Effects of ischemic postconditioning and long non-coding RNAs in ischemic stroke

ABSTRACT

Stroke is a main cause of disability and death among adults in China, and acute ischemic stroke accounts for 80% of cases. The key to ischemic stroke treatment is to recanalize the blocked blood vessels. However, more than 90% of patients cannot receive effective treatment within an appropriate time, and delayed recanalization of blood vessels causes reperfusion injury. Recent research has revealed that ischemic postconditioning has a neuroprotective effect on the brain, but the mechanism has not been fully clarified. Long non-coding RNAs (lncRNAs) have previously been associated with ischemic reperfusion injury in ischemic stroke. LncRNAs regulate important cellular and molecular events through a variety of mechanisms, but a comprehensive analysis of potential lncRNAs involved in the brain protection produced by ischemic postconditioning has not been conducted. In this review, we summarize the common mechanisms of cerebral injury in ischemic stroke and the effect of ischemic postconditioning, and we describe the potential mechanisms of some lncRNAs associated with ischemic stroke.

Graphical abstract

KEYWORDS:

• Ischemic stroke
• ischemic postconditioning
• remote ischemic postconditioning
• long non-coding RNAs
• neuroprotective effect

Introduction

Stroke is the second leading cause of death worldwide [1]. It is an acute disease involving an impaired cerebral blood supply and is characterized by high morbidity, mortality, recurrence rates, disability rates, and economic burden. Acute ischemic strokes caused by intracranial artery occlusion or extracranial carotid artery occlusion account for approximately 85% of all strokes. Cerebral infarction is a disorder of the blood supply to local brain tissue that leads to cerebral ischemia and hypoxia. The mechanism of cerebral ischemic injury is highly complex, and this complexity mainly manifests as inflammatory injury, excitatory amino acid release, intracellular Ca²⁺ overload, free radical formation, protease activation, blood-brain barrier injury, mitochondrial energy disturbance, excessive nitric oxide (NO) levels, excitatory amino acid toxicity, and the formation of glial scars (Table 1). This series of pathological and physiological processes interacts with changes in the temporal and spatial molecular mechanisms caused by cerebral ischemic injury. Hypoxia, neuronal necrosis, and an intense inflammatory response are modulated by many biological processes in ischemic stroke [2,3]. Long non-coding RNAs (lncRNAs) play an important role in cell differentiation, apoptosis, and regulation of gene expression under physiological and pathological conditions. In recent years, an increasing number of studies have focused on the important role of lncRNAs in ischemic stroke and their potential as therapeutic approaches. The effect and mechanism of ischemic postconditioning (IPostC) in ischemic stroke and the potential lncRNAs involved in the physiological and pathological processes of ischemic stroke have not been fully clarified. In this review, we summarize the common mechanisms of cerebral injury in ischemic stroke and the effect of IPostC, and we describe the potential association of lncRNAs with neuronal injury and protective mechanisms in ischemic stroke.

Table 1. Common mechanisms of cerebral injury in ischemic stroke.

Effect on the brain	Related factors	Mechanisms related to cerebral injury	References
Neuroinflammation	1. Pro-inflammatory cytokines: IL-6, IL-8, IL-1β, COX-2, TNF-α; Anti-inflammatory factors: IL-4 and IL-10 2. MAPK, NLRP inflammasome 3. MiRNA, caspase-3 4. ACSL4, rhFGF21, NF-κB, PPAR-γ	1.Regulation of pro-inflammatory and anti-inflammatory cytokines in the ischemic brain. 2. Activation of the MAPK signaling pathway contributes to activation of the NLRP1 and NLRP3 inflammasome proteins in neurons and brain tissues. 3. Circulating miRNA-3552 interacts with caspase-3 to participate in neuroinflammatory responses. 4. ACSL4 and rhFGF21 modulate microglia/macrophage-mediated neuroinflammation via the NF-κB and PPAR-γ signaling pathways.	1. [5–8,15,16] 2. [9,10] 3. [20] 4. [25]
Neuronal apoptosis	Caspase family, TNF-α, TNF-R, Bcl-2 family, CREB, Hes1, FoxP2, p38 MAPK signaling pathway	Regulation of neuronal cell apoptosis in the ischemic brain.	[18–20]
Steady-state balance of Ca^2+	NMDA receptor	Activation of NMDA receptors may result in Ca^2+ influx in neurons and activation of Ca^2+-dependent protease-induced cell death pathways, resulting in neuronal apoptosis after cerebral ischemia.	[21–23]
Oxidative stress	1. ROS 2. Glutathione peroxidase	1. Oxidative stress induced by ROS overproduction is associated with ischemia-reperfusion injury, neuronal apoptosis, inflammatory signaling pathway activation, and blood-brain barrier damage in ischemic stroke. 2. Neuronal glutathione reduction is the result of oxidative stress in the brain caused by ischemia-reperfusion.	[26–29,139]
Autophagy	1. Beclin-1 2. Malat1 3. MEG3	1. Beclin-1 induces autophagy and regulates the survival or death of ischemic neurons. 2. Downregulation of Malat1 inhibits Beclin1-dependent autophagy in ischemic stroke. 3. MEG3 is upregulated and interacts with p53 to mediate neuron autophagy in ischemic stroke.	1. [20] 2. [30] 3. [31]
Energy metabolism disorders	1. NADPH 2. PGC-1α 3. AMPK	1. NADPH diaphorease expression is upregulated in hippocampal astrocytes after ischemic stroke. 2. PGC-1α increases mitochondrial fatty acid oxidation gene expression in ischemic brain tissue. 3. AMPK resists cerebral ischemic injury by activating catabolic pathways and inactivating ATP-consuming processes.	1. [33] 2. [35] 3. [36]
Excessive intracellular free radicals and NO	1. Superoxide dismutase 2. Nitric oxide synthase	1. Superoxide dismutase protects the brain by removing excessive free radicals from cells. 2. Increases in nitric oxide synthase expression and NO production by reactive astrocytes may play a role in delayed neuronal death after ischemic damage.	1. [37] 2. [11]
Excitatory amino acid toxicity	1. Glutamate levels, glutamate transporters (glt-1), gap junction proteins (Cx43), Na^+-K^+-ATPase activity 2. NMDA receptors 3. NO, oxygen free radicals	1. The expression levels of glutamate, glt-1, Cx43, and AMPARs and Na^+-K^+-ATPase activity are related to excitotoxicity and anti-excitotoxicity in ischemic stroke. 2. NMDA receptor antagonists can inhibit the release of excitatory amino acids during ischemic stroke. 3. The neurotoxicity of NO is mainly caused by its reaction with oxygen free radicals.	1. [39,40] 2. [41,42] 3. [43]
Blood-brain barrier damage	1. Aquaporin-4 2. Mitochondrial proteins 3. A Na^+ channel blocker and an antioxidant: AM-36 4. MMP-2/MMP-9	1. Downregulation of aquaporin-4 can increase the integrity of the blood-brain barrier and improve cerebral blood flow after transient MCAO. 2. Nitrite may increase cerebral blood flow via its effect on nitrosylation of mitochondrial proteins. 3. AM-36 exhibits a neuroprotective effect by modulating neutrophil accumulation in the ischemic brain. 4. Activation of MMP-2 or MMP-9 contributes to the breakdown of the blood-brain barrier in ischemic stroke. High serum MMP-9 levels are related to an increased risk of death and disability in acute ischemic stroke.	1. [45] 2. [46] 3. [47,48] 4. [49–51]
Formation of glial scars	Cytokines from neurons and neuroglial cells, such as vimentin, nestin, IL-1β, monocyte chemotactic protein-1, and glial fibrillary acidic protein	Cytokines contribute to the development of reactive gliosis and the formation of glial scars.	[14,52]

IL: interleukin, COX: cyclooxygenase, TNF: tumor necrosis factor, MAPK: mitogen-activated protein kinase, NLRP: nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein, miRNA: microRNA, ACSL: acyl-CoA synthetase long chain, NF-κB: nuclear factor kappa B, PPAR: peroxisome proliferator-activated receptor, NMDA: N-methyl-D-aspartate, ROS: reactive oxygen species, CREB: cAMP-response element binding protein. Malat1: metastasis-associated lung adenocarcinoma transcript 1, MEG3: maternal expression gene 3, NADPH: nicotinamide adenine dinucleotide phosphate, PGC: peroxisome proliferator-activated receptor-gamma coactivator, AMPK: adenosine 5’-monophosphate-activated protein kinase, NO: nitric oxide, MMP: matrix metalloproteinase, MCAO: middle cerebral artery occlusion.

1. Common mechanisms of cerebral injury in ischemic stroke

Neuroinflammation: Studies on the role of inflammation in acute ischemic stroke were initially focused on the reperfusion phase. The existence of an inflammatory penumbra allows the diapedesis of T cells into intact brain regions, where they trigger neuronal cell death by at least two mechanisms, direct cytotoxicity by perforin secretion and interferon-γ secretion [4]. Pro-inflammatory and anti-inflammatory factors are associated with inflammatory responses in cerebral ischemic injury. The expression levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-8, IL-1β, and cyclooxygenase-2 were increased after cerebral ischemia [5–7]. Anti-inflammatory factors, including IL-4 and IL-10, protect the brain from ischemia-reperfusion and inflammation-induced injury. Studies have shown that IL-10 deficiency can exacerbate inflammation in the brain [8]. Activation of the mitogen-activated protein kinase (MAPK) signaling pathway contributes to the activation of the NOD-like receptor protein 1 and 3 inflammasome proteins in neurons in vitro and in mouse brain tissues in vivo following ischemic stroke [9,10]. Neuroinflammation leads to neurodegeneration and brain damage: Neuroinflammation is associated with the activation and release of proinflammatory mediators from resident immune cells in the brain (microglia and astrocytes) and peripherally derived immune cells, such as neutrophils, monocytes, and T cells [11]. Progressive neuroinflammation not only damages brain cells, but also plays a positive role in nerve repair activities after cerebral ischemia. Studies have found that the differentially expressed circulating microRNA (miRNA)-3552 interacts with caspase-3 to participate in neuroinflammatory responses in ischemic stroke [12]. All stages of an ischemic episode, from cessation of cerebral blood flow to late recirculation of ischemic brain tissue, are accompanied by changes in neuroinflammation in the brain [13]. Neuroinflammation increases brain damage, leading to the death of surviving neurons in the previously ischemic brain. Neuroinflammation also contributes to the development of glial scars [11,14]. Microglial activation promotes the release of pro-inflammatory cytokines, such as TNF-α, NO, and reactive oxygen species (ROS), which are harmful to the brain tissue after ischemia [15]. Acyl-CoA synthetase long-chain family member 4 is an important isozyme for polyunsaturated fatty acid metabolism and a novel regulator of neuroinflammation in ischemic stroke [16]. Recombinant human fibroblast growth factor 21 treatment promoted functional recovery by modulating microglia/macrophage-mediated neuroinflammation via the nuclear factor kappa B (NF-κB) and peroxisome proliferator-activated receptor gamma signaling pathways in C57BL/6 mice with middle cerebral artery occlusion (MCAO) [17].

Neuronal apoptosis-related molecules: These molecules mainly include the Bcl-2 family, caspase family, and TNF receptor superfamily, which are involved in the regulation of cell apoptosis after brain injury. Downregulation of the TNF-α and TNF receptor 1 proteins can effectively alleviate neurological impairment after cerebral ischemic injury [18,19]. Within hours of an ischemic stroke, neurons can be permanently damaged or die. Therefore, development of treatment options to save damaged neurons is important [20].

Steady-state balance of Ca²⁺: Ca²⁺ and N-methyl-D-aspartate (NMDA) receptors play a central role in regulating excitotoxic neuronal death after cerebral ischemia. Ca²⁺ homeostasis is necessary to maintain normal physiological functions of cells. After cerebral ischemic injury, Ca²⁺ homeostasis is disrupted, leading to Ca²⁺ pump dysfunction, mitochondrial damage, cell membrane depolarization, release of a large amount of glutamate, and NMDA receptor activation, resulting in Ca²⁺ influx and activation of Ca²⁺-dependent protease-induced cell death pathways [21–23].

Oxidative stress: In response to ischemic stroke, excessive ROS can cause substantial cellular injury and vascular effects. Oxidative stress induced by ROS overproduction is associated with the ischemia-reperfusion injury process in ischemic stroke [24,25]. Neuronal apoptosis, inflammatory signaling pathway activation, and blood-brain barrier damage induced by oxidative stress can aggravate neurodegeneration and cell death during ischemic stroke [26–29].

Autophagy: Beclin-1 is an autophagy-associated protein that strongly induces autophagy and plays a key role in regulating the survival of ischemic neurons [30,31]. Some miRNAs can regulate autophagy by regulating changes in the expression of autophagy-related genes. For example, downregulation of the metastasis-associated lung adenocarcinoma transcript 1 (Malat1) lncRNA inhibits Beclin1-dependent autophagy by regulating miR-30a expression, thus reducing nerve cell death in ischemic stroke [32]. The maternal expression gene 3 (MEG3) lncRNA, which is a cell death promoter, is upregulated and interacts physiologically and functionally with p53 to mediate neuron autophagy in ischemic stroke [20].

Energy metabolism disorders: The sudden interruption or reduction of blood flow and oxygen supply to the brain leads to energy depletion or metabolism disorders during ischemic stroke [33,34]. Increased Ca²⁺ and ROS levels lead to mitochondrial dysfunction, which further aggravates oxidative stress [13,19,33] and is associated with upregulation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorease expression in hippocampal astrocytes during ischemic stroke [33]. Peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α is a cofactor of peroxisome proliferator-activated receptor family transcription factors that leads to increased expression of mitochondrial fatty acid oxidation genes in ischemic brain tissue [35]. Adenosine 5’-monophosphate-activated protein kinase (AMPK), which functions as a cellular energy sensor, can protect against cerebral ischemic injury by activating catabolic pathways and inactivating ATP-consuming processes [36].

Excessive levels of intracellular free radicals and NO: Superoxide dismutase is an important antioxidant enzyme in brain tissue and can protect the brain by removing excessive free radicals in cells [37]. NO plays a crucial role in information transmission and neuroprotection in the central nervous system, but excessive release of NO, as an active gaseous free radical, can lead to neuron damage [11]. Endogenous NO derived from neuronal NO synthase plays a deleterious role in delayed cerebral neuronal death and ischemic reperfusion damage [38].

Excitatory amino acid toxicity: Glutamate levels in brain tissue increase significantly following cerebral ischemia-reperfusion injury. Glutamate causes excitotoxicity leading to neuronal death [39]. The expression levels of glutamate transporters (glt-1), gap junction proteins (Cx43), and glutamate receptors (AMPA receptors) and Na⁺-K⁺-ATPase activity are related to the anti-excitotoxic effects involved in protection against neuronal death and functional recovery after ischemic stroke [40]. NMDA receptor antagonists can inhibit the release of excitatory amino acids during ischemic stroke [41,42]. Additionally, the neurotoxicity of NO is mainly mediated by its reaction with oxygen free radicals [43].

Blood-brain barrier damage: Aquaporin-4 (AQP4) is one of the most widely expressed molecules in the brain, especially in astrocytes [44]. Downregulation of AQP4, which is involved in water homeostasis in astrocytes, can increase the integrity of the blood-brain barrier and improve cerebral blood flow after transient MCAO [45]. Nitrite may increase cerebral blood flow via its effects on nitrosylation of mitochondrial proteins [46]. AM-36, which is a Na⁺ channel blocker and an antioxidant, has a neuroprotective effect by modulating neutrophil accumulation in ischemic brain tissue [47,48]. Matrix metalloproteinases (MMPs) act mainly on extracellular endopeptides and can digest proteins located outside the cell [49]. MMP-2 and MMP-9 damage the neurovascular unit and promote blood-brain barrier disruption [34,50]. Elevated serum MMP-9 levels during acute ischemic stroke are associated with an increased risk of death and major disability [51].

Formation of glial scars: Astrocytes release vimentin, nestin, IL-1β, monocyte chemotactic protein-1, and glial fibrillary acidic protein, which contribute to the development of reactive gliosis and the formation of glial scars after ischemia [14,52].

2. Mechanism by which IPostC triggers endogenous neuroprotection

Acute or delayed cell death after ischemia-reperfusion ultimately leads to the irreversible damage and clinical sequelae observed in stroke patients [53]. IPostC is a temporary, non-lethal ischemia-reperfusion process that protects the primary ischemic organ by producing a non-traumatic ischemic adaptation after ischemia [54]. This process stimulates protective factors, thereby limiting inflammation and delayed cell death [55–57]. IPostC can reduce ischemia-reperfusion injury by regulating a variety of endogenous neuroprotective mechanisms, including reducing ROS production, alleviating inflammatory and cerebral ischemia-reperfusion injury by activating autophagy, inhibiting inflammation of local injured tissues, endoplasmic reticulum stress, and neuronal apoptosis, improving cerebral blood supply, and regulating phosphorylation of the MAPK, protein kinase C, Notch, and PI3K/Akt pathways [58,59]. However, the role of autophagy in IPostC is currently controversial; some research suggests that autophagy eliminates the neuroprotective effect of IPostC [60–62], but other studies have found that autophagy activates the neuroprotective effect mediated by IPostC [30,63]. In the ischemic brain, clearance of damaged mitochondria through autophagy is beneficial for neuronal survival, while nonselective autophagy is harmful [62,64]. Studies have shown that IPostC activates autophagy in rat hearts with ischemia-reperfusion injury and inhibits cardiomyocyte apoptosis [65]. IPostC can activate autophagy to reduce mitochondrial damage and reduce cell death through the AKT/GSK3β pathway and Bcl-2 phosphorylation [63,66]. Recent studies have shown that IPostC is involved in transcription factor EB transnucleation and target gene transcription to promote functional autophagy in cells [67].

IPostC, including in situ ischemic postconditioning (ISPostC) and remote ischemic postconditioning (RIPostC), induces endogenous neuroprotection by producing transient, non-fatal ischemia during reperfusion [68,69]. RIPostC is an intervention that involves repetitive and transient cycles of occlusion and reperfusion of blood flow to a remote organ, such as inducible ischemia of a hind limb, which protects the brain against subsequent ischemic injury. Furthermore, RIPostC is an attractive therapeutic strategy in terms of cost and ease of intervention delivery because it is performed simply by inflating a blood pressure cuff on an arm or leg [70]. ISPostC involves repetitive and transient cycles of occlusion and reperfusion of arterial flow to primary organs. However, the brain is more sensitive and intolerant to ischemia than other organs, and ISPostC may trigger another ischemic event to induce ischemia in brain tissue, which may cause irreversible damage to ischemia-sensitive brain tissue [71]. This drawback has limited the clinical application of ISPostC.

In recent studies, RIPostC has been shown to produce protective effects in the myocardium, kidney, skeletal muscle, and brain after ischemia. Limbs are often used for RIPostC intervention because of their strong ischemia tolerance and because the degree of ischemia can be easily observed [71–73]. IPostC reduced the brain infarct size in a rodent model of acute focal ischemia-reperfusion brain injury [68]. Exosomes secreted by cells can transport signaling molecules to distant cells or organs, thus achieving signal regulation and communication [74]. Previous studies have shown that exosome treatment reduces the cerebral infarction volume, degree of cerebral edema, and expression of NMDA receptors in brain tissues of ischemia-reperfusion injured rats [75]. Exosomes can alleviate the excitotoxicity of glutamate, and organs subjected to RIPostC can secrete exosomes to mitigate glutamate excitotoxicity in the central nervous system [76]. In a rat model involving 100 min of focal brain ischemia, RIPostC could reduce the infarct size when initiated within 30 min of reperfusion [77]. The serum of a limb subjected to RIPostC showed reduced ROS levels and neutrophil activation via the MyD88-TRAF6-MAPK pathway [78]. RIPostC can inhibit ROS production and excessive intracellular Ca²⁺ accumulation to attenuate direct tissue injury and remove a major stimulus that opens the mitochondrial permeability transition pore during reperfusion [79].

The neuroprotective mechanisms of RIPostC are highly complex and have not been fully elucidated. In addition to attenuation of reperfusion injury [80], RIPostC has anti-inflammatory [81], anti-edema [69,73,82], angioneurogenic [83], anti-platelet, and vasodilatory (enhancement of collateral microvascular circulation) effects on ischemic stroke [84,85] that are mediated through numerous neurohumoral chemical messengers, including those released from endothelial-derived exosomes [86]. The neuronal isoform of NO synthase has also been shown to participate in the neuroprotective effect of RIPostC [87]. RIPostC protects the brain against cerebral ischemia-reperfusion injury by upregulating endothelial NO synthase through the PI3K/Akt pathway following focal cerebral ischemia in rats [85,88]. RIPostC effectively improved blood-brain barrier permeability by upregulating MMP-9 expression and downregulating claudin-5 expression in a rat model of hypoperfusion caused by severe carotid stenosis [80,89]. The neuroprotective mechanism of RIPostC is complex, and it often does not involve a single target but results from combined actions on multiple targets and multiple pathways (Table 2) [82]. Therefore, it is of great importance to systematically explore the roles of specific lncRNAs associated with the brain protection produced by RIPostC, which could be used as potential targets for preclinical studies of ischemic stroke.

Table 2. The neuroprotective mechanisms of IPostC in ischemic stroke.

Effect on brain	Neuroprotective mechanism	References
Exosomes	Exosomes can alleviate the excitotoxicity of glutamate and reduce the cerebral infarction volume, cerebral edema, and NMDA receptor expression in the ischemic brain.	[74–76]
Limitation of inflammation and delayed cell death	IPostC can stimulate protective factors to alleviate inflammation and delayed cell death.	[55–57,80,81]
Attenuation of reperfusion injury	IPostC can reduce ischemia-reperfusion injury by regulating a variety of endogenous neuroprotective mechanisms, including reducing ROS production, alleviating inflammation, activating autophagy, endoplasmic reticulum stress, and neuronal apoptosis, and improving the cerebral blood supply.	[58,59,81,82]
Regulation of autophagy	Autophagy eliminates or activates the neuroprotective effects mediated by IPostC, including regulation of the inflammatory reaction, ROS production, endoplasmic reticulum stress, neuronal apoptosis, and phosphorylation of the MAPK, PKC, Notch, and PI3K/Akt pathways.	[58–63]
Anti-edema effects	IPostC effectively reduces brain edema after reperfusion by inhibiting AQP4 expression.	[69,73,83]
Enhancement of collateral microvascular circulation	RIPostC can enhance collateral microvascular circulation.	[84–86]
Nitric oxide synthase	1. The neuronal isoform of nitric oxide synthase participates in the neuroprotective effect of RIPostC. 2. RIPostC protects the brain against cerebral ischemia-reperfusion injury by upregulating eNOS through the PI3K/Akt pathway.	1. [87,88] 2. [85]
Alleviation of blood brain barrier disruption	RIPostC can effectively improve blood-brain barrier permeability by activating matrix metalloproteinase-9 and inhibiting claudin-5.	[80,89]
Inhibition of ROS production and excessive intracellular Ca^2+ accumulation	RIPostC can reduce ROS levels and neutrophil activation through the MyD88-TRAF6-MAPK pathway.	[78,79]

IPostC: ischemic postconditioning, NMDA: N-methyl-D-aspartate, ROS: reactive oxygen species, MAPK: mitogen-activated protein kinase, PKC: protein kinase C, PI3K: phosphoinositide 3-kinase, AQP4: aquaporin-4, RIPostC: remote ischemic postconditioning, eNOS: endothelial nitric oxide synthase.

3. Effect lncRNAs on ischemic stroke

Non-coding RNAs regulate important cellular and molecular events through a variety of mechanisms and affect the prognosis of ischemic stroke [90,91]. LncRNAs have been demonstrated to be key gene regulators in many diseases [92–94]. LncRNA is a major type of non-coding RNA with a length of more than 200 nucleotides. Studies have found that lncRNAs are involved in a wide range of biological processes, including genome imprinting, transcription, and post-transcriptional regulation [95–97]. LncRNAs can also function as miRNA sponges by competitively binding miRNAs to regulate mRNA expression [98]. LncRNA is closely related to the expression of neighboring mRNA, suggesting that lncRNAs may exert their function by regulating a predicted mRNA target [99,100].

A summary of some well-studied lncRNAs and their effects on a variety of pathophysiological processes in ischemic stroke is presented in Table 3. The antisense non-coding RNA in the INK4 locus (ANRIL) lncRNA was significantly increased in rats with cerebral infarction, and it regulated the caspase recruitment domain 8 gene expression level and promoted angiogenesis and inflammation through the NF-κB and vascular endothelial growth factor (VEGF) signaling pathways [101–103]. Activation of the lncRNA calcium/calmodulin dependent protein kinase II delta associated transcript 1 (C2dat1) increased calcium/calmodulin dependent protein kinase II delta expression in C57BL/6 mice with ischemic brain injury and then activated the NF-κB signaling pathway to inhibit the inflammatory response [91,104] Upregulation of lncRNA ENSG00000226482 expression activated the adipocytokine signaling pathway, resulting in acute ischemic stroke [105].

Table 3. LncRNAs associated with ischemic stroke.

LncRNA	Mechanisms in ischemic stroke	References
ANRIL	Regulation of the expression level of the antisense ANRIL lncRNA and promotion of angiogenesis and inflammation through the NF-κB and VEGF signaling pathways.	[101–103]
C2dat1	Activation of C2dat1 lncRNA can increase calcium/calmodulin dependent protein kinase II delta expression and then activate the NF-κB signaling pathway to inhibit the inflammatory response.	[91,104]
H19	The circulating expression level of lncRNA H19 is significantly upregulated in the plasma of stroke patients, OGD/R SH-SY5Y cells, and MCAO/R rat brains.	[106–109]
Malat1	Silencing Malat1 results in increased expression of pro-inflammatory cytokines, and upregulation of Malat1 lncRNA can alleviate ischemic cerebrovascular and brain parenchymal damage. Malat1 lncRNA affects AQP4 expression by competitively binding miR-145 to promote cerebral ischemia-reperfusion injury.	[110] [111]
MEG3	The expression of MEG3 lncRNA is significantly increased in MCAO mouse brain tissue. Inhibition of MEG3 reduces the cerebral infarction volume and degree of cerebral edema. MEG3 lncRNA accelerates the ischemic stroke process by inhibiting miRNA 424–5p to regulate MAPK pathway activation.	[20] [112]
SNHG12	SNHG12 lncRNA targets miR-199a to reduce cell death and inflammation or targets miR-150 to de-repress its target gene, VEGF, in OGD-injured mouse BMECs. SNHG12 lncRNA is upregulated in mouse neuronal cells during ischemia. SNHG12 lncRNA is upregulated at 24 hours of reperfusion in the post-stroke adult mouse cortex.	[114–117]
FosDT	Inhibition of FosDT lncRNA promotes post-stroke neuroprotection via abrogation of REST-mediated gene silencing. FosDT lncRNA is associated with decreased induction of inflammatory genes, decreased apoptosis, mitochondrial dysfunction, and oxidative stress in the ischemic brain.	[86,87] [120]
NEAT1	NEAT1 lncRNA upregulates HOXA1 to alleviate cell apoptosis and promotes cell viability in neonatal mice with hypoxic-ischemic brain damage. NEAT1 knockout can significantly reduce brain injury by inhibiting microglial activation and release of pro-inflammatory cytokines.	[121] [122]
HOTAIR	Silencing of HOTAIR lncRNA improves the recovery of neurological function after ischemic stroke via the miR-148a-3p/KLF6 axis. HOTAIR lncRNA mediates OGD/R-induced cell injury and angiogenesis in an EZH2-dependent manner.	[123,124]
ENSG00000226482 ENSG00000260539	The ENSG00000226482 and ENSG00000260539 lncRNAs are associated with the PPAR signaling pathway and cell adhesion molecules.	[125]
MIAT	Ischemic stroke patients with higher MIAT lncRNA expression levels have a relatively poor prognosis.	[136]
UCA1	A high UCA1 lncRNA expression level is associated with worse accumulated recurrence and survival in ischemic stroke patients.	[138]

IPostC: ischemic postconditioning, lncRNA: long non-coding RNA, ANRIL: antisense non-coding RNA in the INK4 locus, NF-κB: nuclear factor kappa B, VEGF: vascular endothelial growth factor, C2dat1: calcium/calmodulin dependent protein kinase II delta associated transcript 1, OGD/R: oxygen-glucose deprivation/reoxygenation, MCAO: middle cerebral artery occlusion, Malat1: metastasis-associated lung adenocarcinoma transcript 1, AQP4: aquaporin-4, MEG3: maternal expression gene 3, miR: microRNA, MAPK: mitogen-activated protein kinase, SNHG12: small nucleolar RNA host gene 12, FosDT: Fos downstream transcript, NEAT1: nuclear-enriched abundant transcript1, HOXA1: homeoboxA1, HOTAIR: Hox transcript antisense intergenic RNA, EZH2: enhancer of zeste homolog 2, PPAR: peroxisome proliferator-activated receptor, MIAT: myocardial infarction-associated transcript, UCA1: urothelial carcinoma-associated 1, BMECs: brain microvascular endothelial cells.

LncRNA H19 promotes cell death and autophagy in ischemic stroke. The plasma expression level of lncRNA H19 is of potential clinical diagnostic value for stroke, and the circulating lncRNA H19 expression level was significantly upregulated in stroke patients compared with that in healthy controls [106]. LncRNA H19 was upregulated in oxygen-glucose deprivation (OGD)/reoxygenated SH-SY5Y cells and rat brains subjected to MCAO/reperfusion. Inhibition of H19 expression had a significant protective effect against apoptosis and autophagy in SH-SY5Y cells [107–109].

Expression of lncRNA Malat1 was significantly increased in endothelial cells subjected to OGD and brain tissues of MCAO mice. Silencing of Malat1 in cerebral microvascular endothelial cells and ischemic stroke model mice resulted in increased expression of the pro-inflammatory cytokines CMP-1, IL-6, and E-selectin. Upregulation of lncRNA Malat1 can alleviate ischemic cerebrovascular and brain parenchymal damage and has anti-apoptotic and anti-inflammatory effects in the cerebral microvasculature [110]. LncRNA Malat1 is involved in brain tissue damage induced by ischemia-reperfusion injury by affecting AQP4 expression via competitively binding miR-145 in ischemic stroke [111].

LncRNA MEG3 directly binds to the p53 protein at the endogenous RNA position and regulates the expression of lysyl oxidase 12/15. MEG3 expression was significantly increased in OGD-induced HT22 cells and MCAO mouse brain tissue. Inhibition of MEG3 also reduced the cerebral infarction volume and the degree of cerebral edema in MCAO mice [20]. MEG3 accelerates the ischemic stroke process by inhibiting miRNA 424–5p, which targets semaphorin 3A and activates the MAPK pathway [112].

A recent study found that lncRNA 1810034 contributes to protection of the brain against cerebral ischemic stroke and inhibition of neuroinflammation by suppressing the NF-κB pathway in microglial cells [113]. The small nucleolar RNA host gene 12 (SNHG12) lncRNA protects against OGD-induced injury by activating the AMPK-silent information regulator of transcription 1 (Sirtuin 1) pathway via targeting miR-199a to reduce cell death and inflammation in neurons. It can also target miR-150 to de-repress its target gene, VEGF, and promote capillary-like tube formation in OGD-injured mouse bone marrow microvascular endothelial cells [114,115]. SNHG12 was also upregulated in mouse hippocampal cells during ischemia [116]. SNHG12 was upregulated at 24 hours of reperfusion in the post-stroke adult mouse cortex compared with the sham group [117].

Fos downstream transcript (FosDT) is an ischemia-induced lncRNA that preferentially binds to the activated RE1 silencing transcription factor (REST) complex in response to increased cell death and brain damage in ischemic stroke [118]. Knockdown of FosDT resulted in the de-repression of the REST target genes glutamate ionotropic receptor AMPA type subunit 2, NF-κB2, and glutamate ionotropic receptor NMDA type subunit 1, resulting in reduction of the infarct size and improvement of motor function in adult rats with transient MCAO [119]. A recent study found that the improved functional outcome after stroke in rats with knockout of FosDT lncRNA was associated with decreases in inflammatory gene induction, apoptosis, mitochondrial dysfunction, and oxidative stress [120].

The nuclear-enriched abundant transcript1 (NEAT1) lncRNA upregulates homeoboxA1 (HOXA1) to alleviate cell apoptosis and promotes cell viability in neonatal mice with hypoxic-ischemic brain damage [121]. Studies have shown that NEAT1 lncRNA is highly expressed in rats subjected to MCAO, and NEAT1 knockout can significantly reduce brain injury by reducing the number of activated microglia and their release of pro-inflammatory cytokines [122]. The lncRNA Hox transcript antisense intergenic RNA (HOTAIR) can competitively bind miRNA-148a-3p through a competing endogenous RNA mechanism to upregulate Kruppel-like factor 6 (KLF6) expression, inhibit the signal transducer and activator of transcription 3 pathway, promote cell apoptosis and the inflammatory response, and aggravate nerve injury after ischemic stroke [123]. The HOTAIR lncRNA mediates OGD/reoxygenation-induced cell injury and angiogenesis by the HOTAIR/enhancer of zeste homolog 2 axis in human brain microvascular endothelial cells [124]. A recent study found that 228 upregulated and 16 downregulated differentially expressed lncRNAs may be involved in acute cerebral infarction in adult rats. Among them, lncRNA ENSG00000226482 was associated with the peroxisome proliferator-activated receptor signaling pathway, and lncRNA ENSG00000260539, which interacts with cell adhesion molecules, might be a promising indicator for the early diagnosis of acute stroke [125].

The expression characteristics of lncRNAs in the brain tissue of rats with ischemic stroke reveal the key cellular events and various molecular mechanisms of ischemic stroke [99,126,127]. Significant changes in lncRNAs after stroke have a substantial influence on the stability of mRNA expression [128]. LncRNAs can regulate the expression of genes related to ischemic brain injury by interacting with chromatin-modifying proteins [118,119]. Previous studies have shown that lncRNA expression profiles are extensively altered during acute reperfusion after focal cerebral ischemia in adult rats [129–131], and these significantly altered lncRNAs may be related to epigenetic variation after ischemic stroke [132,133].

Exploring the effects of lncRNAs at the systematic expression pattern level in ischemic stroke is of great importance for developing new therapeutic strategies for ischemic stroke. Previous microarray studies have analyzed the lncRNA expression characteristics after ischemia-reperfusion injury and ISPostC. Compared with those in the ischemia-reperfusion injury group, 2292 lncRNAs were upregulated and 1848 lncRNAs were downregulated in the ISPostC group [78]. Our previous study found that after RIPostC intervention in ischemic stroke model rats, the degree of brain edema was alleviated, lncRNA 017702 expression was upregulated, and pro-inflammatory gene expression was downregulated [134]. At present, few studies have examined the regulatory effect of lncRNAs in RIPostC. Some studies have found that lncRNAs may be involved in the neuroprotective effects of IPostC on ischemic stroke, but the effects and mechanisms of lncRNAs in RIPostC after ischemic stroke are still unclear.

Therapeutic potential of lncRNAs for ischemic stroke

LncRNAs affect various signaling pathways of cellular processes, such as inflammation, cell apoptosis, cell death, autophagy, angiogenesis, and microglial activation. In recent years, studies have shown that some lncRNAs may be regarded as biomarkers for prognosis and diagnosis. Expression of the NEAT1 lncRNA was increased in patients with acute ischemic stroke, and increased NEAT1 expression was correlated with worse recurrence-free survival [135]. A multivariate analysis revealed that ischemic stroke patients with high lncRNA myocardial infarction-associated transcript (MIAT) expression levels had a relatively poor prognosis [136]. A recent study found that compared with atrial fibrillation patients with ischemic stroke, atrial fibrillation patients without ischemic stroke had significantly lower serum ANRIL lncRNA levels. The serum ANRIL level exhibits good predictive value for ischemic stroke in patients with atrial fibrillation, and its increased expression is correlated with worse recurrence-free survival [137]. The urothelial carcinoma-associated 1 (UCA1) lncRNA might serve as a candidate prognostic biomarker for acute ischemic stroke patients. UCA1 expression was elevated in acute ischemic stroke patients with recurrence or death within 2 years compared with that in patients without recurrence or death within 2 years, and a high UCA1 expression level was associated with worse accumulated recurrence-free survival in ischemic stroke patients [138]. A recent study performed a meta-analysis and various bioinformatics analyses to further explore the biological role and molecular mechanisms of lncRNAs to provide a theoretical basis for the prognosis of ischemic stroke [125].

LncRNAs are promising targets for diagnosis, treatment, and prognosis assessment of ischemic stroke. Although some studies have confirmed the regulatory mechanism of single lncRNAs related to nerve injury and repair after ischemic stroke, systematic studies of the role of many lncRNAs in the pathological process of ischemic stroke and their interaction mechanisms have not been performed. Future studies will need to focus on the functional mechanism of the systematic and comprehensive lncRNA regulatory network associated with ischemic stroke and further explore and verify promising therapeutic markers for ischemic stroke. A large amount of work remains in this area. However, the potential clinical implications are important.

Conclusion

This review summarizes the mechanisms of cerebral injury in ischemic stroke and the neuroprotective effect of IPostC and describes some lncRNAs associated with regulation of ischemic stroke. LncRNAs have high biological value in the diagnosis and prognosis of ischemic stroke and are expected to become new therapeutic targets. Exploring the functions and mechanisms of these lncRNAs will contribute to the development of promising treatment strategies for ischemic stroke.

Ethical statement

No ethics approval was required for this study as it involved no human participants or animals.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Natural Science Foundation of China, China[82160263], the Research Innovation Team of Yunnan Province, China [2019HC022], Yunnan Applied Basic Research Projects, China [202101AY070001-253, 202001AY070001-057, 2019FE001-175, 2020ZDKFKT004] and the Ten Thousand Person Plan for Famous Doctors of Yunnan Province, China [YNWR-MY-2018-015].