Preclinical Evidence and Possible Mechanisms of Rhodiola rosea L. and Its Components for Ischemic Stroke: A Systematic Review and Meta-Analysis

Background: Rhodiola rosea L. has long been used as traditional medicines in Europe and Asia to treat a variety of common conditions and diseases including Alzheimer’s disease, cardiovascular disease, cognitive dysfunctions, cancer, and stroke. Previous studies reported that Rhodiola rosea L. and its components (RRC) improve ischemia stroke in animal models. Here, we conducted a systematic review and meta-analysis for preclinical studies to evaluate the effects of RRC and the probable neuroprotective mechanisms in ischemic stroke. Methods: Studies of RRC on ischemic stroke animal models were searched in seven databases from inception to Oct 2021. The primary measured outcomes included the neural functional deficit score (NFS), infarct volume (IV), brain water content, cell viability, apoptotic cells, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells, B-cell lymphoma-2 (Bcl-2) level and tumor necrosis factor-α (TNF-α) level. The secondary outcome measures were possible mechanisms of RRC for ischemic stroke. All the data were analyzed via RevMan version 5.3. Results: 15 studies involving 345 animals were identified. Methodological quality for each included studies was accessed according to the CAMARADES 10-item checklist. The quality score of studies range from 1 to 7, and the median was 5.53. Pooled preclinical data showed that compared with the controls, RRC could improve NFS (Zea Longa (p < 0.01), modified neurological severity score (mNSS) (p < 0.01), rotarod tests (p < 0.01), IV (p < 0.01), as well as brain edema (p < 0.01). It also can increase cell viability (p < 0.01), Bcl-2 level (p < 0.01) and reduce TNF-α level (p < 0.01), TUNEL-positive cells (p < 0.01), apoptotic cells (p < 0.01). Conclusion: The findings suggested that RRC can improve ischemia stroke. The possible mechanisms of RRC are largely through antioxidant, anti-apoptosis activities, anti-inflammatory, repressing lipid peroxidation, antigliosis, and alleviating the pathological blood brain barrier damage.


INTRODUCTION
Ischemic stroke, a common neurological disease, has been the major cause for the central nervous system dysfunction with a relative high mortality and morbidity in clinical practice (Benjamin et al., 2017;Benjamin et al., 2018). The burden of stroke will increase greatly during the next 20 years because of the aging population, especially in developing countries (Donnan et al., 2018). Cerebral ischemia causes several pathological processes, such as inflammation, oxidative stress, cell apoptosis, ion imbalance, and calcium overload (Jayaraj et al., 2019) leading to neurologic deficits in ischemic stroke. Unfortunately, intravenously recombinant tissue plasminogen activator (rtPA) is so far the only Food and Drug Administration (FDA)-approved thrombolytic agent for treating ischemia stroke within the golden hour 4.5 h of stroke onset (National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group., 1995;Sandercock et al., 2012). Due to the narrow therapeutic window, several contraindications and the incidence of hemorrhagic transformation, rtPA remains largely underutilized (Medcalf, 2011). Moreover cerebral ischemia/ reperfusion injuries can also lead to severe adverse reactions (Jickling et al., 2014). In spite of the substantial research and development efforts, the available therapeutic options remains insufficient for acute ischemic stroke. Owing to the limitations of the current available treatments, complementary and/or alternative medicine is thus increasingly sought to treat stroke worldwide.
An objective and quantitative systematic review of preclinical studies is a type of secondary research, may identify confounding factors across animal studies (Ritskes-Hoitinga et al., 2014). Systematic reviews are a powerful approach to offer credible evidence and be favourable for selecting the appropriate drug administration for future clinical trials (van Luijk et al., 2013). However, the current evidence of RRC for ischemic stroke still lack systematic analysis. Therefore, in the present study we conduct a preclinical systematic review of RRC on ischemia stroke to further reveal the basis of action and the neurochemical modulatory mechanism of RRC in animal model of ischemia stroke.

Search Strategy
A comprehensive search was performed to identify experimental studies evaluating the effects of RRC for ischemia stroke from databases, including PubMed, embase, CBM, Web of Science, National Knowledge Infrastructure (CNKI), Wanfangdatabase and VIP information database. All searches were electronically searched from the inception up to Oct 2021. Studies about assessing the effectiveness of RRC for ischemic stroke in animals were identified. Our literature search strategy was as following: (Rhodiola OR Rhodiola rosea OR Roseroot OR Rhodioloside OR Salidroside) AND (Ischemic stroke OR Cerebral ischemic injury OR Cerebral infarction OR Brain infraction).

Eligibility Criteria
Experimental studies evaluating the effect of RRC for ischemic stroke were selected. Two authors independently screened the titles and/or abstracts according to the search strategy. Then, we assessed the full-text articles for eligibility. Studies were included if they met the following criteria: 1) Animal models were established for ischemic stroke; 2) RRC as monotherapy was administrated in the experimental group, regardless of its mode, dosage, and frequency. 3) The primary measured outcomes were neural functional deficit score (NFS), infarct volume (IV), brain water content, cell viability, apoptotic cells, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells, B-cell lymphoma-2 (Bcl-2) level and tumor necrosis factor-α (TNF-α) level. The secondary outcome measures were mechanisms of RRC for ischemic stroke; and 4) The control group received vehicle or no adjunct intervention.

Exclusion Criteria
The prespecified exclusion criteria were as follows: 1) the targeting disease was not ischemic stroke; 2) RRC were used as combination; 3) the article was a clinical or in vitro study; 4) the study was a case report, clinical trial, review, abstract, comment, editorial, duplicate publication or in vitro study, and 5) lack of the control group.

Data Extraction
Two independent reviewers assessed the articles and the following details were extracted: 1) the first author, publication year; 2) individual data from each study, including animal species, gender, samples for individual comparison, and weight; 3) type of animal model; 4) type of anesthetic; 5) intervention characteristics from both treatment and control groups, including drug, timing for initial treatment, dosage, mode, and frequency; 6) outcome measures and its corresponding pvalue. For each comparison, the mean value and standard deviation from each treatment and control group of every study were extracted. If the data were demonstrated graphically, we tried to contact the author for further information or digital ruler software was applied. Otherwise we only performed qualitative analysis. The data of highest dose was selected when the treatment group included various doses of the target drug. The result of the last time point was included when the data were expressed at different times.

Quality Assessment
Two authors independently assessed the methodological quality of the included articles according to the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) 10-item checklist (Sena et al., 2007): 1) peer-reviewed publication; 2) statements of temperature control; 3) randomization to treatment or control group; 4) blinded induction of model; 5) blinded assessment of outcome; 6) use of anesthetic without significant intrinsic neuroprotective activity; 7) appropriate animal model; 8) sample size calculation; 9) compliance with animal welfare regulations; and 10) declaration of potential conflict of interests. Each study was given an aggregate quality score based on one-point awarding for each item. Discrepancies were resolved by discussion or consultation with corresponding author.

Statistical Analysis
The pooled analyses were performed using RevMan 5.3 software. All outcome measures were considered as continuous data. To estimate the effect of RRC on ischemic stroke, the random effects model and standard mean difference (SMD) with 95% confidence intervals (CIs) were calculated. Heterogeneity among individual studies was assessed via I 2 statistics test. If probability value was less than 0.05, the difference was considered statistically significant.

Others
One study (Atochin et al., 2016) found that RRC could improve neurological deficit in McGraw scale. One study (Zuo et al., 2018) showed that RRC improved neurological deficits in Ludmila Belayev test, For grid test, Beam walk test, and Wire grip test compared with the control.

Summary of Results
To our knowledge, it is the first preclinical systematic review to assess the efficacy of RRC for cerebral ischemic stroke. In the present study, 15 studies with 345 animals showed that RRC significantly improved NFS and reduced IV in cerebral ischemia animal models. Thus, RRC exerted the potential neuroprotective function for ischemic stroke, mainly through anti-inflammatory, anti-apoptosis, and anti-oxidative and alleviating the pathological BBB damage. However, given methodological weaknesses, the overall available evidence from the present study should be interpreted cautiously. Thus, the conclusions in the present study should be partially treated with caution.

Limitations
There are several limitations in the primary studies. Firstly, only Chinese and English literatures were searched, which may cause selection bias as studies published in other languages were absent . Secondly, no study had used an animal with  co-morbidities, such as hypertension, diabetes or hyperlipidemia (Heusch, 2017), which would be more relevant models for human pathology (Guyatt et al., 2011). Thirdly, the studies had methodological deficiencies. None of these studies reported the blindness of ischemia induction, allocation concealment, randomization to treatment group or control group and sample size calculation, which are the core criteria of study design. Thus the analysis may result in overestimated effect   Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 736198 10 size (Ospina et al., 2005;Higgins and Green, 2012). Thereby, the results in the present study should be interpreted with caution.

Implications
The damage inflicted on the neuron during ischemic stroke is a complex process, involving multiple factors. The main mechanisms of injury are 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 deficitse. Thus, neuroprotective drugs generally work through one or combined aspects of the above targets. The present study showed RRC could exert potential neuroprotective effects in experimental for ischemic stroke indicating that RRC are candidates for ischemic stroke treatment and can be used for further clinical trials. The possible mechanisms of RRC for cerebral ischemia injury are summarized as follows: 1) alleviating the pathological BBB damage; 2) repressing lipid peroxidation; 3) antioxidant through increasing the activity of SOD, HO-1, Nrf2, GSH-Px and GST and decreasing the concentration of MDA and ROS; 4) anti-inflammatory via decreasing the expression of proinflammatory cytokines such as TNF-α, IL-1β, IL-1, IL-2 and IL-6; 5) anti-apoptotic via increasing the levels of Bcl-2, decreasing the levels of Bax, caspase3, C-Fos, GFAP, p53, decreasing the activity of LDH and reducing TUNEL positive cells; 6) neuroprotective effect via regulating BDNK-mediated PI3K/Akt pathway, through calpain1/PKA/CREB pathway and through modulating monoamine metabolism; 7) inhibiting reactive astrogliosis and glial scar formation, probably through Akt/GSK-3β pathway. To summarize, the possible mechanisms of RRC for ischemic stroke are through antioxidant, lipid peroxidation, anti-apoptosis, antiinflammatory, improving blood vessel endothelium differentiation, and cerebral metabolism. A recent review  illustrated that Rhodiola rosea L. and its components, particularly salidroside has strong antioxidant activity through regulating mitochondrial biogenesis, repressing ROS production, increasing the activity of the antioxidant enzymes (such as GSH-Px and SOD), and via various signaling pathways (AMPK, PI3K/ Akt, Mitochondria-dependent, Nrf2). In addition, another review (Pu et al., 2020) showed that Rhodiola rosea L. and its compounds have immune-regulation effects through some inflammatory mediators, such as IL-6, TNFα, IL-1β, and NO, and signaling pathways, such as NF-κ B, AP-1, and STAT3. In the present study, the mechanisms are consistent with the evidences.
Preclinical animal research plays a critical role in human diseases understanding (Murphy and Murphy, 2010). However, original preclinical research is often conducted with    a poor methodological quality, which is considered as a hindrance to the translation of animal research into effective preclinical drug treatments for human disease (Baginskait, 2012;Moher et al., 2015). The systematic review can dentify defects in study design, integrate preclinical evidence and guide potential clinical translation (Macleod et al., 2005;De Vries et al., 2014). In the present analysis, the average CAMARADES score of the included studies ranged from 1/10 to 7/10. The main flaws are lacking of sample size calculation, poor blinding in model induction and outcome assessment. Inadequate sample size can miss the real intervention effect in an experiment, while excessive sample size will result in wasting resources and raising animal ethical issues (Arifin and Zahiruddin, 2017;Chen et al., 2019). Poor blinding in outcome assessment could result in a 27% overestimation of the mean reported effect size (Holman et al., 2015) Additionally, all the animal experiments are conducted in healthy animals which lack the comorbidities, such as diabetes, hypertension and hyperlipidemia. Reporting guidelines set detailed predetermined standards to make biomedical research report more complete and transparent, and enhancing their value in scientific exploration and    (Kilkenny et al., 2012) is a reporting guidelines, which are organized into twenty sections, providing recommendations on Introduction, Methods, Results, and Discussion. The ARRIVE guidelines were recommended to be utilized when designing and reporting animal research on RRC for ischemic stroke which can provide guidance on the complete and transparent reporting of in vivo animal researches, helping to improve the quality of further researches (Karp et al., 2015). Thus, we suggest that further animal researches should follow up the reporting guidelines, increasing the value of clinical trials and further application. Furthermore, the following factors need to be considered: 1) method by which sample size was determined should be appropriately detailed; 2) experimental animals have relevant comorbidities, which are like human pathology under the clinical conditions; 3) primary outcome should be closer to clinical practice.

CONCLUSION
This study showed that RRC exerted potential neuroprotective effects in ischemic stroke largely through anti-oxidative, antiinflammatory, antigliosis, anti-apoptotic, neuroprotective, and alleviating the pathological BBB damage mechanisms. In addition, this systematic review provides an experimental evidence-based suggestion that RRC may be a promising candidate for clinical trials.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.