Does glutathione have an effect on brain damage in preterm infants?

 With the continuous development of neonatal intensive care technology, the survival rate of preterm infants, especially very low birth weight infants, has greatly increased. The incidence of brain injury in preterm infants has increased accordingly, and as a major cause of long-term neurological development of preterm infants, its main causes include hypoxia and ischemia, infection, hypoglycemia, mechanical ventilation, and hyperbilirubinemia, etc.1 The mechanisms of brain injury in preterm infants include excitotoxic injury and free radical injury, of which free radicals are the most important. 

The mechanisms of brain damage in preterm infants include excitotoxic damage and free radical damage, in which free radicals include reactive oxygen species (superoxide anion, hydroxyl radical, hydrogen peroxide, etc.) and reactive nitrogen species (nitric oxide and its derivatives). When the production of free radicals increases, the antioxidant reserve will be depleted rapidly, and the cytosolic proteins, lipids and DNA will be oxidized, resulting in a series of physiological and pathological reactions, i.e. oxidative stress. Preterm infants are particularly sensitive to oxidative stress, which often leads to severe brain damage. In order to resist oxidative stress damage, the body has formed a series of defense barriers in the course of continuous evolution. Among them, glutathione scavenges oxygen free radicals under normal conditions, and can play an antioxidant role by reacting directly with toxic metabolites or by reducing hydrogen peroxide to water and oxygen through the action of related enzymes[2] . In addition, glutathione acts as a metal-binding protein to maintain the homeostasis of the intracellular environment and normal cellular metabolism[3] . The effects of glutathione synthesis and metabolism on brain injury in preterm infants are summarized.

 

1 Main enzymes involved in glutathione synthesis and metabolism

Several key enzymes are involved in the synthesis and metabolism of glutathione. The glutamate/cystine transporter (xCT) in the cell membrane transports cystine into the cell for the synthesis of glutathione and cysteine-containing proteins. Inside the cell, glutamate cysteine ligase (GCL) takes up cystine to synthesize glutathione, which maintains the cell's antioxidant capacity. The enzyme glutathione peroxidase oxidizes glutathione to oxidized glutathione, which is reduced to water by hydrogen peroxidase. The oxidized glutathione is reduced to glutathione by catalase, and the oxidized glutathione is reduced back to glutathione by reduced coenzyme/II quinone oxidoreductase 1-dependent oxidoreductase. This synthesis and metabolism of glutathione is one of the important antioxidant barrier functions of the body.

 

During glutathione synthesis, xCT plays an important role in transferring extracellular cystine into the cell for glutathione synthesis and intracellular glutamate out of the cell for neurotransmitter signaling. xCT also plays an important role in the development of neuronal cells. In addition, xCT plays an important role in neuronal cell development, and astrocytes partially inactivated by xCT lose their ability to proliferate in vitro. Immature neurons and oligodendrocyte precursor cells cultured in vitro expressed significantly lower levels of xCT than mature cells, suggesting that immature neuronal cells are more susceptible to damage than mature neuronal cells [4]. xCT expression on neuronal cells varies widely.

 

Studies have shown that the catalytic subunit of xCT in mouse neuronal cells is expressed in neurons, oligodendrocytes, and microglia, but not in astrocytes, and that inhibition of xCT expression leads to glutathione depletion, neuronal degeneration and demyelination, and disruption of cellular homeostasis [5]. Another study found that mouse xCT is mainly expressed on astrocytes but not on neurons, and up-regulation of xCT expression on the cell membrane can promote the uptake of cystine by astrocytes, which can increase the production of glutathione, and thus make peripheral neuronal cells resistant to oxidative stress[6] . Arsenic-treated microglia competitively inhibit xCT, causing an extracellular cystine/glutamate imbalance, i.e., a decrease in cystine and an increase in glutamate in the culture solution, leading to death of peripheral immature neuronal cells, and supplementation with N-acetylcysteine improves neuronal survival [7]. The above evidence suggests that xCT plays an important role in cell proliferation, maintenance of homeostasis and promotion of neuronal cell protection.

 

GCL is the rate-limiting enzyme for glutathione synthesis and consists of a catalytic subunit and a regulatory subunit, through which glutathione is synthesized from cystine uptake to maintain cellular antioxidant capacity and achieve neuroprotective effects [8]. The amount of glutathione synthesized by this enzyme is controlled by a number of factors, including the level of GCL, the ratio of the two subunits, L-cystine, the substrate for synthesizing glutathione, and the inhibition of GCL by the negative feedback of intracellular glutathione levels[9] . In animal studies, inhibition of GCL gene expression in rats resulted in impaired mitochondrial glutathione homeostasis, mitochondrial dysfunction, and a reduction in neuronal cells[10] . Lack of GCL in neuronal cells leads to a significant reduction in glutathione production, which in turn increases the sensitivity of the cells to hypoxic injury [11]. Pehar et al. [12] found that knockdown of the GCL regulatory subunit in mouse astrocytes resulted in a reduction of 80% in the level of total glutathione, which led to a reduction in the protective effect on neurons. Some studies have also shown that when the activity of GCL continues to decrease, glutathione levels also continue to decrease, but the level of oxidative DNA damage is higher; overexpression of GCL can significantly increase glutathione levels, inhibit oxidative DNA damage, and thus inhibit the migration and growth of tumor cells [13]. polymorphisms exist in the gene for GCL and affect its transcription and expression levels, especially when cells are under oxidative stress, the polymorphisms of this gene can be detected. When cells are under oxidative stress, the polymorphisms in this gene alter the homeostasis of intracellular glutathione, which ultimately leads to the susceptibility of cells to oxidative stress damage. Therefore, glutathione inhibitors are commonly used in laboratories to inhibit the action of GCL and thereby reduce glutathione synthesis, and GCL knockouts are often used in animal models of glutathione deficiency. In addition, GCL also plays a key role in the process of cell development and maturation. Knockout mouse embryos fail to mature and undergo massive apoptosis, but the addition of glutathione or N-acetylcysteine to the culture medium promotes the development of the blastomeres.

 

2 Nuclear Transcription Factor Red Lineage 2 Related Factors Regulate Antioxidant Stress

The nuclear factor erythroid 2-related factor 2 (Nrf2) is a basic leucine zinc-based transcription factor that acts as a receptor for exogenous toxic substances and oxidative stress, and as a central regulator of cellular antioxidant responses. It mediates the initiation of downstream encoded antioxidant proteins and detoxification enzymes to counteract the damage of oxidative stress, which is an important cellular defense mechanism.

 

Under normal physiological conditions, Nrf2 binds to Kelch-like epichlorohydrin-associated protein 1, which inhibits it, in the cytoplasm of the cell, so that Nrf2 becomes an adaptive substrate for E3 ubiquitin ligase, which promotes the ubiquitination of Nrf2 and its degradation by the 26S proteasome. When the number of free radicals and toxic substances increases, electrophilic substances modify the cysteine residues of Kelch-like epichlorohydrin-associated protein 1, causing a conformational change of Kelch-like epichlorohydrin-associated protein 1, which leads to the dissociation of Nrf2 from Kelch-like epichlorohydrin-associated protein 1, and then Nrf2 increases in stability and transfers to the nucleus to bind to the antioxidant response element in the antioxidant specific promoter region, and the antioxidant response element is then detached from the nucleus. At this time, Nrf2 stability increases and translocates to the nucleus where it binds to antioxidant response elements in the promoter region of antioxidant-specific genes and initiates the expression of detoxification enzymes and antioxidant genes, which is the most common mode of Nrf2 activation, and this activation is known as the Nrf2-antioxidant response element pathway; in addition, Nrf2 can be activated indirectly through phosphorylation by the pathways of the schizogen-activated protein kinase, protein kinase C, and phosphatidylinositol-3-kinase, and so on.

 

Nrf2 regulates the expression of many cytoprotective proteins, such as xCT, GCL, heme oxidase, glutathione peroxidase, and reductase-independent CoA/II quinone oxidoreductase 1, which are involved in the regulation of glutathione synthesis and metabolism, and can effectively block neurotoxicity due to glutathione deficiency and impaired utilization of glutathione, and thus protect the body from the effects of reactive substances and toxic substances [14 ]. This can protect the body from active substances and some toxic substances[14] . In an animal experiment simulating cerebral ischemia-hypoxia/reoxygenation injury, inhibition of Nrf2 degradation and enhancement of its binding activity to antioxidant components were shown to be beneficial to the recovery of cerebral neuronal cells from ischemia/reperfusion injury[15] . During oxidative stress injury, tertiary butylhydroquinone, as an activator of Nrf2, exerts its antioxidant function by increasing glutathione levels, increasing Nrf2 stability, and inhibiting the ubiquitination of Kelch-like epichlorohydrin-associated protein 1 to reduce neuronal apoptosis [16-17]. In addition, it has been found that the neuroprotective effect of calcium up-regulation of glutathione levels is mediated by Nrf-2, and that knockdown of the Nrf2 gene eliminates this protective effect [18]. Clinically, the Nrf2 activating drug fumaric acid dicarboxylate also has an indirect effect on delaying neuronal death by increasing Nrf2 activity[19] . Nrf2 and its activators also have neuroprotective effects by regulating the gene expression of xCT [20]. However, whether the lack of xCT attenuates the neuroprotective effect induced by Nrf2 needs to be further investigated.

 

It has been found that microRNAs (miRNAs) are closely related to Nrf2 and antioxidant proteases [21-22]. miRNAs are endogenous, non-coded, single-stranded, small molecular RNAs of 21-25 nt in plants and animals. In animal cells, most miRNAs prevent post-translational translation by binding to the 3′ untranslated region of the messenger RNA of target genes, thus regulating gene expression. In a miR-144 overexpression model, miR-144 not only increased the oxygen free radicals in neuroblastoma SH-SY5Y cells, decreased cellular activity, glutathione and antioxidant enzymes, but also decreased the expression of GCL catalytic subunit, GCL regulatory subunit, and Nrf2 [21]. Some scholars used real-time quantitative polymerase chain reaction analysis and found that miR-27a, miR-28-3p, and miR-34a expression levels in left ventricular cells were higher than those in other organs after myocardial infarction, and that these miRNAs increased oxidative stress by inhibiting Nrf2 activity, which led to heart failure [22]. The above studies suggest that miRNAs can reduce the antioxidant capacity of cells by negatively regulating Nrf2. Some researchers found that miR140-5p directly acts on the 3′ untranslated region of Nrf2 and positively regulates the expression of Nrf2, and the levels of reductase/II quinone oxidoreductase 1 and heme oxidase were also significantly increased, resulting in a significant enhancement of cellular antioxidant capacity through the establishment of the cisplatin-induced acute renal damage phenotype in mice [23]. The in-depth study of miRNA regulation of oxidative stress is likely to provide a new therapeutic approach for the treatment of infections, tumors and autoimmune diseases in the future.

 

3 Role of astrocytes in the antioxidant barrier

Astrocytes play a very important role in combating oxidative stress, and their impaired function is an important cause of primary and secondary damage to neurons and other nerve cells [24]. Astrocytes are the most numerous cells in the human brain, and different degrees of neurological injury activate astrocytes, altering their morphology and expression of functional proteins, and affecting peripheral neuronal cells to form glial scars. In the early stages of neurologic injury, activated astrocytes promote neuronal survival in a variety of ways, but as the injury progresses, they inhibit neuronal regeneration.

 

An important protective mechanism of astrocytes in nerve injury is that they synthesize glutathione to scavenge toxic products (oxygen radicals, iron, oxidized lipids, etc.) produced by the injury, thereby inhibiting oxidative stress damage[2] . This detoxification process requires sufficient levels of glutathione in astrocytes, and under certain conditions, different detoxification processes may interfere or even compete with each other for glutathione; impaired synthesis, recycling, and export of glutathione in astrocytes may affect glutathione-dependent detoxification, resulting in damage to astrocytes and reduced antioxidant protection of other brain cells [25]. It has been shown that when neurons were co-cultured with astrocytes, glutathione increased 1.5-fold and 5-fold at 12 and 24 h, respectively, which prevented the growth of glutathione in neurons and astrocytes. It was shown that glutathione increased 1.5-fold and 5-fold at 12 and 24 h, respectively, when neurons were co-cultured with astrocytes, and it could block the neuronal death and damage induced by rotenone and paraquat in the fetal rat cerebral cortex [26].

 

In addition, co-cultured astrocytes can increase glutathione levels through the orexin-A receptor 1/protein kinase Cα/extracellular signal-regulated kinase 1/2/glutamate transporter pathway, which enhances resistance to hypoxic hypoglycemic injury [27]. It has also been shown that adenosine phosphate-activated protein kinase selectively regulates the expression of the GCL regulatory subunit in astrocytes and promotes glutathione synthesis, thereby protecting neurons[8] . The cerebroprotective effect of astrocytes on neonates is also manifested in their uptake of glutamate, which reduces the toxic effect of glutamate on neonatal brain excitability; under the action of pyruvate carboxylase, astrocytes are able to synthesize neurotransmitters and promote normal neuronal development [28]. It can be seen that astrocytes are the main cells synthesizing glutathione in the brain, and they play an important protective role for the surrounding brain cells. However, there are relatively few studies on the regulation of glutathione synthesis and metabolism by immature astrocytes and their response to injury and neuroprotective mechanisms, which need to be further explored.

 

4 Effect of glutathione levels on the brain of preterm infants

There is no clear conclusion as to whether glutathione levels in the brains of preterm infants differ from those of term infants or adults. An autopsy of a large sample showed that glutathione levels in the brains of newborns were similar to those of older adults, but were higher in adults, presumably because adults are subjected to more oxidative stress, which maintains higher levels of glutathione.29 A study of maternal blood at different gestational weeks and umbilical cord blood at birth monitored the levels of oxidative products, glutathione and other glutathione in the brains of preterm infants. In a study that monitored oxidative products and glutathione peroxidase in maternal blood at different gestational weeks and in umbilical cord blood at birth, oxidative enzymes and antioxidant enzymes were negatively correlated, and it was hypothesized that the antioxidant system, in which glutathione is involved, ensures the maintenance of pregnancy[30] .

 

In premature and damaged neonatal rats, glutathione levels are lower than in normal full-term rats and apoptosis is more severe [31]. This may be due to the fact that glutathione metabolism is influenced by a number of metabolic enzymes, and low levels of these metabolic enzymes result in immature glutathione synthesis and a limited ability to maintain a reduced state in response to oxidative stress. Therefore, when the cerebral cortex is subjected to hypoxic injury, the glutathione reserve is significantly reduced, and the levels of oxidized glutathione and lipid peroxides in the brain tissue are elevated. The administration of N-acetylcysteine (a precursor of glutathione synthesis) to neonatal pigs can significantly reduce the levels of inflammatory mediators, interleukin-1β and nuclear factor κB, and restore the tissue glutathione reserve, which can play a neuroprotective role in the cerebral cortex of neonatal pigs[32 ]. The above studies have shown that the levels of interleukin 1β and nuclear factor κB in the cerebral cortex of adult pigs are higher than those of adult pigs. The above studies indicate that the brain of preterm piglets is less capable of synthesizing, metabolizing and utilizing glutathione than that of mature individuals, and that the role of glutathione in the fight against oxidative stress can be improved by increasing the activity of enzymes related to the synthesis and metabolism of glutathione and by supplying N-acetylcysteine.

 

5 Summary

Currently, many scholars at home and abroad have devoted themselves to studying the developmental mechanisms and preventive and therapeutic measures of brain injury in preterm infants, with a view to improving the long-term prognosis of preterm infants with brain injury. However, more and more evidence suggests that the immaturity of the antioxidant barrier that synthesizes and regulates glutathione in the brain of preterm infants may be an important mechanism that predisposes preterm infants to brain injury[7 , 33-34] . The Nrf-2-mediated antioxidant barrier involving glutathione plays an important role in protecting against various external and internal stresses; astrocytes, as the most numerous cells in the brain, also play a key role in brain cell protection. It is believed that with the in-depth study on the synthesis and metabolism of glutathione, it will have a profound impact on the prevention and treatment of brain injury in preterm infants.

 

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