Metabolic disorders are believed to be the first causal factor of CCI. Following cerebral ischemia, partial pressure of oxygen in the brain decreases. Most aerobic oxidation pathways switch to anaerobic glycolysis, and adenosine monophosphate-activated protein kinase (AMPK) is activated. Active AMPK phosphorylates multiple downstream substrate proteins, inhibits the biosynthetic pathway of ATP consumption, and negatively regulates ATP regeneration to restore cellular energy levels as much as possible (Andjelkovic et al., 2019). However, as the duration of ischemia increases, this negative feedback cannot compensate for the loss of mitochondrial energy and downregulation of the expression of proteases involved in oxidative phosphorylation complexes, such as nicotinamide adenine dinucleotide dehydrogenase and cytochrome oxidase, which reduce ATP synthesis (He et al., 2012).

Mitochondrial permeability transition pore (MPTP) is a non-specific voltage-dependent special protein complex that crosses the mitochondrial outer membrane and controls mitochondrial permeability (Halestrap, 2009). In the physiological state, MPTP is switched off. However, the MPTP is open during ischemia, which is triggered by Ca²⁺ overload and elevated oxidative stress in the mitochondrial matrix (Kushnareva and Sokolove, 2000; Zhao et al., 2019). The opening of the MPTP leads to an increase in mitochondrial permeability, which allows solutes such as water, macromolecules, and ions to freely enter the mitochondrial matrix, resulting in mitochondrial swelling, outer membrane rupture, and the release of large amounts of reactive oxygen species (ROS) (Krasnikov et al., 2005). In addition, increased mitochondrial permeability also leads to the loss of membrane potential, which in turn lowers cellular mitochondrial ATP levels, enhances intracellular Ca²⁺ concentration, and activates the endogenous apoptotic pathway, thereby inducing neuronal damage caused by ischemia and hypoxia (Zorov et al., 2000; Broughton et al., 2009).

Nuclear respiratory factor 2 (Nrf2) is a key transcription factor of antioxidants (Hannan et al., 2020). When cells undergo oxidative stress, Nrf2 is activated, enters the nucleus, binds to promoters of antioxidant response genes and promotes their transcription and expression (Yen et al., 2016). These genes include superoxide dismutase, glutathione peroxidase, and glutathione S-transferase, which scavenge free radicals and other oxidative substances, reducing damage from oxidative stress in cells (Yang et al., 2019). URB597 alleviates ischemic cerebrovascular disease by activating the Nrf2 pathway, reducing mitochondrial oxidative stress and inflammation (Wang et al., 2022).

When entering equilibrium with the antioxidant system, ROS cause minimal damage under typical circumstances (Takizawa et al., 1998). However, after CCI, the activity of the respiratory chain enzyme complex is inhibited, mitochondrial respiratory dysfunction occurs, and excessive ROS (Yu et al., 2014). Excessive ROS damage to proteins, mtDNA, and lipids leads to apoptosis, neuroinflammation, and destruction of the blood-brain barrier in the ischemic brain (Shirley et al., 2014). Excess ROS levels induce apoptosis through lipid peroxidation. In rats with cerebral ischemia, 4-hydroxyacetone, a by-product of lipid peroxidation, increases and induces axonal damage and oligodendrocyte apoptosis (Mccracken et al., 2000; Matsuda et al., 2009). ROS can also lead to cell apoptosis by releasing cytochrome c (Cyt c), improving mitochondrial permeability and activating the NF-κB/MAPK/JNK pathway (Kim et al., 2006, 2010). In particular, Cyt c is a soluble protein anchored to the inner mitochondrial membrane, and upon release from mitochondria, it triggers a cascade of apoptotic signaling, which typically peaks after ischemia in Cyt c release (Hüttemann et al., 2011; Tajiri et al., 2016). Mammalian target of rapamycin (mTOR) is an important cell signal transduction pathway, which is involved in the regulation of cell growth, metabolism and autophagy (Saxton and Sabatini, 2017). Activation of mTOR signaling pathway can inhibit ROS production. Ethidium bromide, for example, induces mitochondrial clearance through the autophagy pathway (Luo et al., 2013). However, inhibition of mTOR with rapamycin preserved mitochondrial membrane potential and reduced the production of ROS (Nacarelli et al., 2014). In addition, sertraline is a selective serotonin reuptake inhibitor (SSRI) that regulates AMPK-mTOR signal-mediated autophagy by targeting the mitochondrial voltage-dependent anion channels 1 protein (VDAC1) (Hwang et al., 2021). To sum up, there is a complex interaction between mTOR, ROS and mitophagy. Most importantly, Autophagy is regulated in the nervous system by activating ROS and mediating the Akt-mTOR signaling pathway (Gao et al., 2019; Liu et al., 2019a,b).

After CCI, cells are unable to maintain a negative membrane potential due to the lack of ATP, and neuronal depolarization results in an influx of calcium ions into the cell (Gouriou et al., 2011). An excessive increase in calcium ion concentration activates the mitochondrial calcium uniporter (mCU) in cells, which changes mitochondrial permeability, impairs its ability to generate ATP, and leads to the release of proapoptotic factors (Gouriou et al., 2011). Preclinical research is currently being conducted on medications that block mCU, such as Ru360 (García-Rivas et al., 2006). Even partial inhibition of calcium uptake prevents mitochondrial depolarization, the opening of large mitochondrial channels, and cytochrome c release.

Hexokinase is a six-carbon sugar phosphorylase involved in glycolysis, from which ATP is produced (Tan and Miyamoto, 2015). Furthermore, after CCI, the levels of VDAC, especially VDAC1, have been found to decrease and the interaction between VDAC1 and hexokinase has been reduced. These changes may result in a reduction in ATP/ADP exchange and affect the transport of small-molecule metabolites required for oxidative phosphorylation to mitochondria, thus inhibiting respiration and affecting mitochondrial energy supply and mitochondrial-mediated apoptosis (He et al., 2012).

Mitophagy is initiated during neuronal apoptosis following CCI through the BNIP3-Cyt c-related pathway and parkin-mediated signaling (Su et al., 2018). After CCI induction, Parkin and BNIP3 expression increased, and Cyt c was released from the mitochondria into the cytoplasm; however, the first two phenomena were significantly attenuated after treatment with the autophagy inhibitor 3-MA. Similarly, URB597 (an orally biocompatible inhibitor of fatty acid amide hydrolase) treatment significantly reversed the increase in Beclin-1, parkin, and BNIP3 protein expression and the decrease in autophagy-related proteins after CCI (Su et al., 2018). Autophagy consists of three major sequential steps: sequestration, transport, and degradation (Mizushima, 2007). During degradation, autophagosomes and their cargo are degraded by lysosomal hydrolases, and lysosomal dysfunction can lead to accumulation of autophagosomes (Mizushima et al., 2008). Some researchers have argued that this accumulation should be treated as an abnormally excessive form of autophagy (Su et al., 2018). Further, the beneficial effects of URB597 on chronic ischemic brain injury occur by inhibiting impaired autophagic degradation and disruption of the Beclin-1/Bcl-2 complex, followed by severing BNIP3-Cyt c and parkin-mediated mitophagy; this ultimately prevents abnormal excessive autophagy and mitophagy (Su et al., 2018).

The biological function of PPAR depends on the coactivation of PPAR-γ coactivator 1α (PGC-1α) (Haemmerle et al., 2011). PGC-1α is a master transcription factor in the regulation of antioxidant enzymes, clearance systems, and mitochondrial biogenesis (Kaarniranta et al., 2020). Once activated by phosphorylation or deacetylation, PGC-1α activates the transcription of NRF 1 and 2, which regulates mitochondrial transcription factor A (TFAM) (Li et al., 2017). TFAM then translocates to the mitochondrial matrix and stimulates mtDNA replication and mitochondrial gene expression (Tang, 2016). The upstream transcription factor Sirt1 regulates PGC-1α by increasing its expression and decreasing its acetylation (Iwabu et al., 2010). Nicotinamide adenine dinucleotide (NAD), a substrate of Sirt1 that regulates Sirt1 expression, improves cognitive function and reduces neuroinflammation in in vivo and in vitro CCI models (Mouchiroud et al., 2013). Furthermore, these therapeutic effects were associated with mitochondrial protection and inhibition of ROS by activating the Sirt1/PGC-1α pathway (Zhao et al., 2021).

Akt activation enhanced GSK-3β phosphorylation, leading to mTOR activation, and the autophagic protein Beclin-1 expression was significantly downregulated, inhibiting cell cytodestructive autophagy (Wang R. C. et al., 2012; Liu et al., 2015). Akt phosphorylation prevents Bax translocation to mitochondria and inhibits Cyt c release as well as destructive autophagy, attenuating CCI-induced neuronal injury (Sadidi et al., 2009; Castillo et al., 2011). Meanwhile, ERK activation upregulates Bcl-2 expression, which negatively regulates destructive autophagy through a combination of Beclin-1 and Bax (Subramanian and Shaha, 2007). Activation of the γ-aminobutyric acid B receptor (GABA_B) can attenuate CCI-induced increases in atg5 and atg7 expression and inhibit cytodestructive autophagy and neuronal apoptosis (Lindqvist et al., 2014). Baclofen-induced ERK1/2 phosphorylation can accelerate cytoprotective autophagy by moderately increasing the expression of Beclin-1. Activation of GABA_B receptors improves the surface expression of the GABA_A receptor α1 subunit, leading to the downregulation of astrocytes and neurons surface and mitochondrial expression, which in turn enhances cytoprotective autophagy (Liu et al., 2015).

The opening of mitoK_ATP channels is related to potassium uptake from the mitochondrial matrix and maintains the volume of the mitochondrial matrix by reducing the Ca²⁺ load. Reduced mitochondrial Ca²⁺ load can inhibit MPTP opening, prevent ROS production in the mitochondria, and inhibit excitatory oxidative stress and cell death (Fornazari et al., 2008). mitoK_ATP consists of two subunits, Kir6.1, 6.2, and SUR1 or SUR2 (Zhou et al., 2010). Chronic intermittent hypobaric hypoxia (CIHH) can upregulate the protein expression of Kir6.2 and SUR1 in the mitochondria of the hippocampal CA1 region induced by ischemia, thus improving learning and memory dysfunction induced by ischemia in the hippocampal CA1 region. Additionally, CIHH alleviates delay neuronal death (DND) by maintaining mitoK_ATP activity, thus inhibiting Cyt c-induced apoptosis (Zhang et al., 2016).

Short-chain fatty acids (SCFAs) produced by bacteria include acetate, propionate, and butyrate. These SCFAs can penetrate the blood-brain barrier and have a considerable impact on the brain due to their effects on numerous neuronal functions and gut-brain signaling pathways. FMT and SCFAs significantly altered Ndufb2 and Atp5mc1 levels, indicating that electron transport chain (ETC) complexes I and V are the main sites for the regulation of oxidative phosphorylation. FMT and SCFAs alleviate mitochondrial dysfunction by increasing acetate, acetyl-CoA, and ATP contents, as well as the activities of complexes I and V of mitochondrial ETC (Su et al., 2022).

Carfilzomib is a proteasome inhibitor that is used to treat multiple myeloma. It forms a covalent irreversible bond with the LMP2 and LMP7 catalytic subunits of the 20S proteasome, which are two intracellular receptors (Sin et al., 1999). Carfilzomib prevents defects in BCL/adenovirus E1B interacting protein 3-like (BNIP3L) degradation and mitophagy deficiency (Wu et al., 2021). Defective mitophagy caused by BNIP3L deletion has significant implications for ischemic neuronal injury. This is because restored BNIP3L has been observed to reduce cerebellar infarct volume, alleviating ischemic brain injury (Wu et al., 2021).

Butylphthalide is a chemical component of celery oil. Superoxide dismutase (SOD) activity increased, malondialdehyde levels decreased, and ATPase activity increased in the hippocampal mitochondria of CCI rats after therapy, significantly improving learning and memory. Pathological results provided additional evidence that injection reduced mitochondrial ultrastructural destruction. Butylphthalide injection has a protective effect on the structure and function of mitochondria in brain tissue, which may be related to its influence on mitochondrial oxidative damage and energy metabolism dysfunction (Gao, 2009).

Pinocembrin is a flavonoid found in propolis that can potentially strengthen the central nervous system. A decrease in transmembrane potential during hypoxia greatly affects mitochondrial function, producing excessive ROS (Nohl et al., 2005). In animal experiments, the expression of Cyt c oxidase in the hippocampus of rats in the 2VO group decreased significantly; meanwhile, mitochondrial membrane potential levels decreased. Pinocembrin significantly reversed these phenomena (Guang and Du, 2006). Cyt c oxidase is a metabolic indicator of neuronal oxidative activity; therefore, this raises the possibility that pinocembrin shields the rat’s brain mitochondria. In addition, pinocembrin can greatly reduce the degree of mitochondrial swelling, increase the mitochondrial membrane potential, and protect the mitochondrial structure and ROS production, which may explain why pinocembrin protects mitochondria from oxidative stress (Guang and Du, 2006).

Rapamycin is a popular allosteric mTOR inhibitor that binds directly to the mTOR complex and promotes autophagy in several eukaryotes. PINK1, Parkin, and LC3B expression levels have been reported to increase after rapamycin treatment in animal studies, stimulating mitophagy and preventing mitochondrial dysfunction and neuronal apoptosis. Together with experimental treatment control of MHY1485 (an mTOR activator) and the initial notion that the mTOR pathway increases autophagy, it also affects the expression of PI3K, AKT, and mTOR (Bartolomé et al., 2017). These findings suggest that rapamycin exerts its neuroprotective effects by suppressing the PI3K/AKT/mTOR signaling pathway, which increases autophagy (Zheng et al., 2021).

The cannabinoid receptor agonist WIN55212-2 (WIN) and the fatty acid amide hydrolase inhibitor URB597 were administered to counteract the effects of CCI on JNK phosphorylation, lowering the Bcl-2/Bax ratio and caspase-3 activation, all of which are involved in controlling neuronal survival. Moreover, WIN and URB597 inhibit neuronal death induced by JNK-dependent Bcl-2 signaling and improve mitochondrial membrane dysfunction by increasing the selective JNK inhibitor SP600125 (Su et al., 2015).

In 2010, the US Food and Drug Administration approved FTY720, a sphingosine-1-phosphate receptor agonist with potent anti-inflammatory properties, as the first oral medication for the treatment of multiple sclerosis (Wang et al., 2020). Moreover, recent studies have shown that it effectively reduces mitochondrial dysfunction and spatial memory impairment (Wang et al., 2020). FTY720 protects the brain from damage by lowering oxidative stress and neuroinflammation and enhancing synaptic function. According to a study in 2VO animals, FTY720 can improve hippocampal mitochondrial function and enhance ATP synthase activity. ATP levels and ATP synthase activity in the hippocampus are increased, suggesting that FTY720 could reduce CCI-induced mitochondrial dysfunction (Zhang et al., 2020). However, p62 expression, which is crucial for the transfer of ubiquitylated substrates to autophagosomes, and SIRT3, the primary regulator of mitochondrial activity, did not show an effect after the intervention (Zhang et al., 2020).

Traditional Chinese medicine has been reported to improve the activity of the ETC complex, decrease calcium overload following excitability toxicity, and restore the self-regulation function of mitochondria by focusing on mitochondrial dysfunction. This preserves the integrity of mitochondrial structure and function, promotes the reconstruction of energy metabolism, and ultimately improves brain injury and cognitive impairment (Wang et al., 2023). For example, the Zuogui pill and Naoxin capsule improve mitochondrial structure and function and reduce ROS accumulation by improving mitochondrial respiratory chain enzyme complexes (Yu et al., 2014; Feng et al., 2022). Xiaoxuming decoction and baicalein have significantly improved oxidative phosphorylation and mitochondrial membrane potential (He et al., 2009; Wang Y. H. et al., 2012). The Shenma Yizhi decoction and Bushen-Yizhi formula can improve mitochondrial dysfunction by regulating the expression levels of various proteins (Sun et al., 2021; Xiao et al., 2023).