Astragaloside Ⅳ Improves Cardiac Function Via Angiogenesis In Mice After Myocardial Infarction

BackgroundAstragalus membranaceus is a traditional Chinese herb which has lots of beneficial effects on human body, such as enhancing immune function, protecting liver injury, stimulating angiogenesis, enhancing diuresis and lowering blood pressure. It also has been widely used in the management of cardiovascular diseases. Astragaloside IV (AS-IV) is one of the major active compounds of Astragalus membranaceus. AS-IV exerts various beneficial effects on cardiovascular system, including anti-inflammatory, anti-apoptotic and anti-fibrosis activity. Meanwhile, AS-IV can improve cardiac function in rat and mice after myocardial infarction (MI), but the underling mechanisms are still uncertain and need to be further studied.MI and its end stage, especially heart failure, are the major causes of cardiac mortality and morbidity. After MI, the preservation of cardiac function is largely dependent on revascularization in the board zone of infarction. However, decreased myocardial capillary density and insufficient angiogenesis are often observed in this area. Microvascular rarefaction and enlarged perivascular space trigger cardiomyocyte hypoxia and necrosis, which then cause further myocardium injury and enlarged infarct area. Therefore, proangiogenic therapies which target promoting reperfusion of ischemic myocardium may be a potential strategy to attenuate adverse left ventricular remodeling and improve cardiac function.AS-IV has proangiogentic effect on endothelial cell proliferation and migration in vitro. In skin recovery of wound, AS-IV can accelerate the healing process by stimulating angiogenesis. Our previous findings showed that AS-IV can modify vascular aort function in rat. AS-IV also can reverse mitochondrial function injured by Angiotensin-II stimulation in cultured vascular smooth muscle cells. Whether AS-IV could stimulate angiogenesis in myocardium and thereby improve cardiac function remains further exploration.ObjectiveThe present study was designed to investigate whether AS-IV can improve cardiac function of MI mice and the underlying mechanism.Materials and Methods1. MI models preparation. MI moldel was established by permanent ligation of left anterior descending branch (LAD) under the microscope. C57/BL6 mice underwent this operation at 8-10 weeks of age and 23-25 g of weight.2. Astragaloside IV injection preparation. Astragaloside IV was dissolved in hydroxypropyl-beta-cyclodextrin (HPBCD) completely to prepare the injection of Astragaloside Ⅳ at 1mg/ml.3. The experimental groups and treatment strategy. Mice successfully revivaled from MI operation were randomly distributed into CTL group (n=12) or AS-IV group (n=12). Mice without permanent ligation of LAD was enrolled into Sham group (n=6). 10mg/kg AS-Ⅳ were injected intraperitoneally everyday until 14 days after the operation.4. Cardiac function. Two weeks after MI operation, transthoracic two-dimensional echocardiography was applied to measure left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) of the mice.5. Cardiac remodeling detection:LV mass and left ventricular internal diameter at end-diastolic (LVIDd) were detected by ransthoracic two-dimensional echocardiography. Morphological measurement included body weight (BW), heart weight (HW) and left ventricular weight (LVW).6. Effects of AS-IV on angiogenesis in MI micea) Detection of vessel density in central infarct zone, board zone and remote zone by immunofluorescence of CD31;b) Detection of angiogenesis and arteriogenesis in terminal infarct zone and board zone by double-immunofluorescence of VEGFR3/CD31and SOX17/CD31;c) Detection of D11-Notch signaling pathway in board zone by double-immunofluorescence of D114/CD31and Notcl/CD31;d) Detection of endothelial proliferation in board zone by double-immunofluorescence of Ki67/CD3.Results1. Compared with Sham-operated mice, MI mice showed significant lower LVEF and LVFS levels (41.3% vs 62.6%,20.3% vs 33.3%, respectively)? indicating impaired cardiac function and HF. Two weeks treatment of AS-IV effectively enhanced the cardiac function. Significant improvements of LVEF and LVFS levels (47.0% vs 41.3%,23.1% vs 20.3, respectively) were observed in AS-IV treated mice.2. Ventricular aneurysm and significantly increased LV mass (115.2mg vs 88.7mg) and LVIDd (4.45mm vs 3.55mm) were observed in CTL mice. AS-IV treatment significantly reduced LV mass (102.6mg vs 115.2mg) and LVIDd (3.99mm vs 4.45mm) when compared with that of CTL mice.3. Morphometric analysis showed that untreated MI mice had significant higher HW/BW ratio (5.8mg/g) and LVW/BW ratio (4.3mg/g) compared with non-infarct mice. Two weeks treatment of MI mice with AS-IV (10mg/kg) remited cardiac hypertrophy and significantly decrease HW/BW ratio (5.0mg/g) and LVW/BW ratio (3.7mg/g).4. Compared with left ventricular anterior wall of non-infarct mice, MI mice showed reduced vessel density in both central infarcted zone and board zone. Treatment of MI mice with AS-IV significantly increased the vessel density in the board zone.5. Compared with the corresponding regions of sham-operated heart, newly formed vessels and regenerated arteries were significantly increased in the terminal zone of infarction. AS-IV treatment stimulates more generation of vessels and arteries.6. After MI, more new vessels were observed in the board zone of MI mice than that in the corresponding regions of sham-operated heart (31.7% vs 16.7%). Compared with MI mice, AS-IV treatment led to more generation of new vessels (51.5% vs 31.7%). Compared with mice in Sham group, the number of regenerated arteries was similar in the board zone of MI mice. AS-IV treatment significantly increased the number of regenerated arteries when compared with mice in CTL group.7. Compared with left ventricular anterior wall of non-infarct mice, endothelial D114 signaling pathway was receded in the board zone of infaction (31.1% vs 65.6%). This reduction was significantly reversed by AS-IV treatment (45.2% vs 31.1%). There is no significant difference of Notchl expression ratio in Sham-operated mice, untreated mice and AS-IV treated mice (79.9%,73.8% and 72.8%, respectively).8. Proliferative endothelial cells were significantly decreased in the board zone of infarction when compared with the sham-operated mice. Treatment of MI mice with AS-IV for two weeks significantly reversed this decrease.ConclusionsIn the present study, AS-IV attenuate cardiac remodeling and improve cardiac function in mice suffered from MI. The enhancement of angiogenesis and arteriogenesis by AS-IV treatment may be one of the underlying mechanisms.