Effect and Mechanisms of Quercetin for Experimental Focal Cerebral Ischemia: A Systematic Review and Meta-Analysis

Quercetin, a naturally occurring flavonoid, is mainly extracted from tea, onions, and apples. It has the underlying neuroprotective effect on experimental ischemic stroke. A systematic review and meta-analysis were used to assess quercetin's efficacy and possible mechanisms in treating focal cerebral ischemia. Compared with the control group, twelve studies reported a remarkable function of quercetin in improving the neurological function score (NFS) (P < 0.05), and twelve studies reported a significant effect on reducing infarct volume (P < 0.05). Moreover, two and three studies showed that quercetin could alleviate blood-brain barrier (BBB) permeability and brain water content, respectively. The mechanisms of quercetin against focal cerebral ischemia are diverse, involving antioxidation, antiapoptotic, anti-inflammation, and calcium overload reduction. On the whole, the present study suggested that quercetin can exert a protective effect on experimental ischemic stroke. Although the effect size may be overestimated because of the quality of studies and possible publication bias, these results indicated that quercetin might be a promising neuroprotective agent for human ischemic stroke. This study is registered with PROSPERO, number CRD 42021275656.


Introduction
Stroke is recognized as one of the significant causes of death and disability, of which ischemic stroke is the primary type [1]. According to the World Health Organization (WHO) report, about 15 million people worldwide suffer strokes each year [2]. Pharmacologic thrombolysis and endovascular thrombectomy are currently available treatments for ischemic stroke. However, both treatments in clinical usage are limited due to the strict timing criteria and contraindications, and their benefits diminish with delays in therapy initiation [3]. Therefore, existing ischemic stroke treatment methods must be reformed, and new pharmacological treatment modalities should be explored.
Flavonoids are widely found in plant and vegetable diets. They are reported to have antiviral, anti-inflammatory, heart protection, antidiabetes, anticancer, antiaging, and other biological activities [4][5][6][7][8][9]. Quercetin (Figure 1), the predom-inant dietary flavonoid, is mainly found in tea, onions, and apples. A previous review showed that quercetin protects cells against oxidative stress damage in various organs during ischemia-reperfusion (I/R) [10]. Quercetin can improve blood-brain barrier dysfunction [11] and reduce neuronal apoptosis after cerebral I/R injury [12]. The randomized, double-blind, placebo-controlled clinical trial reported that quercetin could decrease ambulatory blood pressure (ABP) in patients with hypertension [13], indicating that it has the effect of protecting cardiovascular and cerebrovascular diseases. Another clinical trial also revealed that quercetin possesses a protective effect on central hemodynamic parameters and myocardial ischemia in patients with stable coronary heart disease [14]. Furthermore, the oral clearance of quercetin is high, with an average terminal half-life of 3.5 h in healthy humans [15], suggesting that it may be a candidate drug for clinical application. These studies indicated that quercetin has an excellent therapeutic effect on cardiovascular and cerebrovascular diseases such as ischemic stroke, which should be supported by the pooling data from preclinical studies.
The systematic review is a type of secondary research that gathers all primary research that meets prespecified qualification criteria for solving a specific research problem, minimizing bias [16]. It can provide convincing evidence and help choose the optimal drug administration requirements in clinical trials. However, to date, there has not been a systematic review to investigate the compliance of experimental studies of quercetin on ischemic stroke models. Here, we conducted a preclinical systematic review to appraise the effectiveness and mechanism of action of quercetin in the treatment of ischemic stroke in animal models.

Methods
This systematic review and meta-analysis were carried out according to the methods of Wang et al. [17].

Database and Search
Strategy. The following databases were searched: PubMed, Web of Science, Chinese Biomedical Literature Database (SinoMed), China National Knowledge Infrastructure (CNKI), WanFang Database, and VIP Database. We collected all studies from inception to Aug 2021. Our search terms were as follows: "Quercetin AND ischemic(a) stroke OR cerebral ischemic(a) injury OR cerebral ischemic(a) reperfusion OR cerebral infarct OR middle cerebral artery occlusion (MCAO)." There are no restrictions on the country or language of publication.

Inclusion Criteria.
All animal experiments assessing the effect of quercetin on focal cerebral ischemia were chosen, regardless of animal species, age, and sex. The following screening criteria should be satisfied: (1) quercetin was administered to an animal model of focal cerebral ischemia, regardless of the dosage, route, method, and treatment schedule; (2) animal models of focal cerebral ischemia involved temporary or permanent middle cerebral artery occlusion (MCAO); (3) the intervention group only used quercetin treatment; and (4) control animals received no treatment or vehicle. The primary outcome indicators included NFS and infarct volume; the second outcome indicators were BBB permeability and brain water content.

Exclusion Criteria.
The following were exclusion criteria: (1) the study was a viewpoint, review, case report, abstract, in vitro experiment, ex vivo study, or human study; (2) nonfocal cerebral ischemia models such as chronic cerebral ischemia, global cerebral ischemia, traumatic models, or hypoxia-ischemia; (3) non-quercetin-based interventions, quercetin modifications, and combinations with other compounds and treatments; (4) no control group; and (5) no statement of sample size.

Data
Extraction. The detailed information from included studies was extracted by two independent reviewers, as outlined below: (1) first author, publication year, focal cerebral ischemia model, and anesthesia method; (2) animal species, sex, and weight; (3) the dose, strategy, and frequency of administration of quercetin and the control groups; (4) the data of mean value and standard deviation of NFS, infarct volume, BBB permeability, and/or brain water content; and (5) the timing and sample size for outcome assessments were also extracted. The highest dose data were extracted when the treatment group contained various dose subgroups. If data are derived from different time points, the result at the peak time point is extracted. When data is only displayed as a graph, we contact the author for detailed information. If no response is received, we will use the graphic digitizer software to measure the value or exclude it.
2.6. Statistical Analysis. The Review Manager (version 5.3) software was used for statistical analysis. The estimate of the pooled effect sizes is calculated by the standardized mean difference (SMD) using a fixed-effects model without statistical evidence of heterogeneity (P ≥ 0:1, I 2 ≤ 50%). The random-effects (RE) model is applied with statistical heterogeneity (P < 0:1, I 2 > 50%). The statistical significance is P < 0:05, and the 95% confidence interval for all results is computed. The publication bias was evaluated by the funnel plot and the Egger test.

Study Selection.
A total of 397 studies were found through database search, and 98 unique studies were identified after removing 299 duplicate and irrelevant studies. After screening the titles and abstracts, 7 studies were excluded for at least one of the following reasons: opinions, comments, and abstracts. The remaining 91 studies were read in detail, of which 75 studies were removed subsequently for the following reasons: (1) in vitro studies; (2) not focal cerebral ischemia model; (3) administration of modification of quercetin; (4) combination with other compounds; (5) lack of outcome indicators; (6) no statement of sample size; and (7) no statistical outcome. Finally, 14 studies were selected for quantitative analysis after excluding two  Oxidative Medicine and Cellular Longevity articles for which no available data can be obtained. Figure 2 shows the screening process.

BBB Permeability.
Two studies reported BBB permeability based on the Evans blue assay. It failed for pool analysis because one study measured the content of EB in the ischemic hemisphere, and another measured Evans blue's content in the ischemic cortex. However, they all confirmed the outstanding effects of quercetin for meliorating the BBB permeability induced by focal cerebral ischemia (P < 0:05).

Publication
Bias. The Egger test and funnel plot were used to assess publication bias for NFS (Bederson criterion) and infarct volume (%). As shown in Figure 6, there was no significant publication bias in NFS (P = 0:14), whereas there may have publication bias in infarct volume (P = 0:009).

Summary of Evidence.
A total of fourteen studies were included, and the period of included studies is from 2011 to 2021. The main findings of the present systematic review showed that quercetin could improve NFS, reduce the infarct volume, and protect BBB integrity during focal   [29] √ √ √ √ √ √ 6 Park et al. [30] √ √ √ √ √ 5 Fan et al. [31] √ √ √ √ √ 5 Park et al. [32] √ √ √ √ √ √ 6 Yang et al. [20] √  Oxidative Medicine and Cellular Longevity cerebral ischaemia, suggesting that quercetin exerted the neuroprotection for acute ischemic stroke. The neuroprotective mechanisms of quercetin are mainly mediated by its antioxidant, anti-inflammatory, antiapoptosis properties, and resistance to calcium overload.

Strengths and Limitations.
The strength of the present study is that this is the first meta-analysis to evaluate the effects of quercetin against focal cerebral ischemia. In this meta-analysis, we use six databases with many terms and keywords to increase the number of searches and ensure the extensive retrieval of published articles. Moreover, we clearly defined animal models and outcome indicators before the meta-analysis, which reduced bias in selecting the included studies. Our results provide the practical value for the evidence-based transformation of animal data from the laboratory to the bedside. The present study also has some limitations. First of all, all the databases we searched are in English or Chinese, which might lead to selection bias because studies published in other languages may be excluded. Second, no studies reported the negative effect of quercetin on infarct volume, which may be a pivotal contributor to publication bias, as positive results and large sample sizes are always easier to publish than negative results and small sample sizes. The effective method for avoiding publication bias is to include unpublished studies and trial registries [33]. Third, the methodological quality of the included studies is average. The included studies lacked blinded assessments, no sample size calculations, animals without relevant comorbidities, and no testing on successfully modeled animals. Therefore, some conclusions in the present study should be referenced critically.

Implications.
High-quality methodologies of studies are the elements of drugs translating from animal research into clinical trials for human disease [34]. The methodological quality of these studies was moderate, especially none of the included studies estimated sample size. Insufficient sample size can blunt the actual effect of intervention in the experiment, while the vast sample size can lead to animal waste and induce ethical concerns about animals. Only three     studies were blinded to evaluate the experiment results, resulting in overestimating of treatment effects of quercetin. Animals with relevant comorbidities were not used in these studies, which do not truly represent human pathology under clinical conditions. Animal Research Reporting of In Vivo Experiments (ARRIVE) [35] with a 20-item list of introductions is recommended as the criterion for further studies on quercetin to treat ischemic stroke, which might considerably improve the quality of the methodology.

The Primary Mechanism of Quercetin for Ischemic
Stroke. The neuroprotective mechanisms of quercetin against ischemic stroke are summarized as follows: (1) Oxidative stress plays a vital role in the pathogenesis of ischemic stroke [36]. The excessive reactive oxygen species (ROS) induce oxidative stress during cerebral ischemia injury [37]. Quercetin could reduce oxidative stress [19,20,26,28,31] by decreasing the content of MDA [26,28,31], heightening the activity of SOD [19,26,31] and GSH [19]. These studies suggested that quercetin can significantly inhibit oxidative stress and reduce the neurotoxicity of free radicals.

Conclusion
Quercetin can reduce cerebral infarction volume, nerve function deficit, BBB permeability, and brain edema and protect cerebral ischemia injury through various signaling pathways. Other undisclosed molecular mechanisms deserve further study.