The neuroprotection of hyperbaric oxygen therapy against traumatic brain injury via NF-κB/MAPKs-CXCL1 signaling pathway

It is well known that hyperbaric oxygen (HBO) therapy achieves neuroprotective effects by suppressing or relieving neuroinammatory responses. However, its underlying therapeutic mechanisms are not yet fully elucidated. Based on our previous studies, we further investigated whether HBO therapy exerts neuroprotective effects in vivo by regulating the NF-κB/ MAPKs-CXCL1 inammatory pathway. A rat model of traumatic brain injury (TBI) was established by controlled cortical impact (CCI). The cellular distribution of CXCL1 and CXCR2 was observed by double immunouorescence labeling. The neurological function of TBI rats was assessed by modied neurological severity scores and Morris water maze methods. TUNEL staining was performed to observe apoptosis of neuronal cells in the injured cortical area. The changes in neural function, neuronal apoptosis, and expression of CXCL1, CXCR2, NF-κB, and MAPKs (ERK and JNK) were observed in TBI rats treated with CXCR2 antagonist, ERK, JNK, and NF-κB inhibitor or HBO therapy. mediates the interaction of activated astrocytes and neurons, exacerbating secondary injury after TBI. HBO therapy exerts neuroprotective effects by regulating the NF-κB/ MAPKs (JNK and ERK)- CXCL1 inammatory pathway to control neuroinammation after TBI, which provides the theoretical and experimental basis for the clinical application of HBO therapy in the treatment of TBI.


Introduction
The yearly incidence of traumatic brain injury (TBI) is estimated at 50 million cases worldwide, and thus TBI is a major global health challenge [1]. The high rates of TBI-associated morbidity and mortality in China, a large developing country, has become a major public health concern [2]. TBI includes primary and secondary injuries. Compared to primary injuries, secondary injuries last longer and are the focus of scienti c research and clinical treatment for TBI. Secondary injuries include neuroin ammation, disruption of the blood-brain barrier, abnormal brain metabolism, and excitotoxicity. Neuroin ammation is a major pathogenic mechanism leading to secondary brain injuries following TBI. It has been reported in the literature that the expression of various in ammatory factors, such as IL-1β, CCL2, CXCL1, IFN-γ, TNFα, IL-6, IL-1α, IL-10 and IL-8, is altered after TBI. These factors, involved in the molecular regulatory mechanisms of neuroin ammatory response, are classi ed into two major groups: pro-in ammatory and anti-in ammatory factors [3][4][5][6]. Moreover, it has been shown that neuroin ammation modulation may be a favorable option for the alleviation of secondary brain injuries and to ameliorate the outcome of TBI [7][8][9].
The use of hyperbaric oxygen (HBO) for the treatment of TBI has been controversial, mainly because of uncertainty of its e cacy, complications associated with HBO therapy, and the fact that the therapeutic mechanisms have not been completely clari ed [10][11][12][13]. Numerous animal studies and clinical practice have demonstrated that HBO treatment can reduce secondary injury after TBI. In 2018, a neurosurgery clinical team from the University of Minnesota published a review on the treatment of acute TBI with HBO that collected and analyzed 30 clinical and animal research articles. Their results showed clear e cacy of HBO in the treatment of acute TBI [14]. Preliminary studies of our group also con rmed that HBO treatment can reduce secondary injury after TBI, and its mechanism of action may be related to the reduction of cerebral edema, inhibition of astrocyte proliferation, improvement of brain metabolism, and inhibition of neuroin ammation [15][16][17].
It has been reported in the literature that HBO treatment achieves neuroprotective effects by inhibiting or relieving neuroin ammatory responses, and its mechanism of action may be related to the altered expression of various in ammatory factors such as IL-1β, IL-6, CCL2, IL-10, and TNF-α [13,[18][19][20].
Consistent with the literature, we found that the expression of several chemokines and their effector receptors are increased after TBI and promote neuroin ammation by regulating neuronal-glial interactions [4,15,21]. Through in vitro studies, our group found that HBO therapy can downregulate the expression of chemokines CXCL1 and CCL2 and affect in ammatory responses by inhibiting the LPSinduced NF-κB/ MAPKs (JNK and ERK)-CCL2/CXCL1 in ammatory pathway [17]. In the present study, we further examined whether HBO therapy exerts neuroprotective effects by modulating the NF-κB/ MAPKs-CXCL1 in ammatory pathway through in vivo studies.

Animals and surgery
The rat TBI model was established by the cortical controlled injury (CCI) method described previously [22,23]. All experimental treatments were in accordance with the Chinese "Guidelines for the Care and Use of Laboratory Animals". Healthy adult male Sprague-Dawley rats weighing 230-250 g were purchased from the Experimental Animal Center of Nantong University (Nantong, China). All rats were placed in an environment with constant temperature and humidity and could drink and eat freely in a 12 h day and night cycle (the grouping of rats used for each experiment is shown in Table 1). After intraperitoneal anesthesia with 0.4 mL/100 g chloral hydrate, the rat's head was xed on a stereotaxic device, a hole with a diameter of 6 mm was drilled in the right parietal bone (3.0 mm behind bregma and 3.0 mm centered on the right side of the sagittal suture), the skull was lifted to expose the dura mater, and a pneumatic impact device (TBI 0310, Precision Systems and Instrumentation, USA) was employed to model moderate TBI. The impact parameters of velocity were 4.0 m/s, depth, 3.0 mm, and dwell time, 15 ms. The sham group underwent the same craniotomy, but without impingement.

Assessment of Neurological Injury
Modi ed neurological severity scores The modi ed neurological severity scores (mNSS) method was used to evaluate neurological function [24]. Brie y, the aspects of the test included motor (6 points), sensory (2 points), balance beam test (6 points), lack of re exes and abnormal activity (4 points). If the animal did not complete the required movements, one point was awarded. The higher the total score obtained by the animal, the more severe the nerve damage.

Morris water maze test
The Morris water maze (MWM) was used to assess cognitive function by observing the mean escape latency and number of platform crossings [25,26]. The water maze experiments were conducted in a quiet environment with water temperature maintained at 20 ± 1°C. Before modeling, all rats had been acclimatized for 2 consecutive days to exclude the effects of visual and motor dysfunction on the experiment. In the position navigation test, each rat was subjected to 4 trials per day, 120 s of each trial, 30 s of rest, and 5 min of rest between each trial. Each rat was randomly placed in the water from four positions facing the pool wall, and the platform was hidden 2 cm below the water surface. The escape latency of each rat during the trials was recorded and averaged over the four trials as the average escape latency. If an animal failed to nd the platform within 120 s, the escape latency was recorded as 120 s. In the spatial exploration test, after the platform was removed, rats were placed in the pool from the quadrant farthest from the original platform and swam freely for 120 s. The number of times rats crossed the platform was recorded.
Drug Treatment SD male rats were randomly divided into ve groups: Sham, TBI, TBI vehicle, TBI low-dose, and TBI highdose. The CXCR2 antagonist SB225002 was purchased from Tocris (Bristol, UK). The ERK, JNKand NF-κB inhibitors, PD98059, SP600125, and BAY117082, respectively, were purchased from Calbiochem (Merck, Darmstadt, Germany). The antagonist and the three inhibitors were dissolved in dimethyl sulfoxide (DMSO) and diluted with PBS to a low dose of 2.5 μg/10 μL and a high dose of 25 μg/10 μL, respectively.
The TBI +vehicle group was treated with DMSO and PBS. One hour after TBI, different concentrations of antagonist and three different concentrations of inhibitors were injected into the cortex in the center of the injured area using a 10 μL Hamilton microsyringe for approximately 10 min (1 μL/min), after which the needle was left in place for approximately 5 min and then gently and slowly withdrawn [27,28]. The TBI vehicle group was injected in the same way. All rats were injected continuously for 3 days.
HBO Therapy SD male rats were randomly divided into four groups: sham, sham +HBO, TBI, and TBI +HBO. The rats in the sham +HBO group and TBI +HBO group were subjected to continuous HBO therapy once a day [15].
Rats were placed in a hyperbaric chamber and the pressure in the chamber was slowly increased to 0.2 MPa for about 15 min, then maintained at 0.2 MPa for about 60 min, and nally slowly decreased to atmospheric pressure for 15 min before moving the rats out of the hyperbaric chamber. During treatment, the oxygen concentration in the chamber was kept above 95%.

Real-time uorescence quantitative PCR
Total RNA was extracted from the cerebral cortex of the injured area using Trizol reagent (Invitrogen).
Total RNA (1 μg) was reverse transcribed into cDNA according to the manufacturer's instructions (Takara, Shiga, Japan). After addition of SYBR green I, RT-qPCR analysis was performed in a real-time detection system (Rotor-Gene 6000, Hamburg, Germany). The primers used are listed in Table 2. The PCR ampli cation program was as follows: rst pre-denaturation at 95°C for 3 min, followed by 40 thermal cycles at 95°C for 10 s, 60°C for 30 s, and nally 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s to generate the melting curve. The melting curves were used to ensure that there were no nonspeci c products. Quantitative analysis was performed by the 2 -ΔΔCT method.
Enzyme-Linked Immunosorbent Assay (ELISA) The rat CXCL1 ELISA kit was purchased from Hangzhou MultiSciences (Lianke) Biotech (EK396/2-96, Hangzhou, Zhejiang, China), and the rat CXCR2 ELISA kit was purchased from Cloud-Clone systems (SEC006Ra, Katy, TX, USA). Protein solution from the damaged area of the cerebral cortex was added to 1.5 mL EP tubes containing 250 μL protein lysate and homogenized. The lysis reaction proceeded for 30 min, and the supernatant was collected by centrifugation. The total protein concentration was measured by BCA protein assay (Pierce, Rockford, IL, USA). The sample volume was 100 μg per well, and ELISA was performed according to the manufacturer's instructions. The absorbance of each well was measured at 450 nm and the target protein concentration was calculated based on a standard curve.

Western Blot Analysis
Protein samples for western blot analysis were prepared in the same way as for ELISA. Protein samples (30 μg) were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% BSA for 2 h at RT to avoid non-speci c binding. Following incubation with antibodies, such as p-ERK (9101, rabbit, 1:1000, Cell Signaling, Boston, USA), p-JNK (4688, rabbit, 1:1000, Cell signaling, Boston, USA), p-NF-κB (3033, rabbit, 1:1000, Cell signaling, Boston, USA), or GAPDH antibody (MAB374, mouse, 1:10,000, Millipore, Billerica, MA, USA), at 4°C overnight, the membranes were further incubated with IRDye 800CW antibody for 2 h in the dark at RT. Images were captured using the Odyssey Imaging System (LI-COR Bioscience, Lincoln, NE), and grayscale values were analyzed using Image J software (NIH, Bethesda, MD, USA).

TUNEL Staining
Samples for TUNEL staining were prepared in the same way as described for immuno uorescence double staining. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick 3' end labeling (TUNEL) using an apoptosis detection kit (Vazym), according to the manufacturer's instructions. After completion of apoptosis staining, nuclei were stained with DAPI. The stained slides were examined with a Nikon uorescence microscope and images were obtained with a CCD Spot camera. TUNEL-positive cells in the cerebral cortex of the injured area were counted in each slide using Image J software (NIH, Bethesda, MD, USA).

Statistical Analysis
All data were expressed as MEAN ± SEM. Image J was used to count the number of TUNEL-positive cells and immuno uorescence double-label staining. For western blotting, ImageJ was used to measure the grayscale values of speci c bands. The relative expression levels of p-ERK, p-JNK, and p-NF-κB were standardized to the level of GAPDH. A two-way ANOVA was used to analyze the escape latency and mNSS scores after HBO therapy, and Bonferroni post-test to compare replicate means by row. Multi-group comparisons were performed using one-way ANOVA with post hoc Bonferroni correction. All data were analyzed using GraphPad Prism 5.0 (San Diego, CA, USA).

Results
CXCL1 is predominantly expressed in cerebral cortex astrocytes after TBI To visualize the cellular localization of CXCL1 in the cortex, we performed immuno uorescence double staining of CXCL1 with the astrocyte marker GFAP, the neuronal marker NeuN, and the microglia marker IBA-1, respectively. As shown in Figure 1, about 69% of CXCL1 was co-labeled with GFAP, about 30% with NeuN, and about 1% with IBA-1. The results suggested that CXCL1 was mainly expressed in astrocytes in the cortex of the injured area after TBI. Figure 1. CXCL1 is expressed in astrocytes in the cortex of the injured area after TBI. CXCL1 is co-labeled with astrocyte marker GFAP (A-C). Some co-localization is visible with neuronal marker NeuN (D-F), and to a lesser extent the microglial marker IBA-1 (G-I). Co-labeling rate of CXCL1 with astrocytes is about 69%, with neurons 30%, and with microglia about 1% (J) (Bar=20 μm).
CXCR2 is mainly expressed in cerebral cortex neurons after TBI To con rm the cellular localization of CXCR2 in the cortex, we employed immuno uorescence double staining of CXCR2 with the cellular markers GFAP, NeuN, and IBA-1, respectively. Figure 2 shows that about 85% of CXCR2 was co-labeled with the neuronal marker NeuN, 13% with the microglial marker IBA-1, and 2% with the astrocyte marker GFAP. The results indicated that CXCR2 was mainly expressed in neurons in the cortex of the injured area after TBI. Upregulated CXCL1 and CXCR2 mRNA and protein expression in rat cerebral cortex after TBI The expression level of CXCL1 and CXCR2 mRNA and protein were measured by RT-qPCR and ELISA, respectively, in the peri-injured cortex at 1, 3, 7, and 10 days after TBI. As shown in Figure 3A, the mRNA expression levels of CXCL1 and CXCR2 peaked on the rst day and then decreased compared with the sham group. The protein expression of in ammation-related factors CXCL1 and CXCR2, as shown in Figure 3B, peaked on the rst and third day, respectively, and then showed a decreasing trend compared with the sham group. sham, mRNA expression of CXCL1 and CXCR2 after TBI peaks on the rst day and then decreases. All values are expressed as mean ± SEM (n 6). (B) Compared to sham, expression of CXCL1 and CXCR2 protein after TBI peaks on the rst and third day, respectively, and then decreases. ***p <0.001, *p <0.05.
Up-regulated p-ERK, p-JNK, and p-NF-κB is upregulatedexpression in rat cerebral cortex after TBI The expression trends of p-ERK, p-JNK, and p-NF-κB in the cortex of the injured area were assessed by western Blot at 1, 3, 7, and 10 days after TBI. As shown in Figure 4, compared with the sham group, p-ERK and p-JNK peaked on the third day after TBI, whereas p-NF-κB showed a decreasing trend after peaking on the rst day after TBI. Increased apoptosis of cortical neurons in the injured area after TBI in rats TUNEL staining was employed to detect apoptosis at 1, 3, and 7 days after TBI. Figure 5 shows that the number of TUNEL-positive cells increased at 1, 3, and 7 days after TBI compared with the sham group and showed a decreasing trend after reaching a peak on the rst day.

CXCR2 antagonist improves neurological function in TBI Rats
To evaluate the effect of CXCL1-CXCR2 on the neurological function of TBI rats, mNSS was scored after cortical injection of CXCR2 antagonist in the injured area of TBI rats. As shown in Figure 6, the mNSS of rats after TBI was signi cantly higher than that of the sham group with impaired neurological function. The mNSS score of the TBI +high dose group was lower than that of the TBI+vehicle group after successive 3 days local injection of CXCR2 antagonist in the brain injury area, indicating that downregulation of CXCR2 expression improves neurological function. Figure 6. Improvement of neurological function in TBI rats after application of high dose of CXCR2 antagonist. mNSS scores decrease in TBI rats after application of high-dose CXCR2 antagonist SB225002. Values are expressed as mean ± SEM. ***p <0.001, vs. TBI vehicle; ###p <0.001, vs. sham.

CXCR2 antagonist improves cognitive function in TBI Rats
To evaluate the effect of CXCL1-CXCR2 on the cognitive function of TBI rats, the number of platform crossings and the average escape latency were observed after cortical injection of CXCR2 antagonist in the injured area of TBI rats. As shown in Figure 7, compared with the sham group, the escape latency of TBI rats was signi cantly prolonged and the number of platform crossings was reduced. The escape latency of the TBI +high dose group was lower than that of the TBI + vehicle group, while the number of platform crossings was higher. CXCR2 antagonist inhibits neuronal apoptosis in TBI rats.
To evaluate the effect of CXCR2 antagonist on the apoptosis of cortical neurons in the brain injury area of TBI rats, we performed TUNEL staining. As shown in Figure 8, the number of TUNEL-positive cells in the cerebral cortex of TBI +high dose rats was signi cantly reduced compared with that in the TBI +vehicle group. To verify whether ERK, JNK, and NF-κB regulate the expression of CXCL1/CXCR2, the expression changes of CXCL1/CXCR2 were observed after application of their inhibitors PD98059, SP600125, and BAY117082, respectively. As shown in Figure 9, the mRNA expression of CXCL1 and CXCR2 decreased signi cantly after 3 days of continuous injection of high doses of ERK, JNK, and NF-κB inhibitors compared with the TBI +vehicle group, indicating that ERK, JNK, and NF-κB could regulate CXCL1 and CXCR2.

HBO improves neurological function in TBI rats
The effect of HBO therapy on the neurological function of TBI was observed by mNSS score. We found that after HBO treatment, the mNSS score of the TBI +HBO group was signi cantly lower than that of the TBI group at 3, 7, and 10 days after TBI, and HBO therapy could improve the neurological function of rats after TBI , shown in Figure 10. Figure 10. HBO therapy improves neurological function in TBI rats. mNSS scores are lower in after TBI+HBO than TBI alone at 3, 7, and 10 days after TBI. Values are expressed as mean ± SEM. **p < 0.01, *p < 0. 05.
HBO treatment improved the cognitive function of rats after TBI The Morris water maze was used to assess the effect of HBO on cognitive function in rats. In the locomotor navigation test, as shown in Figure 11A, the escape latency was signi cantly shorter in the TBI + HBO group than in the TBI group on days 5 and 6. In the 7-day spatial exploration trial after TBI, the number of platforms traversed by the TBI +HBO group was signi cantly higher than that of the TBI group. From these results, we can see that HBO treatment can improve the cognitive function of rats after TBI. Figure 11. HBO treatment improves cognitive function in TBI rats. (A) In the locomotor navigation test, the escape latency is signi cantly lower after TBI+HBO than in TBI on days 5 and 6. (B) In the exploration test, the number of platform crossings is higher after TBI+HBO than TBI alone. Values are expressed as mean ± SEM. ***p < 0.001, *p < 0. 05.

HBO treatment inhibits neuronal apoptosis in rats with TBI
We evaluated the effect of HBO treatment on neuronal cell apoptosis in the cerebral cortex of rats with TBI. In Figure 12, the results show that the number of TUNEL-positive cells in the cerebral cortex of the TBI +HBO group was signi cantly reduced compared with the TBI group, indicating that HBO treatment could inhibit apoptosis of neuronal cells in TBI rats.

HBO treatment downregulates CXCL1 and CXCR2 mRNA and protein expression after TBI in rats
To verify whether HBO treatment regulates expression of CXCL1/CXCR2, the mRNA and protein contents of CXCL1 and CXCR2 were detected by RT-qPCR and ELISA, respectively, after 3 days of continuous HBO therapy. In Figure 13, the results show that the mRNA and protein expression of CXCL1 and CXCR2 were signi cantly decreased compared with the TBI group, indicating that HBO treatment could down-regulate the expression of CXCL1 and CXCR2. HBO treatment down-regulates the expression of p-ERK, p-JNK, and p-NF-κB after TBI in rats To verify whether HBO therapy regulates CXCL1/CXCR2 expression by modifying ERK, JNK, and NF-κB, we tested the expression level of p-ERK, p-JNK, and p-NF-κB proteins by western blot after 3 days of continuous HBO treatment. In Figure 14, the results show that the levels of p-ERK, p-JNK, and p-NF-κB were signi cantly lower in the TBI +HBO group compared with the TBI group, suggesting that HBO treatment inhibits expression of CXCL1 and CXCR2 by downregulating the expression of p-ERK, p-JNK, and p-NF-κB. Figure 14. HBO treatment down-regulates expression of ERK, JNK, and NF-κB. p-ERK, p-JNK, and p-NF-κB expression is signi cantly decreased after continuous HBO treatment for 3 days compared to TBI alone. Values are expressed as mean ± SEM. **p < 0.01; *p < 0.05.

Discussion
Neuroin ammation is an important early reversible process underlying secondary injury following TBI, and thus a promising therapeutic target. Activation and proliferation of glial cells (astrocytes and microglia) in damaged brain can harm nerve cells by releasing cytokines and chemokines [29].
Chemokines are divided into pro-in ammatory and anti-in ammatory factors, and after the occurrence of TBI, chemokines help attract a wide range of immune cells to the site of injury [30]. Numerous such in ammatory mediators have been detected in both TBI animal models and patients, including CCL2, CCL3, CXCL1, CXCL2, CXCL10, CXCL12, CCR2, CCR5, CXCR4, and CX3CR1 [9,21,[31][32][33][34][35]. These chemokines act as chemoattractants for peripheral blood leukocytes that can produce further damage through direct cellular toxicity. In TBI, CCL2/CCR2 is involved in disease progression, and CCL2 is one of the chemokines that is signi cantly upregulated within 24 h of injury in TBI animal models [4]. CCL2 has also been detected in cerebrospinal uid, serum, and brain tissue from TBI patients, and animal studies showed that down-regulation of CCR2 expression signi cantly improved neurological function in TBI rats [36][37][38][39]. CXCL12/CXCR4 signaling is also involved in the pathological progression of TBI [35,40]. On days 3 and 7 after TBI, upregulation of CXCR4 expression has been observed in the cortex surrounding the injured area, and intracranial injection of CXCL12 induced angiogenesis, improved edema, reduced blood brain barrier permeability, and decreased the number of apoptotic cells [41].
The cytokine CXCL1 contributes to neuroin ammation primarily through binding to CXCR2 [42,43]. In TBI model rats, local expression of CXCL1 peaked within 4 h after injury and was sustained for hours thereafter [4]. Joanna and colleagues reported that choroidal epithelial cells induced neutrophil in ltration after TBI by secreting CXC-family chemokines [44]. In this study, we found that the number of apoptotic cells increased in the cortical vicinity of the injured area after TBI, and neurological function was impaired. The expression of CXCL1 and CXCR2 increased after TBI, and cell localization experiments revealed that CXCL1 was mainly expressed in astrocytes, while CXCR2 was expressed in neurons. The decrease in neuronal apoptosis and improvement in neurological function after application of an CXCR2 antagonist suggested that CXCL1-CXCR2 mediated the interaction between activated astrocytes and neurons after TBI, aggravating secondary injury. Collectively, these results suggest that CXCL1 signaling induces a sustained intracerebral in ammatory response in the hours and days following TBI. Our previous studies showed that during in ammation, CXCL1 expression in primary cultured astrocytes increased through the NF-κB, ERK, and JNK signaling pathways, and that CXCL1 binding to its receptor mediated the in ammatory response, thereby playing an important role in TBI [17]. We further found that the expression of p-ERK, p-JNK, and p-NF-κB increased after TBI, while ERK, JNK, and NF-κB inhibitors decreased the expression of CXCL1 and CXCR2, suggesting that ERK, JNK and NF-κB can exert neuroin ammatory effects by regulating downstream CXCL1 after TBI in rats. Thus, the NF-κB/ MAPKs (JNK and ERK)-CXCL1 in ammatory pathway has a role in post-TBI neuroin ammation.
HBO treatment exerts neuroprotective effects by modulating multiple neuroin ammatory pathways after neurological injury. After spinal cord injury in rats, HBO treatment inhibits the high-mobility group protein B1/ NF-κB and Toll-like receptor 2 signaling pathways, thus reducing secondary injury caused by in ammation and promoting the recovery of neurological function [45,46]. Some animal studies have shown that HBO treatment reduced the expression of caspase-3, TNF-α, IL-6, and IL-1β by inhibiting the Toll-like receptor 4/NF-κB signaling pathway after TBI in rats, thus reducing the secondary injury caused by in ammation after TBI and promoting the recovery of neurological function [20]. IL-10 plays an important role in the neuroprotective effects of HBO on TBI, and IL-10 de ciency leads to increased brain damage after TBI and decreased suppression of neuroin ammation by HBO [18]. HBO therapy can reduce neuronal apoptosis in the acute phase of TBI by regulating the Akt/GSK3β/β-catenin pathway [12]. We showed in a previous study that HBO therapy modulates the NF-κB/MAPKs -CXCL1 signaling pathways to suppress in ammation induced by lipopolysaccharide in primary astrocytes of neonatal rats [17]. In the present study, we found that HBO therapy could downregulate the expression of p-ERK, p-JNK, p-NF-κB, CXCL1 and CXCR2, while reducing neuronal apoptosis and improving the motor, sensory, and cognitive neurological functions of TBI rats, ultimately alleviating secondary injury. In summary, the results of this study suggest that HBO therapy can play a neuroprotective role by regulating the NF-κB/ MAPKs (JNK, and ERK)-CXCL1 in ammatory pathway to modulate neuroin ammation.

Conclusion
Post-TBI CXCL1-CXCR2 mediates the interaction of activated astrocytes and neurons, exacerbating secondary injury. HBO therapy can exert neuroprotective effects by regulating the NF-κB/ MAPKs (JNK, and ERK)-CXCL1 in ammatory pathway to modulate neuroin ammation after TBI. This study provides theoretical and experimental evidence for the clinical use of HBO in the treatment of brain injury.

Consent for publication
Not applicable.

Availability of data and materials
All data used during the current study available from the corresponding author on reasonable request.

Con ict of Interest
The authors declare that they have no con icts of interest related to this study.