportion of TMPG 3 in ANI group, rhBNP and ANI+rhBNP group were higher than that in control group (P= 0.039). No significant differences were observed about the level of CK-MB at baseline on admission between the two groups. The peak of CK-MB in ANI group、rhBNP and ANI+rhBNP group were significantly lower than that in CON group. Interaction analysis showed that anisodamine and rhBNP may have collaborative effects in reducing the myocardial infarction areas. No significant differences were observed about the LVEF at baseline on admission between the two groups. Echocardiography at 1 week after pPCI showed that, compared to control group, LVEF was higher in other three groups (P=0.004). At the end of 3 months follow up, MACEs occurred in four groups had no significant differences.Conclusion:Preventively intracoronary administration of anisodamine combined with rhBNP could improve myocardial perfusion and cardiac function in STEMI-PCI, anisodamine and rhBNP may have collaborative role in reducing myocardial infarction area.Part V Effect of rhBNP on renal function in STEMI-HF patients with mild renal insufficiency undergoing primary PCIObjectives:This study aims to investigate the effect of recombinant human brain natriuretic peptide (rhBNP) on renal function and contrast-induced nephropathy (CIN) incidence in ST-segment elevation myocardial infarction and heart failure (STEMI-HF) patients with mild renal insufficiency undergoing primary percutaneous coronary intervention (PCI).Methods:A total of 116 participants were randomized into rhBNP (rhBNP, n=57) and Nitroglycerin group (NIT, n=59), receiving intravenous rhBNP or nitroglycerin from admission to 72h after PCI. Renal function was assessed by serum creatinine (SCr), estimated glomerular filtration rate(eGFR), Cystatin-C(Cys-C) and β2-microglobulin before and after primary PCI, and calculated the incidence of CIN within 72h after PCI.Result:There were no significant differences in SCr, eGFR and β2-microglobulin between two groups (P>0.05, respectively). Compared with NIT group, the total urinary volume within 72 h was higher while the level of Cys-C at 24 and 72h after PCI were lower in rhBNP group. rhBNP was associated with a decline in incidence of CIN (12.28% vs.28.81%, P<0.05). No differences were detected in mortality and re-hospitalization in 3 months between two groups.Conclusion:The incidence of renal injury was not different between rhBNP and nitroglycerin in STEMI-HF patients with mild renal insufficiency. However, infusion of rhBNP was associated with a decline in incidence of CIN.