The role and possible mechanism of uric acid in promoting nerve injury repair through anti-oxidative stress after traumatic brain injury

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(1.Department of Emergency,the Affiliated Hospital of Zunyi Medical University,Zunyi Guizhou 563099,China; 2.Department of Neurosurgery,the Southwest Hospital of Army Medical University,Shapinba Chongqing 400038,China)

[Abstract] Traumatic brain injury (TBI) has been becoming one of the leading public health problems as a result of its high mortality and disability rate.Targeting the pathophysiological mechanism of TBI is a pivotal link to develop therapeutic methods to promote neurological rehabilitation of TBI patients.The category of pathologic injury post-TBI could be divided into primary damage and secondary injury.The effective treatment for controlling primary damage mainly relies on surgery.While,various pathologic mechanisms cause secondary injury that results in loss of neural cells.Oxidative stress is one of the common pathologic phenomena during the progression of secondary injury.Therefore,exploring approaches aiming to mitigate oxidative stress might be an effective intervention relieving secondary injury to promote neuroregeneration after TBI.Uric acid (UA),one of the in vivo antioxidants,not only exerts neuroprotection through alleviating oxidative stress,but also might possess the ability of promoting the proliferation of neural stem cell (NSC) that participates in neural repair after TBI,thereafter improving the neurological recovery.Here,we preliminarily reviewed the effect of UA on secondary injury of post-TBI and the possible underlying mechanisms.The aim of this review was to broaden the use of UA in the treatment of TBI.

[Key words] traumatic brain injury; uric acid; oxidative stress; nuclear factor erythroid 2-related factor 2; neural stem cell

Traumatic brain injury (TBI) is one of the worldwide public health problems with more than 10 million cases every year.Based on the Glasgow Coma Score (GCS),TBI can be classified as mild,moderate or severe type with an associated permanent disability rate of 10%,60% and 100%.The overall mortality rate of TBI is 20% -30%[1].By 2030,the incidence of TBI-related disability would be two to three times higher than Alzheimer’s Diseases (AD) and cerebrovascular diseases[2].Recent years,due to both the increased awareness of TBI and improved treatment guidelines,TBI-related mortality rates has decreased,while disability rates have increased and the number of people with disabilities has increased significantly.The increasing number of disabled people caused by TBI has brought serious vitiation to their families and the society.In order to improve the current situation of high disability rate,the research on promoting neurorehabilitation has becoming the hot issue for researchers related to TBI.

The severity of TBI determines the therapeutic strategies.Mild patients only need to inpatient for observation,while severe patients need intensive care or emergency craniotomy,which determines by severity of TBI.However,surgery could only improve the survival rate of patients,but not the disability rate,indicating that the poor prognosis is closely related to secondary injury except for primary injury after TBI.At present,the therapeutic strategies targeting secondary injury for TBI are divided into acute stage and chronic phase.The treatments for acute stage include postural position (head elevation),hypertonic treatment,prevention of epilepsy,sedation,hypothermia treatment and surgery (craniotomy and pressure removal of hematoma).The therapeutic strategies for chronic phase include hyperbaric oxygen[3],noninvasive brain stimulation[4]and behavioral therapy.With the continuous improvement of TBI diagnosis,treatment guidelines and expert consensus,the survival rate of patients has been improved to a certain extent,but the disability rate is still high.This situation has prompted researchers to decipher the factors and targeted intervention which will boosting neurorehabilitation and exploiting the approaches facilitating neuro-repairment after TBI in order to promote the recover rate of patients' neurological function depends on the pathophysiological characteristics of TBI.The pathophysiologic damage induced by TBI is composed by primary and secondary injury resulting in temporary or permanent neurological loss.The effective treatment for controlling primary injury mainly relies on surgery.Secondary injury usually occurs minutes to days after the primary injury and involves a cascade of molecular,chemical,and inflammatory reactions that cause further damage to brain including Wallerian degeneration of axons,mitochondrial dysfunction,excitotoxicity and apoptotic cell death of neurons and glia[5]which is the result of oxidative stress damage.Brain tissue is particularly vulnerable to oxidative stress damage due to its high oxidative metabolic activity with low antioxidant capacity and less repair mechanisms.

Oxidative stress is a state of homeostasis,which is mainly characterized by the accumulation of reactive oxygen species (ROS).These substances include hydrogen peroxide (H2O2),superoxide anion (O2),hydroxyl (OH-),and peroxy(ROO-) radicals.Oxidative stress damage is caused by the increase of oxygen free radicals and the inability of protective mechanisms such as antioxidants to remove these free radicals[6].TBI induces the release of excitatory neurotransmitters,such as glutamate and aspartate,leading to increase of the concentration of free intracellular calcium ions( Ca2+),and excess Ca2+promotes the production of ROS and nitric oxide (NO).After TBI,the production of ROS increased due to the disturbance of mitochondrial electron transport chain,the activation of phospholipase and cyclooxygenase,and the conversion of xanthine dehydrogenase to xanthine oxidase.Activated microglia,infiltrating neutrophils,and macrophages after TBI can also generate ROS via Nicotinamide phosphate adenine dinucleotide oxidase (NOX)[2].The increase in the concentration of oxygen free radicals will damage several macromolecules,including DNA,proteins and lipids,and ultimately whole cells.At the same time,apoptosis regulatory pathway proteins are activated and then promotes apoptosis[7-8],which can not only reduce the number of original nerve cells,but also affect the survival of new nerve cells.Meanwhile,oxidative stress can also inhibit the proliferation of neural stem cells(NSCs)[9].Antioxidant stress is an effective treatment.Investigations have proved that reducing ROS production and increasing ROS degradation could reduce cortical contusion volume,protecting the medulla,and improve neural function in TBI animal models[10-11].

Strategies for anti-oxidative stress therapy are used to decrease ROS production,eliminating ROS,and increase antioxidants.Uric acid (UA),one of the antioxidants,may be involved in the process of neurorehabilitation following TBI.

1 UA as a natural antioxidant in the body may promote the repair of central nervous system damage

UA,also known as 2,6,8-trioxypurine or 2,6,8-trihydroxyuria heterocyclic ring,is the final product of purine nucleotide metabolism in the body and is eliminated from the body by urine.As an important antioxidant,UA accounts for 60% of the total plasma antioxidant capacity in vivo[12].The reference interval for serum uric acid (sUA) is 208-428 μmol/L for men and 155-357 μmol/L for women,while there is currently no relevant value for the concentration of uric acid in brain.Previous study has shown that UA can prolong the life and increase the brain's tolerance to cerebral ischemia[13].Another study has suggested that UA is an independent risk factor affecting the occurrence and prognosis of acute stroke[14-15].However,from a clinical trial of stroke,it was concluded that short-term administration of UA is safe[16].

The current study suggests that UA may act as a protective factor.Within the normal range,sUA > 210 μmol/L can reduce the severity of stroke patients and reduce the complications of hospitalized patients[17],and when sUA level is <236 μmol/L,it may aggravate the recurrence of cerebral hemorrhage after stroke[18].However,in a survey of a larger case of ischemic stroke,it was found that both low and high sUA levels were associated with an increased risk of post-stroke cognitive impairment (PSCI)[19].When sUA = 297 μmol/L,the incidence of PSCI is in lowest rate[20].In the ischemic stroke model,UA inhibits microglia activation through HMGB1-TLR4-NF-κB axis to alleviate oxygen-glucose deprivation/reoxygenation injury[21].Additionally,UA can also upregulate the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) to initiate anti-oxidative effect,and increase the expression of brain-derived nerve growth factor (BDNF) to exert neuroprotection[13,22].In neurodegenerative diseases,endogenous UA holds the potential of protecting dopaminergic neurons from oxidative damage.On the contrary,low level of UA exacerbates morphological,neurochemical,and functional lesions of the dopaminergic nigrostriatal pathway[23].

The current research results affirm the protective effect of uric acid on brain injury,but excessively high levels of uric acid are a risk factor,and only a certain serum concentration can be maintained to protect brain.When the concentration of sUA exceeds the normal level,it is easy to damage peripheral blood vessels,and long-term hyperuricemia is easy to form gout.

Studies have shown that the content of uric acid in the brain of TBI rats is gradually increased,and the serum concentration of it is decreased[24].The content of uric acid in the cerebral cortex of TBI mice increased approximately 10-folds when 24-48 hours after injury[25].In a test of patients with postoperative tumor and severe TBI,it was found that the concentration of uric acid in the patient’s brain first increased and then decreased[26].In patients with TBI,the decline in sUA levels persisted up to 7 days after injury[27].

sUA levels are associated with prognosis in TBI patients.In a survey of severe TBI patients,patients with sUA levels below 200 μmol/L had a relatively high mortality rates[28].However,within the normal range,patients with higher sUA levels are more likely to regain consciousness while sUA within the normal range,patients with higher sUA levels were more likely to regain consciousness,but not happened among female patients for unknown reasons[29].In a dialysis monitoring of severe TBI patients,patients with poor prognosis had higher uric acid concentration,but the small sample size of this study could not accurately reflect the relationship between uric acid concentration and prognosis.In a study of the cerebrospinal fluid and blood of TBI patients,genetic variants of the UA transporter ABCG2 were found to be associated with outcomes after severe TBI[30].Coincidently,previous study has delineated that administration of 16 mg/kg UA obviously reduce neuronal damage and neurological deficits in TBI mice by inhibiting oxidative stress in neurons and blood vessels,thereafter improving the neurological score of the mice with TBI[24].Hence,the change of UA concentration after TBI demonstrates that UA is possibly involved in neurorehabilitation post-TBI.

Uric acid concentration will change after TBI,decreasing serum concentration but increasing brain concentration.The mechanism may be related to the excessive ROS generated in the brain after TBI to stimulate the brain’s anti-oxidative stress response.The specific mechanism needs to be further studied.After an appropriate dose of uric acid intervention,the neurological deficits in mice were improved,suggesting that uric acid promotes the repair of neurological function after TBI.

2 Multiple neuroprotective effects of UA promoting neurorehabilitation after TBI

2.1 Anti-oxidative stress Nrf2 Anti-oxidative stress Nrf2 a nuclear transcription factor,is the main antioxidant regulator,and heme Oxygenase-1 (HO-1) is downstream of Nrf2.UA increases the downstream HO-1 by up-regulating the expression of Nrf2,and at the same time increases the expression of GSH,thereby reducing the production of ROS.When astrocytes exposed to oxidative stress in vitro,UA can resist oxidative stress and protect astrocytes from injury by increasing the expression of Nrf2 and GSH[31].Meanwhile,previous study certifies that 300 μmol/L UA can increase cell viability through decreasing oxidative stress injury resulting from oxygen and glucose deprivation in neurons in vitro[32].

2.2 Anti-excitatory toxicity Anti-excitatory toxicity Glutamate is an excitatory neurotransmitter,and excessive glutamate in the synaptic cleft mediates excitotoxicity to neurons.High-affinity,Na+-dependent glutamate transporters are the main gates removing glutamate from the extracellular space[33].UA regulates the expression of glutamate transporter in astrocytes to protect neurons[34].In addition,calcium overload is also a major cause of neuroexcitotoxicity.UA can reduce nerve excitotoxicity by delaying the increase of intracellular calcium ion concentration[35].

2.3 Promoting the proliferation of vascular smooth muscle cells (VSMCs) Promoting the proliferation of vascular smooth muscle cells (VSMCs) Neurorehabilitation not only includes the regeneration of neural network,but also vascular reestablishment.As aforementioned the role of UA in protecting neural cells from oxidative stress,previous investigation indicates that UA can promote the proliferation of vascular smooth muscle cells (VSMCs) by activating p38 MAPK,p44/42 MAPK and PDGFR-β,thereafter restoring the blood supply in lesions[36].Furthermore,high concentration of UA can down-regulate the expression of vascular endothelial growth factor (VEGF),a pro-angiogenic mediator that can increase the permeability of the blood brain barrier (BBB),reduces BBB leakage,and improves the integrity of the endothelial cell barrier after TBI[37].

2.4 Increasing the expression of neurotrophic factors Increasing the expression of neurotrophic factors Neurotrophic factor is another important element to enhance the proliferation of NSC.Research has proven that UA bears the ability of increasing the expression of BDNF,which reinforces NSC expansion[22].Meanwhile,the elevated BDNF enhances the survival of NSCs and directs NSCs differentiation into neurons after CNS injury[38].

2.5 Enhancing the regeneration of injured myelin sheath Enhancing the regeneration of injured myelin sheath Demyelination is another common phenomenon post-TBI[39].Previous study has shown that UA promotes the proliferation of Schwann cells to promote the regeneration of demyelinating lesions and promoting the recovery of nerve function[40].

3 Uric acid may participate in TBI damage repair by promoting stem cell regeneration

Stem cell therapy,including transplantation of exogenous stem cells and activation of endogenous stem cells,is one of the effective strategies for neurorehabilitation after TBI.For exogenous stem cell transplantation strategies,the source for stem cell could be derived from embryonic stem cells (ESCs),bone marrow mesenchymal stem cells (BMSCs),induced pluripotent stem cells (iPSCs) and adipose-derived stem cells (ASCs).Previous studies have demonstrated that neuronal dysfunction is obviously improved in mice treated with exogenous stem cell transplantation in TBI animal models[41-43].Furthermore,the neurological function of patients is dramatically potentiated with transplantation of mesenchymal stem cells (MSCs) in short-term observation,without toxic and side effects in TBI patients.However,the long-term effects and safety need to be confirmed by further studies because of limited samples and lack of control[44].Previous research has represented that exogenous MSCs rarely differentiates into neurons,and the improvement of neurological prognosis may be related to the enhanced secretion of soluble nutrient factors,increased expression of survival signals and elevated release of exosomes[45].

In mammalian embryonic stage,NSCs exist in the striatum,hippocampus,cerebral cortex,retina,spinal cord,olfactory bulb and the ventricular zone and subventricular zone of the lateral ventricle.In adult,NSCs could be found only the subependymal area and the sublayer of granulosa cells in the dentate gyrus of the hippocampus,NSCs in other areas are mostly in a relatively quiescent state,but they can be activated and proliferated after brain trauma[46].Replacing lost neurons by activating the proliferation of endogenous neural stem cells and promoting their differentiation into corresponding neural cells is an ideal therapeutic option,but the ability to proliferate is limited for reasons related to oxidative stress after TBI.Studies have confirmed that excessive ROS can inhibit the proliferation of stem cells[9].A variety of neuro-regenerative factors have been identified to promote NSCs proliferation,including growth factors,statins,erythropoietin,and even antidepressants such as imipramine[47].However,the proliferation potential is still limited,which may be associated with oxidative stress in the lesion after TBI.The results of the study demonstrate that anti-oxidative stress treatment can improve cortical contusion volume,protect the medulla,and improve neurological function in an animal model of TBI.

Nrf2 is a nuclear transcription factor that not only regulates the oxidative stress response but also promotes the proliferation of endogenous NSCs.Nrf2 can increase the viability of NSCs and promote the proliferation and differentiation of NSCs.In the Nrf2 knockout mouse model,the number of NSCs in the mouse from birth to adulthood is less than that in the control group[48].Meantime,up-regulating the expression of Nrf2,HO-1 and NQO1 can reduce the damage of oxygen and glucose deprivation and promote the proliferation of NSCs[49].Experiments show that uric acid can regulate the expression of Nrf2 to protect nerve function.It is reasonable to believe that UA promotes the proliferation of NSCs by activating Nrf2,and participates in the repair of neural network after TBI.

4 Perspectives

The high disability rate is the main research focus of TBI.Oxidative stress can damage protein structure,affecting its function,leading to lipid peroxidation,damaging the double-stranded structure of DNA,which ultimately aggravate the neurological dysfunction of TBI.UA,one of the in vivo antioxidants,not only exerts neuroprotection through alleviating oxidative stress,but also might possess the ability of promoting the proliferation of neural stem cells (NSCs) that participates in neural repairment after TBI,thereafter improving the neurological recovery.Long-term hyperuricemia can damage peripheral blood vessels and lead to gout.Studies have shown that only maintaining an appropriate concentration of sUA is beneficial for TBI patients.At present,the duration of uric acid use varies,with the longest duration of use in animal models being 14 days,while in clinical trials the duration of uric acid use is 24 hours.In terms of the dosage of uric acid,the in vivo experiment tends to be 16 mg/kg,and the concentration in the in vitro experiment is 300 μmol/L.The specific optimal treatment course,dosage and serum concentration of uric acid may need further research.The detection of uric acid concentration is convenient and feasible.Clinically,uric acid antagonists (probenecid,allopurinol,febuxostat,etc.) can be used according to the monitoring results to maintain the sUA level within the ideal range,which can avoid adverse effects of long-term hyperuricemia.The effects of UA after TBI are multifaced including anti-oxidative stress,anti-excitatory toxicity,promoting the proliferation of VSMCs,increasing the expression of neurotrophic factors,enhancing the regeneration of injured myelin sheath,and accelerating the proliferation of NSCs.While the underlying mechanism has not yet been elucidated.Considering that NSCs participates in neural repairment after TBI,deciphering the role of UA in proliferation of NSCs and the underlying mechanism is worthy of investigating .The aim of this review is to provide a potential therapeutic target to UA for the treatment of TBI.