Insight into Crosstalk Between Mitophagy and Apoptosis/Necroptosis: Mechanisms and Clinical Applications in Ischemic Stroke

Ischemic stroke is a serious cerebrovascular disease with high morbidity and mortality. As a result of ischemia-reperfusion, a cascade of pathophysiological responses is triggered by the imbalance in metabolic supply and demand, resulting in cell loss. These cellular injuries follow various molecular mechanisms solely or in combination with this disorder. Mitochondria play a driving role in the pathophysiological processes of ischemic stroke. Once ischemic stroke occurs, damaged cells would respond to such stress through mitophagy. Mitophagy is known as a conservatively selective autophagy, contributing to the removal of excessive protein aggregates and damaged intracellular components, as well as aging mitochondria. Moderate mitophagy may exert neuroprotection against stroke. Several pathways associated with the mitochondrial network collectively contribute to recovering the homeostasis of the neurovascular unit. However, excessive mitophagy would also promote ischemia-reperfusion injury. Therefore, mitophagy is a double-edged sword, which suggests that maximizing the benefits of mitophagy is one of the direction of future efforts. This review emphasized the role of mitophagy in ischemic stroke, and highlighted the crosstalk between mitophagy and apoptosis/necroptosis.

Due to its increasing incidence, stroke has become one of the leading causes of disability and death worldwide [1,2] . To date, it has been reported that ischemic stroke is the main type of stroke, which accounts for 70%-80% of total stroke events, seriously lowering the quality of life [3] . Thus, a number of researchers have contributed to the field of ischemic stroke, targeting the pathophysiology and various molecular mechanisms for the treatment of ischemic stroke [4][5][6] . The initial event of ischemic stroke is the sudden decrease in blood flow and oxygen supply to the cerebrum [7] . Insufficient blood flow deprives brain neurons of glucose and oxygen, and destroys cell homeostasis, leading to excitotoxicity, oxidative stress and inflammation, and neuronal cell death extensively occurs [8] . In order to minimize the irreversible neuronal death, a lot of therapeutic strategies have been provided, which include intravenous thrombolysis, tissuetype plasminogen activator (tPA), and endovascular therapy [9,10] . Furthermore, the expansion of the time window for reperfusion treatment or emergency reperfusion appears to be an effective intervention for acute ischemic stroke, to date [11] . However, limited by the short treatment window (intravenous thrombolysis within 4.5 h), these strategies also appear to be less effective, and a large number of neurons undergo function loss or even death. For example, it has been reported that the reperfusion process after thrombolysis can cause brain tissue infarction [12] . Thus, clearer pathophysiology of ischemic stroke may provide a means to establish therapeutic guidelines.
As the "energy factory" of cells, mitochondria play an important role in almost all cells for their energy homeostasis [13] . Some studies have revealed that ischemia-reperfusion (I/R) injury can cause mitochondrial dysfunction, which induces the progressive deterioration of neurons [14] . That is, mitochondrial dysfunction works as a decisive step in I/R injury, and this can be described, as follows: (1) breakdown of mitochondrial dynamics (including ATP/ADP decrease, the production of active oxygen increases, and the clearance decreases); (2) the mitochondrial membrane permeability transition pore (mPTP) continues to open [including cytochrome c (cyt c) and mitochondrial DNA, these are released into the cytoplasm] [15,16] . All these can lead to neuronal death. Therefore, increasing attention has been given to mitochondria, and it has been suggested that the effective treatment prospect of ischemic stroke is to target mitochondria. For mitochondrial dysfunction, a self-regulatory mechanism would be activated to remove the dysfunctional mitochondria, which is termed as "mitophagy" (also known as mitochondrial autophagy) [17,18] . After mitochondrial damage, this will be triggered by the initiator, such as parkin [19] . Therefore, when neurons are exposed to external stress, mitophagy is initiated to remove damaged mitochondria, and maintain cellular homeostasis. Notably, mitophagy is not a single signaling pathway, but a complex network between mitophagy and apoptosis/necroptosis, and this has received more attention than one crucial pathway in the field of ischemic stroke [20] . The present review focuses on the crosstalk between mitophagy and apoptosis/ necroptosis after ischemic stroke, with emphasis on the improvement of mitochondrial-based ischemic stroke treatment.

THE MECHANISM OF MITOPHAGY AND ITS FUNCTION IN SCHEMIC STROKE
Mitophagy is a type of selective autophagy that can remove dysfunctional mitochondria and maintain cell homeostasis [21,22] . It is known that mitochondria are important organelles, which are mainly responsible for providing energy for cells [23] . Once the mitochondria are damaged, there would be some harmful factors released into the cytoplasm, such as reactive oxygen species (ROS) and other oxidants, resulting in an awful case in cells. Even worse, cyt c, a mitochondrial intermembrane space protein, would be released under severe mitochondrial damage [6,15,24] . Cyt c triggers the caspase cascade, leading to apoptosis [25] . Interestingly, mitophagy can contribute to the rapid degradation of damaged mitochondria, which in turn can prevent cell apoptosis [26][27][28] . Mitophagy starts with the formation of the phagophore, which is a membrane structure isolated from the endoplasmic reticulum (ER) [29] . The recognition of target mitochondria by the phagophore occurs through microtubule-associated protein 1 light chain 3 α (LC3) adapters, in an ubiquitin-dependent and independent pathway, and through the direct interaction of LC3 with its receptors [30] . Overall, the use of mitophagy can be divided into two major pathways: ubiquitin-mediated pathway and receptor-mediated pathway.

Molecular Pathways Involved in Mitophagy 1.Ubiquitin-mediated Mitophagy
Ubiquitinmediated mitophagy is considered as the canonical signaling pathway, which is regulated by two proteins, PTEN-induced kinase 1 (PINK1) and parkin [31] . In 2008, a study revealed that loss of mitochondrial membrane potential can trigger the recruitment of parkin to mitochondria. In addition, it was also found that parkin can promote the degradation of damaged mitochondria through autophagy [32] . Furthermore, PINK1, a serine/ threonine kinase, has been reported to be able to regulate parkin [33] . Under normal physiological conditions, PINK1 is imported into the mitochondria through the translocase of the outer mitochondrial membrane complex of the outer mitochondrial membrane (OMM), and into the translocase complex of the inner mitochondrial membrane (IMM), where this is cleaved by the mitochondrial processing peptidase and rhomboid protease presenilin-associated rhomboid-like protein [34,35] . The normal expression level of PINK1 is relatively low, but this will accumulate in the OMM during mitochondrial damage, increasing the mitochondrial ROS, and inducing the depolarization and accumulation of misfolded proteins [36] . The accumulated PINK1 is activated and autophosphorylated, and in turn, this phosphorylates ubiquitin on serine 65, thereby recruiting parkin from the cytoplasm to the mitochondrial membrane. Studies have shown that PINK1 Ser228 and Ser402 residues are autophosphorylated upon decrease in mitochondrial membrane potential, and that this autophosphorylation is essential for parkin recruitment onto damaged mitochondria [37] .
Parkin is an E3 ubiquitin ligase. When parkin is recruited to the OMM and activated, this drives OMM proteins to undergo ubiquitination and degradation, thereby driving mitophagy [38] . Importantly, although activated PINK1 can recruit parkin to the OMM, studies have shown that even without PINK1, parkin can be recruited to depolarized mitochondria to drive the mitophagy [39] . Parkin transfers to mitochondria, and ubiquitinates the proteins on the OMM, such as mitofusin 1/2 and voltage-dependent anion channel 1 (VDAC1) [39] (fig. 1). However, for the Dmn/Drp1 knockout model, Yamade et al reported that the ubiquitination of mitochondrial proteins required SQSTM1/p62, but not the ubiquitin E3 ligase PRKN/ parkin, during mitophagy [40] .
PINK1-mediated phosphorylation leads to parkin activation and the ubiquitination of substrates on damaged mitochondria, which is the mitophagy signaling pathway [41,42] . Mitofusin, Miro and VDAC have been identified as parkin substrates [43,44] . Other OMM proteins that undergo parkin-mediated ubiquitylation have been identified by mass spectrometry, suggesting that parkin can ubiquitylate a large number of proteins on the surface of mitochondria [45] . Hence, parkin appears to have low substrate selectivity. Such a unique specificity appears to be optimal for parkin to achieve the efficient and quick ubiquitylation of dysfunctional mitochondria [46] . Even under steady state, a small amount of ubiquitin is attached to proteins on the surface of mitochondria. When PINK1 phosphorylates the ubiquitin, the resultant phosphor ubiquitin recruits parkin from the cytosol, and activates it on depolarized mitochondria to generate more ubiquitin chains and ubiquitin substrates [47] .

Receptor-mediated Mitophagy
An alternative pathway of mitophagy is through mitophagy receptor signaling. These receptors generally contain a LC3 interaction region (LIR) domain, which is used to directly bind to LC3, and phagocytosed by autophagosomes [48] .
At present, a series of receptors have been identified in mitophagy in mammals, such as BCL2 interacting protein 3 (BNIP3), BINIP-like (BNIP3L, also known as NIX) and FUN 14 domain-containing 1 (FUNDC1). BNIP3L is a homology of BNIP3, and has 55% identical amino acid sequence [49] . The C-terminal transmembrane domain of NIX and BNIP3 is inserted into the OMM, while the N-terminal is exposed to the cytoplasm [49] . A number of studies have revealed that NIX and BNIP3 play an important role in mitophagy. NIX and BNIP3 can increase the production of ROS and trigger mitophagy. Furthermore, NIX or BNIP3 competitively binds to Bcl2 to dissociate the Bcl2-Beclin1 complex, and activate the autophagy or mitophagy [50] . A study reported that the glucocorticoid downregulation of BNIP3L/NIX directly guides the glucocorticoid receptor to bind to the PGC1α promoter, and downregulate its expression and nuclear translocation, thereby selectively reducing the NIXdependent mitophagy [51] .
Another important mitophagy receptor, FUN 14 domain-containing 1, is an OMM protein. FUNDC1 contains a C-terminal domain inserted into the IMM and an N-terminal domain exposed to the cytoplasm, as well as a typical LIR sequence. The LIR sequence is located in the 50 amino acid residues exposed to the N-terminus of the cytoplasm [52,53] . FUNDC1 is mainly recruited to mitochondria-mediated ischemia-induced mitophagy by interacting with LC3 [54] . The mass spectrometry results revealed that FUNDC1 has two states: phosphorylation and dephosphorylation. The phosphorylation and dephosphorylation are respectively mediated by protein kinase and phosphatase, and the process is reversible [55] . FUNDC1 controls the interaction with LC3 by regulating its phosphorylation level. Under physiological conditions, Src and casein kinase 2 (CK2) are responsible for the phosphorylation of FUNDC1 at Tyr18 and Ser13, respectively. This phosphorylation event inhibits the interaction of FUNDC1 and LC3 [56,57] . Phosphoglycerate mutase  2) [58] .

Molecular Mechanism and Function of Mitophagy in Ischemic Stroke 1.2.1 Diverse Roles of Mitochondria in Mitochondrial Dynamics
Mitophagy plays a vital role in the balance of mitochondrial dynamics and the balance of mitochondrial fission, and fusion is closely correlated to maintaining mitochondrial homeostasis, cell stability and cell survival [59] . The mitochondrial fission process includes mitochondrial contraction and division, while the mitochondrial fusion process includes mitochondrial joining and tethering. Fission can remove damaged mitochondria, such as damaged proteins, unstable membranes, and mutated or damaged mitochondrial DNA (mtNDA) [21,60,61] . Conversely, fusion helps maintain the integrity of matrix metabolites and mtDNA, and balance membrane components [62] . The balance of mitochondrial division and fusion is critical for the survival of neurons. Studies have revealed that Drp1-mediated mitophagy is triggered after hypoxic/ ischemic stress, and that the inhibition of mitophagy can rescue the loss of neurons [63] .
Mitochondrial fission is mainly regulated by Drp1, Fis1, Mff and Mid49/51 [64] . Drp1 is mainly located in the cytoplasmic matrix, and cannot directly bind to the mitochondrial membrane. Receptors, such as Fis1, Mff and Mid49/51, are needed to help Drp1 translocate to the OMM, and play its role. As a key molecule in mitochondrial fission, Drp1 has been given attention by most researchers [65] . Studies have revealed that the use of Drp1 inhibitor mdivi-1 can effectively improve nerve function damage, reduce mitochondrial damage, and protect neurons [66] . However, the effectiveness and specificity of mdivi-1 remain to be questioned by researchers [67] . Therefore, the identification of molecules that target Drp1 remains promising for the discovery of effective intervention targets. Flippo et al reported that in AKAP1 -/mice, the mitochondrial fission increased, but the total expression level of Drp1 did not change [68] . On the other hand, the phosphorylation level of Drp1 pS637 decreased, while the phosphorylation level of Drp1 pS616 increased. Furthermore, the phosphorylation of S616 increases the activity of Drp1, while the phosphorylation of S637 reduces the activity of Drp1, indicating that AKAP1 deletion controls the survival of neurons through the inhibition of the levels of Drp1 and pS637 [68] .
The fusion of mitochondria is mainly mediated by mitofusin 1 (MFN1), mitofusin 2 (MFN2), and optic atrophy 1 (OPA1). MFN1 and MNF2 are located on the OMM, and mediates the fusion of the OMM through homotypic or heterotypic interactions, or GTPase hydrolysis, while the fusion of the IMM is mediated by OPA1. After ischemic stroke, OPA1 would be cleaved into two isoforms: long optic atrophy 1 (L-OPA1) would decrease, and short optic atrophy 1 would increase. Lai  al reported that after I/R, the overexpression of L-OPA1 significantly reduced OGD/R-induced neuronal death and mitochondrial morphological damage [69] . Recent studies have also revealed that the small molecule echinacoside promotes the process of mitochondrial fusion by targeting CK2 to promote the transcription of MFN2 [70] . The small molecule echinacoside, as a new molecule that targets CK2, provides a target for the future treatment of ischemic stroke. It has been reported that under stress conditions, mitochondrial ubiquitin ligase 1 deficiency can increase the activity of MFN2, trigger mitochondrial hyperfusion, and act as an ER-Mito tethering antagonist. Decreased ERmito coupling leads to the increase in cytoplasmic Ca 2+ activation of calcineurin, and the induction of Drp1dependent mitochondrial fission and mitophagy [71] .

Diverse Roles of Mitochondria in Ischemic Stroke
Increasing evidence has shown that mitophagy is beneficial to the pathological process after ischemic stroke, suggesting that this is an important therapeutic target [72] . As mentioned above, mitophagy is mainly mediated by the PINK1/parkin signaling pathway after ischemic stroke [36] . Specifically, after ischemic stroke, the mitochondria depolarizes and the membrane potential is lost, and these produce some substances that are harmful to cells, such as ROS [52] .
As an E3 ubiquitin ligase, parkin is known to ubiquitinate various mitochondrial outer membrane proteins, and regulate mitophagy by ubiquitinating mitochondrial outer membrane proteins. Necroptosis was found to be involved in cardiac I/R, and it was revealed that parkin attenuates myocardial injury by inhibiting the mPTP opening through catalyzing the Cyclophilin D (CypD) ubiquitination in the necrotic cascade reaction [73] . In addition, studies have revealed that parkin deficiency increases hepatic I/R injury [74] , suggesting that parkin plays an important role in I/R injury.
Phosphorylation promotes the activation of PINK1, and activated PINK1 recruits parkin to induce it to transfer to the mitochondria, thereby initiating mitophagy [33] . Studies have revealed that electroacupuncture is effective in treating ischemic stroke. In the middle cerebral artery occlusion (MCAO) model of rats, it was found that the electroacupuncture treatment of cerebral I/R injury can protect brain neurons through PINK1/parkin-mediated mitophagy [75] . Sphingosine kinase 2 protects brain neurons by activating the BCL2 interacting protein 3 (BNIP3) signal, and activating mitophagy [76] . tPA is the most commonly used thrombolytic drug in clinical practice, but its therapy mechanism remains unclear [77,78] . Recently, a report clarified that the lack of endogenous tPA can significantly aggravate brain damage, apoptosis and mitochondrial damage. Furthermore, it was found that the exposure of neurons to tPA can reduce the severity of damage and protect mitochondria [79] . Moreover, a further study revealed that the protective effect of tPA can be accomplished through the regulation of FUNDC1-mediated mitophagy [54] . Peroxynitrite (ONOO -)-mediated mitophagy represents an important pathogenic mechanism of ischemic stroke. ONOO -mediated mitophagy is accomplished by recruiting Drp1 to mitochondria, and activating PINK1/parkin [80] . Studies have reported that rehmapicroside, a natural compound of medicinal plants, can reduce the infarct area of MCAO in rats [81] . In vivo and in vitro experiments have also revealed that rehmapicroside reduces O 2 and ONOO -, upregulates Bcl2, and downregulates BCL associated X (Bax), caspase 3 and cleaved caspase 3, as well as PINK1, parkin and p62. Rehmapicroside prevents PINK1, parkin and Drp1 from entering the mitochondria to activate the mitophagy induced by cerebral I/R injury in rats [81] .

The Co-network in Mitophagy-apoptosis in I/R Injury
The mitophagy-mediated elimination of mitochondria play a driving role in a series of cases, such as cell differentiation, embryonic development, inflammation, and various cell deaths (including apoptosis, necroptosis, etc.) [16,20,62] . For apoptosis, mitochondria mediated apoptosis has been regarded as a late event of ischemic stroke [82] . The cyt c and other substances released from the mitochondria to the cytoplasm initiate endogenous apoptosis [83] . Mitochondrial apoptosis is correlated to the continuous opening of the mPTP. If the mPTP remains open, cyt c would be released from the mitochondrial membrane inner space to the cytoplasm, and cyt c would bind to the pro-apoptotic protease activator 1 (APAF-1), and promote its oligomerization. The caspase recruitment domain (CARD) of APAF-1 combines with the CARD domain of pro-caspase 9 to form a complex [84,85] . Pro-caspase 9 is activated as caspase 9 to cleave procaspase 3 and pro-caspase 7, in order to execute their apoptotic function [84][85][86][87] (fig. 3).
Mitophagy is a selective autophagy, and an important form of mitochondrial quality control. Cells can escape from death by clearing the damaged mitochondria away through mitophagy [88] . Abundant evidence has shown that proper mitophagy can help neurons survive, while excessive mitophagy can aggravate I/R injury [89] . For example, in the MCAO model of SD rats, the knockdown of peroxiredoxin 6 (PRDX6, an antioxidant protein) aggravated the I/R injury, and increased the expression of mitochondrial autophagy-related proteins and apoptosis-related proteins [90] . However, this injury can be reduced by the knockdown of PINK1, indicating that the knockdown of PRDX6 can aggravate cerebral I/R injury by enhancing the mitophagy mediated by the PINK1/parkin signaling pathway [90] . Stilbene glycoside promotes mitophagy and inhibits the apoptosis of ischemic neurons by promoting the expression of sirtuin (SIRT) and adenosine monophosphate-activated protein kinase (AMPK) [91] . Uncoupling protein (UCP2) is a member of the IMM protein. The deletion of UCP2 enlarges the area of brain congestion, increases the number of necrotic and TUNEL-positive cells, and upregulates the expression of PINK1, Beclin 1 and LC3. At the same time, this downregulates the expression of p62. It has been shown that UCP2 can aggravate I/R injury by promoting mitophagy and apoptosis [92] . In addition to brain I/R injury, in acute kidney injury induced by I/R, the phosphorylation level of AMPKα at Thr172 is significantly reduced. The use of AMPKα activator C24 can protect cells from apoptosis [93] . Consistent with the results reported by this article, by activating AMPK and promoting mitophagy, the damage of ischemic stroke can be reduced. This shows that the protection of ischemic stroke by activating AMPK is a possibility for future treatments [94] .
When parkin is activated by PINK1, this ubiquitinates many substrates, such as VDAC1. According to research reports, parkin ubiquitinates VDAC1 in two different ways to control the autophagy and apoptosis process of cells, respectively. Monoubiquitinated VDAC1 inhibits cell apoptosis, and polyubiquitinated VDAC1 promotes mitochondrial autophagy [95] . This means that the effect of the PINK1parkin pathway on apoptosis and mitophagy occurs through the VDAC1 antagonistic pathway [62] . This is the first evidence that verifies that different types of VDAC1 ubiquitination mediated by parkin can lead to different cellular outcomes. It is known that parkin can ubiquitinate various OMM proteins, except for VDAC1. However, it remains to be determined whether the phenomenon of other proteins is similar to VDAC1, and whether this mechanism exists in ischemic stroke. In addition, a study revealed that the expression of Cyc t VDAC1 is reduced in the vulnerable hippocampal CA1 subfield of rats after global ischemia [96] . This suggests that the reduction of VDAC1 may have been caused by the parkin ubiquitination. BNIP3 is a pro-apoptotic BH3 protein, and has been considered as a regulator of mitophagy. When the BNIP3 gene was silenced, the interaction of BNIP3 with LC3 was reduced, thereby inhibiting mitophagy and reducing cell apoptosis. Furthermore, autophagy markers, such as Beclin 1 and lysosome-associated membrane glycoprotein 2 (LAMP2), and the ratio of LC3-Ⅱ/Ⅰ increased, indicating that the decrease in apoptosis may be correlated to the increase in general autophagy [97] . According to previous studies, I/R injury can lead to a significant increase in the expression of BINP3 and its homologue NIX, indicating that I/R injury induces mitophagy. Dexamethasone (DXMS) is the most widely used glucocorticoid in clinical practice. Higher doses of DXMS can cause cell apoptosis [98] . A recent study reported that under hypoxic conditions, mitophagy-related protein hypoxia-inducible factor-1α (HIF-1α) and BNIP3 protein levels increases. After using DXMS, the expression of BNIP3 was downregulated and the cell apoptosis increased, while the overexpression of HIF-1α significantly increased the expression of BNIP3 and reduced the apoptosis induced by DXMS [99] .

The Co-network in Mitophagy-necroptosis in I/R Injury
Necroptosis is the regulated form of cell death, and this is mainly regulated by receptorinteracting protein kinase 1 (RIPK1), receptorinteracting protein kinase 3 (RIPK3), and mix lineage kinase domain-like protein (MLKL) [100][101][102] . In I/R injury, RIPK1 is phosphorylated, and the downstream molecule RIPK3 is phosphorylated, which promotes MLKL activation. Then, the activated MLKL executes death commands [103][104][105] . Recently, studies have shown crosstalk between I/R injuryinduced necroptosis and mitochondrial dysfunction [105,106] . For example, the RIPK3-mediated activation of Ca 2+ /Calmoduline-dependent protein kinase Ⅱ (CaMKⅡ) promotes mPTP opening [107] and RIP3-PGAM5-Drp1 mediates the mitochondria fission [108] . In addition, studies have shown that in an in vitro model, cardiomyocytes and brain cells were more severely damaged after I/R injury in PGAM5-knockout mice [20] . As a mitochondrial membrane protein, PGAM5 is an important protective gene in ischemic injury, and it is also the anchor of the RIP1-RIP3-MLKL complex in mitochondria. Furthermore, research has revealed that PGAM5 is essential for PINK1-dependent mitophagy. PGAM5 independently promotes mitophagy to protect cells from necroptosis [20] . The continuous opening of the mPTP mediated by RIPK3 is the key to regulate the necroptosis under different stimuli [98,109] . Mounting evidence has revealed that the activation of mitophagy can inhibit the opening of the mPTP [110] . It has been reported that RIPK3 can reduce the level of parkin phosphorylation, and reduce the interaction between parkin and LC3 induced by hypoxia damage, thereby inhibiting mitophagy and promoting mPTP opening. The knockout of RIPK3 can reverse this phenomenon via the AMPK-parkin-mitophagy signal axis [110] (fig.  3).

THERAPEUTIC TARGET FOR MITOPHAGY-DEPENDENT ISCHEMIC STROKE
As mentioned above, mitophagy exerts a vital role in the mitochondria and ischemic stroke. A number of studies have contributed in providing a wide range of strategies to improve neuronal protection during ischemic stroke, based on the molecular mechanism, which includes intravenous thrombolysis, tPA, and endovascular therapy [111,112] .
For example, tPA is the most important clinical thrombolytic drug for ischemic stroke, and has a neuroprotective effect [113] . The knockout of endogenous tPA can significantly aggravate the brain damage, and increase the neuronal apoptosis and mitochondrial damage. Mitochondrial damage in I/R injury is correlated to the endogenous reduction of tPA. Cyt c is released into the cytoplasm, triggering a caspase cascade reaction and neuronal apoptosis. At the same time, the mitochondrial damage would lead to the degradation of FUNDC1, preventing FUNDC1 from binding to free LC3, thereby inhibiting mitophagy. After adding exogenous tPA, the AMPK phosphorylation level and FUNDC1 both increase, and FUNDC1 binds to LC3 through the LIR domain, leading to the autophagy double-layer membrane wrapped mitochondria, and inducing mitophagy [54] . Although this is the only clinical thrombolytic drug approved by the U.S. FDA, a large number of problems need to be solved, such as determining how tPA responds to I/R injury and the mechanism, including how tPA enters into neurons and the downstream mechanism of FUNDC1. Ligustilide (3-butylidene-4, 5-dihydroisodenzofuranone; LIG) is the main active ingredient in the traditional medicine Angelica sinensis. DL-3-n-butylphthalide, which is the first innovative drug with independent intellectual property rights in China, has been widely used in the treatment of ischemic stroke in clinic, and has a similar structure to LIG [94] . This study revealed that LIG attenuates the injury of ischemic stroke via the activation of AMPK, and the promotion Drp1 to mediate mitochondria fission [94] . In addition, ligustilide has a wide range of pharmacological properties, including anticancer, anti-inflammatory, anti-oxidant and neuroprotective activities [114] . Rehmapicroside ameliorates cerebral I/R injury by attenuating the peroxynitrite-mediated mitophagy activation [81] . Tetrahydrocurcumin (THC) epigenetically ameliorates the mitochondrial dysfunction in brain vasculature during ischemic stroke [115] . Methylene blue promotes mitophagy by maintaining the mitochondrial membrane potential (MMP) at a relatively high level. This contributes to the decrease in necrosis, and the improvement in neurological function, thereby protecting against acute cerebral ischemic injury [116] (table 1). It has been found that some traditional Chinese medicine ingredients and compounds can treat ischemic stroke through mitophagy. However, it remains a challenge to find the target of traditional Chinese medicine, and the research at this stage remains on the phenotype [75] .

CONCLUSION AND PERSPECTIVES
Mitochondria plays a driving role on tissue homeostasis to provide energy for the normal life activities of cells through the production of ATP. A vital process, known as mitophagy, can work as an emergency strategy after a series of stimuli damage. To date, a fuller molecular mechanism of mitophagy has been performed, which is closely correlated to the control of the quality of mitochondria involved in various diseases. Recent studies have revealed that mitophagy can aggravate neuronal death [81] . Among these, focus has been given on the field of ischemic stroke, in which the mitophagy was significantly downregulated, while the apoptosis and necroptosis were upregulated. Promoting mitophagy can alleviate I/R injury, and reduce neuronal apoptosis and necroptosis. In addition, promoting mitophagy can reduce apoptosis to a certain extent, but this may promote its general autophagy process, indicating that mitophagy and general autophagy also have a crosstalk. Thus, the mitophagy pathway is a complex, but practical, integrated network, and there is a necessity to focus on this overall crosstalk.
Although a number of studies have proved that mitophagy can exert a neuroprotective effect on ischemic stroke, in the complex molecular network of the human body, it remains unclear how mitophagy works on neuron protection. For example, a more comprehensive explanation should be provided in promoting PINK1/parkin and BNIP3/NIX, and Drp1mediated mitophagy could alleviate I/R injury, while promoting ONNO --mediated mitophagy, which promotes I/R injury. More research is needed to improve the molecular network diagram of mitophagy. In addition, after I/R injury, it remains to be determined how these molecules work at different time points after ischemia, such as AMPK, FUNDC1 and BNIP3. Promoting mitophagy can reduce the apoptosis or necroptotic pathways, and determine whether other cell apoptosis pathways are involved. The intersection of mitochondria and cell death is also a problem worth exploring. Answering these questions would not only be of great significance to our understanding of the protection of neurons through mitophagy in ischemic stroke, but also provide new theoretical support for the treatment of ischemic stroke.

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