Molecular chaperones and hypoxic-ischemic encephalopathy

Cong Hua, Wei-na Ju, Hang Jin, Xin Sun,, Gang Zhao,

1 Department of Neurosurgery,e First Hospital of Jilin University, Changchun, Jilin Province, China

2 Department of Neurology,e First Hospital of Jilin University, Changchun, Jilin Province, China

Molecular chaperones and hypoxic-ischemic encephalopathy

Cong Hua1, Wei-na Ju2, Hang Jin2, Xin Sun2,＊, Gang Zhao1,＊

1 Department of Neurosurgery,e First Hospital of Jilin University, Changchun, Jilin Province, China

2 Department of Neurology,e First Hospital of Jilin University, Changchun, Jilin Province, China

How to cite this article:Hua C, Ju WN, Jin H, Sun X, Zhao G (2017) Molecular chaperones and hypoxic-ischemic encephalopathy. Neural Regen Res 12(1):153-160.

Open access statement:is is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Hypoxic‐ischemic encephalopathy (HIE) is a disease that occurs when the brain is subjected to hypoxia, resulting in neuronal death and neurological de fi cits, with a poor prognosis.e mechanisms underlying hypoxic‐ischemic brain injury include excitatory amino acid release, cellular proteolysis, reactive oxygen species generation, nitric oxide synthesis, and in fl ammation.e molecular and cellular changes in HIE include protein misfolding, aggregation, and destruction of organelles.e apoptotic pathways activated by ischemia and hypoxia include the mitochondrial pathway, the extrinsic Fas receptor pathway, and the endoplasmic reticulum stress‐induced pathway. Numerous treatments for hypoxic‐ischemic brain injury caused by HIE have been developed over the last half century. Hypothermia, xenon gas treatment, the use of melatonin and erythropoietin, and hypoxic‐ischemic preconditioning have proven e ff ective in HIE pa‐tients. Molecular chaperones are proteins ubiquitously present in both prokaryotes and eukaryotes. A large number of molecular chaperones are induced after brain ischemia and hypoxia, among which the heat shock proteins are the most important. Heat shock proteins not only maintain protein homeostasis; they also exert anti‐apoptotic e ff ects. Heat shock proteins maintain protein homeostasis by helping to transport proteins to their target destinations, assisting in the proper folding of newly synthesized polypeptides, reg‐ulating the degradation of misfolded proteins, inhibiting the aggregation of proteins, and by controlling the refolding of misfolded proteins. In addition, heat shock proteins exert anti‐apoptotic e ff ects by interacting with various signaling pathways to block the activation of downstream effectors in numerous apoptotic pathways, including the intrinsic pathway, the endoplasmic reticulum‐stress mediated pathway and the extrinsic Fas receptor pathway. Molecular chaperones play a key role in neuroprotection in HIE. In this review, we provide an overview of the mechanisms of HIE and discuss the various treatment strategies. Given their critical role in the disease, molecular chaperones are promising therapeutic targets for HIE.

nerve regeneration; hypoxic-ischemic encephalopathy; molecular chaperones; excitatory amino acid; cellular proteolysis; oxygen radicals; in fl ammation; apoptosis; reviews; neural regeneration

Accepted: 2016-12-26

Introduction

Hypoxic‐ischemic encephalopathy (HIE) is a disease that occurs when the brain is subjected to hypoxia and ischemia. Neonates su ff er from HIE most frequently due to birth as‐phyxia. HIE can also result from pathological conditions, such as cardiac arrest, the most common cause of HIE in adults (Chan et al., 2014). Other causes of HIE include shock, cerebrovascular events, diffuse cerebral vasospasm, severe intracranial hypertension, carbon monoxide (CO) poisoning, and status epilepticus (Yang et al., 2016).e ce‐rebral ischemia and hypoxia in HIE perturbs energy metabo‐lism, leading to neurodegeneration and neurological de fi cits, resulting in a poor prognosis. It is a debilitating neurological disease in desperate need of e ff ective treatment.

Although asphyxia in newborns and cardio‐cerebrovascu‐lar events in adults both give rise to HIE, their pathogeneses di ff er substantially. In general, in neonates, the cessation of respiration initially causes hypoxemia, leading to a reduction in cardiac output, which fi nally results in cerebral ischemic and hypoxic injury (Liu et al., 2015). In comparison, adults primarily su ff er brain ischemia as a result of cardiac arrest or cerebrovascular disease, and cerebral hypoxia is secondary to the reduced regional cerebral blood fl ow (Biagas, 1999). Furthermore, the severity of brain injury caused by hypoxia and ischemia varies according to the maturity of the neuron. A previous study demonstrated that the immature brain has a stronger capacity to resist hypoxia and ischemia than the mature brain (Wang et al., 2009), although the mechanisms underlying this ability remain unknown. The mechanisms underlying the death of immature and mature neurons during hypoxia and ischemia are greatly di ff erent (Zhu et al., 2009). Immature neurons can initiate the intrinsic apoptotic machinery upon ischemia, while this ability weakens gradu‐ally as the brain matures (Hu et al. 2000a, b; Liu et al., 2004a, b; Blomgren et al., 2007). Studies are needed to compare the responses of the brain at di ff erent maturities to hypoxia and ischemia. Such studies should provide molecular targets for the treatment of hypoxic‐ischemic brain damage in adultsand neonates.

Parcellier et al. (2003) found that heat shock proteins (HSPs), which are ubiquitous and highly conserved proteins that are induced in response to a wide variety of physiolog‐ical and environmental insults, are induced in HIE. These proteins, which play essential roles in cellular housekeeping, help cells survive otherwise lethal conditions.

In this review, we describe the mechanisms of HIE and the various treatment approaches, with a focus on the molecu‐lar chaperones, which are promising therapeutic targets for brain injury in HIE.

Mechanisms of Hypoxic-Ischemic Brain Injury

The processes leading to neural injury after hypoxic‐isch‐emic brain injury include excitatory amino acid (EAA) release, cellular proteolysis, free radical generation, nitric ox‐ide (NO) synthesis, in fl ammation, and abnormal protein ag‐gregation (Li et al., 2015; Yao et al., 2016; Zhao et al., 2016). Hypoxic‐ischemic insult to the brain leads to neuronal de‐polarization and massive EAA (e.g., glutamate [Glu]) release, as well as a reduction in the activity of neurotransmitter reuptake pumps on presynaptic astrocytes (Zanelli et al., 2015).is leads to the accumulation of Glu in the synaptic cle, which in turn triggers the opening of N‐methyl‐D‐as‐partate (NMDA) receptor channels and calcium channels, leading to excessive calcium in fl ux into neurons.is calci‐um in fl ux activates nitric oxide synthetase (NOS), leading to abnormally high NO synthesis. NO reacts with oxygen free radicals generated by mitochondria upon reoxygenation fol‐lowing hypoxia, attacking enzymes associated with oxidative phosphorylation and electron transport (Blanco et al., 2017). Calcium also activates other enzymes, including calpains and esterases (Zhong et al., 2016). Together, these processes cause the necrosis and apoptosis of neuronsviathe activa‐tion of cell death cascades (Johnston et al., 2011).

EAA release

Glu homeostasis is dependent on Glu transporters, includ‐ ing EAA transporters (EAATs) and cystine/Glu antiporters. EAATs play a principal role in the transport and elimination of Glu, preventing the excessive accumulation of the neu‐rotransmitter in the synaptic cleft (Rothstein et al., 1996). However, during cerebral hypoxia and ischemia, EAATs may release Glu, resulting in its accumulation in the intercellular space (Chen et al., 2005).

Cellular proteolysis

During ischemia and hypoxia, the calcium overload impairs cellular homeostasis and leads to the activation of hydrolytic enzymes that degrade proteins (Willis et al., 2016). Many recent studies have focused on calpain (a calcium‐dependent cysteine protease).e activation of calpain by the calcium in fl ux plays an important role in EAA neurotoxicity (Siman et al., 1989). When neurons are stimulated by EAA, cal‐pain‐1 is activated and cytoskeletal proteins are hydrolyzed (Rosenkranz et al., 2012). Furthermore, calpain is involved in cell death (Blomgren et al., 2001) and participates in the endogenous apoptosis pathway (Choi, 1992). In global brain ischemia models, inhibition of calpains protects against hip‐pocampal dysfunction and neuronal cell death (Bevers et al., 2010).

ROS generation

ROS are extremely reactive molecules that include NO, superoxide, peroxynitrite and the hydroxyl radical. NOS is strongly activated by the influx of calcium, and NO reacts with superoxide produced by mitochondrial stress to gen‐erate the highly toxic peroxynitrite anion, which in turn causes the nitration of tyrosine residues, protein damage and organellar dysfunction through lipid peroxidation (Beckman et al., 1990). NO also inhibits the activity of cytochrome ox‐idase, thereby a ff ecting mitochondrial respiratory function.is leads to further increases in peroxide and peroxynitrite levels (Blomgren and Hagberg, 2006; Robertson et al., 2009).

NO synthesis

Animal experiments show that NOS is induced during isch‐emia and hypoxia. Inhibiting the activity of NOS reduces iron deposition and NO generation, thereby reducing the death of neurons (Lu et al., 2015). NO plays a dual role in hypoxic‐ischemic brain injury. In the pathological state, NO perturbs neurotransmitter release, impairs protein synthesis and induces membrane damage. However, NO also appears to play a neuroprotective role. Rapidly increasing endothelial nitric oxide synthase (eNOS) activity elevates NO produc‐tion and accelerates cerebral blood fl ow aer hypoxic‐isch‐emic brain injury. Indeed, Yamamoto et al. (1992) inhibited NOS activity using N‐nitro‐L‐arginine methyl ester (a com‐petitive inhibitor), resulting in an increase in infarct area in rats with hypoxic‐ischemic brain injury.

In fl ammation

In fl ammation is an important component of the excitotoxic cascade. Hypoxic‐ischemic brain injury activates in fl amma‐tory cells and increases ROS production and the expressionof in fl ammatory mediators, such as interleukin (IL)‐1β and IL‐18 (Bhalala et al., 2015). Oxidative stress is a common feature of all inflammatory cascades in hypoxic‐ischemic brain injury (Leuc et al., 2015). During in fl ammation, ac‐tivated astrocytes, microglia and endothelial cells play a neu‐roprotective role.e accumulation of immune cells and the release of ROS, chemokines and cytokines leads to blood‐brain barrier damage, cerebral edema, neuronal cell death, and hemorrhagic transformation (Dirnagl et al., 1999).

Protein misfolding and aggregation

Proper polypeptide folding is essential for normal protein conformation and function. When the newly synthesized polypeptide chain is misfolded, hydrophobic groups might be exposed on the surface, resulting in the aggregation of the protein (Gi ff ard et al., 2004). Protein aggregation is toxic to cells (Taylor et al., 2002). Molecular chaperones help prevent protein aggregation, and they also facilitate protein degrada‐tion through the ubiquitin‐proteasome system (Hershko and Ciechanover, 1998). In pathological conditions, abnormal proteins exhaust the cell’s capacity to keep them soluble and to degrade them, and may result in their aggregation (Bence et al., 2001). Aer hypoxia or other severe stress, unfolded or misfolded proteins aggregate in the endoplasmic reticulum, blocking protein synthesis. Normal protein conformation is important for cellular homeostasis. Protein aggregation in‐hibits the functioning of the proteasome, further disrupting cell function. Protein aggregation is a feature of excitotoxic neuronal injury.

Several studies have shown that protein misfolding, ag‐gregation and destruction of organelles are the main neu‐ropathological changes aer hypoxic‐ischemic brain injury (Salminen et al., 2016; Zhao et al., 2016). Recent transmis‐sion electron microscopy (TEM) studies have revealed the presence of massive electron dense deposits in neurons undergoing delayed neuronal death aer cerebral ischemia. These deposits represent aggregates of unfolded and mis‐folded proteins (Giffard et al., 2004; Hu et al., 2001, 2004; Liu et al., 2004a, b, 2005a, b, 2010; Zhang et al., 2006; Ge et al., 2007). Protein misfolding and aggregation in neurons are important pathogenetic features of neurodegenerative dis‐eases, suggesting that the aggregation of misfolded proteins is the pathological basis of neuronal degeneration (Hardesty et al., 1999; Frydman, 2001).

Why does protein misfolding and aggregation occur af‐ter hypoxic‐ischemic brain injury? Only if the polypeptide chain folds correctly into its final 3‐dimensional structure can it perform its normal biological functions.e folding of nascent polypeptide chains occurs as they exit the ribosome, in a process called co‐translational folding (Siesjo and Siesjo, 1996; Hardesty et al., 1999; Frydman, 2001; Hartl and Hay‐er‐Hartl, 2002). The hydrophobic groups of nascent poly‐peptide chains are exposed, which can, under the in fl uence of hydrophobic forces, easily lead to misfolding.e normal co‐translational folding process requires the following (Ito and Nagata, 2016) (Figure 1): (1) molecular chaperones, which assist the normal folding process; (2) auxiliary pro‐ teins; and (3) energy. Under normal circumstances, when newly synthesized peptide chains misfold and aggregate, they are immediately degraded by the ubiquitin‐proteasome system or by the autophagy pathway. Any abnormal protein folding or degradation may lead to protein aggregation and delayed neuronal death.

Molecular chaperones, which regulate the folding pro‐cess, can identify and shield hydrophobic groups of un‐folded proteins and prevent their aggregation under nor‐mal conditions. Molecular chaperones and their auxiliary proteins play important roles in the regulation of protein folding, transportation, assembly and degradation (Weiss et al., 2016). Heat shock protein 70 (HSP70) and its auxiliary protein, HSP40, play important roles in protein synthesis. The HSP70 family includes HSC70 and inducible HSP70 in mammalian cells. Under normal conditions, HSC70 is a major auxiliary protein in the co‐translational folding pro‐cess. Together with HSP40, HSC70 identifies the nascent peptide to assist its correct folding and transportation. When the synthesis of a domain of the polypeptide chain is completed, HSC70 dissociates and enters the next cycle, continuing to assist protein synthesis.is process is ade‐nosine triphosphate (ATP)‐dependent. If the intracellular ATP level falls below 80% of normal, such as aer ischemia and hypoxia, intracellular ATP‐dependent regulatory ac‐tivities terminate or slow down, including co‐translational folding, ubiquitin‐proteasome‐mediated degradation and autophagy (Siesjo and Siesjo, 1996; Huang et al., 2016). eventually leading to the aggregation of misfolded nascent peptide chains. Several recent studies have shown that the accumulation of nascent peptides after hypoxic‐ischemic brain injury results in abnormal protein aggregation, irre‐versible protein synthesis dysfunction and irreversible or‐ganelle damage (Figure 2).

Apoptosis

Mitochondrial (intrinsic) apoptotic pathway

Under mitochondrial stress, the pro‐apoptotic protein Bax increases the permeability of the mitochondrial outer mem‐brane, thereby releasing cytochrome c, Smac/Diablo, HtrA2/ Omi and apoptosis‐inducing factor from the intermembrane space into the cytoplasm, leading to the formation of apop‐totic bodies and DNA fragmentation (Parcellier et al., 2003; Johnston et al., 2011).

In hypoxic‐ischemic brain injury, nuclear factor kappa B (NF‐κB) mediates microglial activation and the release of inflammatory factors (such as IL‐1β and IL‐6, IL‐8, tumor necrosis factor (TNF)‐α, TNF‐β, macrophage in fl ammatory protein (MIP)‐1 and MIP‐2) (Marcinkiewicz et al., 1995; Szaflarski et al., 1995; Hagberg et al., 1996; O’Neill and Kaltschmidt, 1997; Vodovotz et al., 1999; Bando et al., 2003; Johnston et al., 2011). Activation of the cell surface Fas re‐ceptor by its cognate ligands results in the sequential activa‐tion of caspase‐8 and caspase‐3, DNase activation, and DNA fragmentation (Johnston et al., 2011).

Endoplasmic reticulum stress-mediated apoptotic pathway

Hypoxic‐ischemic brain injury leads to protein aggregation. High abnormal protein levels produce endoplasmic reticu‐lum stress, which results in cell membrane dysfunction, the accumulation of intracellular calcium, and the induction of CHOP.is leads to a reduction in the expression of the anti‐apoptotic protein bcl‐2, which increases the permea‐bility of the mitochondrial membrane, initiating the mito‐chondrial apoptotic pathway (Johnston et al., 2011). At the same time, dysfunction of the endoplasmic reticulum causes the activation of caspase‐12, which sequentially activates caspase‐9 and caspase‐3, eventually leading to nuclear DNA cleavage and the induction of apoptosis (Morishima et al., 2002).

For nearly half a century, much attention has been given to treatment of hypoxic‐ischemic brain injury associated with HIE. With advances in our understanding of pathogenesis, an increasing number of treatments have been developed.

Hypothermia

The temperature of the brain is closely related to cerebral metabolic rate and cerebral blood fl ow. Each 1° Celsius re‐duction in body temperature decreases brain metabolic rate by 6—7% (Nel et al., 2009). In addition to reducing oxygen and energy consumption, protecting the blood‐brain barrier and alleviating cerebral edema, hypothermia decreases EAA release (Kim et al., 2011), glutamate antiporter expression, the generation of NO and free radicals, phosphorylation of the NMDA receptor, in fl ammation and apoptosis (Chao et al., 2010).

Figure 2e relationship between protein synthesis, AP and molecular chaperones.

Hypothermia has been widely used to treat neonatal HIE. It also exerts neuroprotective effects on acute ischemic stroke, although there is a lack of adequate clinical research evidence (Han et al., 2015). A meta‐analysis study compar‐ing the therapeutic effects of hypothermia with systemic support treatment shows that hypothermia can increase the survival rate of children with moderate to severe HIE and promote the development of the nervous system (Tagin et al., 2012). Hypothermia therapy has also been shown to be neuroprotective after recanalization for ischemia‐reperfu‐sion injury (Han et al., 2015).

Medical treatment

Developing treatments for hypoxic‐ischemic brain injury requires the continued exploration of the underlying patho‐genesis.e following drug categories have been developed: anti‐excitotoxic (sedative hypnotic agent, inert gas xenon, magnesium sulfate), anti‐inflammatory and anti‐oxidant (cromolyn sodium, N‐acetylcysteine, minocycline, mela‐tonin), erythropoietin (EPO), and growth factors (nerve growth factor, insulin‐like growth factor‐1, brain‐derived neurotrophic factor) (Johnston et al., 2011). Here, we discuss several promising drug treatments for hypoxic‐ischemic brain injury.

Inert xenon gas

The inert gas xenon is an NMDA receptor antagonist. It plays a role in neuroprotection by interfering with the exci‐totoxic cascade, and it also acts on ion channels, reducing the release of neurotransmitters (Johnston et al., 2011). Xenon can easily traverse the blood‐brain barrier and exert a rapid effect (Baumert et al., 2016). Animal experiments have shown that xenon inhalation combined with hypother‐mia therapy for hypoxic‐ischemic brain injury doubled the therapeutic e ffi cacy relative to monotherapy (Johnston et al., 2011). Dingley et al. (2014) published the results of a clinical trial on the treatment of neonatal HIE using xenon com‐bined with hypothermia, demonstrating its clinical e fficacy (Johnston et al., 2011).

Melatonin

Melatonin can be used as a free radical scavenger. Addition‐ally, it can reduce inflammatory factor release and activate antioxidant enzymes, such as glutathione peroxidase, glu‐tathione reductase and superoxide dismutase (Lee et al., 2007). Aly et al. (2015) used hypothermia in combination with melatonin to treat neonatal asphyxia in the perinatal stage and showed that melatonin can ameliorate brain injury, compared with hypothermia alone.

Erythropoietin (EPO)

EPO can play a neuroprotective role through a variety of mechanisms. Sun et al. (2005) demonstrated that EPO binds with the EPO receptor of astrocytes and microglial cells, and has an anti‐in fl ammatory e ff ect. Sakanaka et al. (1998) found that EPO inhibits NO‐mediated apoptosis and reduces ex‐citotoxic damage. Additionally, EPO promotes repair after injury by regulating neuronal genesis and di ff erentiation (Lee et al., 2007). A pilot clinical study in 2015 demonstrated that EPO combined with hypothermia therapy for the treatment of hypoxic‐ischemic brain injury in neonates was safe and e ff ective (Lee et al., 2007).

Hypoxic-ischemic preconditioning

Hypoxic‐ischemic preconditioning refers to a protective therapeutic strategy, in which the subject is exposed to a short‐term hypoxic‐ischemic stress prior to an upcom‐ing long period of hypoxic‐ischemic insult. In a study by Gustavsson et al. (2005), rats were exposed to 8% oxygen (hypoxia) for 3 hours before hypoxic‐ischemic brain injury. Brain injury was substantially alleviated and long‐term cog‐nitive function was also greatly improved in rats subjected to hypoxic‐ischemic preconditioning, compared with control rats (Gustavsson et al., 2005). The neuroprotective mecha‐nisms of hypoxic‐ischemic preconditioning remain unclear. However, NMDA receptors, glutamate receptors, ATP‐de‐pendent potassium channels and G‐protein‐coupled adenos‐ine receptors appear to be involved in the neuroprotection conferred by hypoxic‐ischemic preconditioning (Lee et al., 2007). Hypoxic‐ischemic preconditioning also increases en‐dogenous anti‐oxidant and anti‐apoptotic capacity, increases brain glycogen and slows down energy depletion following hypoxic‐ischemic insults (Brucklacher et al., 2002).

Molecular Chaperones and Hypoxic-Ischemic Brain Injury

Molecular chaperones

HSPs

HSPs were first discovered in Drosophila exposed to high temperature, accounting for their name. HSPs are induced by the heat shock response (HSR).e HSR is an adaptive re‐sponse, characterized by changes in gene expression (Bruck‐lacher et al., 2002), following exposure to heat stress or other stressors, such as ischemia or hypoxia. HSPs prevent protein misfolding and aggregation (Brucklacher et al., 2002). More recent studies have shown that other stress states, such as nutritional deficiencies, hypoxia and chemical toxicity, in‐duce HSP expression as well. For this reason, HSP is also considered a stress protein. Under stress conditions, such as oxidative stress and in fl ammation, proteins are prone to misfold and aggregate.e aggregation of proteins can lead to many diseases, such as type 2 diabetes mellitus, cardiovas‐cular disease and neurodegenerative diseases (Brucklacher et al., 2002). HSPs assist in the proper folding of newly syn‐thesized proteins, allowing for a proper fi nal 3‐dimensional structure. Furthermore, HSPs can help refold denatured pro‐teins, unfold misfolded and unstable proteins, and assist in their degradation. Molecular chaperones are oen classi fi ed according to their molecular weight and homology. Molec‐ular chaperones are classified into several groups: Hsp110, Hsp90, Hsp70, Hsp60, the small HSPs and ubiquitin.

HSPs help maintain protein homeostasis after ischemia and hypoxia

The HSP90 family members include HSP90α, HSP90β and glucose regulated protein 9 (GRP9). Numerous HSP90 members are expressed under physiological conditions, among which 1—2% are in the cytoplasm. Under stress con‐ditions, HSP90 expression is greatly increased (Parcellier et al., 2003), helping proteins such as steroid receptors, tyrosine kinases and serine/threonine kinases acquire their proper fi nal conformation.e HSP70 family includes stress induc‐ible HSP70, constitutive HSC70, mitochondrial HSP75 and GRP78. Under physiological conditions, HSP70 proteins assist in the proper folding of newly synthesized polypep‐tides, mediate the assembly of macromolecular complexes and the transmembrane transport of proteins (Brucklacher et al., 2002). Under stress conditions, HSP70 assists in the proper folding of unfolded and misfolded proteins and helps in their degradation (Brucklacher et al., 2002). Gupta et al. (2010) showed that HSP70 activates IRE1α in the endoplas‐mic reticulum, enhances expression of transcription factor XBP1 and regulates the expression of unfolded protein re‐sponse‐related genes, helping cells adapt to ER stress. HSP40is an important accessory factor for HSP70, which promotes ATP hydrolysis (Brucklacher et al., 2002).

Nearly 60% of HSP60 proteins are in the mitochondrial ma‐trix, and 15—20% are found in the cytoplasm. Together with HSP10, HSP60 promotes the proper folding of proteins in the mitochondria, and degrades misfolded and denatured pro‐teins in a process that consumes ATP (Parcellier et al., 2003).

Small HSP family proteins are ATP‐independent molecu‐lar chaperones.e human genome encodes 10 small HSPs that range in molecular weight from 12 to 42 kDa. Small HSPs can bind to unfolded or misfolded proteins and pre‐vent their aggregation (Brucklacher et al., 2002).

Anti-apoptotic e ff ects of HSPs after hypoxic-ischemic brain injury

After hypoxic‐ischemic brain injury, overexpression of HSP70 prevents the release of cytochrome c from mitochon‐dria and the activation of casepase‐9 by binding to Apaf‐1, thereby blocking the caspase‐dependent apoptotic pathway (Brucklacher et al., 2002). Overexpression of HSP70 can also block the caspase‐independent apoptotic pathway by binding to apoptosis‐inducing factor (AIF) (Brucklacher et al., 2002). Furthermore, overexpression of HSP70 upregulates the expression of cellular Fas‐associated death domain‐like in‐terleukin‐1β converting enzyme inhibitory protein (cFLIP), inhibits caspase‐8, and blocks the Fas receptor‐mediated ex‐trinsic apoptotic pathway (Brucklacher et al., 2002). Further‐more, overexpression of HSP70 inhibits NO synthetase and suppresses the production of oxygen radicals, such as NO, OH−, ONOO−and O2−(Brucklacher et al., 2002). HSP70 can inhibit JNK, activate IRE1α, promote the production of tran‐scription factor XPB1 and inhibit the endoplasmic reticulum stress‐mediated apoptotic pathway (Brucklacher et al., 2002).

Glucose regulated proteins (GRPs), such as GRP78, are a family of highly conserved proteins that help cells cope with endoplasmic reticulum stress. Under hypoxic‐ischemic conditions, cellular energy depletion causes the accumulation of unfolded and misfolded proteins in the endoplasmic retic‐ulum, triggering the unfolded protein response and increasing GRP78 expression (Brucklacher et al., 2002). GRP78 prevents aggregation by binding to the denatured proteins, and it also binds transiently to newly synthesized polypeptides by nonco‐valent bonds to facilitate the proper folding of newly synthe‐sized proteins. It is also involved in the folding and extension of proteins and the assembly of protein complexes. Addition‐ally, GRP78 helps in the elimination of abnormal proteins by transporting them to the protein degradation system in the en‐doplasmic reticulum. Furthermore, GRP78 inhibits caspase‐9 and caspase‐12 to block the endoplasmic reticulum‐mediated apoptotic pathway (Brucklacher et al., 2002).

The anti‐apoptotic function of HSP90 is controversial.e function of HSP90 varies based on the apoptotic induc‐ers. In the monocytic cell line U937, overexpressed HSP90 increases apoptosis in cells exposed to cycloheximide and TNF‐α (Brucklacher et al., 2002). However, in cells exposed to staurosporine, overexpressed HSP90 inhibits the for‐mation of apoptotic bodies by binding to Apaf‐1, thereby inhibiting apoptosis (Brucklacher et al., 2002). In addition, overexpressed HSP90 can bind to phosphorylated serine/ threonine kinase Akt/PKB to protect against inactivation by dephosphorylation. Phosphorylated Akt/PKB promotes the phosphorylation of the pro‐apoptotic proteins Bax and caspase‐9, and blocks the mitochondrial apoptotic pathway (Brucklacher et al., 2002).

GRP94 is a member of the HSP90 family. It inhibits the ac‐tivation of caspase‐3 and calpain, maintains intracellular cal‐cium homeostasis, and blocks the caspase‐3‐dependent apop‐totic pathway to protect neurons (Brucklacher et al., 2002).

HSP27 is a small HSP. Overexpressed HSP27 inhibits the production of oxygen radicals (Brucklacher et al., 2002), stabilizes actin microfilaments in the cells (Brucklacher et al., 2002), inhibits the release of cytochrome c, blocks the formation of apoptotic bodies, and suppresses the activation of caspases, thereby inhibiting the mitochondrial apoptotic pathway (Brucklacher et al., 2002). In addition, phosphorylat‐ed HSP27 can directly interact with Fas death domain‐asso‐ciated protein to inhibit the Fas‐mediated apoptotic pathway (Brucklacher et al., 2002). Hsp27 exhibits neuroprotective e ff ects in the nervous system (Kato et al., 1995; Badin et al., 2006; An et al., 2008), for example, in rat models of cerebral ischemic preconditioning (Currie et al., 2000; Dhodda et al., 2004).e inhibition of Hsp27 degradation mediated by autophagy‐lysosome pathway was reported to be necessary for neuronal survival aer transient global cerebral ischemia (Zhan et al., 2016). Zhan et al. (2016) also showed that post‐translational modi fi cation of Hsp27 was signi fi cant in medi‐ating neuroprotection aer hypoxic post‐conditioning.ese findings offer a solid foundation for targeting an inherent regulatory mechanism of Hsp27 for therapeutic development and broke a new path to exploring the neuroprotective role of hypoxic post‐conditioning against ischemic brain injury.

Conclusion and Future Perspective

HIE occurs when the brain is subjected to hypoxic/ischemic conditions. HIE results in neuronal death and neurological de fi cits and has a poor prognosis. Many treatment methods for hypoxic‐ischemic brain injury have been developed over the past half century. Among these, the therapeutic use of HSPs is the most promising. HSPs play a key role in neuro‐protection by maintaining protein homeostasis and by regu‐lating apoptosis. Neurons can be protected by up‐regulating the expression of HSPs or reducing the degradation of HSPs using a protein synthesis inhibitor such as cycloheximide. In the near future, HSPs may be used clinically to prevent or treat HIE in the early stage to reduce brain injury.

Author contributions:HJ performed the analysis with constructive discussion; WNJ revised the paper; XS conceived and designed the work;CH and GZ wrote the paper. All authors approved the fi nal version of this paper.

Con fl icts of interest:None declared.

Plagiarism check:This paper was screened twice using CrossCheck to verify originality before publication.

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Copyedited by Patel B, Raye W, Li CH, Song LP, Zhao M

Gang Zhao, M.D., Ph.D. or Xin Sun, M.D., Ph.D., 1035722011@qq.com or sjnksunxin@163.com.

10.4103/1673-5374.199008

＊< class="emphasis_italic">Correspondence to: Gang Zhao, M.D., Ph.D. or Xin Sun, M.D., Ph.D., 1035722011@qq.com or sjnksunxin@163.com.

orcid: 0000-0001-5472-7828 (Gang Zhao)