Ginsenoside-Rb1 for Ischemic Stroke: A Systematic Review and Meta-analysis of Preclinical Evidence and Possible Mechanisms

Background Ischemic stroke is the most common type of stroke, while pharmacological therapy options are limited. Ginsenosides are the major bioactive compounds in Ginseng and have been found to have various pharmacological effects in the nervous system. In the present study, we sought to evaluate the effects of Ginsenoside-Rb1 (G-Rb1), an important ingredient of ginsenosides, and the probable neuroprotective mechanisms in experimental ischemic strokes. Methods Studies of G-Rb1 on ischemic stroke animal models were identified from 7 databases. No clinical trials were included in the analysis. The primary outcome measures were neurological function scores, infarct volume, evans blue content and/or brain water content (BWC). The second outcome measures were the possible neuroprotective mechanisms. All the data were analyzed by Rev Man 5.3. Result Pooled preclinical data showed that compared with the controls, G-Rb1 could improve neurological function (Zea Longa (n = 367, P < 0.01); mNSS (n = 70, P < 0.01); Water maze test (n = 48, P < 0.01); Bederson (n = 16, P < 0.01)), infarct area (TTC (n = 211, P < 0.01); HE (n = 26, P < 0.01)), as well as blood-brain barrier function (BWC (n = 64, P < 0.01); Evans blue content (n=26, P < 0.05)). It also can increase BDNF (n = 26, P < 0.01), Gap-43 (n = 16, P < 0.01), SOD (n = 30, P < 0.01), GSH (n = 16, P < 0.01), Nissl-positive cells (n = 12, P < 0.01), Nestin-positive cells (n = 10, P < 0.05), and reduce Caspase-3 (n = 36, P < 0.01), IL-1 (n = 32, P < 0.01), TNF-α (n = 72, P < 0.01), MDA (n = 18, P < 0.01), NO (n = 44, P < 0.01), NOX (n = 32, P < 0.05), ROS (n = 6, P < 0.05), NF-κB (P < 0.05) and TUNEL-positive cells (n = 52, P < 0.01). Conclusion Available findings demonstrated the preclinical evidence that G-Rb1 has a potential neuroprotective effect, largely through attenuating brain water content, promoting the bioactivities of neurogenesis, anti-apoptosis, anti-oxidative, anti-inflammatory, energy supplement and cerebral circulation.


INTRODUCTION
Globally, stroke is the second most common cause of death and the most common cause of long-term disability (Kassebaum et al., 2016;Feigin et al., 2017), causing a huge burden with approximately 10.3 million new cases of stroke and 113 million disability-adjusted life years every year (Pandian et al., 2018). The two major mechanisms causing brain damage in stroke are ischemic and hemorrhagic, where ischemic stroke occurs more frequently according to the epidemiological research (Meyers et al., 2011;Venketasubramanian et al., 2017).
Ischemic stroke, in most instances, is caused by a transient or permanent occlusion (either by an embolus or by local thrombosis) in cerebral arteries, which leads to a sharp reduction in cerebral blood flow (CBF) (Dirnagl et al., 1999). In order to maintain normal physiological activity, the brain requires substantial amounts of oxygen and glucose as brought by CBF. Immediately after ischemia, neurons are deprived of oxygen and energy, and become unable to preserve normal transmembrane ionic gradients and homoeostasis . Cerebral ischemia initiates several pathologic processes, including oxidative and nitrative stress, inflammation, apoptosis, ion imbalance, calcium overload, and energy depletion (Terasaki et al., 2014;Jayaraj et al., 2019), leading to neurovascular unit dysfunction and neurologic deficits in ischemic stroke. In the central area of an ischemic stroke, CBF is severely in deficit and cells die rapidly. But in the ischemic penumbra, an area of damaged but not yet dead brain tissue after focal cerebral ischemia, CBF deficit is milder which makes it a salvageable area (Lo, 2008). Therefore, therapeutic candidates that can prompt restoration of CBF and improve neurological dysfunction have become an important research hotspot.
Intravenously recombinant tissue-type plasminogen activator (tPA) has been approved by the Food and Drug Administration as the effective pharmacological therapy for acute ischemic stroke since 1996 (National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995;Sandercock et al., 2012;Berge et al., 2016). However, only a minority of acute ischemic stroke patients can receive it because of the narrow therapeutic time window and several contraindications to its use, especially the risk of a fatal symptomatic intracranial hemorrhage (Demaerschalk et al., 2016). Beyond the beneficial effects, artery recanalization can also lead to severe adverse reactions, for example, cerebral ischemia/reperfusion injuries (Jickling et al., 2014). Given the present dilemma, it is necessary to seek other novel pharmacological treatment modalities.
Ginseng is a famous herb medicine and has been used as a tonic to improve a wide variety of disorders for millennia (Lee et al., 2015). In modern time, it is continuously and widely used worldwide (Kim, 2012). Ginsenosides, which are responsible for the pharmacologic effects, are extracted from Ginseng and often divided into two different groups: the 20 (S)-protopanaxatriol group (ginsenosides Re, Rf, Rg1, Rg2, and Rh1) and the 20 (S)protopanaxadiol group (ginsenosides Ra1, Ra2, Ra3, Rb1, Rb2, Rb3, Rc, Rd, Rg3 and Rh2) (Ong et al., 2015;Jin et al., 2019). Randomized, double-blind, placebo-controlled, multicenter clinical trials have reported that ginsenosides could significantly improve the overall distribution of disability scores on the modified Rankin scale after acute ischemic stroke. It could also improve scores on the National Institutes of Health Stroke Scale with similar mortality and adverse event rates when compared with placebo (Liu et al., 2009;Liu et al., 2012). Ginsenoside-Rb1 (G-Rb1) (the chemical structure is shown in Figure 1) is regarded as the major active ingredient (Lee et al., 2015), which has been proven to cause a wide range of biological activities, especially in regards to its neuroprotective role, probably through anti-inflammatory, anti-oxidative, antiapoptosis, anti-stress and anti-depressive effects in vivo and in vitro (Kim, 2012;Kim et al., 2013;Ong et al., 2015;Rokot et al., 2016;Wang et al., 2018).
Animal experiments are often conducted to test the effects of a drug before proceeding to clinical trials. However, there are some defects in animal experiments. For one thing, it is not feasible to imitate all aspects of human ischemic stroke in an animal model because of the complex pathophysiology and its heterogeneous nature. For another, most studies are conducted in young animals without any comorbidities, which is different from human stroke as it particularly affects elderly people with the additional risk factor of cerebrovascular diseases (Fluri et al., 2015). Thus, the results of animal experiments are dependent on the type of animal model used (Roberts et al., 2002). The transient or permanent middle cerebral artery occlusion (MCAO) model is one of the closest models to human ischemic stroke, which is characterized by reliable and well-reproducible infarcts and has been applied in the majority of studies on the pathophysiological processes or neuroprotective agents of ischemic stroke (Fluri et al., 2015).
An objective and quantitative systematic review is a type of secondary research, which collates all primary studies that fit prespecified eligibility criteria to solve a specific research problem, which can minimize bias (Higgins et al., 2011). Systematic reviews can offer credible evidence and be helpful for selecting the appropriate drug administration for clinical trials (van Luijk et al., 2013). But up until now, there has been no systematic review to pool the therapeutic effects of G-Rb1 in preclinical studies. Thus, in the present study, we aimed to evaluate the effects and mechanisms of G-Rb1 monotherapy for focal ischemic stroke in animal models using a systematic review approach.

MATERIALS AND METHODS
This systematic review and meta-analysis is reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement (Moher et al., 2010) and our previous study .

Search Strategy
A comprehensive search was performed to find studies evaluating the effects of G-Rb1 treatment on animal models of ischemic stroke from PubMed, Web of Science, the Cochrane Library, Embase, VIP information database, Wanfang Data Information Site, and the Chinese National Knowledge Infrastructure. All of the searches were conducted from the inception to September 27 th , 2019 without language or publication status restrictions. The following search strategy was used for PubMed and was modified to suit other databases: (ginseng OR ginsenoside OR Ginsenoside-Rb1 OR G-Rb1) AND (stroke OR "middle cerebral artery occlusion" OR "middle carotid artery occlusion" OR MCAO OR "brain infarct*" OR "brain ischemi*" OR "cerebral infarct*" OR "cerebral ischemi*"). The Chinese databases were also searched by the above search terms, translated into Chinese.

Inclusion Criteria
We included controlled studies evaluating the effects of G-Rb1 treatment on experimental ischemic stroke. To prevent bias, the following inclusion criteria were prespecified: (1) focal cerebral ischemia, induced by transient middle cerebral artery occlusion (MCAO) or permanent MCAO; (2) no restriction on animal species, as well as gender, age, weight and sample size; (3) the experimental group was administered by G-Rb1 monotherapy, while the control group received a vehicle, saline, or positive control drug or no treatment. There was no restriction on dosage, mode and time of initial treatment. The primary outcome measures were neurological function score (NFS), infarct volume (IV), evans blue content and/or brain water content (BWC). The secondary outcome measures were possible mechanisms of G-Rb1 for cerebral ischemic stroke.

Exclusion Criteria
The prespecified exclusion criteria were: (1) clinical articles, case reports, comments, reviews, abstracts and in vitro studies; (2) using global models, traumatic models or hypoxic-ischemic models; (3) not using a cerebral ischemia model; (4) the intervention for the experimental group was a combination of G-Rb1 and another drug; (5) lacking a control group.

Data Extraction
According to the previous studies Cascella et al., 2018), the following details were collected from the included studies independently by two authors: (1) name of the first author and the publication year; (2) the characteristics of animals including animal species, gender, number and weight; (3) type of anesthetic; (4) treatment information, including the establishment of an ischemic stroke model and the method of administration; (5) outcome measures and timings for outcome assessments. If the experimental group used different doses, only the highest dose was extracted. If outcomes were presented at different time points, only the last time point was extracted. If the data were expressed graphically, we extracted it by use of digital ruler software. The mean value and standard deviation were extracted from the data of experimental and control groups of each comparison.

Risk of Bias Assessment
The Collaborative Approach to Meta-Analysis and Review of Animal Data in Experimental Studies (CAMARADES) 10item quality checklist was proposed by Macleod et al., (2004) for studying the efficacy of candidate drugs in experimental stroke. Two investigators independently assessed the methodological quality of the included studies based on the checklist as follows: (1) publication in a peer-reviewed journal; (2) statement of control of temperature; (3) randomization to treatment or control; (4) blinded induction of ischemia; (5) blinding of outcome assessment; (6) no obvious intrinsic neuroprotective effect of anesthetic; (7) appropriate animal model such as aged, diabetic, or hypertensive; (8) sample size estimation; (9) compliance with animal welfare regulations; (10) declared any potential conflict of interest. Each item of the 10-item scale contributed one point, and each study was given an aggregate quality score. Any disagreements were settled through consultation with the corresponding author.

Statistical Analysis
The mean difference (MD) with 95% confidence intervals (CI) was used as a summary statistic when outcome measures in all studies applied the same scale. On the contrary, standardized mean difference (SMD) with 95% CI was used when the same outcome index was measured in various ways. Statistical heterogeneity was assessed by the I-square test. A randomeffects model test was conducted when there was significant heterogeneity (I 2 > 50%). Otherwise, the fixed-effects model test was adopted. Sensitivity analysis and subgroup analysis were carried out to find the origin of heterogeneity. A probability value of less than 0.05 was considered statistically significant. Data analyses were performed by Rev Man 5.3.
According to the pre-established inclusion and exclusion criteria, studies included in this systematic review generally show that: the baseline status of experimental animals remained consistent in each independent study; the animal subjects had the same disease model of focal cerebral ischemia that was induced by middle cerebral artery occlusion; all animal subjects in experimental groups received G-Rb1 monotherapy while those in control groups received normal saline or no treatment.
Summary of the methodological quality was presented in Table 3.
Bederson score: Dong et al. (2017) found that Bederson scores were lower in the G-Rb1 group than in the control group (n = 16, P < 0.01). Jiang et al. (2013) showed a similar result.
Others: One study (Fan, 2006) showed a beneficial effect of G-Rb1 for reducing IV according to MRI scans (n = 8, P < 0.05). One study with 2 comparisons (Zhang et al., 1998) did not mention the measuring method and showed no significant difference of G-Rb1 compared with the control (P > 0.05).
Meanwhile Gao et al. (2010) (n = 10, P < 0.05), evaluating Nestin-positive cells, reported an obvious effect of G-Rb1 on increasing the number of neural precursors cells.

Summary of Results
This is the first preclinical systematic review to evaluate the efficacy of G-Rb1 for experimental cerebral ischemia/reperfusion injury. Twenty-eight studies with 937 animals were selected. The quality of the included studies was generally moderate. The evidence available from the present study showed that G-Rb1 substantially reduced IV and improved NFS and BWC in focal cerebral ischemia animal models. Thus, G-Rb1 exerted a  potential neuroprotective function, largely through the promotion of neurogenesis; anti-apoptosis, anti-oxidative and anti-inflammatory effects; and the improvement of energy metabolism and cerebral circulation.

Limitations
Clinically, ischemic stroke usually occurs in elderly patients or those with hypertension or diabetes. However, only one study structured an experimental ischemic stroke model in aged rats and two studies in hypertensive rats. Several other methodological weaknesses also existed in the primary studies. Nineteen (76%) studies claimed randomization, but only 4 (16%) trials provided specific information as to the randomization generation. Blinded assessment of outcomes is necessary to minimize performance and detection bias, but only 9 (36%) studies mentioned a masked assessment of outcomes. An adequate sample size is important for study design, but only one study described a sample size calculation. Lacking a formal sample size calculation results in dubious statistical analysis validity.

Implications
Ginseng is a famous herbal medicine and has been deeply researched. Ginsenosides, the major active pharmacological constituents of ginseng, are usually divided into the 20 (S)protopanaxatriol group (ginsenosides Re, Rf, Rg1, Rg2, and Rh1) and the 20 (S)-protopanaxadiol group (ginsenosides Rb1, Rb2, Rc, Rd, Rg3 and Rh2) (Ong et al., 2015;Jin et al., 2019). G-Rb1 is considered to be the major ginsenoside (Kim, 2018), which is enriched in the roots and is also present in stems and leaves (Yu et al., 2019). A recent study illustrated that in the human body, G-Rb1 shows a longer half-life and a higher plasma concentration, compared to other ginsenosides, when taking red ginseng extract for 2 weeks, suggesting a stable absorption and slow elimination process (Jin et al., 2019). In terms of nonclinical pharmacokinetic behavior, the bioavailability of Rb1 in rats is about 1.18-4.35% with slowed renal excretion when the oral dose is about 10-104 mg/kg. (Won et al., 2019).
G-Rb1 has been proven to be linked to a wide range of biological activities, such as its neuroprotective role and antitumor activities. In the present study, we reviewed the possible neuroprotective mechanisms of G-Rb1 on experimental ischemic stroke and summarized as follows: i. Reduction of brain edema Brain edema is significantly associated with the poorer functional outcomes of ischemic stroke. Aquaporin-4 is a significant water channel protein mainly expressed in astrocytes throughout the central nervous system (CNS), especially in foot processes at the blood-brain barrier (BBB) (Verkman et al., 2017). A recent study showed that G-Rb1 can significantly reduce the c o nt e nt o f A Q P 4 in th e i s c h em ic p en u m b r a , hippocampus, and striatum , and reduce brain edema. Additionally, matrix metalloproteinases have been shown to be strongly associated with brain edema after BBB disruption (Chen et al., 2015), while cerebral vessel formation and stabilization can alleviate brain edema (Lu, 2010), which is important for the development of new therapeutic approaches. ii. Promotion of neurogenesis G-Rb1 had a positive effect on neurogenesis probably through increasing BDNF and Gap-43 levels (Jiang et al., 2013;Gao, 2017). BDNF is highly expressed in the CNS and contributes to the maintenance of neurons. BDNF/TrkB system and its downstream intracellular signaling pathways, such as ERK-, Akt-, and PLCg-pathways, are necessary conditions for neuron survival and synaptic plasticity (Numakawa et al., 2017). Axonal growth cones guide and promote the growth of axons during nervous system development and regeneration in areas where GAP-43 is abundantly expressed (Kusik et al., 2010). In addition, G-Rb1 promotes the growth of neurons and axonal branches probably through activating the cAMP-PKA-CREB signaling pathway (Gao, 2017) or inhibiting the NgR/ PhoA signaling pathway (Ren, 2005). iii. Anti-apoptosis Apoptosis is an essential part of the pathogenesis of acute and/or chronic neurodegenerative diseases, for example, ischemic stroke, which can be responsible for neuronal death and irreversible cerebral dysfunction (Khoshnam et al., 2017). Inhibition of apoptosis could alleviate cerebral injury in stroke models (Radak et al., 2017). Caspase is the general name of the cysteine proteases family, which dominates the apoptosis process. Caspase-2, -8, -9, -10, -11, and -12 are regarded as initiator Caspases that are closely related to pro-apoptotic signals. Once initiator Caspases are activated, downstream effector Caspases, such as Caspase-3, begin to cleave targeted cellular proteins and perform apoptosis (Kuranaga and Miura, 2007). G-Rb1 was reported as contributing to the reduction of Caspase-3 (Fan, 2006;Gao et al., 2010;Zeng et al., 2014;Lv et al., 2016) and causing an anti-apoptosis effect. iv. Anti-oxidative activity Oxidative stress plays an important role in the pathogenesis of ischemic stroke (Luo et al., 2018). Free radicals can be excessively produced-particularly in the ischemic areas-promoting lipid peroxidation, protein breakdown and DNA damage, which leads to cellular apoptotic neuronal damage (Milanizadeh et al., 2018). G-Rb1 plays an antioxidative role through increasing the activity of SOD (Zeng et al., 2014;Dai et al., 2016) and GSH levels (Dong et al., 2017) and decreasing the concentration of MDA (Zeng et al., 2014;Dai et al., 2016;Dong et al., 2017), NO (Lv et al., 2016;Dong et al., 2017;Liu et al., 2018) and the activity and expression of NOX (Chen et al., 2015;Dong et al., 2017). The pooled data indicated that G-Rb1 significantly inhibited oxidative stress reactions and reduced the neurotoxicity of free radicals. v. Anti-inflammation The present study showed that G-Rb1 exerts anti-inflammatory effects by decreasing the expression of pro-inflammatory cytokine IL-1 (Jiang et al., 2013;Liu et al., 2013;Chen et al., 2015), IL-6 (Zhu et al., 2012;Yu et al., 2013;Liu et al., 2018) and TNF-a (Zhu et al., 2012;Jiang et al., 2013;Yu et al., 2013;Liu et al., 2018) as well as the expression of HMGB1 . In addition, the NF-kB signaling pathway (Zhu et al., 2012;Zeng et al., 2014;Lv et al., 2016;Liu et al., 2018) could be the mechanism through which this occurs. Inflammation is a well-recognized pathological event which can be responsible for secondary brain tissue damage following ischemic stroke (Rajkovic et al., 2018). The proinflammatory cytokines induce nerve tissue damage in the ischemic area of the brain largely through down-regulation of microcirculation, enhancing the pro-thrombotic processes and releasing other neurotoxic cytokines (Cieslak et al., 2013). HMGB1 is a well-researched nonhistone DNA-binding protein located in the nucleus. Once there are infections or tissue injuries, HMGB1 can be upregulated by immune cells or necrotic cells (Lotze and Tracey, 2005). In cerebral ischemia/reperfusion processes, it triggers delayed inflammation and exacerbates neuron damage . On the other hand, the NF-kB family, consisting of transcription factors, and its associated signaling pathway play a complex but crucial role in the regulation of immune response (Tilborghs et al., 2017). vi. Improvement of energy metabolism Neurons have higher energy expenditure and lower reserves compared to other cell types (Belanger et al., 2011). After cerebral ischemia a lack of glucose and oxygen, and consequently adenosine triphosphate, is one of the major events that results in energy failure (Hu et al., 2017). Numerous interactions of transporters, enzymes, and intracellular signaling processes within the neurovascular unit at the BBB participate in glucose transport, where GLUT1 and GLUT3 are regarded as the major glucose transporters (Patching, 2017). In the present study, G-Rb1 increased the expression of GLUT1 and GLUT3 in cerebral ischemic penumbra indicating that it can maintain the energy supply of the injured brain, which may be one of the mechanisms of its protective effect (Tao, 2004;Li et al., 2005). vii. Improvement of cerebral circulation In cerebral ischemic stroke, clinical evidence shows that early revascularization is a critical process for rescuing salvageable tissue (Wardlaw et al., 2014;Goyal et al., 2016). In the present study, 3 studies that were investigated found that G-Rb1 has a positive effect on improving cerebral blood flow (Dai et al., 2016;He et al., 2016;Lv et al., 2016) probably by activating A2a receptors and the cAMP-PKA-KATP signaling pathway in vascular endothelial cells and vascular smooth muscle cells.
More details were summarized in Table 4 and Figure 11. In addition to the mechanisms mentioned above, the specific molecular mechanism needs further research.
In the present study, chloral hydrate is the most common anesthetic used in 16 (64%) included studies. Although it has relatively few effects on cardiovascular function and no neuroprotection in stroke studies (Ozden and Isenmann, 2004), chloral hydrate has raised questioned regarding its prolonged recovery, mutagenic effects, and carcinogenic effects in animal use. Currently, isoflurane has been suggested as a replacement for chloral hydrate, because it is easy to administer and to titrate, has a rapid onset and recovery period, an adequate and reproducible anesthesia depth, minimal cardiac depression, and ethical considerations (Maud et al., 2014). Although previous studies demonstrated that anesthetics including ketamine, isofl urane, and halothane had potential neuroprotective effects (Kapinya et al., 2002;Xiong et al., 2003;Kitano et al., 2007b;Wang et al., 2008;Deng et al., 2014;Tang et al., 2015), isoflurane can be an appropriate anesthetic agent when establishing the MCAO model (Kitano et al., 2007a).
Ischemic stroke animal models are an indispensable tool for identifying the mechanisms of ischemic stroke and developing new agents for stroke therapy. Currently, stroke experiments are mainly carried out in small animals, such as mice, rats and rabbits (Fluri et al., 2015). Rodents are one of the most commonly used animals in stroke studies for the following advantages: the cerebrovascular system and physiology of the rat is similar to that of humans (Yamori et al., 1976); its moderate body size allows easy monitoring of physiological parameters; its small brain size is makes it ideal for fixed procedures, for example, in vivo cryocapture for biochemical analysis (Ponten et al., 1973); there is a relative homogeneity within strains (Strom et al., 2013); and most of all, it is easy to conduct reproducible studies. In addition, large animal models, such as non-human primates, are more similar to human anatomy and pharmacodynamics (van Hout et al., 2016); however, it has weaknesses as they are more expensive, difficult to manipulate, and raise accompanying ethical issues. Thus, in future research, we should select an ideal model that usually is a biological representative of human disease, inexpensive, reproducible, easily manipulated, and ethically sound according to the experimental purpose.
There are some differences between animal and human ischemic strokes. For one thing, the anatomy of the brain and cerebral vessels varies between species, which results in different patterns of ischemic damage (Sommer, 2017). For another, ischemic stroke in humans preferentially affects elderly patients with multiple risk factors of cerebrovascular diseases (such as diabetes mellitus, hypertension, hyperlipidemia, obesity), suggesting that the heterogenicity of human stroke requires complex interventions (Fluri et al., 2015). Experimental stroke is usually performed on young, healthy, male rodents and under highly standardized and well-controlled conditions, which might be beneficial for reducing infarct volume (Dirnagl, 2016;Becker, 2016). In addition, anesthesia can be another influence factor. It is reported that anesthetics play a role in affecting blood pressure, CBF and metabolism. Moreover, anesthetics may have neuroprotective effects, thus modulating some aspects of post-ischemic processes (Koerner and Brambrink, 2006).
Blood hypercoagulability and thromboembolism play important roles in the pathophysiology of cerebrovascular disease. A recent study evaluated a new-developed "nanoplatelet" (tP-NP-rtPA/ZL006e) in rats that can deliver thrombolytics and neuroprotectants sequentially to the site of the thrombus and ischemic penumbra, for a maximized combinational effect (Xu et al., 2019). Meanwhile, G-Rb1 was found to have anticoagulant and antianemia effects in rodents with cancer , which could be a potential neuroprotective agent from the perspective of pathophysiology. Converting experimental results into clinically effective treatment methods creates a bottleneck, especially in the research of cerebrovascular diseases. Thus, preclinical research process must be rigorously designed and closely follow the preset protocols, which could strengthen the internal validity of experimental studies (Zinsmeister and Connor, 2008). Stroke Therapy Academic Industry Roundtable has given suggestions to improve the quality of preclinical studies and improve the rate of clinical translation (STAIR, 1999). As for preclinical systematic reviews, according to Moher et al., there would be around a 40% magnification of treatment efficacy statistically when low-quality trials are included in the pool (Moher et al., 1998). Thus, we recommend classic CAMARADES 10-item checklist (Macleod et al., 2004), which is an international collaboration established in 2004, aiming to support meta-analyses of animal data, especially for experimental ischemic stroke.

CONCLUSION
Pooled data from the present study demonstrated that G-Rb1 can reduce IV, attenuate NFS and BWC, and thus have potential neuroprotective effects in the animal model of ischemic stroke, largely through enhancing neurogenesis, anti-apoptosis, anti- oxidation, anti-inflammation, improving energy metabolism and improving cerebral circulation. Although some factors such as study quality and methodological flaws may undermine the validity of positive findings, this systematic review provides an experimental evidence-based approach to translate new therapies for ischemic stroke.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/ Supplementary Material.