Annexin A1 conveys neuroprotective function via inhibiting oxidative stress in diffuse axonal injury of rats

Diffuse axonal injury (DAI) is a critical pathological facet of traumatic brain injury (TBI). Oxidative stress plays a significant role in the progress of DAI. Annexin A1 (AnxA1) has been demonstrated to benefit from recovery of neurofunctional outcomes after TBI. However, whether AnxA1 exhibits neuronal protective function by modulating oxidative stress in DAI remains unknown. Expression of AnxA1 was evaluated via real-time PCR and western blotting in rat brainstem after DAI. The neurological effect of AnxA1 following DAI through quantification of modified neurologic severity score (mNSS) was compared between wild-type and AnxA1-knockout rats. Brain edema and neuronal apoptosis, as well as expression of oxidative factors and inflammatory cytokines, were analyzed between wild-type and AnxA1 deficiency rats after DAI. Furthermore, mNSS, oxidative and inflammatory cytokines were assayed after timely administration of recombinant AnxA1 for DAI rats. In the brainstem of DAI, the expression of AnxA1 remarkably increased. Ablation of AnxA1 increased the mNSS score and brain water content of rats after DAI. Neuron apoptosis in the brainstem after DAI was exaggerated by AnxA1 deficiency. In addition, AnxA1 deficiency significantly upregulated the level of oxidative and inflammatory factors in the brainstem of DAI rats. Moreover, mNSS decreased by AnxA1 treatment in rats following DAI. Expression of oxidative and inflammatory molecules in rat brainstem subjected to DAI inhibited by AnxA1 administration. AnxA1 exhibited neuronal protective function in the progression of DAI mainly dependent on suppressing oxidative stress and inflammation.


Introduction
Diffuse axonal injury (DAI) is a pivotal and common pathological facet of traumatic brain injury (TBI) [1], which is a public health issue with a high rate of mortality, co-morbidities, and long-term disabilities [2].DAI is caused by sudden rotated acceleration and deceleration leading to immediate tearing of the axonal cytoskeleton throughout white matter [3].The development of DAI is not only due to primary axotomy from mechanical forces but also from secondary axotomy as a result of a progressive molecular and cellular cascade of pathologic alteration after initial traumatic damage.As the axonal transport is disrupted, pathological gradual accumulation of transport products causes axonal swelling [4], cerebral axonal disconnection [5], and Wallerian degeneration, which finally leads to cognitive, motor, and sensory deficits [6].Nevertheless, since some axons are not permanently injured in the early process, only their function is disrupted, so researchers regard this as a potential time window for therapeutic intervention.Initial events that contribute to the progression of DAI include oxidative stress, inflammation, apoptosis, and so on [7].In the early process of post-trauma, oxidative stress is confirmed as playing a pivotal role in the genesis of DAI and is linked to the effects of a plethora of molecular and cellular changes [2].Excess intracellular Ca 2+ influx caused by mechanical disruption induces the generation of reactive oxygen species (ROS) in the mitochondria, triggering oxidative stress in the axons [8].Numerous production of free radicals in the axons leads to peroxidation and toxicity of lipids, protein, and DNA resulting in cellular and tissue degeneration [9].In addition, oxidative stress initiates and exacerbates neuroinflammation contributing to the mechanism of cell apoptosis, which can also further promote ROS generation.Importantly, the involvement of cytokines in neuroinflammation such as TNF-α, IL-1β, IL-6, and adhesion molecules ICAM-1 has been demonstrated in the damaged diseases of the central nervous system [10].Furthermore, oxidative stress can directly disrupt the balance between neurotransmitters, leading to excitotoxicity and neuronal cell death.Thus, inhibiting oxidative stress in DAI may potentially improve multiple aspects of the response of the brain to trauma.
Recent studies have demonstrated that annexin A1 (AnxA1) is a 37-kDa calcium-dependent phospholipidlinked protein that is known to be involved in oxidative stress, inflammation, and immune response in multiple conditions.Knockdown of AnxA1 enhanced the oxidative stress and inflammation in cigarette smoke extract (CSE)-induced bronchial epithelial cells, a chronic obstructive pulmonary disease in vitro model [11].In addition, AnxA1 peptide Ac2-26 significantly suppressed the activity of NADPH oxidase and malondialdehyde to decrease oxidative stress, lung injury, and epithelium apoptosis in acute respiratory distress syndrome rats [12].Besides, in the kidney following ischemic injury, AnxA1 limited ROS production and increased antioxidant enzymes to abrogate oxidative stress and attenuate tubular cell death, which finally preserves the kidney against ischemia [13].Furthermore, Ac2-26 significantly ameliorated neuronal apoptosis reduced the volume of cerebral infarct, and improved the neurological function of mice on cerebral I/R (ischemic/reperfusion) injury [14].
On the basis of these observations and previous studies, rescuing axons not permanently damaged in the early process is of great significance for DAI therapeutic intervention.Importantly, exogenous recombinational AnxA1 improved neuron functional outcomes through ameliorating inflammation and BBB (blood-brain barrier) disruption in mice after an animal model for TBI [15].In addition, given that focal lesions of DAI mainly involve the midline of the brain including the brainstem and corpus callosum.Thus, in this project we further set out to investigate the effect of AnxA1 on neuronal function after DAI, and whether AnxA1 conveys its neuroprotective role via modulating the oxidative stress in the early process of DAI.The study will provide key insights into the role of AnxA1 and its modulation mechanism in the early development of DAI.

Animal
Sprague-Dawley rats (male, weight 250-300 g, 8-10 weeks of age, n = 84), AnxA1 knockout (AnxA1 -/-) and wild-type (AnxA1 +/+ ) rats were purchased from the Experimental Animal Center of Xi'an Jiaotong University (Shaanxi, China).The animals were housed and fed in a room under SPF conditions and kept at 20 °C to 23 °C under 12:12-h light-dark cycles.Rats were euthanized with dioxide and anesthetized with ketamine (100 mg/ kg) and xylazine (10 mg/kg).All experimental procedures involving animals were in strict agreement with the Guidelines for the Care and Use of Laboratory Animals from the National Institutes of Health and all methods are reported in accordance with ARRIVE guidelines.All animal studies were approved by the Institutional Animal Care and Use Committee of the Medical College of Xi'an Jiaotong University.

Rat model of DAI and treatment
A DAI rat model was established using a lateral head rotation device as described in previous studies [16][17][18].After weighing, all rats were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg).The head of the rat was positioned and fixed horizontally in a rotating injury device by two lateral ear bars, a head clip, and an anterior teeth hole, with the body 30° oblique to the laboratory table.After the trigger was started, the injury device rapidly rotated the rat head, involving sudden acceleration and deceleration, through a 90° laterally in the coronal plane.Primary coma was tested in all injured rats.The control group (sham group) only underwent the same anesthesia and fixation to the device without injury.Rats who died from injuries were excluded (one out of 10 modeled rats were excluded) and would be replaced by new ones.Rats were held in an electric blanket to maintain body temperature until complete recovery from anesthesia.Administration of human recombinant AnxA1 (rhAnxA1) (R&D Systems, Minneapolis, MN, USA, 3770-AN) (1 µg/kg) was performed through the tail vein at 30 min after DAI.

Neurological assessment
Neurological dysfunction was assessed through the modified neurological severity score (mNSS) test at different time points (1, 3, and 7 days post-DAI) [19].mNSS test is comprised of motor function, sensory function, and reflexes.Function was graded on a scale from 0 to 18 points.A higher score implies worse neurological impairment (normal score is 0; maximal deficit score is 18).mNSS was applied to evaluate the neurological deficits of rats before sacrificing by two researchers who were blinded to group information (AnxA1 +/+ group and AnxA1 -/-group).

Evaluation of brain edema
Brain water content was measured at 1, 3, and 7 days after DAI between AnxA1 +/+ group rats and AnxA1 -/-group rats [15].Rat's brain was dissected immediately after sacrifice and promptly removed for weighing to determine the wet weight.The brain was dried at 72 °C for 3 days to re-weigh the dry weight.At last, brain water content was analyzed according to [(wet weight − dry weight)/wet weight] × 100%.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
Apoptosis of the brainstem at 1, 3, and 7 days after DAI was tested by TUNEL assay using an In Situ Cell Death Detection Kit (Invitrogen, Carlsbad, CA, USA, C10625) according to the manufacturer's instructions.After rats were euthanatized, the entire brains, excluding the olfactory bulbs, were collected and fixed in a 4% PFA solution.The fixed brain tissues were embedded in paraffin.5μm paraffin-embedded sections were de-paraffinized, rehydrated, and permeabilized.Permeabilized brain sections at the pons level of the brainstem were incubated with the TUNEL reaction mixture solution in a 37 °C incubator for 1h avoiding the light.Sections were counterstained with hematoxylin for 1 min after washing under running water.Sections were dehydrated in graded ethanol, cleared in xylene, and coverslipped with fluorescent mounting medium (Invitrogen).At last, sections were imaged under the microscope (Carl Zeiss Microscopy LLC, Thornwood, NY, USA).Apoptotic cells in the brainstem, identified by a dark brown stain, were counted by using Adobe Photoshop software with two independent researchers blinded to group information.

Measurement of superoxide anion for brainstem
Superoxide (O 2− ) anion is one form of ROS, which is the main contributor to triggering oxidative stress.Production of superoxide anion in brainstem homogenates was measured according to the approach of Babior et al. (1973), based on the reduction of cytochrome c.After the rats, the brainstem was dissected immediately under a surgical microscope.Brainstem tissues were homogenized in freshly prepared Tris-KCl buffer (1.15% KCl and 0.05 M Tris, pH = 7.4), using a Potter-Elvehjem homogenizer fitted with a Teflon pestle.The 2 mL reaction mixtures contained 25 μl of homogenate and 0.05 mM cytochrome c in Tris-KCl buffer (pH = 7.4).Reaction mixtures were incubated at 37 °C for 15 min, and then tubes were placed on ice to terminate reactions.The mixtures were centrifuged at 700g for 10 min, and the supernatant fractions were collected for subsequent spectrophotometric measurement at 550 nm under a Spectronic-20 spectrophotometer.After averaging the measurements and background subtraction, the values were normalized by protein concentration for each brainstem sample.

Quantification of glutathione in brainstem tissue
Antioxidative substance total glutathione (GSH) in brainstem tissue was assayed using the Glutathione Fluorometric Assay Kit (BioVision, China, ab65322) according to the manufacturer's protocol.Freshly obtained brainstem tissues were sonicated in ice-cold GSH assay buffer.De-protonation was conducted using perchloric acid followed by centrifugation at 13 000g × 2 min.Reduced GSH in the supernatant was assayed using o-phthalaldehyde, which reacts specifically with reduced GSH (not GSSG), with the generation of a fluorescent signal.Samples and standards were read on a fluorescence plate reader equipped with Excitation/Emission at 340/420 nm using a microplate reader (BioVision).

Statistical analysis
Results were expressed as the mean ± SEM.Prism software was used for statistical analyses.Significant differences between samples were analyzed with a two-tailed Student's t-test.P < 0.05 was considered as statistically significant.

Endogenous AnxA1 molecule was activated in rat brainstem after DAI
Previous studies showed a significant increase in the AnxA1 level in the injured cortex of mice at different time points after the controlled cortical impact model, an animal model for TBI [20].In addition, DAI is characterized by focal lesions in the midline of the brain such as the brainstem and corpus callosum.Therefore, in the first set of experiments, we investigated the involvement of AnxA1 in the brainstem after DAI induced by TBI.
As shown in Fig. 1a, an obvious increase of AnxA1 mRNA level in rat brainstem at different time points (1 day, 3 days, and 7 days) post-DAI was observed compared to sham control.Besides, western blotting results showed that DAI remarkably increased AnxA1 protein levels in the brainstem compared to the normal control brainstem (Fig. 1b).Quantification of western blotting band intensities were consistent with band picture results (Fig. 1c).These mRNA and protein data suggested that AnxA1 may be an important regulator in the pathogenesis of DAI.

AnxA1 deficiency exacerbated neurological outcomes and brain edema of rats after DAI
The above results demonstrated an increased expression of AnxA1 in the DAI brainstem, which revealed the involvement of AnxA1 in the pathogenesis of the DAI.In addition, given that the previous literature indicated exogenous AnxA1 administration improves the neurological outcomes following the animal TBI model, we investigated the potential influence of AnxA1 deficiency on the neurological function of rats after DAI.The modified Neurological Severity Score (mNSS) system, a reliable and well-accepted approach for quantitative evaluation of neurological function, was applied to assess the degree of injury.As shown in Fig. 2a, compared to wild-type rats, AnxA1 knockout rats showed higher scores at 1 day, 3 days, and 7 days after DAI, suggesting that deficiency of AnxA1 sharply impairs the neurological function for rats subjected to DAI.
Brain edema, induced by DAI, is a critical pathological facet resulting in dysfunction of neurons.Therefore, brain water content was measured for DAI rats with AnxA1 deficiency at different time points.Figure 2b showed that the brain water content increased remarkably in AnxA1 -/-rats at day 3 and day 7 post-DAI compared to AnxA1 +/+ rats post-DAI.This result indicated that AnxA1 protects the neuron against the DAI.

AnxA1 deficiency significantly increased the apoptotic index in the rat-injured brainstem
Apoptosis, a serious pathological process for neuronal injury, was demonstrated to significantly increase after DAI in a previous study.So next the extent of apoptosis for DAI rats with AnxA1 deficiency was performed via TUNEL assay.The results showed that rats subjected to DAI in the AnxA1 -/-group had more apoptotic cells and neuronal loss than the wild-type rats at 1 day, 3 days, and 7 days post-DAI, which implied that AnxA1 plays a potential benefit for neuronal survival in the process of DAI (Fig. 2c).

AnxA1 deficiency increased DAI-stimulated oxidative stress in rat brainstem after DAI
The above results demonstrated an upregulation of AnxA1 in the brainstem subjected to DAI and improved neuron survival in rats with DAI, which revealed the involvement of AnxA1 during the pathogenesis of the axonal injury induced by TBI.Previous studies demonstrated that oxidative stress evoked by TBI boosts neuroinflammation and contributes to neuronal degeneration and cell death in the brain after injury.AnxA1 has not only been confirmed to exert neurological benefit but also has been demonstrated to correlate with oxidative stress in several disease conditions, including a chronic obstructive pulmonary disease in vitro model induced by CSE [11] and ischemic kidney injury [13].Thus we set out to investigate the effect of AnxA1 on expression of oxidative stress factors in DAI.Quantification of the level of superoxide, a critical component of ROS, was performed in brainstem tissues of rats with AnxA1 deficiency.Compared to wild-type rats, AnxA1 -/-rats showed higher levels of superoxide at 3 days after DAI (Fig. 3a).
GSH is the major cellular antioxidant to counteract ROS.At the 3 days after DAI, the GSH level robustly reduced in the brainstem of AnxA1 -/-rats compared with AnxA1 +/+ rats (Fig. 3b).NADPH oxidase 2 (Nox2), the primary producer of ROS, is the major contributor to oxidative stress.In AnxA1 -/-rats, the DAI brainstem generated significant mRNA accumulation of Nox2 as compared to wild-type brainstem at the 3 days after DAI (Fig. 3c).These results implicated that the deficiency of AnxA1 in brainstem subjected to DAI exacerbates the induction of oxidative stress mediators.

AnxA1 deficiency promoted inflammatory molecule expression in the brainstem of DAI rats
In addition to the effects of AnxA1 on the expression of oxidative stress molecules in DAI, we explored the influence of AnxA1 on inflammation after DAI.We evaluated the mRNA and protein levels of typical inflammatory cytokines, including TNF-α, IL-1β, and ICAM-1, involved in the process of DAI.The mRNA level of TNFα, IL-1β and ICAM-1 significantly increased in the rat Annexin A1 deficiency exacerbated neurological outcomes and apoptotic index of rats after DAI.(a) Modified neurologic severity score (mNSS) was detected for rats at different time points (1 day, 3 days, and 7 days after diffuse axonal injury).Compared to wild-type DAI rats, annexin A1 deficiency rats have higher mNSS score, especially at 3 days and 7 days after DAI.(b) Quantitation of brain water content at different time points (1 day, 3 days, and 7 days) after DAI.Compared to wild-type DAI rats, the brain water content of AnxA1 -/-DAI rats is much higher than the AnxA1 +/+ rats subjected to DAI, especially at 3 days and 7 days after DAI.(c) TUNEL staining was detected in the injured brainstem of AnxA1 +/+ rats and AnxA1 -/-rats at 1 day, 3 days, and 7 days after DAI, respectively.There was more apoptotic index in the injured brainstem of AnxA1 -/-rats compared with wild-type DAI rats.The images were taken under a 10 × 10 microscope.Pre: before the DAI.The values are represented as mean ± SEM. *P < 0.05, AnxA1 +/+ DAI group vs. AnxA1 -/-DAI group, n = 5 for each group.Scale bar: 50 μm.

Recombinant AnxA1 downregulated oxidative stress and inflammatory factors in rat brainstem after DAI
A previous study revealed that human recombinant AnxA1 ameliorated inflammation and improved functional outcomes in mice subjected to TBI triggered by controlled cortical impact [20].Our study results indicated a deficiency of endogenous AnxA1 exacerbated neuronal injury and aggravated the oxidative stress and inflammation in DAI.In order to investigate whether AnxA1 exerts its neuronal benefit through modulating oxidative stress and inflammation, we further evaluated the effect of exogenous AnxA1 on oxidative stress and inflammation after DAI.After modeling, animals were treated with a concentration of rhAnxA1 (1 μg/kg) by tail vein for 30 min after DAI modeling for one time, and controls were injected with 0.9% NaCl.As shown in Fig. 5a, levels of superoxide, a pro-oxidative factor, in brainstem tissues significantly decreased in rat brainstem subjected to DAI with immediate rhAnxA1 administration, compared to DAI rats with 0.9% NaCl administration.Besides, rhAnxA1 treatment robustly increased the level of antioxidant factor GSH in rat brainstem at the 3 days after DAI (Fig. 5b).Real-time PCR results showed rhAnxA1 treatment significantly decreased Nox2 expression in DAI brainstem at the 3 days after DAI (Fig. 5c).In addition, the inflammatory molecules TNF-α and IL-1β downregulated by rhAnxA1 in brainstem subjected to DAI (Fig. 5c).

Recombinant AnxA1 protected the neurological function of rats against DAI
In light of the above results that exogenous AnxA1 ameliorated oxidative stress and inflammation after DAI, we next investigated whether exogenous AnxA1 improves neuronal functional outcomes following the DAI.Recombinant AnxA1 was injected through the tail vein at 30 min after DAI modeling.mNSS was quantified for rats at different time points (1 day, 3 days, and 7 days) after DAI.Compared to vehicle group DAI rats, treatment with rhAnxA1 led to a significant decrease in the score in treatment groups, especially at 3 days and 7 days after DAI (Fig. 6).

Discussion
TBI is a major source of health loss and disability worldwide.Globally, the annual incidence of TBI is variably estimated at 27 to 69 million.Many suffered individuals live with significant permanent disabilities, resulting in a major socioeconomic burden [21].DAI, a significant pathological facet of TBI, is resulted from the direct impact of the shearing forces, which cause disruption, retraction, and edema of the axons.These alterations impede the normal transport of proteins and electrolytes resulting in secondary axonal damage [22].The degree of injury and the duration of loss of consciousness and coma directly correlate with the extent of axonal pathology.While most of the suffered brain appears structurally normal and only a minority of axons are affected even in severe TBI, DAI can cause collective impairment that leads to disruption of signal transfer and plays critical implications for neurological function [23].Nevertheless, some axons are not permanently injured during the Annexin A1 deficiency increased DAI-stimulated oxidative stress in the brainstem of rats.(a) Superoxide levels in the brainstem of rats subjected to DAI significantly increased in annexin A1 deficiency rats compared to wild-type rats.(b) Annexin A1 deficiency remarkably downregulated glutathione (GSH) levels in rat brainstem at 3 days after DAI.(c) mRNA level of annexin A1 was not detected in annexin A1-deficient DAI rats compared with wild-type DAI rats.mRNA level of pro-oxidative factors Nox2 increased in annexin A1-deficient DAI rats compared with wild-type DAI rats.All assays were performed at 3 days after DAI.The values are represented as mean ± SEM. *P < 0.05 vs. wild-type DAI group, n = 5 for each group.period of DAI, and only their function is disrupted.Therefore, this early period is regarded as an optimal time window for DAI therapy.According to this theory, timely administration is crucial to rescuing the function of the axoplasmic transport.
A recent study indicated that an imbalance between the production and removal of ROS is disputed in the early time after DAI [24], and further triggers and aggravates inflammation, apoptosis, and calcium-mediated damage [25].AnxA1 is demonstrated to suppress oxidative stress in multiple diseases and exit neuronal benefits in nervous system conditions.In the current study, our objective was to gain insights into the potential effect of AnxA1 in the progression of DAI.In addition, we had a particular interest in studying whether AnxA1 plays its role in modulating oxidative stress and inflammation.Together, current results clearly demonstrated that modulating AnxA1 may represent an extra approach to suppressing oxidative stress and inflammation, further attenuating neurological impairment in the progress of DAI.
Recent literature demonstrated that endogenous AnxA1 increased in the injured cortex of mice at different time points after a controlled cortical impact model, an animal model for TBI [20].Significant insights in therapy for axonal pathology have arisen through the establishment of a lateral head rotation device using sudden acceleration and deceleration [16].Given that the brainstem is vulnerable to mechanical rotated acceleration damage [26], so we detected the AnxA1 expression in the brainstem of the DAI rat.In agreement with these previous results, our research results showed that both protein and mRNA levels of AnxA1 are upregulated in the DAI brainstem, especially on the first day following DAI.Considering the previous study results and current results, we can see that AnxA1 significantly increased and activated in the early pathogenesis of axonal injury.
Neurological function is the ultimate prognostic outcome for brain trauma.The neuronal benefit of AnxA1 is studied in various conditions, such as ischemic retina diseases [27], nociception in dorsal root ganglion neurons [28], and epileptogenic processes [29].Therefore we established AnxA1 knockout rats and evaluated the neurological effect of AnxA1 on rats after DAI.The results demonstrated that AnxA1 deficiency aggravated neurological dysfunction at 3 days and 7 days after DAI, which is consistent with previous research data that AnxA1 has a neuroprotective effect.
Brain edema, induced by axonal mechanical damage, is a critical pathological element that contributes to the dysfunction of neurons.DAI can also induce brain edema, and uncontrolled brain edema increases intracranial pressure and leads to brain herniation, which results in poor prognosis [30].The edema was more serious, and the extent of axonal injuries was more severe [31].Recent studies indicated brain edema formation is associated with glia dysfunction, increase aquaporin-4 expression and aquaporin-4 translocation to the blood-spinal-cord-barrier [32,33].In our study, brain water content results showed that AnxA1 deficiency increases brain edema at 3 days and 7 days after DAI, which indicated that AnxA1 significantly attenuates brain edema.The measurement of brain water content also indicated AnxA1's beneficial effect on brain edema which might contribute to the neuroprotective properties of AnxA1.Previous studies mentioned targeting glial cells regulatory mechanisms could help decrease edema to protect the neural function in neurodegenerative diseases [34,35], which confirmed that the decrease of brain edema is great benefit for neural disoders.In addition, considered that dysfunction of axonal transport could induce apoptosis, so we investigated the AnxA1 effect on apoptosis.The result showed that AnxA1 decreased the apoptosis in the brainstem in DAI.All these results indicated that AnxA1 plays a beneficial role in DAI.
Based on these findings, we then wanted to determine the effects of AnxA1 on neuron cell death.Neuronal apoptosis can be resulted from disrupted axon transport.A recent study showed that AnxA1 administration can alleviate neuronal apoptosis in acute ischemic stroke [14].In agreement with previous data, our results indicated that AnxA1 deficiency increases neuronal cell death.From these data, we concluded that AnxA1 is a critical molecular in the progression of DAI, alleviates neuronal dysfunction and protects neurological outcomes against diffused axonal damage.Recombinant annexin A1 protected the neurological function of rats against DAI.mNSS was quantified for rats at different time points (1 day, 3 days, and 7 days) after DAI.Recombinant annexin A1 was injected through the tail vein at 30 min after DAI modeling.Compared to vehicle group DAI rats, rats treated with recombinant annexin A1 showed lower mNSS score, especially at 3 days and 7 days after DAI.All assays were performed at 3 days after DAI.Values are represented as mean ± SEM. * P < 0.05 vs. vehicle group, n = 5 for each group.
stress is a key mechanism that contributes to axonal deterioration and neuronal death in the development and progression of DAI, which can trigger a cascade of events that exacerbate axonal damage.Increases in the defense mechanisms through the use of exogenous antioxidants may be neuroprotective, particularly if they are given within the neuroprotective time window [36].AnxA1, a protein that has been shown to possess antioxidant properties and can help protect cells from oxidative damage in multiple conditions [37], can scavenge ROS and inhibit their harmful effects, thereby reducing oxidative stress.Therefore, we reason that AnxA1 plays a neurological beneficial role via inhibiting oxidative stress in the early development of DAI.As expected, our research showed that AnxA1 deficiency downregulated crucial pro-oxidative molecular superoxide and Nox2, and upregulated the synthesis of primary antioxidative substance GSH, which implied that AnxA1 may be dependent on suppressing oxidative stress to exhibit its neuroprotective effects in the progression of DAI.
Under pathological state, ROS can activate inflammatory pathways, leading to the release of pro-inflammatory cytokines and chemokines that attract immune cells to the site of injury.These immune cells can further exacerbate oxidative stress and axonal damage by releasing ROS and other toxic molecules.For example, a high concentration of TNFα impairs the ability of microglia to eliminate glutamate, and this causes excitatory toxicity and injures neurons [38].In addition, AnxA1 exhibits anti-inflammatory function in various conditions, such as AnxA1 -/-mice showed an expansion of pro-inflammatory macrophages in the cardiac ischemic area [39].In agreement with previous data, our results showed that AnxA1 - /-rat dramatically downregulates the expression of inflammation factors in the brainstem of DAI.Combined with the above figures, these findings suggested that AnxA1 ameliorates axonal deterioration and neuron degeneration relying on inhibiting oxidative stress and inflammation in the development of DAI.
In order to confirm our hypothesis, we further investigated the effect of exogenous AnxA1 on oxidative stress and inflammation in the brainstem following DAI.To achieve optimal outcomes following axonal injury by trauma, timely treatment is essential.Our result showed that the high level of pro-oxidative substances superoxide and Nox2 induced by DAI can be inhibited after timely rhAnxA1 treatment.Besides, antioxidants GSH are enhanced by AnxA1 treatment.On the other hand, pro-inflammatory cytokines TNF-α and IL-1β in the brainstem decreased by timely AnxA1 administration.Importantly, emerging evidence indicated that timely exogenous AnxA1 administration is appreciated for its neuroprotective effects, and detrimental complications of prolonged cerebral hypoxia and ischemia have been attenuated by AnxA1 administration.As expected, our data confirmed that neurological deficits in rats suffering from DAI were ameliorated by the optimal administration of rhAnxA1.All these above findings for the first time suggested that AnxA1 conveys its neuroprotective effect mainly dependent on suppressing the oxidative stress in the murine DAI model.
In conclusion, this current study showed that AnxA1 is significantly increased and activated in the brainstem after DAI.AnxA1 deficiency exacerbates neurological function, neuronal death, oxidative stress, and inflammation.What's more, the administration of rhAnxA1 suppresses oxidative stress and inflammation and improves the neurological outcome.Collectively, we summarized that AnxA1 protects neuronal function in the development of DAI mainly dependent on inhibiting oxidative stress and inflammation, which strongly suggests AnxA1 may be a potent and promising neuroprotective agent against DAI, also in other axonal damage conditions, such as hypoxia/ischemia, infarcts, multiple sclerosis, hypoglycemia.