Traditional Chinese medicine in treating ischemic stroke by modulating mitochondria: A comprehensive overview of experimental studies

Ischemic stroke has been a prominent focus of scientific investigation owing to its high prevalence, complex pathogenesis, and difficulties in treatment. Mitochondria play an important role in cellular energy homeostasis and are involved in neuronal death following ischemic stroke. Hence, maintaining mitochondrial function is critical for neuronal survival and neurological improvement in ischemic stroke, and mitochondria are key therapeutic targets in cerebral stroke research. With the benefits of high efficacy, low cost, and high safety, traditional Chinese medicine (TCM) has great advantages in preventing and treating ischemic stroke. Accumulating studies have explored the effect of TCM in preventing and treating ischemic stroke from the perspective of regulating mitochondrial structure and function. In this review, we discuss the molecular mechanisms by which mitochondria are involved in ischemic stroke. Furthermore, we summarized the current advances in TCM in preventing and treating ischemic stroke by modulating mitochondria. We aimed to provide a new perspective and enlightenment for TCM in the prevention and treatment of ischemic stroke by modulating mitochondria.


Introduction
Stroke is a devastating disease with high disability and mortality rates worldwide (Ajoolabady et al., 2021). Approximately 7,95,000 people suffer from either new or recurrent strokes annually, increasing the economic burden on the family and society (Virani et al., 2020). Most strokes are ischemic, accounting for approximately 87% of all strokes. Ischemic stroke causes brain tissue necrosis due to narrowing or occlusion of the blood supply arteries (carotid and vertebral arteries) and insufficient blood supply to the brain .
In patients with ischemic stroke, a significant decline in focal cerebral blood flow causes glucose and oxygen deprivation (OGD). Mitochondrial dysfunction is an early and initiating event in OGD following ischemia. It is increasingly evident that it plays a critical role in the OPEN ACCESS EDITED BY Yusof Kamisah, Faculty of Medicine Universiti Kebangaan Malaysia, Malaysia onset, development, and pathology of ischemic stroke (Song et al., 2022). During ischemic stroke, OGD causes adenosine triphosphate (ATP) consumption and Na + /K + ATPase pump failure, resulting in neuronal membrane depolarization and excessive glutamate release. Excessive Ca 2+ influx can cause reactive oxygen species (ROS) production and mitochondrial dysfunction, such as an imbalance in mitochondrial dynamics, mitochondrial-induced apoptosis, mitochondrial biogenesis dysfunction, and mitophagy overactivation. These cellular processes eventually lead to neuronal cell death.
The most effective treatment for acute ischemic stroke is reperfusion therapy, which aims to restore blood flow and oxygen levels before neuronal damage occurs. Tissue plasminogen activator (tPA) is the only thrombolytic agent approved by the U.S. Food and Drug Administration for patients with acute ischemic stroke (Alkahtani et al., 2023). However, the narrow treatment window and the risk of complications limit its clinical application Li et al., 2022c). Additionally, tPA can cause mitochondria to produce excessive ROS, exacerbating cell damage (Giorgi et al., 2018). New therapeutic agents are required to address the paucity of stroke management approaches. Increasing evidence suggests that maintaining mitochondrial function is critical for neuronal survival and neurological improvement in ischemic stroke, and mitochondria are the key therapeutic targets in cerebral stroke research (Andrabi et al., 2020;Zhou et al., 2021;Zhong et al., 2022). Therefore, a promising treatment option for ischemic stroke that targets mitochondria is needed.
Chinese herbs and acupuncture are essential components of traditional Chinese medicine (TCM). In clinical trials and basic research, Chinese herbs and acupuncture have demonstrated therapeutic effects in preventing and treating ischemic stroke Song et al., 2022). Further studies reported that Chinese herbs and acupuncture could prevent and relieve cerebral ischemia injury in vivo and in vitro and have neuroprotective effects by modulating the mitochondrial respiratory chain (Zhong et al., 2009), increasing mitochondrial biogenesis (Sun et al., 2021), inhibiting the mitochondrial apoptotic pathway  and attenuating excessive mitophagy (Ting et al., 2017). However, only a few studies have comprehensively reviewed these studies hindering the elucidation of the mechanism of action of TCM and the development of clinical applications. Moreover, the existing review, published 2 years ago, only summarized the effects of Chinese herbs on mitochondrial permeability transition pore (mPTP) overopening-induced ischemic neuron apoptosis. Nonetheless, the regulatory effects of TCM on mitochondria in the treatment of ischemic stroke are multifaceted and acupuncture, an integral aspect of TCM, appears to be overlooked in the treatment of ischemic stroke by restoring mitochondrial function.
Understanding the molecular mechanisms of the mitochondria involved in ischemic stroke is crucial to identify potential interventional targets. Thus, we first discuss the role of mitochondria in ischemic stroke. We subsequently summarized the recent advances in TCM in preventing and treating ischemic stroke by regulating mitochondria. We aimed to provide a new perspective and insight into the use of TCM in treating ischemic stroke by improving mitochondrial structure and function.
2 The role of mitochondria in ischemic stroke 2.1 Ischemic stroke cascade involves mitochondrial function and structure changes 2.1.1 Ischemic stroke cascade involves mitochondrial function changes Mitochondria produce the majority of ATP via the mitochondrial respiratory chain and oxidative phosphorylation to meet the high-energy demands of neurons in the brain that are extremely sensitive to ischemia and hypoxia. Within minutes of the onset of cerebral ischemia, ATP depletion deactivates the Na + /K + ATPase pump, causing excessive glutamate release into the extracellular fluid (Sarmah et al., 2020). Overactivation of glutamate receptors, such as N-methyl-D-aspartate-receptor, αamino-3-hydroxy-5-methyl-4-isox-azolepropionic acid receptor, and kainic acid receptor, results in Ca 2+ influx and accumulation into cells (Hu et al., 2018;Engin and Engin, 2021;Guo and Ma, 2021). A large Ca 2+ influx leads to a series of events ranging from mPTP opening and dissipation of mitochondrial membrane potential (MMP) to the release of cytochrome c (Cyt-c) or apoptosis-inducing factor (AIF), thus activating effector caspases and eventually causing neuronal death (Anzell et al., 2018;Li et al., 2020). Concomitantly, decreased ATP depletes nicotinamide adenine dinucleotide (NAD + ), and the reduced NAD + drives mitochondria to the vicinity of the endoplasmic reticulum to form mitochondria-associated endoplasmic reticulum membranes (MAMs). Moreover, certain MAM-related proteins join mPTP to regulate its opening, an important marker of cerebral cell death during ischemia/reperfusion (I/R).
In addition to energy production, mitochondria are the primary producers of intracellular ROS and are sites of eukaryotic oxidative metabolism. Disrupting mitochondrial electron transport increases ROS generated during cerebral ischemia, particularly during reperfusion . Further, this excess ROS affects mitochondrial function and promotes neuroinflammation and neuronal apoptosis after oxygen-glucose deprivation/ reoxygenation (OGD/R) .

Ischemic stroke cascade involves mitochondrial structure changes
In addition to the function of mitochondria, their structure also plays an important role in the pathophysiological process of ischemic stroke. Mitochondria are highly dynamic cellular organelles that can change the shape, size, position, and integrity of mitochondrial DNA (mtDNA) through highly coordinated fission, fusion, and transport to tactical locations. The imbalance of mitochondrial fission and fusion after stroke may increase mitochondrial fragmentation, cause aberrant mitochondrial morphology, and disrupt mitochondrial homeostasis, leading to mitochondrial dysfunction and ultimately triggering neuronal death . Additionally, mutation of gene-encoded subunits in mtDNA results in increased ROS generation, which makes mtDNA more susceptible to mutations than nuclear DNA . Researchers have reported that the frequency of mtDNA mutations was significantly higher in the brains of patients with ischemic stroke (Luan et al., 2021). In summary, the ischemic stroke cascade involves changes in mitochondrial function and structure, indicating that mitochondrial structure and function play a critical role in the pathogenesis of ischemic stroke.

Mitochondrial biogenesis in ischemic stroke
Mitochondrial biogenesis is a multifaceted process involving the coordinated regulation of mitochondrial and nuclear transcription factors. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a major regulator of mitochondrial biogenesis. During ischemic stroke, PGC-1α is first activated by upstream AMPactivated protein kinase (AMPK) phosphorylation and sirtuin 1 (SIRT1) acetylation (Kaarniranta et al., 2018), which then interacts with downstream nuclear respiratory factor 1/2 (NRF1/2), taking part in the expression of nuclear and mitochondrial respiratory factors. The binding of NRF1 to the promoter of the mitochondrial transcription factor A (TFAM) gene is enhanced under oxidative stress. Activated TFAM promotes mtDNA copying, transcription, and related protein synthesis, ultimately inducing mitochondrial biogenesis (Ryoo and Kwak, 2018). Additionally, two mitochondrial proteins, uncoupling protein 2 and superoxide dismutase 2, both regulated by PGC1-α, play a pivotal role in counteracting the damaging effects elicited by excessive oxidative stress in ischemic stroke (Chen et al., 2011). Peroxisome proliferator-activated receptor gamma agonists can upregulate PGC-1α, NRF1, TFAM, and cytochrome c oxidase subunits I and IV and enhance mitochondrial biogenesis in ischemic stroke (Yang et al., 2018). This indicated that mitochondrial biogenesis exerted a protective effect by enhancing the signal transduction pathways upstream of mitochondrial biogenesis.
Generally, mitochondrial biogenesis plays an important role as an endogenous protective mechanism in ischemic stroke. Therefore, boosting the signal transduction pathways upstream of mitochondrial biogenesis, such as the PGC-1α signaling cascade, may become a novel therapeutic strategy against ischemic brain damage.

Mitochondrial dynamics in ischemic stroke
Mitochondrial dynamics include fission and fusion. Mitochondrial fission allows damaged mitochondria to separate, leading to their subsequent elimination by mitophagy. The production of one or more daughter mitochondria is highly dependent on dynamin-related protein 1 (Drp1). Mitochondrial fusion facilitates the complementation of neighboring mitochondria, enabling the survival of damaged mitochondria (Zhou et al., 2021). It is a two-step process that requires the fusion of outer and inner mitochondrial membranes, mediated by mitofusins-1/mitofusins-2 (Mfn1/2) and optic atrophy 1 (Opa1), respectively.
The interaction between calcium overload, ROS production, and mPTP increases mitochondrial fission and decreases mitochondrial fusion in ischemic stroke (Zhou et al., 2021). Although increased mitochondrial fission during hypoxia may increase mitochondrial energy production, which is beneficial for maintaining neural function after stroke (Quintana et al., 2019), inducing excessive mitochondrial fission is harmful to neurons . Excessive mitochondrial fission affects intracellular calcium homeostasis, exacerbates excitotoxicity, and accelerates neuronal death after ischemic stroke (Zhou et al., 2021). Researchers have observed increased levels of Drp1 in mice subjected to cerebral ischemia and reperfusion injury. After the knockdown of Drp1, oxidative stress, mitochondrial ROS production, and infarct volume decrease, contributing to the survival of neurons in cerebral ischemia . Mitochondrial fusion can repair damaged mitochondria and produce additional energy by upregulating the activity of ATP synthase through mitochondrial cristae remodeling (Cohen and Tareste, 2018). The levels of mitochondrial fusion proteins, such as Mfn-1/Mfn-2 and Opa1, decrease after cerebral ischemia (Rutkai et al., 2019). However, hypoxia-induced apoptosis improved when Mfn-2 was restored .
In summary, inhibiting excessive mitochondrial fission, promoting mitochondrial fusion, and restoring the balance of mitochondrial dynamics are beneficial for ischemic stroke recovery. Maintaining this balance can serve as a target for treating ischemic stroke.

Mitophagy in ischemic stroke
Mitophagy is a type of selective autophagy in which damaged or dysfunctional mitochondria are removed. In ischemic stroke, mitophagy could be predominantly mediated by the PINK1/Parkin pathway, Bcl-2/E1B-19 KD-interacting protein 3 (BNIP3), NIP3-like protein X (NIX, also known as BNIP3L), and FUN14 domain containing 1 (FUNDC1). Shen et al. demonstrated that mitophagy could protect brain cells from ischemic injury during the ischemic phase of stroke . In contrast, mitophagy serves as a doubleedged sword when the brain suffers from reperfusion injury. Activating mitophagy to clear excessively aggregated and damaged mitochondria reduces neuronal damage caused by cerebral I/R injury Wang and Xu, 2020;Wu et al., 2021). However, some studies have shown that inhibiting excessive mitophagy can protect against cerebral I/R injury in middle cerebral artery occlusion (MCAO) rats (Lan et al., 2018;Jakic et al., 2019). Inhibition of excessive mitophagy could exert neuroprotective effects against neuronal death caused by chronic cerebral hypoperfusion (Su et al., 2018).
Mitophagy is important for the pathogenesis of cerebral I/R damage. Regulation of mitophagy could exert neuroprotective effects in ischemic stroke, although some issues regarding its role in ischemic stroke remain unclear. It would be meaningful to explore the role of mitophagy in treating I/R.

Proteins associated with mitochondriadependent apoptosis in ischemic stroke
Apoptosis is a planned or controlled cell death triggered by mitochondrial malfunction through intrinsic and extrinsic pathways.
Mitochondria are associated with many apoptosis-related proteins, suggesting that they are crucial for cell Frontiers in Pharmacology frontiersin.org 03 death following I/R (Yang et al., 2018). Many studies have revealed that B cell lymphoma (BCL-2) family proteins regulate neuronal death in cerebral ischemic stroke (Ader et al., 2019). After I/R, apoptotic members of the Bcl-2 protein family (e.g., Bax and Bak) are inserted into the outer mitochondrial membranes, and MMP is significantly downregulated. Another decisive step in the apoptotic cascade is related to the mPTP. Transient opening of the mPTP in the mitochondrial inner membrane after I/R causes MMP collapse. Several apoptosis-related proteins (e.g., AIF, Cyt-c, endonuclease G [Endo G], the second mitochondrion-derived activator of caspase/ direct inhibitor of apoptosis-binding protein with low pI [Smac/ Diablo]) originating in the mitochondria are released into the cytoplasmic matrix (Zhou et al., 2021). After migration to the cytoplasmic matrix, Cyt-c interacts with apoptosis-activating factor-1 (Apaf-1), deoxyadenosine triphosphate (dATP), and procaspase-9 to form the apoptosome, which then activates procaspase-9 and follows with caspase-9 to cleave and activates caspase-3 . Smac binds to and inhibits inhibitorof-apoptosis proteins (IAPs), which normally inhibit procaspase activation and caspases activity . AIF can trigger caspase-independent chromatin condensation and large-scale DNA breakage  and functions as a mitochondrial effector of apoptotic cell death following translocation from mitochondria to the nucleus (Guida et al., 2019).
Overall, modulating the expression of apoptotic members of the Bcl-2 protein family and preventing translocation of AIF, Cyt-c, and Smac from the mitochondria into the cytoplasmic matrix can serve as targets for the treatment of ischemic stroke.
3 Progress in ischemic stroke prevention and treatment using TCM that regulates mitochondria Based on the above summary, we identified several targets for treating ischemic stroke from the mitochondrial perspective. In clinical and experimental studies, TCM has demonstrated significant efficacy in preventing and treating ischemic stroke. The mechanisms of action of TCM have also been gradually revealed in recent years. Many studies have revealed that TCM exerts therapeutic effects on ischemic stroke by regulating the mitochondria. Therefore, we summarized the literature on acupuncture, herbal extracts, effective TCM compounds, and TCM prescriptions in preventing and treating ischemic stroke and attempted to further clarify the molecular mechanisms of TCM in improving ischemic stroke from the perspective of regulating mitochondria.
3.1 Acupuncture and its molecular mechanisms for regulating mitochondria in ischemic stroke 3.1.1 Acupuncture pretreatment for regulating mitochondria in ischemic stroke The MCAO group exhibited apparent mitochondrial structural abnormalities, including a reduction in mitochondrial volume and number, swelling, vacuolization, formation of autophagosomes and lysosomes, and broken/irregular/disappeared inner membranes and cristae. However, 5-7 consecutive days of electroacupuncture (EA) pretreatment reduced mitochondrial abnormalities, including an increase in mitochondrial volume and number (Sun et al., 2021), less swelling , a relatively integrated membrane and cristae , and a reduction in the number of autolysosomes . Elevated radical generation (Sun et al., 2021), attenuated MMP levels (Mao et al., 2020;Sun et al., 2021;Tian et al., 2022), and reduced citrate synthase (Sun et al., 2021) were detected in the MCAO group 24 h after reperfusion, compared with those in the control group. These trends could be reversed by EA pretreatment. Additionally, researchers reported that five consecutive days of EA pretreatment at the Baihui (DU20) acupoint induced neuronal protection by inhibiting the expression Zhang et al., 2018) and translocation  of mitochondrial Drp1 in rats with focal cerebral IR injury. Meanwhile, EA pretreatment at the DU20 and Shuigou (DU26) acupoints for 5 days was applied to treat cerebral I/R injury in rats and exerted neuroprotective effects by inhibiting the autophagy-related p-ULK1/FUNDC1 pathway (Mao et al., 2020;Tian et al., 2022). EA pretreatment at the DU20 acupoint induced cerebral ischemic tolerance, increased the expression of NRF-1, TFAM, and mtDNA levels, and further promoted mitochondrial biogenesis by activating CB1R-dependent PGC-1α (Sun et al., 2021). Sun et al. found that the release of Cyt-c in the cytoplasm (Cyto-Cyt-c) was reduced in the EA group 24 h after reperfusion compared with that in the I/R mice group induced by MCAO (Sun et al., 2021). Their findings were consistent with another previous study that also found a significant decrease in Cyto-Cyt-c levels in the EA group compared with the IR group at 6, 24, and 48 h after reperfusion .
In summary, EA pretreatment promoted mitochondrial biogenesis 4 h after reperfusion. At 6, 24, and 48 h after reperfusion, EA pretreatment inhibited mitochondrial fission and apoptosis by decreasing mitochondrial Drp1 and Cyto-Cyt-c levels, respectively. Moreover, after 24 h of reperfusion, EA pretreatment reversed mitochondrial structural abnormalities, inhibited the autophagy-related p-ULK1/FUNDC1 pathway, attenuated radical generation, elevated MMP levels, and increased mitochondrial energy metabolism. Specific mechanisms are shown in Table 1; Figure 1A.

The effect of acupuncture after ischemic stroke in regulating mitochondria
In the MCAO group, the neuronal mitochondria became swollen, the mitochondrial cristae and outer membrane were broken, and EA alleviated the mitochondrial structure abnormalities within 24 h after reperfusion . Acupuncture can alleviate cerebral I/R injury by increasing MMP levels and inhibiting nitro/oxidative stress by downregulating oxidase, ROS, and malondialdehyde (MDA) levels and upregulating superoxide dismutase (SOD) (Ting et al., 2017;. In addition, EA at DU20 and DU26 for 30 min decreased the neurological deficit score, improved the respiratory control ratio, and promoted the activities of respiratory enzymes, including succinic dehydrogenase, NADH dehydrogenase, and cytochrome C oxidase, in MCAO rats (Zhong et al., 2009). These findings are consistent with those   (Tian et al., 2015). Tian et al. further pointed out that 3 mA EA could more effectively elevate the activities of succinic dehydrogenase and lactate dehydrogenase compared to 1 mA EA and 5 mA EA in the brain tissue of rats with I/R injury (Tian et al., 2015). Zuo et al. noticed that EA at ZuSanLi (ST36), DU20, and Mingmen (DU4) five times within 48 h after reperfusion could improve cerebral I/R by inhibiting excessive autophagy in neurons (Ting et al., 2017). However, Zhong et al. conducted 7 days of EA treatment at DU20 and Shenting (DU24) after reperfusion and reported that EA could alleviate cerebral I/R injury and improve neural function by promoting BNIP3L mediated autophagic clearance (Zhong et al., 2022). Another study also demonstrated that EA within 24 h after reperfusion decreased the accumulation of damaged mitochondria by increasing Pink1/Parkin-mediated mitophagy clearance to protect cells against neuronal injury in cerebral I/R . Furthermore, within 24 h after reperfusion, EA at DU20 and Fengfu (DU16) increased the expression of anti-apoptotic Bcl-2, Bcl-Xl, and cellular inhibitor of apoptosis-1,-2 (cIAP-1, -2), and decreased the activities of caspase-3, -8, and -9 compared with the untreated rats with MCAO (Kim et al., 2013). Similarly, EA at DU20 and DU16 for 7 consecutive days activated p38 MAPK-mediated anti-apoptotic signaling pathways, which ultimately contributed to the prevention of Smac/DIABLO translocation and subsequent restoration of the X-linked inhibitor of apoptosis protein (XIAP) suppression of caspase-3 in the cortical peri-infarct area (Cheng et al., 2015). Another study also found that EA treatment within 6 h of ischemic stroke could attenuate ischemic brain injury and cellular apoptosis by inhibiting mitochondrial translocation of cofilin and caspase-3 cleavage  ( Figure 1B). In summary, acupuncture treatment and pretreatment could both restore mitochondrial morphology, improve MMP levels, further upregulate mitochondrial energy metabolism, attenuate mitochondrial autophagy, and inhibit mitochondrial-dependent apoptosis. Acupuncture pretreatment promoted mitochondrial biogenesis and inhibited mitochondrial fission. Additionally, acupuncture treatment inhibited oxidative stress, cofilin translocation, and activated mitochondrial autophagy. The detailed mechanisms are shown in Table 1  3.2 Herbal extract and its molecular mechanisms by regulating mitochondria in treating ischemic stroke 3.2.1 Herbal extract pretreatment in regulating mitochondria of ischemic stroke Although the clinical treatment of ischemic stroke with a single herb is rare, in recent years, researchers have reported that the individual application of certain herbs has the potential to treat diseases. Mitochondrial ultrastructure injury was partially improved in cerebral I/R rats after pretreatment with in vitro cultured Bos taurus domesticus Gmelin or Chrysanthemum morifolium Ramat. extracts (Lin et al., 2010;Lu et al., 2020). Pretreatment with herbal extracts (e.g., Astragalus membranaceus (Fisch.) Bge. combined with Panax notoginseng (Burk.) F.H.Chen, Astragalus membranaceus (Fisch.) Bge., and Gardenia jasminoides (Ellis) alleviated nerve injury after cerebral I/R by improving mitochondrial respiration 1, phospholipase C γ 1; Dr5, death receptor 5; MAP2, microtubule-associated protein-2; NADH, nicotinamide adenine dineucleotide; RCR, respiratory control ratio; iNOS, inducible nitric oxide synthase; LDH, lactate dehydrogenase; SDH, succinate dehydrogenase; GSH, glutathione; SQSTM1, Sequestosome-1; VDAC, voltage-dependent anion channel; Tomm20, translocase of outer mitochondrial membrane 20 homolog; NOX, oxidase; 3-NT, 3nitrotyrosine; GPX4, glutathione peroxidase 4; Tf, transferrin; TfR1, transferrin receptor 1, FTH1, ferritin heavy chain 1; GFAP, glial fibrillary acidic protein; mito-Drp1, mitochondrial dynamin-related protein 1; COX IV, cytochrome c oxidase IV; IL-6, interleukin 6; IL-1β, interleukin 1β; TNF-α, Tumor necrosis factor-alpha; p-mTORC1, phosphorylated mTORC1; 4-VO/R, 4-vessel occlusion/reperfusion.
In brief, evidence from in vivo and in vitro studies indicated that herbal extract pretreatment could ameliorate cerebral ischemia by improving mitochondrial ultrastructure, increasing MMP levels, mitochondrial respiration function, and energy metabolism, maintaining mitochondrial dynamic balance, inhibiting mitochondria-related oxidative stress, autophagy, and mitochondria-dependent apoptosis ( Figure 2).
In summary, TCM compounds can alleviate abnormal mitochondrial structure, improve mitochondrial energy metabolism, decrease the expression and translocation of Drp1, reduce oxidative stress, mPTP, and mitochondria-dependent apoptosis, upregulate MMP, and promote the transfer of astrocytic mitochondria to neurons to prevent ischemic stroke. However, mitophagy results remain controversial and require further investigation (Figure 4).
Through in-depth comparative analysis, TCM compounds provided during pretreatment and treatment could alleviate abnormal mitochondrial structure; inhibit oxidative stress, mPTP opening, and Drp1 translocation to the mitochondria; and improve mitochondrial energy metabolism and MMP. TCM compounds provided pretreatment could promote the transfer of astrocytic mitochondria to neurons and potentiate autophagy and also decrease the level of mitochondrial autophagy by inhibiting the AMPK/mTOR pathway. Meanwhile, TCM compounds during treatment could maintain the dynamic balance between mitochondrial fission and fusion, inhibit mitochondrial autophagy by activating the PI3k/Akt pathway, promote mitophagy by activating PINK1/Parkin, inhibit Endo G and AIF release from mitochondria into the cytoplasm, and further attenuate mitochondria-mediated apoptosis. The specific mechanisms of these compounds in vivo and in vitro are shown in Table 3; Figure 3.

TCM prescription pretreatment for regulating mitochondria in ischemic stroke
Ischemic strokes are usually treated using TCM prescriptions owing to TCM's overall concept of TCM and syndrome differentiation-based treatment. In MCAO rats, pretreatment with Xiao-Xu-Ming decoction improved the abnormal mitochondrial ultrastructure (Lan et al., 2018). Pretreatment with Buyang Huanwu decoction prevents H 2 O 2 -induced ultrastructural disruption of mitochondria in human umbilical vein endothelial cells, whereas Guhong injection preconditioning preserves mitochondrial morphology during OGD injury (Shen et al., 2016;Zhou et al., 2021). In human umbilical vein endothelial cells and primary cultured cortical neurons, H 2 O 2 decreased ATP production, MDA levels, and MMP levels while increasing ROS and SOD levels, which could be reversed by pretreatment with Buyang Huanwu Decoction, Zhenbao pill, and YiQiFuMai Powder injection (Shen et al., 2016;Xu et al., 2017;Jia et al., 2021). In brain microvascular endothelial cells and primary cortical neurons, OGD/R induced MMP loss and oxidative stress injury, which could be alleviated by pretreatment with Guhong injection, Xingxiong injection, and Naoxintong capsules Zhou et al., 2021;Zhu et al., 2022). Danhong injection pretreatment improved mitochondrial energy metabolism after OGD/R injury (Orgah et al., 2019 (Lan et al., 2018;Jia et al., 2021). In addition, Zhenbao pill-containing serum represses cell apoptosis by inhibiting autophagy (Jia et al., 2021). Xiao-Xu-Ming decoction inhibited the translocation of Smac/Diablo from the mitochondria to the nucleus, increased the level of cytoplasmic c-IAP1, and further inhibited ischemia-induced neuronal apoptosis (Lan et al., 2014). To date, many in vivo studies (e.g., Ershiwei Chenxiang pills, Pien-Tze-Huang and Xiao-Xu-Ming decoction) (Lan et al., 2014;Zhang et al., 2018;Hou et al., 2020) and in vitro studies (e.g., Buyang Huanwu decoction, Guhong injection, Xingxiong injection, YiQiFuMai powder injection, and Zhenbao pill) (Shen et al., 2016;Zhou et al., 2021;Zhu et al., 2022) have revealed that traditional Chinese prescriptions protect mitochondria from ischemic injury and inhibit the mitochondria-dependent apoptosis pathway. In brief, TCM pretreatment improved abnormal mitochondrial structure, inhibited oxidative stress and mitophagy, improved mitochondrial energy metabolism and MMP, and maintained the dynamic balance of mitochondrial fission and fusion. TCM prescriptions inhibited Smac/Diablo release from the mitochondria into the cytoplasm and further attenuated mitochondria-mediated apoptosis ( Figure 5).
Interestingly, only TCM compounds have been reported to promote the transfer of astrocytic mitochondria to neurons in response to ischemic stroke. Emerging evidence suggests that mitochondria could serve as "help-me" signaling in response to various external stimuli and recruit neighboring cells to rescue injured cells. Removing damaged mitochondria and replacing them with healthy ones is a potential treatment for hypoxia and ischemia-related disorders, especially in the central nervous system, where mitochondria are abundant in the distal axonal synapses and dendritic protrusions. More studies can be conducted exploring the underlying therapeutic mechanism of TCM in treating ischemic stroke from the perspective of mitochondrial transfer.

Conclusion and prospects
In this review, we summarize the molecular mechanisms underlying the involvement of mitochondria in ischemic stroke. Mitochondrial function and structure play important roles in ischemic stroke, serving as crucial targets for TCM in alleviating ischemic stroke, and we have identified some key proteins and signaling pathways, as mentioned above. In addition, some issues require further clarification and improvement in future research. First, the precise molecular mechanisms underlying the effects of TCM on mitochondria in cellular and rat models of ischemic stroke remain incompletely understood. Further research is needed to elucidate the underlying mechanisms of TCM's effects on mitochondrial structure and function in ischemic stroke, using molecular, cellular, and biochemical approaches. Second, the lack of standardized experimental designs and methods may affect the reproducibility and comparability of the results. Standardization of experimental designs and methods, including the quality control of TCM preparations, should be established to ensure the scientific rigor and reliability of future studies. Third, in the field of EA therapy for ischemic stroke, research from the perspective of mitochondria is scarce in comparison to studies on herbal extracts, compounds, and prescriptions. More rigorous and well-designed studies are urgently needed. In addition, it is critical to establish standardized EA stimulation parameters (e.g., frequency, duration, and intensity) to investigate the dose-response relationship and corresponding mechanisms from the perspective of mitochondria in future studies. Fourth, the results of mitophagy, mitochondrial fission, and mitochondrial fusion have been inconsistent among studies. These controversial results might be associated with different experimental models, different stages of ischemic stroke, and intervention modes. Further investigations are required to elucidate this. Fifth, most of the studies summarized above are based on OGD/R or MCAO ischemia models, and most MCAO ischemia models are conducted on young rats or mice. Few studies have used aged animals or models that closely mimic clinical patients who often have hypertension, hyperglycemia, or other disorders. Therefore, it is necessary to investigate the neuroprotective benefits of TCM against ischemic stroke using pseudo-clinical models (e.g., complicated models of multiple coexisting disorders), which will provide a reliable foundation for TCM's clinical application of TCM. Sixth, most in vitro studies mentioned above were limited to a particular type of nerve cells. Neurovascular dysfunction induced by ischemic stroke demonstrates a combined action of multiple nerves in the brain, and investigating only one type of nerve cell is insufficient. Thus, ischemic stroke can be better understood using a cell co-culture model of neurons, microglia, Frontiers in Pharmacology frontiersin.org and astrocytes in vitro. Finally, since Chinese herbs and prescriptions contain various chemical components, the precise underlying mechanisms remain unknown. Further research is required into the molecular targets and active components that contribute to the bioactivity of Chinese herbs and prescriptions in preventing and treating ischemic stroke. Furthermore, we should examine the synergic effects of the constituents and their metabolites, as well as the targeted signaling pathways in post-ischemic brains.
In conclusion, this review summarizes the recent experimental evidence of TCM in preventing and treating ischemic stroke by modulating mitochondria and identifies areas that future research should focus on. In addition, TCM has few side effects and is highly effective and specific; therefore, with adequate research, it will be widely available for ischemic stroke treatment.