A Preclinical Systematic Review and Meta-Analysis of Astragaloside IV for Myocardial Ischemia/Reperfusion Injury

Astragaloside IV (AS-IV), the major pharmacological extract from Astragalus membranaceus Bunge, possesses a variety of biological activities in the cardiovascular systems. Here, we aimed to evaluate preclinical evidence and possible mechanism of AS-IV for animal models of myocardial ischemia/reperfusion (I/R) injury. Studies of AS-IV in animal models with myocardial I/R injury were identified from 6 databases from inception to May, 2018. The methodological quality was assessed by using CAMARADES 10-item checklist. All the data were analyzed using Rev-Man 5.3 software. As a result, 22 studies with 484 animals were identified. The quality score of studies ranged from 3 to 6 points. Meta-analyses showed AS-IV can significantly decrease the myocardial infarct size and left ventricular ejection fraction, and increase shortening fraction compared with control group (P < 0.01). Significant decreasing of cardiac enzymes and cardiac troponin and increasing of decline degree in ST-segment were reported in one study each (P < 0.05). Additionally, the possible mechanisms of AS-IV for myocardial I/R injury are promoting angiogenesis, improving the circulation, antioxidant, anti-inflammatory and anti-apoptosis. Thus, AS-IV is a potential cardioprotective candidate for further clinical trials of myocardial infarction.


INTRODUCTION
Acute myocardial infarction (AMI) was one of the leading causes of morbidity and mortality worldwide (Dariush et al., 2016). Acute interruption of coronary artery led to cardiomyocyte ischaemia and apoptosis (Luo et al., 2015). Invasive vascular reconstructions such as percutaneous coronary intervention and coronary artery bypass grafting can improve coronary perfusion (Richard, 2011), and thus they were widely adopted after weighing the risks of invasive diagnostics and the benefits in terms of diagnostic accuracy, risk stratification and assessment of the risks related to revascularization (Damman et al., 2015). Although treatment is usually directed at prompt restoration of flow in the occluded artery, reperfusion may trigger further injury beyond that induced by ischaemia alone (Maria et al., 2016). Such ischaemia/reperfusion (I/R) injury can markedly reduce the benefits of reperfusion therapies employed in myocardial infarction (MI) (Yellon and Hausenloy, 2007).
Astragaloside IV (AS-IV) (Figure 1) is one of the major and active components isolated from Astragalus membranaceus Bunge for tonifying Qi, and is a lanolin alcohol-shaped tetracyclic triterpenoid saponin with high polarity. Recent experimental studies (Ren et al., 2013;Li et al., 2017) demonstrated that AS-IV had pleiotropic anti-ischemic properties against focal cerebral ischemia/reperfusion injury, cardiovascular disease, pulmonary disease, liver fibrosis and diabetic nephropathy. AS-IV has multiple pharmacologic effects, including regulation of the calcium balance, antioxidative stress, anti-inflammatory, antiapoptosis antifibrotic, antidiabetes, immunoregulation, and cardioprotective effect via numerous signaling pathways (Schmidt et al., 2007;Ren et al., 2013;Li et al., 2017). In addition, systemic review of animal studies plays a critical role in drug development and the clarification of physiological and pathological mechanisms of clinical research (Rob et al., 2014). Thus, we conducted a preclinical systematic review to evaluate the effectiveness and the mechanisms of AS-IV for experimental MI.

Database and Literature Search Strategies
Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA)statement was followed (Stewart et al., 2015). Experimental studies assessing the effects of AS-IV in animal models of MI were identified from PubMed, EMBASE, Science Direct, Web of Science, wanfang data Information Site, Chinese National Knowledge Infrastructure (CNKI), and VIP information database by searching for all published articles from inception to May, 2018. The following key words were used: "astragaloside (MeSH Terms) OR astragaloside (Title/Abstract)" AND "myocardial infarction OR myocardial ischemia OR myocardial ischemia/reperfusion injury OR myocardial I/R injury, " Moreover, reference lists of potential articles were searched for additive studies.

Eligibility Criteria
We included studies of the effect of AS-IV in animal models of MI. To prevent bias, inclusion criteria were prespecified as follows: (1) experimental MI was induced by transient left anterior descending coronary artery (LAD) ligation or permanent LAD ligation or isoproterenol (ISO); (2) the treatment group was received AS-IV as monotherapy in any dose. Interventions for control group were isasteric and nonfunctional liquid (normal saline) or no treatment; (3) the primary outcome measures were MI size and/or left ventricular ejection fraction (LVEF) and/or shortening fraction (FS) and/or the level of ST-segment depression cardiac and/or enzymes and/or cardiac troponin T (cTnT) and/or cardiac troponin I (cTnI). The second outcome measures were mechanisms of AS-IV for myocardial I/R injury. Prespecified exclusion criteria were treatment with AS-IV conjunction with other compounds or AS-IV based prescriptions, non-myocardial ischemia model, no control group, duplicate publications, and no available data.

Data Extraction
Two independent authors extracted the following details from included studies: (1) publication year and the first author's name, model of MI (transient or permanent); (2) the characteristics of animals used including animal number, species, sex, weight, age, and any comorbidity; (3) model of myocardial I/R, and the anesthesia methods for model preparation; (4) the information of treatment group, including therapeutic drug dosage, method of administration, duration of treatment, and the same information of control group; (5) mean value and standard deviation of outcomes. If outcomes were performed at different time points, only the final test was included. If the experimental group of animals received various doses of the drug therapy, only the data of highest dose of the drug was included. If the data for metaanalysis were missing or only expressed graphically, we tried to contact the authors for further information, and where a response was not received, we measured data from the graphs using digital ruler software or exclude. For each comparison, we extracted data of mean value and standard deviation from each experimental and control group of every study.

Quality Assessment
We evaluated the methodological quality of the included studies using the collaborative Evidence-Based Complementary and Alternative Medicine approach to meta-analysis and review of animal data in experimental infarction (CAMARADES) 10-item quality checklist (Malcolm et al., 2004) with minor modification (Yu L. J. et al., 2017). One point was awarded for each of (1) publication in a peer-reviewed journal; (2) statement of temperature control; (3) random allocation to groups; (4) allocation concealment; (5) blinded assessment of outcome; (6) use of anesthetic without significant intrinsic cardioprotective activity; (7) appropriate animal model (aged, diabetic, or hypertensive); (8) sample size calculation; (9) compliance with animal welfare regulations; (10) statement of potential conflict of interests.

Statistical Analysis
All CI were considered as continuous data, and then an estimate of the combined effect sizes utilizing mean difference (MD) or standard mean difference (SMD) with the random effects model was given. In the present meta-analysis, the results using the random effects model were presented because heterogeneity between multistudies has to be taken into account. I 2 statistic was used to assess heterogeneity. The significance of differences among groups was assessed by partitioning heterogeneity and by using the X 2 distribution with degrees of freedom (df), where equals the number of groups. Probability values 0.05 were considered significant. All analyses were performed with Revman version 5.3 by the Cochrane Collaboration.

Study Characteristics
A total of 484 animals were included in the 22 studies, of which 234 were in the experimental group and 230 were in the control group. Eight studies Wang et al., 2012;Gong and Sun, 2013;Tu et al., 2013;Lu et al., 2015;Yu et al., 2015;Cheng et al., 2016; were published in English and 14 studies Guan et al., 2010;Liu et al., 2010;Cui et al., 2013;He et al., 2014;Liu and Yi, 2014;Qu et al., 2014;Zhang et al., 2014;Huang et al., 2015;Ma and Wang, 2015;Sun et al., 2015;Li and Yang, 2016; FIGURE 2 | Summary of the process for identifying candidate studies.

Summary of Evidence
In this preclinical systematic review, we assessed the efficacy of AS-IV for myocardial I/R injury according to 22 studies with 484 animals. The evidence available from present study revealed that AS-IV exerted potential cardioprotective function in acute MI largely through promoting angiogenesis, improvement of the circulation, antioxidant, anti-apoptosis and anti-inflammatory.

Limitations
First, no study provided calculation of sample size and blindness of model establishment and outcome measurement that is pesearch. Second, the deficiency of negative studies might lead the effecacy to be overestimated. Thus, the dominance of positive studies might lead the efficacy to be overestimated. Third, MI generally occurs in patients with other complications, such as old age, diabetes, hypertension, and hyperlipidemia (Blankstein et al., 2012); However, only 5 studies (Liu et al., 2010;Qu et al., 2014;Huang et al., 2015;Sun et al., 2015;Yu et al., 2015) selected appropriate animal model. Fourth, 3 studies (Liu et al., 2010;Gong and Sun, 2013; adopted female animals, it cannot be ignored that the heart protection of estrogen has been reported both in clinical and preclinical studies (Menazza et al., 2017).

Implications
The high-quality preclinical studies are crucial to transform preclinical data to clinic (Ramirez et al., 2017). Thus, we suggest that further design of the studies should refer to the arrival guidelines (Carol et al., 2010) and use appropriate animals, random allocation, blinded induction of model, and blinded assessment of outcomes to improve the accuracy of the results. The molecular and biological mechanisms of the cardioprorective effects of AS-IV have not been fully elucidated. The present study showed that AS-IV exerted the cardioprorection and the possible mechanisms are summarized as follows: (1) promoting angiogenesis and improving MVD Li and Yang, 2016; through increasing the expression of VEGF  and basic fibroblast growth factor (bFGF) ; (2) inhibition of apoptosis through down-regulating the expression of caspase-3 (Liu and Yi, 2014;Lu et al., 2015;Ma and Wang, 2015;Sun et al., 2015), and increasing the expression of Bcl-2 and reducing the expression of Bax protein in the cardiac myocytes Tu et al., 2013;Liu and Yi, 2014;Lu et al., 2015;Ma and Wang, 2015;Cheng et al., 2016;Wang et al., 2018); (3) improvement of the coronary flow by enhancing the expression of NO via up-regulating the expression of endothelial nitric oxide synthase (eNOS) Liu et al., 2010); (4) upregulating HIF-1α  and enhancing SOD-induced antioxidant via attenuating chondriokinesis to reduce the release of MDA Guan et al., 2010); otherwise, reducing the reactive oxygen species (ROS) to decrease myocardial cell lysis by regulating the PI3K/Akt/mTOR pathway (Zhang et al., 2014); (5) protecting against energy metabolism disorder through reducing the concentration of calcium in cardiac myocytes (Wang et al., 2012(Wang et al., , 2018Tu et al., 2013;Lu et al., 2015;Sun et al., 2015); (6) anti-inflammatory through inhibiting the expression of TNF-α  and NF-κB Lu et al., 2015;Cheng et al., 2016); (7) upregulating Notch1/Jagged1 signaling  which may be involved in infarct healing and cardiac repair Gude and Sussman, 2012). As mentioned above, cardioprotective mechanism of AS-IV for myocardial I/R injury was largely through promoting angiogenesis, improvement of the circulation, antioxidant, anti-apoptosis and anti-inflammatory.
It is well known that animal experiments have contributed to our understanding of effecacy and mechanisms for diseases (Hackam and Redelmeier, 2006). The present study showed AS-IV significantly decreased the MI size and cardiac enzymes, decreased cardiac troponin and increased the decline degree in ST segment. Therefore, it provides a preclinical evidencebased approach to develop AS-IV for acute MI. However, the translation of preclinical experiment which results in a prediction of the effectiveness of treatment strategies in clinical trials is still challenging (Hackam, 2007). The application of excessive drug doses and the timing of drug administration in animal models, which are inapplicable for human disease, are considered to be two of the main reasons for the failure to translate from animal models to human (Baker et al., 2014). In the present study, doses of AS-IV and timing for initial administration in animal models were inconsistent among the 22 included studies. Thus, we suggest further studies to determinate the optimal gradient doses and timing of administration in animal models of myocardial I/R injury. After that, given the huge gap between the animal studies and the clinical trials, the rigorous RCTs of AS-IV are needed.

CONCLUSION
We have provided a first comprehensive systematic review of AS-IV on animal studies and the findings indicate that AS-IV exerted potential cardioprotective function in acute myocardial I/R injury largely through promoting angiogenesis, improvement of the circulation, antioxidant, anti-apoptosis and anti-inflammatory.