In the pathophysiological process of IS, inflammatory thrombi lead to cerebral vascular occlusion, inflammatory response, and severe nerve damage after ischemic events (De Meyer et al., 2022). Early platelet adhesion and activation are key factors for the development of IS inflammatory thrombosis. The main receptors that mediate platelet adhesion are glycoprotein (GP) VI and integrin α2β1, both of which bind to the GPIbα subunit of collagen and the GPIB-IX-V complex, which interact with the von Willebrand factor (vWF) (Poulter et al., 2017; Constantinescu-Bercu et al., 2022; Feitsma et al., 2022). After endothelial injury, vWF interacts with GPIbα, thus causing platelets to decelerate on the fixed vWF (Constantinescu-Bercu et al., 2022; Kanaji et al., 2022) and thereby contributing to platelet aggregation. The use of GPIbα-vWF inhibitors restores vascular patency by specifically breaking down the outer layer of the occlusive thrombus (Le Behot et al., 2014). Subsequently, platelet activation induces a conformational change in the GPIIb/IIIa surface receptor and its affinity to fibrinogen and vWF, thus promoting platelet aggregation (O'Brien and Salmon, 1990).

In addition, vWF was found in different samples of thrombus extracted from IS patients; the thrombus contained 20.3% ± 10.1% vWF on average (Denorme et al., 2016). In a middle cerebral artery occlusion (MCAO) rat model, cerebral infarct size and fibrinogen deposition were significantly increased in platelet-only vWF chimeric rats (Verhenne et al., 2015). Interestingly, vWF can be cleaved by metalloprotease ADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif member 13. ADAMTS13 effectively dissolves anti-tissue-plasminogen activator (t-PA) thrombus within 5–60 min of MCAO occlusion (Denorme et al., 2016). Furthermore, caADAMTS13, a ADAMTS13 variant, significantly reduced residual vWF, fibrin, and platelet aggregation as well as neutrophil recruitment in the middle cerebral artery (MCA) (South et al., 2022).

However, thrombosis not only involves simple platelet aggregation, but also includes leukocyte-platelet complexation (Li et al., 2015; Pircher et al., 2019; Schrottmaier et al., 2020). This may be because basic diseases, such as hyperlipidemia and hyperglycemia, stimulate hematopoietic cells in the bone marrow to produce a large number of white blood cells in the circulating blood (Stumvoll et al., 2005; Zhou et al., 2016; Vekic et al., 2019). Neutrophils are closely related to thrombosis in IS patients with COVID-19 (Genchi et al., 2022). Neutrophils account for the majority of leukocytes in IS thrombi, followed by macrophages and T cells (Heo et al., 2020). This difference is partly due to their proportion in the circulating blood under physiological conditions; however, IS is also related to the level of activation of various white blood cells. Thrombus formation is a series of complex events that occur sequentially in the vascular system, including endothelial activation, neutrophil extracellular trap (NET) formation, vWF secretion, blood cell adhesion, aggregation, and activation (Yang J. et al., 2020). Genetic deletion of PKM2 in bone marrow hematopoietic cells reduces NET after cerebral ischemia/reperfusion, which further reduces fibrinogen, platelet deposition, and inflammatory cytokines in the brain (Dhanesha et al., 2022). Similarly, rats lacking CD84 on their platelets or T cells showed reduced cerebral thrombosis and milder nerve damage after MCAO (Schuhmann et al., 2020). In contrast, endothelial CD69 deficiency increases fibrinogen and vWF levels in ischemic tissue and exacerbates nerve injury (Brait et al., 2019). Thus, inhibition of inflammatory thrombi formation is one of the goals of IS prevention.

Activation of immune cells, including neutrophils, T cells, and microglia, is involved in brain tissue repair after IS (Ma et al., 2017). Subsequently, neutrophils are attracted along a concentration gradient of chemokines in areas of ischemia to release pro-inflammatory factors, ROS, proteases, and matrix metalloproteinases (MMPs) (Wang et al., 2007), thus leading to the disruption of the BBB and exacerbation of neurological damage (Rosell et al., 2008). Similarly, in the acute phase of IS, Th1 and Th17 cells degrade tight junction proteins (TJ) by secreting IFN-γ, IL-17, and IL-21, thereby further disrupting the integrity of the BBB (Gelderblom et al., 2012; Clarkson et al., 2014). T cells and their isoforms have also been associated with repair and functional improvement in late brain injury (Liesz et al., 2009). During ongoing inflammation, activated M1 microglia phagocytose astrocyte ends and disrupt the integrity of the BBB by secreting various vascular proteins (Wang et al., 2018; Haruwaka et al., 2019).

Oxidative stress and mitochondrial dysfunction are important factors for the development of cerebral ischemic injury (Lo et al., 2003). Mitochondria are central to ROS production and cell death (Lo et al., 2003). Cerebral ischemia induces a cascade of excessive ROS production. Excess ROS leads to lipid peroxidation (LPO), exacerbates oxidative damage to proteins and nucleic acids, and contributes to neuronal apoptosis and BBB destruction (Allen and Bayraktutan, 2009; Kleinschnitz et al., 2010; Casas et al., 2017; Sun et al., 2018). Immune activation and oxidative stress also contribute to programmed neuronal death in the ischemic zone.

Both hypoxia and ischemia induce autophagy. Shortly thereafter, autophagic vesicles accumulate extensively in the brain tissue (Tuo et al., 2021). Mitochondrial autophagy facilitates the maintenance of cellular homeostasis under mild ischemia or hypoxia. In contrast, sustained ischemia-reperfusion (I/R) results in prolonged autophagy, thus promoting neuronal cell damage or even death (Zhang et al., 2013). Similarly, neuronal apoptosis is the main mechanism through which I/R injury induces cell death. The balance between anti-apoptotic Bcl-2 and pro-apoptotic Bax protein expression is critical for the regulation of apoptosis (Culmsee and Plesnila, 2006). ROS production and mitochondria-dependent apoptosis play an important role in neuronal death following I/R injury (Jordan et al., 2011; Wang et al., 2014). After IS, a series of molecular events induced by oxidative stress overlap with iron sagging/oxidation processes; these have common molecular targets, such as LPO and glutathione (GSH) depletion (Seiler et al., 2008; Choi et al., 2013; Yang et al., 2014). Iron death is dependent on excessive iron accumulation, with the core process being LPO (Cao and Dixon, 2016). In a rat model of MCAO, GSH inhibited iron death by driving glutathione peroxidase 4 (GPx4) expression, thereby protecting neurons and reducing core ischemic areas (Karuppagounder et al., 2016; Alim et al., 2019).

The activation of peripheral immune cells promotes platelet aggregation. Pre-treatment of activated T cells with quercetin blocks IL-12-induced JAK-STAT tyrosine phosphorylation, thereby reducing T cell proliferation and Th1 differentiation (Muthian and Bright, 2004). Quercetin has a similar effect on neutrophils. NETs are closely associated with inflammatory thromboses. Quercetin does not directly affect NET formation, but inhibits it in peripheral blood polykaryotic cells by downregulating TNF-α production in lipopolysaccharide (LPS) peripheral blood monocytes (Yuan et al., 2020; Jo et al., 2021). During inflammation, LPS delays the spontaneous apoptosis of neutrophils, while quercetin accelerates this process (Liu J. J. et al., 2005; Yuan et al., 2020). This is associated with a reduction in the expression of inflammatory cytokines, activation of PKCa, and enhancement of CD95-mediated apoptosis in neutrophils (Russo et al., 2003). Quercetin effectively protects LDL from neutrophil-mediated modification at physiological concentrations (1 µM) and inhibits myeloperoxidase (MPO) oxidative damage (Loke et al., 2008) (Figure 2). Subsequently, quercetin downregulates the TLR-NF-κB signaling pathway, reduces the activities of COX, 5-LOx, NOS, MPO, and CRP, inhibits ldL-induced adhesion molecule expression, and ameliorates endothelial dysfunction in atherosclerosis (Bhaskar et al., 2016). Quercetin also reduces the activity of neutrophil MPO and inhibits the production of HOCl, a powerful oxidant, to protect endothelial cells from oxidative damage (Lu et al., 2018). In contrast, Suri et al. (2008) found that quercetin did not exert excessive influence on neutrophils, but only reduced the calcium response induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP).

The key factor for the occurrence of IS is the formation of an inflammatory thrombus; thrombolysis significantly alleviates brain injury. Quercetin, a natural flavonol compound, can significantly reduce diabetes-induced platelet aggregation (Mosawy et al., 2014), which may be related to the inhibition of compact platelet granule exocytosis (Mosawy et al., 2013b). Similarly, quercetin inhibits agonists (ADP, collagen, and thrombin) as well as induces platelet aggregation and granule secretion (Liang et al., 2015). Quercetin also binds to the GPIIb/IIIa platelet receptor, thus inhibiting the aggregation-promoting properties of calcium ion carriers and avoiding an increase in platelet-derived particles; these improve hemorheology (Zaragozá et al., 2021) and reduce thrombosis after carotid artery injury induced by FeCl3 in C57BL/6 rats (Mosawy et al., 2013a). Quercetin also effectively blocked in vivo FeCl3-induced arterial thrombosis and reduced IS infarct volume by inhibiting glycoprotein VI (GPVI)-mediated platelet signal transduction (Oh et al., 2021).

The binding of collagen to GPVI leads to receptor aggregation, which stimulates tyrosine phosphorylation and thus causes platelet aggregation (Gibbins et al., 1996; Poole et al., 1997). Quercetin inhibits platelet activation by inhibiting various components of the GPVI signaling pathway (e.g., collagenous tyrosine phosphorylation) (Hubbard et al., 2003; Hubbard et al., 2006; Wright et al., 2010), which may be a key factor for improving nerve injury in IS. In clinical trials, platelet aggregation was inhibited 30 and 120 min after oral quercetin administration, with a corresponding reduction in collagen-stimulated platelet tyrosine phosphorylation (Hubbard et al., 2004). Quercetin also inhibited platelet aggregation when collagen was stimulated at concentrations between 0.5 and 1.0 μg/ml, with IC50 values below 3 μM (Hubbard et al., 2003). Thus, these results support the clinical transformation of quercetin.

In contrast, Lee et al. (2013) found that quercetin did not reduce PT, aPTT, or platelet aggregation in experimental rats. They revealed that its downregulation of mitogen-activated protein kinase (MAPK) activation restricted tissue factor expression, thereby prolonging the time for atherothrombosis development. In endothelial cells, quercetin transcription induces the human t-PA gene by requiring a specific Sp1 (b) element within the proximal promoter region, which is mediated by the P38 MAPK-dependent signaling pathway (Pan et al., 2008).

Protein disulfide isomerase (PDI), a thiol isomerase secreted by vascular cells, is required for thrombosis. Quercetin-3-rutinoside prevents thrombosis in a PDI-dependent manner in experimental rats (Jasuja et al., 2012). Oral administration of 1,000 mg isoquercetin can reduce the plasma concentration of D-dimer by 21.9% and inhibit the activity of PDI in the plasma, thereby exerting an antithrombotic effect (Zwicker et al., 2019). This was related to the reduction of platelet-dependent thrombin content by blocking the production of platelet factor Va (Stopa et al., 2017). Intravenous administration of quercetin significantly attenuated TNF-α levels and prothrombin activity in a rabbit model of LPS-induced DIC (Yu et al., 2013) (Figure 2). Nonetheless, oral quercetin did not prevent thrombo-embolic stroke in an earlier Dutch cohort study (Knekt et al., 2000). This finding may be related to the use of dietary quercetin content as an intervening factor. Table 2 summarizes the role of quercetin and its derivatives in limiting thrombosis.

In an in vitro ischemia model, quercetin-treated cells showed improved tolerance to oxygen-glucose deprivation (OGD) or oxygen glucose recovery (ROG) (Lee et al., 2016). Quercetin administration reduced the corrected total infarct volume and edema percentage by 43.6% and 48.5%, respectively, along with a significant behavioral recovery effect (Lee et al., 2015). Quercetin and Kolaviron pre-treatment significantly improved the I/R-induced changes in brain water content. Significant remission of cerebral infarction was observed in the Kolaviron and quercetin treatment groups (Akinmoladun et al., 2015), which might be linked to the role of quercetin in the Sirt1/nuclear factor-erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling cascade (Yang et al., 2021). Compared to free quercetin or quercetin-carrying exosome (quercetin-EXO) therapy, treatment with quercetin/mAb gAP43-EXO dramatically reduced infarct size and improved neurological recovery in MCAO reperfusion-induced rats (Guo et al., 2021) (Figure 3). Simultaneously, quercetin improved the IS-associated motor and sensory deficits in the dorsal striatum, which may be related to the upregulation of MC4R-mRNA expression (Ulya et al., 2021). More intuitively, quercetin showed 6.79 2 ± 0.41 right turn in rats in the permanent MCAO model and 9.31 ± 0.33 right turn in the control group. Most rats in the treatment group showed mild to moderate neuromotor deficits (p < 0.0001) (Ahn and Jeon, 2015). In addition, the infarct volume of rats in the control and treatment groups was 26.35 ± 2.25% and 14.87 ± 1.75%, respectively (Park et al., 2020). Quercetin can improve cognitive function in rats with ischemic injury. In the Morris water maze (MWM) test, quercetin therapy restored spatial learning deficits by increasing the time and amount of access to the central region (Le et al., 2020). By boosting the number of new Olig2+ oligodendrocyte progenitors in the subventricular zone, quercetin alleviated hypoxia/ischemia (HI)-induced cognitive impairment (Qu et al., 2014). Compared with the control group, I/R rats pre-treated with quercetin (20 mg/kg) for 7 days showed a significant reduction in cognitive impairment as well as improvement in motor capacity, cerebral edema, and infarct volume (p < 0.001) (Viswanatha et al., 2018; Viswanatha et al., 2019). Table 3 summarizes the neuroprotective effects of quercetin on IS.

IS significantly induced endogenous neurogenesis in the dentate gyrus of the hippocampus. However, newborn neurons are difficult to differentiate into mature neurons (Arvidsson et al., 2002; Doeppner et al., 2011). Quercetin maintains isocitrate dehydrogenase levels in MCAO animal models and helps to preserve neuronal cell energy production, thereby reducing IS-induced neuronal cell damage (Shah et al., 2018). Moreover, quercetin attenuates the decrease in PP2A subunit B expression caused by glutamate treatment, thus further reducing neuronal cell death (Park et al., 2019). Through the Sirt1/Nrf2/HO-1 signaling pathway, quercetin restores the normal structure of hippocampal neurons in I/R mice with severe neuronal injury (Yang et al., 2021). Quercetin also reduces the activity and pathophysiology of the following processes: protein tyrosine and serine/threonine phosphatase in rat cortical tissue, oxygen-glucose deprivation/reoxygenation (OGD/R) in hippocampal slices and neuronal/glial cell lines, phosphorylation of ERK and Akt, and I/R-induced hindbrain damage (Wang et al., 2020). Quercetin treatment can also significantly increase the activity of SHSY5Y cells and E18 mouse cortical neurons (Lee et al., 2016), enhance the expression of synaptophysin in PC12 cells in the OGD model, and promote neurite growth in PC12 cells (Orbán-Gyapai et al., 2014). Three days after reperfusion, oral administration of nano-encapsulated quercetin reduced the activity of iNOS and caspase-3, expanded the number of neurons in the hippocampus, and prevented neuronal cell damage (Ghosh et al., 2013) (Figure 3).

The mechanism of quercetin-induced autophagy in cell survival is complex because of the large number of biomolecules involved in this process. Based on the scope of the damage caused by HI, autophagy is used as a pro-apoptotic signal, wherein quercetin can be used as its inducer (Costa et al., 2016). In models of oxidative damage and ischemia, studies have revealed that the protective impact of quercetin is directly linked to the induction of autophagy (Wu et al., 2017). As a result, autophagy is linked to the pro-survival mechanism of quercetin in IS-induced brain injury and other related events (Wang et al., 2011; Zhi et al., 2016; Granato et al., 2017; Liu et al., 2017) (Figure 3). Quercetin has a protective effect against MCAO-induced neuronal cell apoptosis and likewise induces autophagy-mediated neuronal PC12 cell survival (Ahn and Jeon, 2015; Park et al., 2018). In IS, if myeloid cells lack an autophagy response, inflammatory glial cells would thus play a significant role in neuronal cell apoptosis by increasing ischemia; this happens in compensation for the reduced activity of myeloid cells (Kotoda et al., 2018). In this case, autophagy protected the neurons from ischemia-induced cell death. Surprisingly, quercetin, like many other polyphenols, induces autophagy (Pallauf and Rimbach, 2013). Quercetin plays a role in cellular survival by activating autophagy in brain myeloid cells (Chang et al., 2017). Furthermore, in MCAO-induced ischemia, quercetin altered the apoptosis/autophagy interaction and its linkage with the nuclear factor kappa B (NF-κB) signaling pathway by upregulating ubiquitin carboxy-terminal hydrolase L1, which is a related gene enzyme, at almost double the rate (Chirumbolo et al., 2019).

Quercetin upregulates the intracellular Ca²⁺ concentration in the cerebral cortical and hippocampal neurons of MCAO rats. It also regulates the gene expression of Bcl-2, Bax, and caspase-3, thereby preventing apoptosis (Park et al., 2020). Another study found that quercetin reduced HI-induced cortical cell death by blocking the neuro-inflammatory response mediated by the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway (Wu et al., 2019). The anti-apoptotic activity of quercetin may be due to its ability to suppress inflammatory genes in BV2 microglia (Mrvová et al., 2015). In addition, quercetin has also been reported to improve I/R-induced cognitive deficits as well as inhibit neuronal apoptosis by increasing p-Akt and decreasing p-ASK1, P-JNK3, cleaved caspase-3, and FADD protein expressions (Pei et al., 2016). Furthermore, quercetin not only inhibits acid toxicity mediated by acid-sensing ion channels, but also improves neuronal apoptosis in focal cerebral ischemia by reducing caspase-3 and PARP expression through the PI3K/Akt pathway (Park et al., 2018). After local cerebral ischemia, human umbilical cord mesenchymal stem cells (HUMSCs) transplanted with quercetin can reduce pro-inflammatory cytokines IL-1B and IL-6, increase anti-inflammatory cytokines IL-4 and IL-10, inhibit the expression of apoptosis factor caspase-3, and promote the recovery of nerve function (Zhang et al., 2016). Furthermore, isorhamnetin (30-methoxy-3,40,5, 7-tetrahydroxy flavanone), a quercetin metabolite, has been demonstrated to lower blood pressure and endothelial dysfunction in spontaneously hypertensive rats (Sánchez et al., 2006; Sanchez et al., 2007). In the methylglyoxal-binding OGD model, isorhamnetin inhibited caspase 8 activation and decreased Fas and FasL expression, thereby lowering the activation and ability of NF-κB to perform an anti-mitochondrial-dependent apoptotic role (Li W. et al., 2016) (Figure 3).