Mechanisms of immune response and cell death in ischemic stroke and their regulation by natural compounds

Ischemic stroke (IS), which is the third foremost cause of disability and death worldwide, has inflammation and cell death as its main pathological features. IS can lead to neuronal cell death and release factors such as damage-related molecular patterns, stimulating the immune system to release inflammatory mediators, thereby resulting in inflammation and exacerbating brain damage. Currently, there are a limited number of treatment methods for IS, which is a fact necessitating the discovery of new treatment targets. For this review, current research on inflammation and cell death in ischemic stroke was summarized. The complex roles and pathways of the principal immune cells (microglia, astrocyte, neutrophils, T lymphocytes, and monocytes/macrophage) in the immune system after IS in inflammation are discussed. The mechanisms of immune cell interactions and the cytokines involved in these interactions are summarized. Moreover, the cell death mechanisms (pyroptosis, apoptosis, necroptosis, PANoptosis, and ferroptosis) and pathways after IS are explored. Finally, a summary is provided of the mechanism of action of natural pharmacological active ingredients in the treatment of IS. Despite significant recent progress in research on IS, there remain many challenges that need to be overcome.


Introduction
Stroke is one of the major causes of death and disability worldwide (1).In the past three decades, the global incidence of stroke has increased by 70%, stroke mortality has increased by 43%, disability adjusted life spans lost due to stroke increased by 32%, and the economic burden of all countries has increased.Ischemic stroke (IS) is a severe insufficiency of the blood supply to the brain caused by thrombosis or embolism in the blood supply to the Microglia, as resident CNS macrophages, play a dual role of neurotoxicity and protection in IS.When brain injury occurs, numerous DAMPs and cytokines are released, and microglia are activated with the death of nerve cells in the central area of the infarct (5); this, together with the activation of macrophages from bloodderived monocytes, constitute the innate immune response, which is the first line of defense (56).During systemic inflammation, microglia are involved in tissue damage and repair, respectively (57).
and macrophages) gene programs over several days, including 3 days proliferation and motility, 7 days axon chemical attraction and ion channel activity, and 14 days extracellular matrix (ECM) recombination.The data also found that (i) phagocyte genes were still induced at 14 days to support the durable repair function of IAM; (ii) there was a high expression of ECM genes and nutrient factors; and (iii) the anti-inflammatory gene set signature was enriched at all stages.These three phenomena indicate that IAM has a long-lasting repair function.RNA-seq did not show significant changes in M1/M2 genes in bone marrow cells, and scRNA-seq further revealed their heterogeneous expression patterns in bone marrow subclusters.This further demonstrates the limitations of the conceptual dichotomy of pro-inflammatory and antiinflammatory phenotypes (60).
The involvement of microglia in tissue damage reaches its peak 3-5 days after ischemic injury.At this stage, microglia play a harmful role, mainly by destroying the blood brain barrier (BBB), aggravating brain edema, and promoting neuronal apoptosis by producing and secreting many inflammatory mediators (61).The release of pro-inflammatory cytokines leads to secondary brain injury, while microglia have been shown to exhibit repair functions for nearly 14 days after injury (62).Microglia play a primarily protective role that promotes the regression of inflammation by secreting IL-4, IL-10, and transforming growth factor (TGF)-b, thereby indirectly preventing inflammation-induced damage to the blood brain barrier (BBB).The regulatory pathways of microglial cell polarization can be divided into four categories: 1) transcription factors; 2) receptors; 3) ion channels; and 4) gene modulators (shown in Table 1).Moreover, microglial phagocytosis is a double-edged sword in immune inflammation and stroke recovery.Microglia have been shown to exhibit phagocytosis, and their ability to clean neuronal debris reduces brain damage after stroke (5).Microglia invade the ischemic site of stroke earlier than macrophages, and they are the main phagocytes for the first three days after stroke (63).After ischemia, microglia infiltrate the injured brain tissue, engulfing living and dead neurons, myelin debris, apoptotic cell debris, endothelial cells, and leukocytes.During pathological cases, microglial phagocytosis can be initiated by specific "eat me" signals on specific cell types and their corresponding receptors (64).
As shown in Table 1, many pathways and mediators have been confirmed to regulate the activation of microglia.For example, NF-kB and IL-4 as regulators promote activation of microglia, respectively, and are the most extensively studied and clearly researched mediators.Some conflicting results have been found in studies on the regulatory effects of TLR and STAT, and further research is necessary to clarify their roles (65).Most of these signaling pathways overlap to varying degrees and do not appear to work independently, but rather synergistically, resulting in an inflammatory maelstrom (66).Many studies have shown that targeting microglia can effectively treat IS (67).Further research on microglial activation signal pathways after IS will help identify effective drugs that inhibit microglial activation and prevent neuroinflammation mediated by microglial activity.Therefore, in future studies, it is crucial to identify appropriate targeted intervention drugs according to the role of microglia at different times.

Dual regulatory effects of astrocytes
Astrocytes, the most abundant neuroglial cells in the brain, are essential housekeeping cells that maintain the central nervous system.Astrocytes play, as do microglia, a dual role in the pathophysiology of IS (68).After IS, damaged cells produce and release cytokines and DAMPs to stimulate receptors of astrocytes and change their phenotype.A few minutes after IS, due to reactive astrocyte proliferation, astrocytes respond to various inflammatory factors (including TGF-a, ciliary neurotrophic factor, IL-1, IL-6, and kallikrein-related peptidase 6) released by ischemic/hypoxic cells, and are subsequently activated and reproduced (69,70).Reactive astrogliosis occurs in the peri-infarct region, and a glial scar is formed to maintain CNS homeostasis and wall off the lesion (71).During this process, astrocytes display cellular hypertrophy, proliferation, and increased expression of intermediate proteins, including glial fibrillary acidic protein (GFAP), vimentin, and nestin (72).After experiencing reactive astrogliosis, astrocytes produce and release pro-inflammatory cytokines (such as IL-6, TNF-a, IL-1a, IL-2b, and IFN-g), chemokines (such as CXCL1/ 10 and CCL2/3/5), important sources of ATP, and free radicals such as NO, superoxide, and peroxynitrites (73).Thus, the activation of microglia and infiltration of white blood cells are enhanced (74).Studies have discussed the two subtypes of reactive astrocytes as A1 and A2.The A1 subtype includes astrocytes induced by IL-1a, TNF-a, and complement component subunit 1q (C1q) secreted by activated microglia.A1 subtype astrocytes induce neuronal and oligodendrocyte death.The A2 subtype can secrete IL-2, IL-10, and TGF-b, thus accelerating the regression of inflammation (75).Additionally, the A2 subtype can play an inflammatory and neuroprotective role by secreting neurotrophic factors, neuropoietic cytokines, and growth factors (76).
The response of astrocytes to Injury is a major determinant of the outcome after stroke.The gene expression of A2 astrocytes dominates the expression of A1 astrocytes (77).The JAK/STAT3 signaling pathway was found to be an important switch controlling many molecular and functional changes in reactive astrocytes in vivo and in vitro (78).However, the roles of all these molecules and pathways need to be further validated in future research (79,80).In addition, the complexity of the multiple roles of complement protein and receptor expression in astrocytes has only recently been studied.Further in vivo and in vitro research is needed to determine astrocyte pathways and effects to determine targeted treatment strategies.

Peripheral immune cells 2.2.1 Recruitment and infiltration of neutrophils
Increased numbers of leukocytes have been found to be a marker of the inflammatory response in IS.Among various types of leukocytes, neutrophils are the first to respond to ischemic brain injury (81).An in vivo study found that neutrophils were found in leptomeninges and cerebral parenchyma 6 hours and 12 hours, respectively, after permanent middle cerebral artery occlusion (pMCAO) (82).Their recruitment reached a peak on days 1-3 and gradually decreased over time (83).Neutrophils produce extensive weblike structures of DNA (neutrophil extracellular traps, NETs) that reached their peak 3-5 days after the transient middle cerebral artery occlusion (tMCAO) (84).These NETs have been associated with inflammation (85).After cerebral ischemia, neutrophils undergo conformational changes due to the presence of many adhesion molecules, which helps them to migrate through blood vessel walls to the brain tissue.Activated microglia and astrocytes release chemokines (such as CXC and CC) to promote neutrophil activation.These chemoattractants bind to the C-C chemokine receptor 5 (CCR5) and C-X-C chemokine receptor 1 (CXCR1) on the surface of neutrophils, making neutrophils the first bloodderived immune cells to migrate to damaged brain tissue (86).Neutrophils are attracted to the ischemic region by chemokines, and then infiltrate damaged brain tissue soon after injury, which aggravates inflammation (87).
Traditionally, neutrophils have been considered the main mediators of harmful inflammatory responses in IS (88).However, a significant amount of evidence suggests that neutrophils can obtain different phenotypes.As in the case of microglia, it is believed that some neutrophil subsets show different characteristics.The response phenotype of neutrophils to the ischemic environment, and the interaction between neutrophils and endothelial adhesion molecules has shifted from protective N2 to the injurious N1 phenotype (89).In vivo and in vitro studies have shown that PPARg and TLR4 mediate the N2 phenotype of neutrophils (90,91).However, research on the functional changes and biomarkers of the N1/N2 phenotypes of neutrophils after IS are not sufficient.This also leads to a shortage of known pathways.Therefore, further research is needed on the role of neutrophils in the inflammatory response after IS.

Conflicting roles of T lymphocytes
T lymphocytes play an important role in the process of nerve damage and repair in the late stage of IS.In the acute phase of IS, T cells chiefly react in an antigen-independent manner and are closely related to the development of the infarct volume.After 3-7 days, the T cell response gradually transforms into antigen-dependent antigen recognition (92).Brain-derived antigens are recognized by T cell receptors (TCRs) on the surface of naïve T cells.Then, T cells migrate to the brain parenchyma through cell adhesion molecules (such as P-selectin, E-selectin, VCAM-1, and ICAM-1) and chemokines.Ultimately, adaptive immune responses exacerbate ischemia-reperfusion (I/R) injury.In vivo studies involving ischemic rats demonstrated that by day 3 after ischemia, many T cells infiltrated the peripheral areas around the lesion and surrounded the infarct center, and the number of T cells increased between days 3 and 7 (93).According to different functions, T cells have multiple types marked by CD3 expression, including CD8 + cytotoxic T lymphocytes (CTL), CD4 + T helper (Th) cells, regulatory T cells (Treg), and gamma delta (gd) T cells (94).The different roles of different types of T cells are already known, but the specific mechanism of T cell function after IS still needs further research.
CD 4+ T cells, as the main effector T cells, regulate brain inflammation by producing cytokines (95).The signals derived from T cell and co-stimulatory T cell receptors and extracellular cytokines determine the phenotype of Th cells.Cytokine signals are received through multimeric receptors and propagated largely through Janus kinase/signal transducer activator of transcription (JAK/STAT) signaling pathways (96).Th cells can be divided into Th1 and Th17 (pro-inflammatory), and Th2 and Treg (antiinflammatory) based on their cytokine secretion profile.Th1 and Th17 cells produce IL-1, IFN-g, IL-17, IL-22, and other cytokines.Th2 and Treg cells produce IL-4, IL-10, and TGF-b (97).Different types of CD4 + T cells have their own specific transcription factors that play a crucial role in their differentiation, maintenance, and function (98).
CD8 + T cells can play a cytotoxic role through antigen recognition of the TCR and subsequent release of granzyme and perforin, forming pores on target cells and inducing apoptosis (99).Selvaraj et al. investigated the role of CD8 + T cells in stroke by establishing a tMCAO mouse model.The results showed that CD8 + T cells had an adverse effect in the chronic phase after stroke.At 30 days, there was an increase in the number of ipsilesional CD8 + T cells, revealing its association with deterioration in mouse functional outcomes (100).In recent years, there have been studies on CD 8+ T cells inducing neuronal apoptosis through the FasL/PDPK1 pathway, but their mechanism of action after IS remains unclear.
gd T cells do not require antigen recognition to activate and are detected in infarcts 6 hours after ischemia.During the onset of IS, gd T cells mainly secrete cytokines such as IL-17, IL-21, IL-22, and IFN-g through receptors to protect the barrier from infection and exacerbate inflammation (101).Arunachalam et al. found that Vg6 + /CCR6 + gd T cell subtypes are the main source of IL-17 (102).However, few studies have paid attention to the signaling pathways present in gd T cells after activation by ligands that bind to their receptors.The low number of gd T cells, difficulty in extraction, and lack of cell lines may be the reasons for this lack of research.

Double-edged sword: monocytes/macrophage
The role of monocytes and macrophages in ischemic stroke is the same as that of microglia.Post-IS, pro-inflammatory monocytes infiltrate the inflamed brain, where they differentiate into macrophages that are morphologically indistinguishable from the local microglia (103).In contrast to the rapid microglial response, macrophages are rarely detected within the first 48 hours.Their level gradually increases, with a peak during the first week after stroke (104).On day 3 after stroke, the phenotypes of monocytes were found to change from the predominantly pro-inflammatory M1 to the anti-inflammatory M2 phenotype, indicating a functional shift from an enhanced immune response to inflammation resolution (105).Transcriptomic analysis of macrophages has shown that infiltrated macrophages on day 5 after stroke promote an effervescent increase and inflammation resolution after ischemic stroke (106).M2 macrophages can be further subdivided.For example, studies have classified macrophages as M2a, M2b, and M2c (107), while other authors have also classified them into an M2d subtype (108).All four M2 macrophage subtypes acquired enhanced phagocytosis and expressed IL-10, contributing to the resolution of inflammation (61).In a mouse model of ischemic stroke, researchers found that inflammatory activity peaked at 72 hours.Microglia produce relatively high levels of reactive oxygen species and TNF, while monocytes are major IL-1b producers.Although microglia show enhanced phagocytosis activity after stroke, monocytes have a significantly higher phagocytosis capacity at 72 hours (104).
Most M2 macrophages derived from monocytes can protect the blood-brain barrier from ischemic damage through vascular remodeling, physical attachment, and regression of inflammation (109).M2a macrophages express various anti-inflammatory and neurotrophic factors, such as arginase 1 (Arg1) and insulin-like growth factor-1.M2c macrophages increase the expression of TGFb, CD163, and sphingosine kinase.However, M2b macrophages increase the production of pro-inflammatory factors, including IL-1b, IL-6, and TNF-a, which may enhance inflammation and increase blood-brain barrier permeability early in IS (110).M2d macrophages secrete VEGF-A and TNF-a, all of which are detrimental to the blood-brain barrier integrity in IS (111).

Crosstalk between microglia and astrocytes
The interaction between activated microglia and astrocyte has a critical role in the process of neuroinflammation (shown in Table 2).In the first 6 hours after cerebral ischemia, microglia are first activated by pathogens or injury through TLR4, and release inflammatory mediators (112,113).At the same time, astrocytes dependently activate TLR2, TLR3, and TLR4 to respond (114).The "molecular signal" (IL-1, TNF-a, and C1q) released by microglia can convert astrocytes into a neurotoxic A1 phenotype.For example, Dr. Ben Barres' lab, using single-, dual-, and triple-gene knockout mice, pioneered the discovery that activated microglia secreting IL-1a, TNF-a, and C1q together are necessary and sufficient to induce A1 astrocytes (75).These neuroinflammatory reactive astrocytes lose many of their stereotypical physiological functions and secrete one or more unknown factors with strong toxicity to neurons and oligodendrocytes (115).Tarassishin et al. showed that human astrocytes and reactive astrogliosis are highly sensitive to IL-1b but unresponsive to lipopolysaccharide (LPS) stimulation.In human astrocytes, IL-1 induced both A1 and A2 responses (116).Glucagon-like peptide-1 receptor (GLP1R) is highly expressed in microglia, and is also expressed in astrocytes and neurons at reduced levels.Some studies have found that GLP1R agonists can directly prevent microglia-mediated astrocyte transformation into the A1 neurotoxic phenotype and have neuroprotective effects (117).The interleukin-1 family member interleukin-33 (IL-33) is produced by developing astrocytes, and it mainly signals to microglia and promotes synaptic phagocytosis of microglia under physiological conditions.IL-33 also drives microglia-dependent synaptic depletion in vivo.The transcriptomes of acutely isolated microglia from IL-33 −/− animals showed 483 significantly altered transcripts, including reduced expression of NF-kB targets (e.g., Tnf, Nfkbia, Nfkbiz, and Tnfaip3) (118).IL-15 is also the mediator of crosstalk between astrocytes and microglia, thus aggravating brain damage after intracerebral hemorrhage.Shi et al. established a transgenic mouse model targeting IL-15 expression in astrocytes and found that the accumulation of microglia near astrocytes in the tissue around the hematoma increased after brain injury.The expression of biomarkers in M1 microglial cells increased significantly (119).

Crosstalk between glial cells and peripheral immune cells
Astrocytes are the bridge between infiltrating T lymphocytes and neurons during cerebral ischemia.In vivo knockdown of interleukin-15 (IL-15) in astrocytes alleviates ischemic brain damage.Decreased levels of CD8 + T cells were also found in mice with knockdown of the IL-15 receptor a or blockade of cell-to-cell contact.Subsequent studies further confirmed the role of IL-15 from astrocytes on T cells.At the same time, a lower number of activated brain infiltrating CD4 + T cells were also found in Il15 −/− mice (120).Astrocytes, gd T cells, and Th17 cells are the main sources of interleukin-17 (IL-17) after IS.The main function of IL-17 involves coordinating local tissue inflammation by upregulating pro-inflammatory and neutrophil-mobilizing cytokines and chemokines.Kang et al. established a mouse model with specific deletions of key components of IL-17 signaling in various immune cells.It was found that astrocytes were crucial in IL-17-mediated white blood cell recruitment (121).Astrocyte-derived CXCL-1 acts as a key mediator of IL-17-initiated neutrophil chemotaxis in stroke.IL-17 secreted by gd T cells has also been reported to attract neutrophils to the site of injury (122).Subsequently, reactive microglia engulf neutrophils in the periphery of ischemic lesions, while the local microglia loss and dystrophy occurring in the ischemic core are associated with the accumulation of neutrophils, first in perivascular spaces and later in the parenchyma (123).Following IS, central nervous system injury can trigger the release of IL-33 from astrocytes.Ito et al. found that many Treg cells accumulated in the brain of mice dependent on IL-33 after IS.The chemokines CCL1 and CCL20 drive penetration into the brain.This helps with neurological recovery in the chronic phase of ischemic brain injury (124).In the MCAO mouse model, IL-33 treatment increased the number of Treg cells in the ischemic brain.IL-33 was shown to increase the levels of anti-inflammatory cytokines in serum and brain tissue (125).IL-33 also enhanced M2 polarization marker expression in microglia.Activation of the IL-33/ST2 axis led to polarization of M2 microglia, which provided protection for ischemic neurons in an IL-10 dependent manner (126).
The crosstalk between M1 microglia and Th1/Th17 cells plays a pro-inflammatory role and contributes to brain injury.The crosstalk between M2 microglia and Th2/Treg cells plays an anti-inflammatory role and helps with brain recovery.M1 microglia secrete IL-12 and TNF-a, which induce Th1 cells, and these two types of cells work together to promote inflammation.The M1 polarization promoted by Th1 cytokines (TNF-a and IFN-g) is associated with classic activation (127).M1 microglia secrete IL-6 and IL-23, which recruit Th17 cells and induce their differentiation (128).Th2 cells secrete IL-4 and IL-10, while Tregs secrete IL-10, further driving M2 polarization, inhibiting inflammation, and promoting tissue repair.IL-33 is suppressed in human stroke, resulting in an insufficient Th2-type response driven.In human T cells, IL-33 treatment induced IL-4 secretion while reducing astrocyte activation and increasing the number of M2 microglia (129).

Cell death in ischemic stroke
After ischemia, hypoperfusion of brain tissue leads to a decrease in oxygen, ATP, and glucose, which leads to cell death over time.Ischemic tissue can be functionally divided into irreversibly injured infarcted core tissue and peripheral ischemic penumbra tissue.The infarct core is composed of dead or dying tissues and is located in the central area of the infarct area.In the penumbra, this depletion hampers cellular physiological functioning but does not induce an irreversible change.Neuronal death in IS involves a variety of cell death pathways.Apoptosis, pyroptosis, necroptosis, and PANoptosis are four key cell death pathways (Table 3; Figure 2).

Pyroptosis
Pyroptosis is a form of regulatory necrosis mediated by caspase-1 and is mainly seen in the ischemic penumbra.Pyroptosis can be divided into inflammatory and non-inflammatory pathways (146).Inflammatory pathway is the main pathway, one is the classical inflammatory pathway mediated by caspase-1, the other is the nonclassical inflammatory pathway mediated by caspase-11.Inflammatory pathway of pyroptosis is an effective inducer of pro-inflammatory pathways in IS, occurring after the assembly and activation of inflammasomes (147).Inflammasomes contain pattern recognition receptors (PRRs), adapter proteins, and caspase family members (148).The adapter protein ASC has a cysteine protease recruitment domain (CARD) and a pyrin domain (PYD) (149).The structural characteristics of ASC provide support for binding of procaspase-1 to receptors.After IS, DAMPs secreted by necrotic cells in the ischemic core region are recognized by PRRs.
Then, procaspase-1 autocrine signaling produces cleaved caspase-1.Cleaved caspase-1 mediates microglial pyroptosis with the release of a large number of pro-inflammatory factors (IL-1 and IL-18) that induces neuronal death (150).Caspase-1 cleaves gasdermin D (GSDMD) into N-GSDMD, which binds directly to the plasma membrane and forms pores, releasing large amounts of cytosolic content to promote inflammation.The non-classical inflammatory pathway is one in which caspase-11 mediates the "non-canonical inflammasome" to participate in IL-1 and IL-18 processing and cell death (108).Studies had shown that ccaspase-11 (mouse-derived) also cleaves GSDMD, leading to focal ptosis under LPS stimulation (151).In addition, caspase-11 has been found to promote inflammation by regulating caspase-1 expression by promoting K + efflux (152).Non-inflammatory pyroptosis pathways are pathways in which caspase-8 is involved.It has been found that catalytic caspase-8 promotes the assembly of ASC-procaspase-1, in which caspase-8 acts as a scaffolding protein (153).In addition, caspase 8, procaspase 1, and cleaved caspase-1 were upregulated in an MCAO/R model (154).Similar to caspase-1, caspase-8 can also

Apoptosis
Apoptosis is the most common form of programmed cell death in multicellular organisms that elicits no inflammatory response.It is the main mechanism of neuronal loss after IS and can be triggered either through the intrinsic or the extrinsic pathway.The intrinsic pathway is caused by DNA damage or endoplasmic reticulum stress, and the extrinsic pathway is mediated by the activation of the death receptor family members (157).
The intrinsic pathway involves a non-receptor-mediated signaling cascade (158).After IS, excitotoxicity produced through the mitochondrial pathway can mediate Ca 2+ overload, leading to cell apoptosis.Glutamate binds to N-methyl-D-aspartate receptors (NMDARs), resulting in an overload of Ca 2+ in neurons (159).Ca 2+ activates the interaction of calpain with the Bcl-2 family proteins.Eventually, proapoptotic proteins are upregulated and mitochondrial permeability transition pores are formed (160), allowing for the release of apoptogens.The Bcl-2 protein family members (pro-apoptotic) regulate changes in the mitochondrial permeability, the release of cytochrome c, and contribute to apoptogen formation by binding with apoptotic protease activating factor-1 (Apaf-1) (161).Finally, activation of caspase leads to degradation of nuclear DNA, thus promoting cell apoptosis (162).
Following IS, the activation of immune cells during inflammation results in the release of a variety of factors (including pro-inflammatory cytokines) that trigger neuronal cell death via the extrinsic apoptotic pathways (163, 164).The extrinsic cell apoptosis pathway is triggered by the ligation of tumor necrosis factor (TNF)-family death receptors on the cell surface during external stimuli (165).After the receptor is bound, it recruits the adapter protein [Fas-associated death domain protein (FADD)] to create a death-inducing signaling complex with procaspase-8, which activates caspase-8 (166).Caspase-8 activates the downstream effector caspase, mediating apoptosis by direct proteolytic cleavage or indirectly by catalyzing the Bcl-2 protein family members (167).Velier et al. established a pMCAO rat model and found that proteolytic processing yielding the active form of caspase-8 was active 6 hours after stroke (168).

Necroptosis
Necroptosis, which is a lytic-programmed cell death with the ability to cause inflammation, is independent of caspase transmission.Similar to apoptosis, necroptosis is triggered by the ligation of specific death ligands to TNF-family death receptors or by pro-caspase inhibitors (169).This process leads to deubiquitination of receptor interacting protein kinase 1 (RIPK1) by the de-ubiquitination enzyme CYLD (170).RIPK1 activates the kinase RIPK3 within a cytoplasmic high molecular weight complex called a necrosome.RIPK3 phosphorylates and activates the mixed lineage kinase domain-like protein (MLKL), forming the homotrimer necrosomes (171).The accumulation of necrosomes leads to increased permeability of plasma membranes and organelles.This leads to membrane damage and subsequent cell death (172, 173).The phosphorylation of MLKL and the formation List of key genes involved in pyroptosis, apoptosis, and necroptosis in ischemic stroke.The genes involved in PANoptosis are marked in yellow.This image was created using BioRender.com.
of necrotic bodies are therefore considered as cellular markers of necrosis (174).After cerebral I/R injury, perivascular M1-microglia secrete TNF-a and its receptor TNFR1 on the endothelium, which serve as the main mediators triggering endothelial necroptosis (175).Necroptosis promotes the release of DAMPs, driving an inflammatory response.In vivo and in vitro studies found that RIPK3 promoted NLRP3 inflammasomes and the IL-1b inflammatory response independently of MLKL and necroptosis (176).In another study, it was found that MLKL signaling also activated NLRP3 inflammasomes and induced IL-1b secretion to promote inflammation.MLKL-induced NLRP3 inflammasome formation and IL-1b cleavage occur before cell lysis (177).

Ferroptosis
Ferroptosis refers to a new form of cell death caused by an increase of iron ion-dependent lipid peroxide (178).It is characterized by the accumulation of iron-regulated lipid peroxidation and caused by an imbalance of lipid metabolism, the depletion of glutathione (GSH), and the abnormal metabolism of iron.Excessive accumulation of iron is the key feature of ferroptosis, and most iron comes from damaged or aged red blood cells.Fe 2+ produced by erythrocyte degradation can be oxidized to Fe 3+ , and Fe 3+ binding transferrin (TF) mediates endocytosis through transferrin receptor (TFR)1 (179,180).After the endocytosis of TF-TFR1, Fe 3+ is released from TF and reduced to Fe 2+ by sixtransmembrane epithelial antigen of the prostate 3 (STEAP3).Finally, unbound iron is easily absorbed by neurons, resulting in intracellular iron accumulation (181).When iron is overloaded, Fe 2+ generates a large number of lipid-active oxygen radicals through the Fenton reaction.Fe 2+ can also participate in the synthesis of lipoxygenase and then catalyzes lipid peroxidation (182).
Lipid peroxidation is a critical process of ferroptosis (183).Ferroptosis shows obvious lipid peroxidation stress and cell membrane damage.Polyunsaturated fatty acid (PUFA)-phospholipid (PL) species are the most sensitive to peroxide because they contain highly active hydrogen atoms in their methylene bridge.In ferroptosis, acyl-CoA synthetase long-chain family member 4 (ACSL4) catalyzes fatty acids to form acyl coenzyme A and promotes fatty acid oxidation or lipid biosynthesis (184).Next, lysophosphatidylcholine acyltransferase 3 (LPCAT3) inserts the composite into the membrane phosphatidylethanolamine (PE).The ferroptotic signal is then activated (185).These lipids can be peroxidized under the catalysis of lipoxygenase (LOX) or under the induction of ROS (OH -) produced in the Fenton reaction.The resulting lipid peroxide can attack the proximal PUFA, causing a chain reaction and ferroptosis (186).
The manifestations of ferroptosis is the depletion of GSH and the inactivation of glutathione peroxidase 4 (GPX4).GSH is a tripeptide containing a sulfhydryl group, and it is composed of glutamic acid, glycine, and cysteine.It can combine with free radicals to repair cell membrane damage caused by lipid peroxide and it can clear ROS (187).GPX4 is a selenium enzyme.It can reduce oxidized lipids (L-OOH) (such as cholesterol and PL containing PUFA) to harmless lipid alcohols (L-OH) by converting GSH into oxidized glutathione (GSSG) (188).Therefore, GSH can also regulate GPX4 activity.In the process of ferroptosis, the accumulation of oxidation-reduction active iron consumes GSH reserves through the Fenton reaction, and then inhibits the activity of GPX4, leading to an overwhelming antioxidant reaction (189).The lack of GPX4 in turn leads to the accumulation of iron.

PANoptosis
Pyroptosis and apoptosis both involve the activation of members of the caspase protease family.Studies have found that the activation of caspase-1 triggers pyroptosis and apoptosis (190).As has already been mentioned, RIPK3 and MLKL are crucial for the occurrence of necroptosis.They can also mediate the formation of NLRP3 inflammasomes and trigger pyroptosis.This widespread crosstalk between pyroptosis, apoptosis, and necroptosis led to a new form of programmed cell death called "PANoptosis".
PANoptosis is an inflammation-regulated cell death pathway.These cell death pathways are interconnected through the shared regulatory proteins called the PANoptosome.The PANoptosome is a cell death-inducing complex that is characterized by pyroptosis, apoptosis, and necroptosis molecules.It was identified as an inducer and regulator of PANoptosis.Christgen et al. found that RIPK1, RIPK3, caspase-8, NLRP3, ASC, and FADD interacted to form PANoptosomes (191).These proteins can be divided into sensors (ZBP1 and NLRP3), adapters (ASC and FADD), and catalytic effectors (RIPK1, RIPK3, caspase-1, and caspase-8) based on their functions (192).Lee et al. found that AIM2 regulated the innate immune sensors pyrin and ZBP1 to drive inflammation signal transduction and PANoptosis.The results confirmed that AIM2 mediated the assembly of multi-protein complexes, known as the AIM2 PANoptosome (193).Another study found a RIPK1 PANoptosome complex in an in vivo model of bacterial pathogen infection, which regulates all three branches of PANoptosis (194).In addition, during influenza virus infection, ZBP1 recruited RIPK3 and caspase-8 to activate ZBP1-NLRP3 inflammasomes.The formation of ZBP1-NLRP3 inflammasomes mediates PANoptosis by assembling the ZBP1 PANoptosome (195).Yan et al. confirmed the existence of PANoptosis in in vitro and in vivo models of ischemic brain injury through researching literature (196).In a following study, they demonstrated the occurrence of PANoptosislike cell death in in vivo and in vitro models of ischemia/reperfusion injury (197).In summary, these studies indicate the presence of PANoptosis in ischemic brain injury.However, more research is needed to broaden our understanding of the basic processes of neuronal cell death and molecular targets, and to identify key molecules that regulate PANoptosis, which will lead to the development of new therapies.

Regulation mechanisms of natural compounds
The pathology of ischemic brain injury is an exceptionally complex pathological process involving a variety of cytotoxic factors and inflammatory cells in the CNS as well as in the peripheral circulatory system.Inflammation and cell death are the two main factors in IS.Inflammation and cell death, which are caused by ischemia, overlap and are interrelated.Due to the complexity of these factors and their interactions, it is very difficult to develop effective treatment methods based on the "one drug, one target" strategy, which leads to adverse outcomes in stroke treatment (198).A substantial number of studies have shown that some natural compounds (such as salidroside, baicalin, astragaloside IV, and curcumin) have protective effects on IS with few side effects (shown in Table 4).

Salidroside
Salidroside (Sal) is the main bioactive component in Rhodiola rosea.In many studies of IS in vitro and in vivo in cells and animals, salidroside has demonstrated strong biological activity.Sal can significantly reduce the brain infarct size and cerebral edema by inhibiting inflammatory signaling.Sal reduces the levels of proinflammatory cytokines and chemokines in tissues or serum, such as TNF-a, IL-2, IL-6, IL-8, IL-1b, MCP-1, and MIP-1a (199).After cerebral ischemia, inflammatory transduction mainly depends on NF-k B, mitogen -ac tivated prot ein kinase s (MA PK), phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt), and phosphoinositide 3-kinase/protein kinase B (PI3K/PKB) signaling pathways.Chen et al. found Sal effectively reduced the levels of IL-6, IL-1b, and TNF-a by blocking the RIP140/NF-kB pathway (200).Sal also significantly inhibited activation of NF-kB, blocked degradation of tropomyosin-related kinase B (IkBa), and reduced p-MAPK levels (JNK, p38 and ERK1/2) (201).In a further study, it was demonstrated that Sal inhibited CD11b and inflammatory mediators through PI3K/Akt/HIF signaling.Sal significantly upregulated HIF subunits (HIF1a, HIF2a, and HIF3a) and the HIF downstream target (erythropoietin).Sal reduced CD14, CD44, and iNOS mRNA (202).Zhang et al. demonstrated that Sal reduced inflammation and brain damage through PI3K/PKB/Nrf-2/NFkB signaling transduction.Sal induced NeuN and inhibited NF-kB p50 subunit and other pro-inflammatory mediators.It prevented a significant decrease in the proportion of p-PKB/PKB in the brain (203).These studies imply that Sal may inhibit inflammatory signaling through the Nrf2, HIF, MAPK, PI3K/Akt, PI3K/PKB, and NF-kB signaling pathways.In addition, Sal acts on immune cells to recover the damage caused by IS.A recent study reported that Sal significantly inhibited the release of inflammatory factors derived from microglia.To study microglia polarization, M1 phenotypic markers (CD16, CD32, iNOS, and CD11b) and M2 phenotypic markers (CD206, Arg1, TGF-b, and YM1/2) were analyzed.The results showed that Sal promoted the transformation of microglia from the M1 phenotype to the M2 phenotype to enhance the phagocytosis of microglia.At the same time, Sal-treated M1 microglia promoted oligodendrocyte differentiation (204).Sal has been shown to effectively reduce VCAM-1, ICAM-1, P-selectin, and E-selectin, as well as neutrophil recruitment in the ischemic brain (205).
Apoptosis is one of the main mechanisms of brain injury, and Sal has been found to have significant anti apoptotic effects.Brainderived neurotrophic factor (BDNF) is a member of the neurotrophic factors.BDNF has a protective effect on ischemic brain injury.Zhang et al. indicated that Sal produced its antiapoptotic effects by regulating the BDNF-mediated PI3K/Akt apoptosis pathway in a DNA-binding-dependent and -independent manners (206).Sal has been shown to inhibit the downregulation of Bcl-2, the upregulation of Bax, and the release of mitochondrial cytochrome c into the cytosol.Sal attenuated the activation of caspase-3, -8, and -9, and ultimately protected cells from apoptosis (207).Another study demonstrated that Sal induced activation of the mitogen-activated protein kinase kinase (MAPKK)/extracellular signal-related protein kinase (ERK) pathway, thereby reducing cell apoptosis (208).Shi et al. showed that Sal decreased the expression of Bax and restored the balance between pro-apoptotic and anti-apoptotic proteins (209).
These studies indicate that Sal has anti-inflammatory and antiapoptotic effects (Figure 3).In addition, Sal also demonstrated excitotoxicity inhibition and anti-oxidant effects, and reduced damage to the BBB.Therefore, as an effective neuroprotective agent, it can be developed as a potential drug for treating stroke.

Astragaloside IV
Astragaloside IV (AS-IV) is one of the main active ingredients from Astragalus (Astragalus membranaceus (Fisch.)Bunge., Leguminosae, Huangqi in Chinese).AS-IV has been shown to significantly reduce neuronal apoptosis.AS-IV can suppress the activation of key factors in the death receptor pathway.AS-IV was found to inhibit mRNA upregulation of Fas, FasL, Caspase-8, and Bax/Bcl-2.AS-IV also inhibited the protein levels of caspase-8, Bid, cleaved caspase-3 and cytochrome C (210).AS-IV can regulate the Nrf2 signaling pathway.Yang et al. found that AS-IV induced Nrf2 through the downstream signaling pathways (MAPK pathway) to prevent cell apoptosis.AS-IV inhibited the CXCR4 receptor and downregulated the activation of the p-JNK/JNK pathway, thereby inhibiting the expression of Bax/Bcl-2 and ultimately increasing Nrf2/Keap1 signaling (211).Another study confirmed that AS-IV regulated cell apoptosis through the PI3K/Akt/GSK-3b pathway (212).The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor.Its activation can increase the intracellular calcium concentration and contribute to cell apoptosis (213).AS-IV alleviated brain injury by inhibiting cell apoptosis induced by CaSR activation (214).Excitotoxicity by glutamate and mitochondrial dysfunction are common causes of cell apoptosis.AS-IV protects the integrity of mitochondria by promoting the combination of Akt and hexokinase II (HK-II).This helps to protect neurons from cell apoptosis and DNA damage (215).The PKA/ CREB pathway regulates mitochondrial activity.AS-IV protects primary neurons from IS-induced apoptosis by regulating the PKA/ CREB pathway and protecting mitochondrial function (216).
AS-IV also promotes the conversion of immune cells to an anti-inflammatory phenotype after IS, thereby reducing brain    The meaning of the symbol "↑" is "upregulated".The meaning of the symbol "↓" is "downregulated"./, not appicable.
Regulatory network of salidroside in ischemic stroke.Post-IS, salidroside regulates inflammation and cell apoptosis.This image was created using BioRender.com.

Baicalin
Baicalin (BA) is a natural flavonoid compound isolated from the dried roots of Scutellaria baicalensis Georgi.BA alleviates the inflammatory reaction.BA was found to inhibit the TLR2/4 signaling pathway during cerebral ischemia, reducing expression of TLR2/4 and NF-kB in rat brain tissue.BA also attenuated the serum levels of TNF-a and IL-1b (222).In subsequent studies, Tu et al. also found that administration of BA after focal cerebral ischemia significantly reduced brain edema and BBB permeability.Overexpression of MMP-9 degraded the tight junction protein occludin, disrupting the integrity of the tight junction of the BBB (223).BA significantly downregulates the expression of MMP-9 protein and mRNA (224).
A growing body of evidence has shown the beneficial roles of BA in stroke management, such as anti-apoptosis.BA significantly inhibited neuronal apoptosis after cerebral ischemia injury in rats.Tu et al. found that BA significantly decreased MPO enzyme activity and iNOS and COX-2 mRNA expression in rat brain tissue, and significantly inhibited the expression of cleaved caspase-3 protein after IS (225).Li et al. showed that BA inhibited the expression of dynein related protein 1 (Drp-1).BA also reduced mitochondrial division and promoted the production of mitochondrial fusion protein 2 (MFN2) in an AMPK-dependent manner (226).Zhou et al. found that BA activated Akt phosphorylation and upregulated glutamate transporter 1 (GLT-1) expression through the PI3K/Akt signaling pathway.This inhibited cell apoptosis and reduced cerebral infarction volume and neuronal loss (227).BA also reduced mitochondrial succinate dehydrogenase (SDH)-mediated oxidative stress and reduced subsequent loss of glutamine synthetase (GS) (228).

Curcumin
Curcumin (CCM) is a compound mainly extracted from Curcuma longa.After IS, CCM can attenuate the inflammatory effect.The MAPK signaling pathway is regulated by TLR4 signaling and plays a key inflammatory role in IS.CCM alleviates inflammation of IS through the TLR4/p38/MAPK pathway.After CCM treatment, the protein levels of TLR4, p-p38, and IL-1 decreased, while the expression of IL-6, TNF-a, and iNOS increased (229).Another study demonstrated that CCM reduced inflammation by reducing levels of pro-inflammatory cytokines.Simultaneously, mitochondrial function was restored through an increase of MMP (230).In addition, CCM has a profound regulatory effect on the microglial response, promoting M2 microglia polarization and inhibiting the microgliamediated proinflammatory response (231).However, further research is needed to confirm the involvement of curcumin and the specific mechanism of microglia phenotype regulation using stroke models.At the transcriptional level, the activation of NF-kB regulates ICAM-1, MMP-9 and caspase-3 expression (232).CCM decreased the expression of NF-kB, and subsequently attenuated the expression of the downstream mediators ICAM-1, MMP-9, and caspase-3 (233).
CCM exerts neuroprotective effects on IS and inhibits cell apoptosis.CCM reduces mitochondrial dysfunction and inhibits apoptosis by maintaining mitochondrial membrane potential and inhibiting the upregulation expression of Bax and downregulation of Bcl-2 (234).Silent information regulator 1 (Sirt1) is a class III group histone deacetylases that can protect the brain from ischemic damage (235).CCM was found to upregulate the expression of Sirt1 and Bcl-2 and downregulate the expression of acetylated p53 (Ac-p53) and Bax.Activating Sirt1 weakened cell apoptosis and promoted the neuroprotective effect of CCM (236).The MAPK signaling pathway regulates the expression of various proinflammatory cytokines and mediates apoptosis after ischemic injury.ERK1/2 and JNK are two of the main effectors of the MAPK signaling pathways (237).Lu et al. found that CCM reduced p-ERK1/2 and increased p-JNK protein levels.CCM also increased the level of flotilin-1 protein, thereby reducing cell death (238).CCM also improved neurofunctional recovery and promoted neurogenesis through Notch signaling after IS (239).
Therefore, based on the anti-inflammatory and anti-apoptosis effects of CCM, it may be a useful and promising neuroprotective agent against acute IS.

Conclusions and perspectives
Immunity, inflammation, and cell death play critical roles in the occurrence and development of stroke.This review summarizes the roles and mechanisms of immune cells and cell death pathways in IS.The immune cells discussed included microglia, astrocyte, neutrophils, T lymphocytes, and monocytes/macrophages.The cell death pathways discussed included apoptosis, pyroptosis, necroptosis, PANoptosis, and ferroptosis.This review also summarized the mechanisms of natural compounds in the treatment of IS.The natural compounds discussed include salidroside, baicalin, astragaloside IV, and curcumin.
Microglia and monocytes/macrophages form the first line of defense, but are involved in damage in the early stages of ischemic stroke.Microglia can induce increased damage to the A1 neurotoxic subtype of astrocytes.Neutrophils are recruited into damaged brain tissue, which can exacerbate inflammation.Subsequently, microglia and monocytes/macrophages show anti-inflammatory and repair functions.After T cells migrate to the brain parenchyma, they differentiate into different functional types.Hence, time-defined treatments targeting different phenotypes of immune cells may provide a clear protective strategy.At the same time, the interactions between immune cells cannot be ignored.The mutual coordination between immune cells is also caused by various inflammatory mediators.After IS, peripheral immune cells and brain immune cells form a complex inflammatory network.Treatments that target only one type of immune cell may be harmful or offset the benefits of another type of immune cell, resulting in an unsatisfactory stroke prognosis.Therefore, therapeutic strategies to modulate the immune system need to be further explored to determine effective treatment measures.
Compared to PANoptosis, the key molecular pathways involved in apoptosis, pyrotosis, ferroptosis, and necroptosis are clearer.Further research is needed on the molecular basis and key pathways of PANopotosis after IS.Further research on the molecular and regulatory mechanisms of PANopotosis will have new impacts on the treatment of IS.For natural compounds in this review, mechanism research of Sal is the most extensive.All the natural compounds included in this review have therapeutic effects on inhibiting inflammation and cell apoptosis in IS.Salidroside, baicalin, astragaloside IV, and curcumin may be effective and promising candidates for the treatment of IS.However, they still have certain limitations, including whether they can show the same effect clinically as in research studies.Future research directions include 1) mechanisms for drugs to enter the central nervous system, 2) the ability to penetrate the blood-brain barrier and distribute widely in the brain, and 3) the side effects of drugs.With further research, the discovery of new drugs will lead to better treatment of IS for the benefit of public health.

FIGURE 2
FIGURE 2 damage.PPARg is a nuclear transcriptional factor that is widely expressed in microglia(217).AS-IV can promote the polarization of M1 microglia to the M2 phenotype, which plays a long-term neuroprotective role in cerebral ischemia/reperfusion injury through the PPARg pathway (218).In addition, AS-IV also promotes angiogenesis by activating the HIF/VEGF/Notch, Wnt, and BDNF-TrkB pathways after IS, increasing cell proliferation, migration, and neovascularization (219-221).

TABLE 1 Continued
2.1.1First line of defense: microglia

TABLE 2
Cytokines in mutual coordination between immune cells.

TABLE 3
Cell death and pathways.

TABLE 4
Regulation mechanisms of natural compounds.

TABLE 4 Continued
References are shown in text.