2.1. Comprehensive literature review

A systematic search was conducted using bibliographic databases and other evidence sources which addressed the search question. The comprehensive literature search involved looking at the eligibility of articles, searching strategies for identification of studies, study selection, and data extraction.

2.2. Formulating search question

The search was performed by identifying the type of evidence needed to answer the search question. A strategy of using the Domain, Determinant, and Outcome (DDO) format was used in the study to obtain relevant answers to the formulated question. The strategy was as follows:

1. Domain – “Stroke” OR “Cerebral Ischemia” OR “Brain Ischemia” OR “Ischemic Stroke
2. Determinant – “Genistein” OR “Soy Isoflavones”
3. Outcome – “Mechanism”

Thus the formulating search question is,

How does genistein display its mechanism in the treatment of ischemic stroke, as demonstrated in in vivo and in vitro studies?

2.3. Eligibility criteria

The inclusion and exclusion criteria for this study is presented and summarized in Table 1.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Eligibility criteria for this study.

https://doi.org/10.1371/journal.pone.0338590.t001

2.4. Search strategy for identification of studies

The identification of studies included all published studies. The Boolean search was performed on each database using the search terms: (“genistein” OR “soy isoflavone”) AND (“stroke” OR “cerebral ischemia” OR “brain ischemia” OR “ischemic stroke). The published literature was thoroughly searched. The database was searched for articles published between January 1, 1999, and October 31, 2025. This review includes the research papers from 1999 onward focusing on genestein’s therapeutic potential in the treatment of Ischemic stroke particularly in in vivo and invitro studies.The period starting from 1999 was chosen because it had significant advancements in understanding genistein’s mechanism of action and target pathways. Hence by focusing on studies from this time frame, the review aims to include more accurate research findings related to genistein and its relevance with ischemic stroke.

2.5. Identification of published articles

Published articles refer to any article that has been published in any publishing journal platform. The first step was to locate all relevant published articles for this research using a computer-based information search. The established databases in this study were Web of Science (WoS), Scopus, PubMed, and Science Direct. The references of the chosen studies were then analyzed manually by all researchers.

2.6. Screening and data extraction

Screening of titles and abstracts was conducted independently by two reviewers (N.Y.M.K. and N.A.). Disagreements were resolved through discussion with two other reviewers (R.S. and A.M.A.). Full-text articles were then retrieved, and eligible studies were uploaded into EndNote for organization. Data extraction was performed by the same two reviewers to ensure inter-rater reliability and minimize data entry errors. The extracted data were organized and tabulated in Microsoft Excel.

2.7. Full text retrieval

Full-text articles of eligible studies were obtained and downloaded from Web of Science (WoS), Scopus, PUBMED and ScienceDirect. Only articles that offer free access were included in this study. Articles without full text were excluded from this study.

2.8. Selection and inclusion of final articles

The selection of final full-text articles was based on predefined eligibility criteria. Disagreements were discussed until consensus was reached among the four reviewers. The finalized articles were manually coded and analyzed using structured data extraction forms and Excel.

2.9. Critical appraisal

A process of critical appraisal was performed by N.Y.M.K and N.A to assess the article quality and appropriateness of study design to the research objective. The critical appraisal of the selected articles was performed by using the modified version of the “McMaster University’s Critical Review Form” [11] to extensively analyse and evaluate the articles in following domains as listed in the review form: study purpose, literature, study design, appropriateness of study design, outcomes, results, and conclusion of studies [12]. Excel files were prepared based on key assessment report checklists to make the appraisal process easier. The researchers conducted critical assessments in duplicate and if there was disagreement, consensus will be reached with other researchers regarding the credibility of the articles.

3.0. Flowchart of review process

The overall process of article identification, screening, eligibility assessment, and final inclusion is illustrated in Fig 1.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Flowchart of the review process.

https://doi.org/10.1371/journal.pone.0338590.g001

3.1. Evidence of genistein’s mechanism in in vivo models

In vivo studies were performed on rat and mouse models, including models of middle cerebral artery occlusion (MCAO), global cerebral ischemia (GCI) and hypoxic-ischemic brain injury. A total of 26 studies from the final included studies contributed to the in vivo evidence in evaluating genistein’s protective mechanisms in ischemic stroke. These are detailed in Table 3. These studies explored the neuroprotective mechanisms of genistein or its more soluble counterpart, GSS, and consistently demonstrated reduced infarct size, improved neurological scores, and modulation of inflammation, oxidative stress, and apoptosis. Some researchers used GSS, a sulfonated version of pure genistein, to improve water solubility and bioavailability.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 3. Summary of key molecular pathways and mechanistic categories of genistein in in vivo models of ischemic stroke.

https://doi.org/10.1371/journal.pone.0338590.t003

3.2. Evidence of genistein’s mechanism in in vitro models

Seven studies examined genistein’s neuroprotective mechanisms using in vitro models, including various neuron-like and cortical cell lines. These are detailed in Table 4. These studies demonstrated genistein’s ability to reduce oxidative stress, regulate apoptotic proteins, and improve cell viability. GSS, though primarily applied in vivo, also showed efficacy in cell-based assays when used.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 4. Summary of key molecular pathways and mechanistic categories of genistein in in vitro models of ischemic stroke.

https://doi.org/10.1371/journal.pone.0338590.t004

3.3. Critical appraisal/ Quality

The quality scores of the included studies ranged from 8 to 10, based on the modified McMaster Review Form. The average quality score was 8.39. Detailed scoring per study is shown in Table 5.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 5. Modified McMaster critical review form for quantitative studies for individual studies.

https://doi.org/10.1371/journal.pone.0338590.t005

3.4. Inter-rater reliability assessment

To measure the inter-rater reliability between these reviewers for the critical appraisal of the final included studies, Cohen’s Kappa statistic was used. Cohen’s Kappa agreement values were calculated to assess the level of agreement between the two reviewers (N.Y.M.K and N.A.). If there were any disagreements, then it was solved by third and fourth reviewers (R.S. and A.M.A.). Cohen’s Kappa value (κ) for the critical appraisal of a total of 31 final eligible articles was reviewed, and the value obtained was κ = 0.829, which was almost perfect agreement. The inter-rater reliability tables were given as S1 Table.

4.1. Anti-apoptotic mechanism of genistein in ischemic stroke

Genistein has been shown to exhibit anti-apoptotic effects on in vivo and in vitro models of ischemic stroke. The mechanisms include regulation of the mitochondria-mediated apoptotic pathway, regulation of the Bcl-2/Bax ratio and caspase activation inhibition. The use of in vivo animal models has demonstrated that genistein is effective in inhibiting apoptosis via mitochondrial and receptor-mediated pathways. Liang et al. [15] demonstrated in a rat model of transient global cerebral ischemia that genistein suppressed mitochondrial ROS and lipid peroxidation, inhibited cytosolic cytochrome c and caspase-3 activity, and reduced TUNEL-positive neurons, indicating that the mitochondria-dependent apoptotic pathway had been suppressed. Mitochondrial integrity and Bcl-2 family proteins are known to play a significant role in regulating caspase-3-mediated neuronal apoptosis during ischemia [36]. Equally, Qian et al. [6] found that genistein dramatically reduced infarct size and oxidative stress markers by inhibiting mitochondrial ROS generation and NF-κB activation, thereby reducing neuronal apoptosis.

Genistein’s anti-apoptotic effect is further enhanced by hormonal and kinase modulation. Wang et al. [22] also reported that in ovarizing mice, genistein stimulated ERK1/2 phosphorylation, increased Bcl-2, decreased Bax, and enhanced neurological performance; which were inhibited by ERK. Similarly, Aras et al. [23] demonstrated that genistein minimized oxidative stress and apoptosis in MCAO rats by increasing the expression of NRF1 and SOD, and decreasing the activity of caspase-3 and −9.

In line with those results, Miao et al. [1] identified that genistein stimulated the Nrf2/NQO1 antioxidant pathway, reduced ROS, and increased neuronal survival, which is in line with the contribution of the Nrf2/HO-1 pathway to alleviating ischemic apoptosis [37]. Lu et al. [3] further added that genistein stimulated PI3K-Akt-mTOR signaling, which increased the phosphorylation of Akt and mTOR and reduced infarct size and neuronal death. The neuroprotective role of PI3K/Akt signaling in stroke has been corroborated in other ischemia models [38]. Li et al. [34] showed that genistein inhibited Wnt/Ca²⁺, reducing ROS and NOX1 and increasing antioxidant enzyme.

These observations are reinforced by complementary in vitro studies, which explain the upstream molecular events. Schreihofer and Redmond [16] observed that in cortical neurons subjected to OGD toxicity or glutamate toxicity, estrogen receptor-dependent PI3K and ERK signaling by genistein inhibited LDH release and caspase activity. Qian et al. [9] validated that genistein prevented H₂O₂-induced neuronal apoptosis through the restoration of Bcl-2/Bax ratio, caspase-9 and −3 inhibition, and the inhibition of NF-κB, JNK and ERK phosphorylation. In models of in vitro studies, genistein steadily decreased neuronal apoptosis by stabilizing mitochondrial and calcium homeostasis. It has increased the anti-apoptotic ratio of Bcl-2/Bax and inhibited caspase-3 activity [25], reduced ROS generation and cleaving of the proteins of the proteolytic enzyme PARP-1 and increased autophagic balance by modulating LC3, avoiding calcium overload and recovered the expression and potassium currents of the AMPA receptor, GluR2 [28]. Collectively, these multi-model results demonstrate the convergent anti-apoptotic mechanism of genistein, including mitochondrial stabilization, control of pro-survival kinase signatures (ERK1/2, PI3K/Akt/mTOR), Ca²⁺ and oxidative homeostasis, and caspase activation. The mechanistic consistency in the in vitro and in vivo whole systems testifies to the strong neuroprotective potential of genistein in ischemic stroke.

Beyond the studies included in this review, additional literature supports genistein’s anti-apoptotic role in other organ systems. In a rat model of polycystic ovarian syndrome, genistein enhanced Bcl-2 expression and suppressed Bax in ovarian granulosa cells [39]. In the cardiovascular system, Hu et al. [40] showed that genistein protected H9c2 cardiomyoblasts from isoproterenol-induced mitochondrial apoptosis by down-regulating pro-apoptotic proteins (Bad, caspase-3, −8, −9) and up-regulating survival pathways including Akt, ERK1/2, and NF-κB. Similarly, Si and Liu [41] reported that genistein reduced TNF-α-induced apoptosis in human aortic endothelial cells by activating the p38β MAPK pathway and upregulating Bcl-2, highlighting its vasculoprotective role. These findings across endocrine, cardiovascular, and vascular models reinforce genistein’s capacity to inhibit apoptosis through multiple signaling mechanisms, supporting its broader therapeutic relevance in diseases characterized by oxidative or stress-induced cellular damage.

4.2. Anti-inflammatory mechanism of genistein in ischemic stroke

Genistein has demonstrated significant anti-inflammatory properties in various in vivo models of ischemic stroke, primarily through its modulation of multiple key signaling pathways. Early evidence from Li and Zhang [14] revealed that genistein inhibited STAT3 phosphorylation and DNA-binding activity in the rat hippocampus following cerebral ischemia/reperfusion, indicating direct blockade of a major inflammatory transcription factor. A soy isoflavone-enriched diet was shown to inhibit NF-κB translocation in ovariectomized rats subjected to tMCAO, leading to the suppression of pro-inflammatory cytokines, including IL-1β, IL-2, IL-13, and CXCL1, and a reduction in microglial activation within the infarct core [20]. In another study using a forebrain ischemia model, genistein attenuated neuroinflammation by inhibiting GR nuclear translocation and promoting its degradation via the Mdm2-ubiquitin pathway, thus preventing GR-induced microglial activation and neuronal injury in the hippocampus [21].

In reproductively senescent mice, genistein reduced neuroinflammation by inhibiting the NLRP3 inflammasome, resulting in decreased expression of Caspase-1 and inflammatory cytokines, such as IL-1β, IL-6, and IL-18, alongside improved neurological outcomes [8]. GSS, a water-soluble derivative, further enhanced anti-inflammatory efficacy by upregulating the α7nAChR, leading to the inhibition of NF-κB and a shift away from pro-inflammatory M1 microglial polarization. This was associated with reduced infarct volume and improved neurological function [7].

Likewise, Wang et al. [31] also demonstrated that genistein increased neuronal GPER, which stimulated PGC-1α and thus inhibited NLRP3 inflammasome assembly in ovariectomized ischemic mice. This cascade decreased caspase-1 and pro-inflammatory cytokine release and yielded smaller infarcts, better neurological outcome and reduced apoptotic neurons- identifying a GPER/PGC-1alpha-dependent anti-inflammatory mechanism between hormonal and mitochondrial regulation in ischemic neuroprotection.

Moreover, GSS also attenuated neuroinflammation via suppression of JAK2/STAT3 signaling and cytokine release, including IL-1β, IL-6, and TNF-α, in MCAO models [4]. In HIBD, genistein activated the Nrf2/HO-1 antioxidative pathway while inhibiting NF-κB, reducing both oxidative stress and inflammation, and promoting neuronal survival [30]. Similar effects were observed with GSS, which downregulated multiple pro-inflammatory cascades including complement, phagosome, and glutamatergic synapse pathways, resulting in decreased neuronal degeneration and neuroinflammation [32]. Notably, long-term treatment with GSS in post-MCAO rats not only suppressed NF-κB but also promoted a beneficial shift in astrocyte polarization from the pro-inflammatory A1 phenotype to the neuroprotective A2 phenotype, along with increased levels of IL-10 and BDNF and improved cognitive and motor recovery [33]. Most recently, Yu et al. [35] demonstrated that GSS prevented NLRP3-mediated pyroptosis following ischemia by reducing the expression of cleaved-caspase-1, GSDMD-N, IL-1β, and IL-18, thereby limiting inflammatory cell death and restoring neurological function.

Collectively, these findings suggest that genistein and its derivatives exert robust in vivo anti-inflammatory effects through inhibition of NF-κB, STAT3, and GR signaling, suppression of NLRP3 inflammasome activation and pyroptosis, and modulation of glial polarization. By simultaneously targeting transcriptional, inflammasome, and glial-cell pathways, genistein offers a multi-targeted strategy for mitigating post-ischemic neuroinflammation.

Beyond the studies included in our systematic review, external mechanistic literature further supports the plausibility of genistein’s anti-inflammatory actions through multiple signaling pathways. These findings are consistent with other in vivo studies showing that genistein suppresses NF-κB signaling and pro-inflammatory cytokine release in models of systemic or organ-specific inflammation, such as high-fat diet-induced liver inflammation [42] and LPS-induced acute lung injury [43], further supporting its potential to mitigate neuroinflammation through similar mechanisms.

Moreover, genistein’s anti-inflammatory effects have been linked to inhibition of the NLRP3 inflammasome and modulation of downstream pathways. In a mouse model of dextran sulfate sodium (DSS)-induced colitis, genistein significantly suppressed NLRP3 inflammasome activation via the TGR5–cAMP signaling axis, leading to reduced IL-1β secretion and improved clinical outcomes [44]. Similarly, in a traumatic brain injury (TBI) model, genistein alleviated neurological deficits and anxiety-like behaviors by downregulating NLRP3 and caspase-1 expression, suggesting a central role in neuroinflammatory regulation [45].

Additionally, genistein has been shown to activate peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear receptor involved in regulating both metabolic and inflammatory genes. For example, in human mesenchymal stem cells, genistein upregulated PPARγ expression, confirming its role as a functional activator of this pathway [46]. Although demonstrated in the context of adipogenesis, this mechanism aligns with PPARγ’s known anti-inflammatory effects in other cell types.

Complementing this, genistein has also demonstrated vascular anti-inflammatory activity through cAMP preservation and inhibition of monocyte adhesion under hyperglycemic conditions. While not directly mediated via PPARγ, these actions are consistent with anti-inflammatory profiles typically associated with PPARγ activation [47].

4.3. Anti-oxidative mechanism of genistein in ischemic stroke

Genistein exhibits strong antioxidative activity in both in vivo and in vitro models of cerebral ischemia, oxidative stress, and hypoxic brain injury. In one of the earliest studies, Trieu and Uckun demonstrated that genistein reduced hydroxyl radical formation and oxidative damage in murine models of familial ALS and photochemically induced stroke, primarily via tyrosine kinase inhibition and estrogen receptor-dependent pathways [13]. Similarly, Liang et al. showed that in rats subjected to transient global cerebral ischemia, genistein significantly attenuated mitochondrial ROS production, reduced lipid peroxidation as evidenced by decreased MDA levels, and suppressed caspase-3 activation, highlighting its role in preserving mitochondrial function under oxidative conditions [15].

Ma et al. further confirmed genistein’s ability to mitigate oxidative stress through estrogen receptor-mediated mechanisms in male and ovariectomized female rats undergoing transient tMCAO [17]. Genistein reduced NADPH oxidase activity, lowered superoxide levels, and upregulated NQO1 and ERCC2, genes critical for antioxidant defense and DNA repair [17]. In mice treated orally with genistein for two weeks, Qian et al. reported a dose-dependent reduction in oxidative stress, as evidenced by decreased levels of MDA and ROS, along with increased activity of endogenous antioxidant enzymes such as SOD and GPx [6].

A mechanistically distinct yet complementary pathway was described by Wang et al., who demonstrated that genistein activated the eNOS/Nrf2/HO-1 axis, promoting the nuclear translocation of Nrf2 and enhancing the expression of HO-1, which collectively contributed to reduced oxidative injury in a rat model of global cerebral ischemia [19]. In another study, Aras et al. demonstrated that genistein administration in MCAO-induced rats led to increased NRF1 expression and SOD activity, reduced MDA levels, and protected against neuronal degeneration by preserving mitochondrial function [23]. In a diabetic stroke model, Rajput et al. noted genistein’s role in inhibiting DPP-4 activity and boosting GLP-1 levels, which subsequently improved mitochondrial integrity, reduced oxidative stress (TBARS), and increased glutathione concentrations [27].

Miao et al. and Li et al. both reported that genistein significantly upregulated the Nrf2/ARE antioxidant signaling pathway, enhanced expression of NQO1, and reduced ROS levels, contributing to improved neurological recovery after ischemic insult [1,30]. Additionally, Xue et al. showed that GSS not only enhanced SOD, CAT, and GPx activity but also regulated the nitric oxide synthase (NOS) system, increasing total and constitutive NOS while suppressing iNOS, thus optimizing redox balance in ischemic brain tissue [29].

In vitro findings further corroborate these results. Qian et al. demonstrated that genistein pretreatment of primary cortical neurons exposed to H₂O₂ reduced ROS levels, suppressed NF-κB and MAPK (JNK/ERK) activation, and increased the Bcl-2/Bax ratio, indicating enhanced cellular resilience to oxidative insult [9]. Banecka-Majkutewicz et al. provided additional mechanistic insights by showing that genistein formed complexes with homocysteine and restored GPx activity, mitigating homocysteine-induced oxidative stress in enzyme-based and bacterial models [24]. In hippocampal HT22 cells undergoing repeated oxygen-glucose deprivation/reoxygenation cycles, Morán et al. reported that genistein preserved cytochrome c oxidase activity, stabilized HIF-1α, reduced PARP-1 cleavage, and maintained mitochondrial function, underscoring its role in protecting against oxidative damage during recurrent ischemia [26].

Collectively, these studies underscore genistein’s multifaceted antioxidative mechanisms. Its ability to inhibit ROS, activate endogenous antioxidant defenses (particularly the Nrf2/HO-1 and Nrf2/ARE pathways), protect mitochondrial integrity, and modulate nitric oxide and redox-sensitive enzymes underlines its therapeutic potential in oxidative stress-related neural injury.

Beyond the studies included in our systematic review, external mechanistic literature further supports the plausibility of genistein’s antioxidative actions in non-neural tissues through diverse pathways. For instance, in a murine model of alcoholic liver disease, genistein administration ameliorated acetaldehyde-induced oxidative stress and liver injury by restoring Nrf2 and HO-1 signaling pathway [48]. Similarly, in mice with diet-induced non-alcoholic fatty liver disease (NAFLD), genistein supplementation reduced hepatic steatosis and oxidative stress, effects attributed to improvements in visceral adipocyte metabolism and antioxidant defenses [49]. In diabetic cardiomyopathy, Gupta et al. [50] demonstrated that genistein significantly reduced oxidative stress, lipid peroxidation, and inflammatory cell infiltration in streptozotocin-induced diabetic rats, highlighting its cardioprotective role through attenuation of oxidative and inflammatory damage. Furthermore, genistein has also been shown to protect against acetaminophen-induced hepatotoxicity in mice by enhancing hepatic sirtuin 1 (SIRT1) expression and activity, thereby augmenting antioxidative defenses [51]. Collectively, these findings from non-neural tissues reinforce genistein’s multifaceted antioxidative mechanisms, highlighting its systemic therapeutic potential beyond the brain.

4.4. Integrated mechanistic interplay in genistein’s neuroprotective role

Several pathways of genistein have been observed throughout the considered literature as neuroprotectants in ischemic stroke. Importantly, it was demonstrated that genistein exhibits a significant overlap in its therapeutic effects on ischemic stroke models, affecting the Nrf2, NF-κB, and NLRP3 pathways. Although there is a difference in the types of models, the three pathways are biologically linked, creating a unified mechanistic structure through which genistein promotes its neuroprotective effects. These pathways, though historically classified as antioxidative or inflammatory responses, are functionally connected in a cascade.

Genistein continues to modulate the redox inflammatory balance by activating Nrf2 and inhibiting NF-κB, subsequently suppressing the NLRP3 inflammasome. Nrf2 interaction in vivo has been observed in global/focal/HIBD models, Nrf2/ HO-1 through eNOS activation with less oxidative damage [19], direct Nrf2- NQO1 upregulation and less ROS/apoptosis in OVX-MCAO rats [1], and simultaneous Nrf2-up/NF-κB-down signaling with less neuroinflammation in neonatal HIBD [30].

NF-κB signaling suppression proved to be one of the key mechanisms in both animal and cellular models. Genistein interfered with mitochondrial ROS-NF-κB feedback in MCAO mice [6], prevented nuclear translocation of NF-κB and cytokine release after dietary soy intake [20] and stimulated α7nAChR to inhibit NF-κB-mediated M1 microglial polarization [7]. NF-κB activity was also attenuated in neonatal hypoxic-ischemic brain injury [32] and in long-term GSS treatment, which transformed astrocytes of the pro-inflammatory A1 phenotype to the neuroprotective A2 state [33]. Additional in vitro evidence also showed that genistein inhibits NF-κB in combination with the JNK and ERK pathways and reinstates the Bcl-2/Bax ratio during the oxidative stressed conditions [9].

The activity of NLRP3 inflammasomes was consistently inhibited at the downstream level in various ischemic models. Genistein blocked microglial NLRP3 activation and downstream effects, caspase-1 and IL-1b, in mice undergoing reproductive senescence [8]; activated neuronal GPER1 signal to inhibit NLRP3 assembly and cytokine maturation, an effect reversed by GPER inhibition [31]; and inhibited pyroptotic cell death by suppressing GPER1-NLRP3 axis after tMCAO [35].

Together, these results outline a mechanistic cascade of events where genistein triggers Nrf2-mediated antioxidant defense mechanism, inhibits NF-κB -mediated inflammation, and suppresses NLRP3-mediated pyroptosis. This tri-pathway cascade, characterized by two-way crosstalk between the oxidative stress response and inflammatory response, points to an integrated signaling axis where enhanced Nrf2 activity attenuates oxidative stress and thereby suppresses NF-κB activation, which in turn prevents NLRP3 inflammasome assembly, underlying the coordinated neuroprotective effects of genistein in models of ischemic stroke [9,10,14,20,23,24,26,29,31].

Reoccurrence of other signaling pathways, including PI3K/Akt, ERK1/2, Bcl-2/Bax and JAK/STAT, can be explained by two major factors. Firstly, genistein has a dual effect, acting as both an estrogen receptor-based signaling molecule and a tyrosine kinase blocker, which enables it to influence several survival-related pathways. Second, although studies are methodologically diverse, including a variety of ischemia models, e.g., MCAO, GCI, or OGD, and different dosages or routes of administration are used, the pathological hallmarks of ischemic injury, such as oxidative stress, inflammation, and apoptosis, are consistent.

These common pathophysiological events naturally intersect on conserved molecular signaling hubs, which are recurrently studied in various studies. Instead of suggesting redundancy, the recurrent finding of these signaling pathways indicates the conserved biopharmacodynamics of genistein and biologically denotes its importance in the ischemic stroke model.

4.5. Dose- and time-dependent modulation of genistein’s neuroprotective mechanisms

General observations without qualitative analysis in the current study indicate that the neuroprotective pathways of genistein, as antioxidative, anti-inflammatory, and anti-apoptotic, are responsive to dose and time-dependent effects.

At low doses (≤5 mg/kg), genistein enhanced its antioxidant defense effect, primarily by stimulating Nrf2. Intermediate doses (5−15 mg/kg) showed an extended effect, with pathways such as the inhibition of NF-κB, NLRP3, the activation of PI3K/Akt-mTOR, and GLP-1 signaling, as well as an increase in the level of Bcl-2, demonstrating both anti-inflammatory and anti-apoptotic effects. Although only a few studies have performed higher doses (>15 mg/kg), additional signaling routes, including JAK/STAT3 and Wnt/Ca2 + , were found to be activated by stronger stimulation, suggesting that more sophisticated cell survival pathways are engaged by more robust stimulation.

In terms of the timing of treatment. earlier treatment before the insult was associated with more significant changes in infarct size and more effective inhibition of oxidative, inflammatory, and apoptotic molecular products. Long-term treatment (> 7 days) promoted the remodelling of neuroglia, such as the switching of astrocyte phenotype [33], suggesting additional benefits in the course of post-stroke recovery.

In line with in vivo evidence, in vitro experiments also indicated concentration and time-based effects. An increase in micromolar concentrations (or 50 µM and above) and exposure times (6–24 hours) resulted in more pronounced oxidative stress and modulation of the apoptotic pathway, further confirming the dose-responsive effect of genistein in experimental models.

In general, these results highlight the importance of optimizing the dosage and timing of genistein delivery to achieve the maximum neuroprotective effects and molecular stability of genistein at various time points following ischemic injury.

4.6. Limitations

Some of the limitations in this systematic review are that many studies mainly rely on preclinical models, which have focused on animal-based in vivo experiments. These models provide information regarding the neuroprotective mechanisms of genistein, but they may not accurately replicate the complex pathophysiology of ischemic stroke in humans. This restricts the immediate applicability of the results in clinical practice. Another limitation is the variation in dosage and administration routes between in vitro and in vivo studies. This complicates the standardization of the therapeutic protocols of genistein. In addition, various studies have varied in the selection of the ischemic model, which can result in varying findings on the effectiveness of genistein when used in different experiments. Also, there is no existing literature on long-term effects, so the long-term effects and possible side effects of genistein are not fully understood. These restrictions indicate that additional clinical studies and standard practices are required to confirm the therapeutic efficacy of genistein on ischemic stroke in humans. Ultimately, numerous studies have focused on molecular pathways, potentially overlooking other critical mechanisms involved in ischemic injury and recovery. Future research addressing these limitations is significant for advancing genistein as a viable therapeutic option for stroke patients.