Astragaloside IV for Experimental Focal Cerebral Ischemia: Preclinical Evidence and Possible Mechanisms

Astragaloside IV (AST-IV) is a principal component of Radix Astragali seu Hedysari (Huangqi) and exerts potential neuroprotection in experimental ischemic stroke. Here, we systematically assessed the effectiveness and possible mechanisms of AST-IV for experimental acute ischemic stroke. An electronic search in eight databases was conducted from inception to March 2016. The study quality score was evaluated using the CAMARADES. Rev Man 5.0 software was used for data analyses. Thirteen studies with 244 animals were identified. The study quality score of included studies ranged from 3/10 to 8/10. Eleven studies showed significant effects of AST-IV for ameliorating the neurological function score (P < 0.05); seven studies for reducing the infarct volume (P < 0.05); and three or two studies for reducing the brain water content and Evans blue leakage (P < 0.05), respectively, compared with the control. The mechanisms of AST-IV for ischemic stroke are multiple such as antioxidative/nitration stress reaction, anti-inflammatory, and antiapoptosis. In conclusion, the findings of present study indicated that AST-IV could improve neurological deficits and infarct volume and reduce the blood-brain barrier permeability in experimental cerebral ischemia despite some methodological flaws. Thus, AST-IV exerted a possible neuroprotective effect during the cerebral ischemia/reperfusion injury largely through its antioxidant, anti-inflammatory, and antiapoptosis properties.


Introduction
Radix Astragali seu Hedysari, milkvetch root (Huangqi), the dried root of Astragalus membranaceus (Fisch.) Bge. var. Mongolicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge., is a famous Traditional Chinese Qi-tonifying herb for a numerous disorders [1]. Huangqi was originally described in the Shennong Bencaojing (Shennong's Classic of Materia Medica), the earliest complete Pharmacopoeia of China written from Warring States Period to Han Dynasty [2]. Specifically, Huangqi has been used to treat stroke in China for thousands of years and elsewhere around the world in recent years. For example, Buyang Huanwu Decoction is a well-known classic herbal prescription for ischemic stroke [3], in which Huangqi is used as a major medicinal herb, that is, the sovereign drug based on traditional Chinese medicine formula theory. The chemical composition of Huangqi mainly includes saponins, polysaccharides, flavonoids, amino acids, and trace elements, and various biological activities have been reported [4]. Currently, more than 200 constituents are being identified [5].

Eligibility Criteria.
We prespecified the eligibility criteria as follows: (1) we included controlled studies of AST-IV Oxidative Medicine and Cellular Longevity 3 for experimental ischemic stroke; (2) the primary outcomes were measured as neurological function score (NFS), infarct volume (IV), and/or blood-brain barrier (BBB) permeability such as Evans blue and/or brain water content (BWC); the second outcome measures were mechanisms of AST-IV for ischemic stroke; (3) animal model of focal cerebral ischemia was induced by temporary middle cerebral artery occlusion (MCAO); (4) AST-IV was used merely in intervention group; (5) control animals received vehicle or no treatment. Prespecified exclusion criteria were as follows: (1) nonfocal cerebral ischemia model such as global, traumatic models, or hypoxicischemic models; (2) permanent MCAO; (3) combined use of any other agents; (4) no control group; (5) duplicate publications.

Data Extraction.
Two independent authors extracted the following details from included studies: (1) the first author's name and publication year, model of ischemic stroke, and the anesthesia methods for model preparation; (2) the specific information of animals for each study, including animal species, number, sex, and weight; (3) the treatment group's information, including therapeutic drug dosage, method of administration, duration of treatment, and the same information of control group; (4) the outcomes' data of mean value and standard deviation were extracted from each study, including NFS, IV, and/or BBB permeability, and timing for outcome assessments. The data of highest dose was included when the treatment group included various doses of the target drug. The result of the peak time point was included when the data were expressed at different times. Some records' published data were only expressed graphically, we made efforts to contact authors for further information, and when a response was not received, the numerical values were measured from the graphs by using digital ruler software.

Quality Assessment.
The study quality score was independently valued with the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) [21] 10-item quality checklist by two authors. One point was given for each of the following criteria: publication in a peer reviewed journal, control of temperature, random allocation to treatment or control, blinded induction of model, blinded assessment of outcome, use of an anesthetic without intrinsic neurogenesis activity, animal model (aged, diabetic, or hypertensive), performing a sample size calculation, compliance with animal welfare regulations, and a statement of potential conflicts of interest.

Statistical
Analysis. NFS, IV, and BBB permeability were considered as continuous data analyzed by using Review Manager (version 5.0). The estimate of the combined effect sizes was calculated by the standardized mean difference (SMD) utilizing the random effects model. 2 statistic was used to assess heterogeneity. Statistical significance was set at < 0.05, and the 95% confidence intervals (CIs) of all results were calculated.

Study Selection.
We identified 272 potentially relevant articles, of which 162 were duplicates. After screening the titles and abstracts, 54 papers were excluded because they were not (1) animal trials, (2) focal cerebral ischemia model, or (3) AST-IV in intervention group. We then read the remaining 56 full-text articles. Among them, 43 articles were deleted because 5 studies used other combined drugs in experimental group, 23 articles did not possess an appropriate outcome, and the remaining 15 studies were other types of publications. Finally, 13 studies were selected. The screening process is summarized in the flow diagram in Figure 2.

Summary of Evidence.
Thirteen studies with 244 animals were selected. The quality of many studies included was moderate. Treatment with AST-IV could reduce the IV and BBB and improve NFS outcomes during cerebral ischemia/reperfusion (I/R), suggesting that AST-IV exerted potential neuroprotection in acute ischemic stroke. Mechanisms of AST-IV for neuroprotective effects are largely mediated by its antioxidant, anti-inflammatory, and antiapoptosis properties. Despite the apparent positive results, we should interpret them with caution because of the methodological flaws.  overestimated. Third, high quality of control experimental study has significant impacts on reported outcome. However, many studies were of methodological flaws, suggesting that the weaknesses existed in the primary study.

4.3.
Implications. Systematic reviews of preclinical animal studies can help improve the methodological quality of animal experiments [22]. Adequate methodological details are crucial to value the quality of a body of evidence and to identify the risk of bias in trials. However, in the present study, the methodology's insufficiency exists in many fields, which potentially leads to an overestimation of treatment effects [23,24]. In particular, appropriate animal model is one of the important aspects to improve the quality of experimental design. All included studies were performed on healthy animals, in which the conditions of clinical stroke patients with morbidity may not accurately be replicated owning to stroke generally occurring in elders with comorbidities such as hyperglycemia or hypertension [25]. In addition, comorbidities can affect efficacy in animal models [26]. For instance, hypertension might attenuate neuroprotective effects [27]. Thus, there was a need to improve methodological standards in the design, conduct, and reporting of preclinical animal studies in the acute ischemic stroke.
Additionally, appropriate animal model with comorbidities such as aging, hyperglycemia, or hypertension should be used in experimental stroke research.
Neuroprotection for ischemic stroke was defined as an innovative strategy for antagonizing the injurious biochemical and molecular events that eventually resulted in irreversible ischemic injury [28]. Although there is identification of over 1000 effective neuroprotectants in animal studies and execution of over 100 clinical trials, successful translation of a neuroprotectant to the routine clinical use for stroke has not yet occurred [29]. Systematic reviews of preclinical stroke studies are useful in identifying the design-related factors such as poor methodological quality, differences in design between animal researches and clinical trials, and publication bias so as to improve the internal and external validity and thereby to increase the predictive value of experimental stroke [22]. Thus, systematic review of preclinical animal studies of stroke can contribute to more evidence-based translation of animal data from the bench to the bedside. The present study showed that AST-IV had potentially neuroprotective effect for acute ischemic stroke in animal models. It provides a preclinical evidence-based approach to the development of new treatments for acute ischemic stroke. Thus, findings of AST-IV from systematic review level in the present study may be collectively used to influence the decision for candidate drugs to be assessed in further clinical trials.
Once the cerebral I/R occurred, two-stage process will be triggered: acute injuries and delayed injuries. Acute injuries are associated with energy metabolism dysfunction, Li Figure 6: The forest plot: effects of Astragaloside IV for reducing the brain water content compared with middle carotid artery occlusion group.
oxidative stress, and the destruction of BBB. During period of I/R, the following inflammatory response and apoptosis exacerbate the injuries [30]. Several signalling pathways of cerebral I/R injury have been studied, including mitogenactivated protein kinase (MAPK) signalling pathways such as c-Jun NH2-terminal kinase 1/2 MAPK and p38 MAPK [31,32], Toll-like receptors (TLRs) signalling pathways such as TLRs/turn activates activated kinase 1/I B kinase complex and TLRs/turn activates activated kinase 1/mitogen-activated protein kinase kinase kinase [33], NF-B signalling pathway [34], phosphatidylinositol 3-kinase/Akt signalling pathway, and extracellular signal-regulated kinase 1/2 signalling pathway [35,36]. Partially based on the previous publications of mechanisms of AST-IV, it can inhibit apoptosis through the transforming growth factor-1/Smad2 signalling pathway [37], reduce oxidative stress via p38 MAPK pathway [38], diminish the myocardial I/R injury in rats by downregulating the TLR4/NF-B signalling pathway [39], and stimulate angiogenesis through the phosphatidylinositol 3-kinase/Akt pathway [40]. The present study indicated that AST-IV could attenuate oxidative stress [12,14,15,[17][18][19] and inhibit inflammatory stress [9-11, 14, 17] and apoptosis [8,14,15] in acute ischemic stroke of animal models by regulating the downstream signalling cascade molecules, proinflammatory mediators, inflammatory mediators, and antiapoptotic regulator. AST-IV exerted a neuroprotective effect during cerebral I/R injury, largely through its antioxidant, antiinflammatory, and anti-apoptosis properties. However, the signalling pathways of AST-IV during cerebral I/R were infrequently and incompletely reported. Thus, it is worth exploring this field in the future.

Conclusion
From a scientific and translational perspective, animal experiment researches should be appropriately designed, well conducted, thoroughly analyzed, and transparently and completely reported. Systematic review of animal studies can help improve the methodological quality of animal experiments and the more evidence-based translation of animal data to the clinic and contribute to the development of replacement, reduction, and refinement of animal experiments. In the present study, the findings demonstrated that AST-IV could improve NFS and IV and reduce the BBB permeability in animal models of focal cerebral ischemia largely through its antioxidant, anti-inflammatory, and antiapoptosis effects. Thus, AST-IV is a potential candidate neuroprotectant in further stroke clinical trials.