Anacardic acid improves neurological deficits in traumatic brain injury by anti-ferroptosis and anti-inflammation

Background: Traumatic brain injury (TBI) is an important cause of disability and death. TBI leads to multiple forms of nerve cell death including ferroptosis due to iron-dependent lipid peroxidation. Anacardic acid (AA) is a natural component extracted from cashew nut shells, which has been reported to have neuroprotective effects in traumatic brain injury. We investigated whether AA has an anti-ferroptosis effect in TBI. Methods: We used the Feeney free-fall impact method to construct a TBI model to investigate the effect of AA on ferroptosis caused by TBI, in which Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, served as a positive control group. We first identified the therapeutic effect of AA on TBI through modified neurological severity score (mNSS) and determined the appropriate concentration. Secondly, we investigated the effect of AA on the expression level of the key protein of ferroptosis by Western blotting and immunohistochemistry. Then the effect of AA on nerve tissue injury and nerve function improvement was verified. Finally, enzym-linked immunosorbent assay (ELISA) was used to verify that AA could reduce inflammation after TBI. Results: We found the intensely inhibitory effect of AA on ferroptosis, which is in parallel with the results obtained after Fer-1 treatment. In addition, AA and Fer-1 mitigated TBI-mediated tissue defects, destruction of the blood-brain barrier, and neurodegeneration. Novel object recognition (NOR), mNSS and water maze test showed that AA could significantly reduce the impairment of neural function and behavioral cognitive ability caused by TBI. Finally, we also demonstrated that AA has not only an anti-ferroptosis effect, but also an anti-inflammation effect. Conclusions: AA can reduce the neurological impairment and behavioral cognitive impairment caused by TBI through the dual effect of anti-ferroptosis and anti-inflammation.


Introduction
In today's society, the disability and death rate of traumatic brain injury (TBI) remain high (Meyfroidt et al., 2017).Patients who have experienced TBI often have residual limb movement and behavioral cognitive impairment, which can seriously affect their quality of life (Haarbauer-Krupa et al., 2021;Ware et al., 2020).TBI often leads to destruction and defect of brain tissue, and further causes secondary brain injury, such as increased cerebral edema, destruction of the bloodbrain barrier (BBB), neuroinflammation and oxidative stress (Jha et al., 2019;Khatri et al., 2018;Corrigan et al., 2016).These pathophysiological changes eventually lead to the death of a large number of nerve cells in the brain tissue, including necrosis and programmed cell death (PCD) such as apoptosis, autophagy and ferroptosis (Hu et al., 2022).
Ferroptosis is a new type of cell death discovered in recent years.It is an iron-dependent oxidative cell death induced by small molecules (Jiang et al., 2021).Distinct from other types of cell death, iron death is characterized by mitochondrial shrinkage, ridge disappearance, iron accumulation and reactive oxygen species (ROS) production, and GPX4 inactivation (Chen et al., 2021).Iron metabolism is closely related to TBI (Tang et al., 2020).After TBI, tissue defects, damage to the integrity of the blood-brain barrier (BBB), and vascular injury can lead to iron deposition in the brain parenchyma (van Vliet et al., 2020).Several proteins maintain iron homeostasis, such as transferrin receptor-1 (TfR1), which regulates iron consumption, and transferrin (Fpn), which regulates iron output (Huang et al., 2022;Guo et al., 2016).After TBI, iron metabolism disorders lead to iron accumulation in iron pools, induce iron-dependent lipid peroxidation, and ultimately lead to neuronal death (Ge et al., 2022;Zhang et al., 2019;Cheng et al., 2016).GPX4 is a glutathione peroxidase that plays a crucial role in the regulation of iron death, and it has a direct detoxification effect on hydroperoxides in membrane lipids (Seibt et al., 2019).The inactivation or decreased expression of GPX4 after TBI will lead to iron catalyzed metabolism disorder of intracellular lipid peroxides, resulting in ROS accumulation on membrane lipids, cell redox imbalance and induction of ferroptosis (Xiao et al., 2019;Fang et al., 2023;Gao et al., 2021).
Anacardic acid (AA), also known as hydrogenated ginkgo acid, is an acid substance isolated from cashew shell extract (Hollands et al., 2016).Previous studies have shown that AA has anti-inflammatory, anticancer, anticonvulsant, antioxidant and neuroprotective pharmacological effects, and can improve body damage by inhibiting inflammation and oxidative stress (Yang et al., 2018;Gao et al., 2022;Gomes Junior et al., 2020;Luiz Gomes et al., 2018).Previous studies have shown that AA improves neurological deficits, reduces nociception, BBB permeability, axonal degeneration, gliosis, microglial activation, and neuronal death (De-Yong et al., 2017;Karen-Amanda et al., 2018).However, whether AA can ameliorate persistent cognitive deficits by alleviating the degree of ferroptosis through alleviating inflammation and antioxidant response after TBI has not been clearly studied.
In order to explore this issue, we first evaluated the expression levels of iron death related proteins at different time points after TBI and the time nodes for significant changes for subsequent studies.Next, we used Ferrostatin-1 (Fer-1), an ferroptosis inhibitor, as a positive control group to investigate the effects of AA on the expression levels of key ferroptosis related proteins and iron deposition in tissues.Finally, the improvement of nerve tissue and function by AA and its mechanism were further explored.

Experimental animals and grouping
The ICR male mice (6-8 weeks, 20-30 g) used in this experiment were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., LTD., and were fed for 7 days in advance for animal experiment.All mice were fed in isolated cages with independent air supply, with room temperature controlled at 22-25 • C, humidity of 70%-75%, regular light, and adequate supply of water and food.All animal experiments were conducted in accordance with the animal welfare policy of The Affiliated Huai'an Hospital of Xuzhou Medical University and were approved by The Affiliated Huai'an Hospital of Xuzhou Medical University.The experimental animals were randomly divided into Sham group, TBI group, TBI + Fer-1 group and TBI + AA group.The Sham group was the sham operation group, only the bone window was opened without trauma.Fer-1 (MedChemExpress, USA) was also though intraperitoneal 30 min post-TBI and the final usage of Fer-1 was 2 mg/kg.AA (100 mg/kg, ip) is administered once 30 min after TBI and then once a day for seven days.Fer-1 and AA were both dissolved in DMSO, and the same dose of DMSO was correspondingly given in Sham group and TBI group to reduce the influence of confounding factors on the experimental results.

Construction of TBI mice model
In this study, Feeney free-fall impact method was used to construct a mice model of TBI (Ma et al., 2019).In simple terms, the steps are as follows: First, mice were anesthetized with isoflurane by an inhalation anesthesia machine (M5209, Changsha Maiyue Biotechnology Co., Ltd.), fixed in a stereoscope, and routinely disinfected with iodophor.Second, the scalp of the mice was incised along the median line of the top of the forehead for about 1.5 cm, exposing the skull and stripping the periosteum.Drilling was then performed with a 2.3 mm diameter cranial rotatory to create a bone window of about 5 mm diameter, exposing the parietal lobe while maintaining the integrity of the dura mater as much as possible.The craniocerebral injury striking device is set for striking.The specific parameters are: weight 20 g, falling height 15 cm, striking depth 1.5 mm, simulating moderate TBI.Finally, properly stop bleeding and tightly seal and scalp, and return to the original cage.The whole process is carried out in accordance with the aseptic operation process, the instruments used are autoclaved in advance, and other instruments and consumables that cannot be autoclaved are continuously irradiated with ultraviolet light for 12 h.

TEM
1mm 3 small pieces of brain tissue within 2 mm around the injury site from normal mice and mice with TBI were rapidly placed in electron microscope fixation solution, fixed at room temperature for 2 h away from light, and then transferred to 4 • C for preservation.Then all specimens were rinsed with 0.1 mol/L phosphate buffered saline (PBS) (PH7.4) for 3 times for 15 min each time.Then they were fixed with 1% osmic acid in 0.1 mol/L PBS at dark room temperature for 2 h.Then, the samples were rinsed with 0.1 mol/L PBS for 3 times for 15 min each time.The tissues were dehydrated with increasing concentration of alcohol and 100% acetone.Then infiltrated slice (60-80 nm).Finally, the uranium lead was double stained, the slices were dried at room temperature overnight and observed under transmission electron microscope for image analysis.

Western blotting
We took protein from the brain tissue of five mice in each group for Western blots, meaning we performed five replicates.After anesthesia, mice were injected with normal saline, corresponding brain tissue was taken into a centrifuge tube, and strong cell lysate containing protease inhibitor was added for 20 min.After being homogenized by the ultrasonic homogenizer, it was left for 20 min, kept at 4 • C in the centrifuge, and centrifuged at a speed of 12,000 g for 15 min.The supernatant was retained and the protein concentration was quantified using the protein quantification kit (UA276918, Thermo Fisher Technologies, USA).Add a certain amount of loading buffer and boil at 95 • C for 10 min to complete sample preparation.Prepare the electrophoresis apparatus, electrophoretic solution and gel, and take the protein sample in turn, then electrophoresis, and stop when the sample runs to the lower edge of the gel.After electrophoresis, the gel was taken for membrane transfer.After the protein was transferred to the polyvinylidene fluoride membrane, the membrane was sealed in 50 ml/L skim milk powder solution for 1-2 h.After closure, it was placed in a cold storage at 4 • C for incubation night.On the next day, wash with PBS buffer for 3 times, 5 min each time, incubate the corresponding secondary antibody at room temperature for 2 h, and then wash with PBS buffer for 3 times again, 10 min each time.Luminescent solution was configured and protein band detection and gray scale analysis were performed by imaging system.The main reagents include the primary antibody TfR1 (1:1000, Proteintech, USA) and GPX4 (1:1000, Proteintech, USA).The secondary antibodies include HRP labeled goat anti-mouse IgG (1:2000, Servicebio, China) and HRP labeled goat anti-rabbit secondary antibody (1:2000, Servicebio, China).

Immunohistochemistry
Immunohistochemical (IHC) assays of TfR1 and GPX4 were performed with reference to previous literature (Chitambar et al., 2018;Zhao et al., 2017).Brain tissue within 2 mm around the injury site was taken from 5 mice in each group for follow-up studies, and the experiment was repeated at least three times in each group.In simple terms, dewaxing the section blocks endogenous peroxidase.Then the slices Y. Liu et al. were placed in 3% hydrogen peroxide solution, incubated at room temperature away from light for 25 min, and the slides were placed in PBS and washed three times on a decolorizing shaker for 5 min each time.Serum sealing: the tissue was uniformly covered with 3% bovine serum albumin (BSA) (Servicebio, China) in the tissue circle, and closed at room temperature for 30 min.TfR1 monoclonal antibody (1:300, Proteintech, USA) and GPX4 polyclonal antibody (1:1000, Proteintech, USA) were added respectively, and staining was performed by immunohistochemical EnVision method.The dyeing steps were carried out in strict accordance with the kit instructions: DAB color development, hematoxylin redyeing, bluing back, ethanol dehydration, xylene transparent, neutral gum sealing, and reading under a white light microscope.Secondary antibody reagents include Goat Anti-Mouse IgG labeled by HRP (1:200, Servicebio, China) and Goat Anti-Rabbit igg labeled by HRP (1:200, Servicebio, China).Image J was used to quantitatively analyze the total signal intensity of the IHC positive area.

Perl's staining
Iron deposition in tissues was determined by Perl's staining.Five mice in each group were used for follow-up studies.First, 2% potassium ferrohydride and 2% hydrochloric acid were mixed in equal proportions, sliced into the mixed solution, stained for 30 min, and washed twice with distilled water.Then, DAB color developing liquid drops were dyed for about 5-10 min, and the degree of color development was controlled under the microscope.Then, the dye solution was poured out and washed with 0.01 mol/L PBS solution for 1 time and distilled water for 3 times.Then stained with hematoxylin for 1 min, washed with tap water, differentiated by hydrochloric acid aqueous solution, washed with tap water, ammonia aqueous solution returned to blue, washed with tap water.Finally, the dewaxed seals were examined under a microscope, and the images were collected and analyzed.Image J was used to quantitatively analyze the area signal intensity of iron-positive regions deposited in tissues.

Lesion degree assessment
In order to determine whether AA and Fer-1 can reduce tissue defects after TBI, HE section staining was performed at the maximum coronal level of brain tissue defects 21 days after TBI (n = 6 mice per group).First, the brain tissue was made into paraffin sections and then dewaxed, and then the sections were placed into hematoxylin dye solution for 3-5 min, washed with tap water, differentiated solution, washed with tap water, returned blue solution, and rinsed with running water.After that, the slices were dehydrated with 85% and 95% gradient alcohol successively for 5 min, and then stained with eosin dye for 5 min.Add anhydrous ethanol (3 times, 5 min each time), xylene (2 times, 5 min each time), and seal with neutral gum.We started the section from the site with the greatest coronary defect, with a thickness of 30 μm.We cut the section with the greatest coronary defect to the normal brain tissue without defect on both sides, and calculated the brain tissue defect volume by NIH Image J software (Bethesda, MD, USA).

Evans blue extravasation assay
BBB permeability was assessed by measuring the exosmosis of Evans blue (EB) dye in brain tissue 3 days after TBI (n = 6 animals per group).Mice were anesthetized 2 h after intravenous injection of EB dye (2%, 2 μL/g) through the tail vein, and intraventricular infusion of PBS through the left ventricle to adequately eliminate the localized dye from sinus bleeding.The brain tissue was first weighed and then each sample was soaked in formamide solution and homogenized at a concentration of 200 mg of tissue per milliliter.37 • C warm bath for 48 h, centrifuge at 5000 rpm for 15 min, divide into two layers, transfer supernatant.Finally, a spectrophotometer (BioTek, Winooski, VT, USA) was used to measure the absorbance of the mixture at 632 nm.

Nissl staining
Nissl staining was used to evaluate the degree of neuronal degeneration in the damaged cerebral cortex within 2 mm around the injury site.
Five mice in each group were used for follow-up studies.Slices (10 μm) dehydrated with gradient alcohol: 75% alcohol for 15 min, 90% alcohol for 25 min.Slices were placed in toluidine blue dye solution (no.G1032, Servicebio, China) for 2-5 min, washed with water, slightly differentiated with 0.1% ice acetic acid, the reaction was terminated by washing with tap water, and the degree of differentiation was controlled under a microscope.After washing with tap water, the slices were dried in the oven.Then put the slices into clean xylene transparent for 10 min, neutral gum seal.Finally, the image was examined with a microscope, and further completed the image acquisition and analysis.

Fluoro-Jade B Staining
Staining of neurons with degenerative changes was examined with Fluoro-Jade B Staining(FJB).The brain tissue within 2 mm around the injury site of 5 mice in each group was sliced.The slices were put into environmentally friendly dewaxing solution I (no.G1128, Servicebio, China)10 min, environmentally friendly dewaxing solution II 10 min, environmentally friendly dewaxing solution III 10 min, anhydrous ethanol I 5 min, anhydrous ethanol II 5 min, anhydrous ethanol III 5 min, and finally washed with distilled water.Using 50% glacial acetic acid as solvent, the FJB solution was prepared at 1:400, and the diluted FJB green fluorescent probe was added and left at 4 • C overnight.DAPI staining for 8 min, rinse with pure water.Dry with a hair dryer, xylene transparent for 1 min, super clean fast dry sealing sheet rubber sealing sheet.Sections were observed with a microscope (Nikon Eclipse C1, Japan).The count of FJB positive cells was calculated and determined using NIH Fiji software (Bethesda, MD, USA).

Behavioral experiments
The modified neurological severity score (mNSS) was applied on day 1, day 3, day 7 and day 14 after the experiment to evaluate the motor (n = 5 mice per group), sensory and reflex functions of mice, and to determine the severity of nervous system injury (Ma et al., 2018).The highest score of mNSS is 18 points, and the higher the score, the more serious the nervous system injury.
The novel object recognition experiment (NOR) was used to explore the ability of mice to perceive and remember new objects in the environment (Zhang et al., 2022a).To put it simply, the mice were first trained in a square black-walled iron box (100 cm in diameter and 50 cm in height) for 3 days.During the training process, an identical object (yellow cuboid) was placed in a symmetrical position 20 cm away from the wall of the box, and the mice were placed from the iron box wall in front of the middle of the two objects and freely explored for 5 min.On day 5, a familiar object (yellow cuboid) was replaced with a new object (white cylinder), and the mice were placed in the iron box to explore freely for 5 min (n = 6 mice per group).The exploration of an object is defined as follows: the mouse nose is <2 cm away from the object or it touches the object with its nose.Sitting or leaning on objects is not considered exploratory behavior.New object recognition rate (NORI) = residence time of new object / (residence time of new object + residence time of old object) × 100%.
Cognitive function was assessed through the Morris water maze (Tucker et al., 2018).All data during the test were recorded and evaluated using a video tracking system (Anhui Zhenghua Biological Instrument Equipment Co., LTD.: Huaibei, China).The pool is 120 cm in diameter and 40 cm in height.The water is colorless and transparent pure water, and the temperature is set at 24 • C. The water maze is divided into four quadrants, and a movable platform is placed 1.0 cm below the surface of the water in the center of the target quadrant.During the first phase of the test (the first 7 days of the water maze test), Y. Liu et al. training was performed 4 times a day using a hidden platform in the water maze (n = 6 mice per group).Facing the wall of the pool, the rats were randomly lowered into the water from one quadrant.Mice that successfully found the platform within 90 s were allowed to stay on the platform for 10 s, while mice that failed to find the platform were guided to the platform and were also allowed to stay for 10 s.In the second phase (day 8 of the water maze test), the platform was removed and the mice were placed in the pool from the opposite quadrant of the platform for a single test.Each mouse swam for 90 s.The number of mouse platform station crossing and the stay time in the target quadrant were recorded.

Enzyme-linked immunosorbent assay (ELISA)
On the 7th day after injury, the brain tissue around the injury site was extracted (n = 6 mice per group), and the homogenate of 10% brain tissue was prepared by adding PBS, and the supernatant was removed after centrifugation at 3000 r/min for 10 min.The expression levels of IL-6, TNF-α, IL-1β and CXCL1 in brain tissue of mice in each group were detected by ELISA.The specific steps are strictly in accordance with the ELISA kit instructions (Bosters Biological Technology, Wuhan, China).

Statistic analysis
Statistical analysis of data was performed with the Student's t-tests or one way ANOVAs by SPSS 20.0.P values <0.05 was considered to be statistically significant.The results were expressed as mean ± SD in the figures (*P < 0.05, **P < 0.01, ***P < 0.001).

Validation of the effect of AA on TBI
In order to confirm the therapeutic effect of AA on TBI and to determine its appropriate concentration, we initially analyzed the effect of AA on neurological deficits.The results showed that mNSS was significantly improved after AA treatment compared with the control group (P < 0.05) (Fig. 1A).This suggests that the neurological function of mice treated with AA is better and the secondary injury is relatively mild.After clarifying the therapeutic effect of AA, the concentration gradient of AA was further studied, and the appropriate concentration was selected for follow-up experiments.Through literature review, it is known that AA concentration at 0-200 mg/kg will not produce toxic and side effects on mice, while AA concentration at 300 mg/kg will produce serious toxic and side effects.Therefore, we selected four concentrations of 10 mg/kg, 50 mg/kg, 100 mg/kg and 150 mg/kg (Carvalho et al., 2013;Junior et al., 2019).The results showed that AA concentration at 10-150 mg/kg had a clear effect on improving nerve function in a concentration-dependent manner, but there was no significant difference in improving nerve function at 100-150 mg/kg (Fig. 1B).In the follow-up studies, the dosage of AA was 100 mg/kg.

Ferroptosis was detected after TBI
In order to confirm the role of ferroptosis in TBI, we compared the ferroptosis-related characteristics of normal brain tissue and TBI brain tissue, including mitochondrial morphology, mitochondrial ridge, membrane density, and expression levels of ferroptosis-related proteins (Qin et al., 2021).Electron microscope results showed that compared with normal brain tissue, the mitochondrial membrane of brain tissue after brain injury was wrinkled and the density increased, and the mitochondrial ridge decreased, indicating that ferroptosis would occur after TBI (Fig. 2A).As a specific marker of ferroptosis, the expression of TfR1 increased significantly after TBI and gradually decreased over time (Fig. 2B, C).GPX4 detoxises lipid peroxidation through glutathione (GSH) and plays an important role in inhibiting ferroptosis, and its expression level decreases after TBI (Fig. 2B, C).In conclusion, TBI can lead to ferroptosis, and the expression levels of TfR1 and GPX4 will change significantly 1 day after TBI.

AA inhibits ferroptosis
After confirming the presence of ferroptosis after TBI and knowing the time-dependent changes in the expression levels of selected ferroptosis related proteins, we chose 24 h after injury to detect the effect of AA on ferroptosis, in which Fer-1was used as a positive control.By western blotting, we found that AA significantly inhibited the expression of TfR1, and its expression was significantly elevated 1 day after TBI (Fig. 3A-C).In addition, AA reversed the depletion of GPX4 caused by TBI, but not as effectively as Fer-1 (Fig. 3A-C).In order to further verify the above conclusions, the expression levels of TfR1 and GPX4 in tissues were further compared by immunohistochemistry. Overall comparison showed that AA inhibited ferroptosis, but its degree of inhibition on ferroptosis was lower than that of Fer-1 (Fig. 3D-G).In addition, ironpositive cells were observed in the sections of the TBI group.However, Fer-1 and AA treatments reduced iron deposition in tissues, respectively (Fig. 3H, I).This further proves that AA has an anti-ferroptosis effect.

AA alleviates brain tissue defects and BBB damage
Usually, after TBI, brain tissue defects will occur in the area of trauma, and the greater the degree of tissue defects in the same functional area, the greater the loss of neural function will be relatively greater (Yao et al., 2019).After 21 days of TBI, the mice were euthanized, and the brain tissue was stained with HE, and the coronal section with the largest defect was selected to compare the degree of brain tissue defect.The results showed that both Fer-1 and AA could reduce brain tissue defects (Fig. 4A, B).BBB damage is also one of the characteristics after TBI, and the permeability of BBB after TBI was detected by EB exosmosis (Shen et al., 2019).BBB permeability in injured hemispheres was significantly reduced after Fer-1 and AA treatment compared with TBI group (Fig. 4 C, D).In addition, AA treatment has a better protective effect on BBB than Fer-1, indicating that AA is more effective in protecting BBB.

AA reduces TBI-induced neuronal loss and neurodegeneration
We examined brain tissue near the injury site.The neurons of normal brain tissue are clear and complete, and the number of irregular cell bodies, wrinkled and deeply stained nuclei of neurons after TBI is significantly increased (Huang et al., 2021).Compared with the control group, treatment with Fer-1 and AA reduced the loss of neuronal cells caused by TBI (Fig. 5A, C).In addition, FJB staining of brain tissue sections indicated that TBI increased the number of degenerative cells compared with normal brain tissue, while the number of degenerative cells decreased in brain tissue treated with Fer-1 and AA (Fig. 5B, D).These results suggest that the number of cells with degenerative changes in brain tissue can be reduced by inhibiting ferroptosis.

AA alleviates neurological deficits after TBI
We then performed behavioral tests on the treated mice.The effect of AA on recognition memory of TBI mice was evaluated by calculating NORI on day 28 after TBI.NORI was significantly lower in the TBI group than in the Sham group, suggesting impaired memory.Compared with the TBI group, the TBI + AA and TBI + Fer-1 groups had a longer time to recognize new objects and a significantly higher NORI (Fig. 6A).This suggests that AA or Fer-1 can mitigate the cognitive impairment caused by TBI in mice.mNSS is a combination of motor, sensory, reflex, and balance tests used to assess the severity of trauma.The higher the score, the more serious the injury (Wang et al., 2022).Moderate injury is considered to be a score between 10 and 15, and all mice scored in this range on the day of the brain injury (Liu et al., 2023).The mNSS scores of the AA and Fer-1 treated groups and mice were lower than those of TBI group, and the scores of the TBI + AA group were lower than those of the Fer-1 + AA group, indicating that inhibition of ferroptosis can improve nerve function and that the improvement of nerve function of AA is greater than that of Fer-1(Fig.6B).
The water maze test was performed on days 14-21 after TBI.Compared with mice in the Sham group, mice in the TBI group showed impaired spatial learning and memory.AA or Fer-1 treatment significantly reduced spatial learning and memory deficits in rats after brain injury (Fig. 6C).After TBI, the number of mice crossing the water platform and the proportion of activity time in the target area were significantly reduced; however, AA or Fer-1 treatment significantly increased the number of mice crossing the water platform and the proportion of activity time in the target area, proving that AA and Fer-1 can reduce the deficits of spatial learning ability and memory ability caused by TBI (Fig. 6D, E).

AA has anti-inflammatory effect in TBI
Our previous studies have shown that AA has better therapeutic effect on TBI than Fer-1 alone, indicating that AA has other effects besides anti-ferroptosis.Studies have shown that AA can inhibit IL-6 and play an anti-inflammatory role (Al-Roub et al., 2021).So we further explored whether AA has an anti-inflammatory effect in TBI.We measured the production levels of pro-inflammatory cytokines (IL-6, TNF-α and IL-1β) and chemokines (CXCL1).Compared with the TBI group and the TBI + Fer-1 group, the TBI + AA group mice had the lowest levels of inflammatory factors, indicating that AA has anti-inflammatory effects, which is another reason why AA alleviates the damage of TBI nerve function.However, by measuring the expression level of IL-6, we found that Fer-1 could inhibit TBI-mediated inflammation to a certain extent, but its effect was not as good as that of AA at the selected time node (Fig. 7A-D).As for the specific mechanism of action between inflammation produced by AA and TBI, we will further explore it in subsequent experimental studies.

Discussion
As we all know, TBI mainly includes primary injury and secondary injury, and there is nothing we can do about primary injury.Like a firefighter fighting a fire, he can only protect what has not yet burned and save what has not yet been reduced to ashes, and most of all surgeries and treatments are aimed at alleviating secondary brain damage (Vella et al., 2017).Edema, inflammation, and programmed cell death are typical pathophysiological processes of TBI (Kaur and Sharma, 2018).Therefore, the treatment of TBI requires comprehensive treatment of its secondary injury.In recent years, ferroptosis has been found to be a form of neocell death, which plays an important role in the occurrence and development of TBI, liver injury, cardiovascular disease, tumor and other diseases (Chen et al., 2022;Zhao et al., 2022;Zhang et al., 2022b).Our results on the changes of TfR1 and GPX4 expression Ferroptosis is involved in secondary injury after TBI, but its exact role in TBI is unknown.Recent studies have found that the ferroptosis pathway is activated after TBI and is involved in nerve cell death (Shen et al., 2020).Ferroptosis is an iron-dependent cell death characterized by excessive accumulation of intracellular reactive oxygen species (ROS), which cannot be inhibited by inhibitors such as apoptosis, pyrodeath and autophagy, but can be inhibited by antioxidants and iron chelating agents (Park and Chung, 2019).Morphologically, ferroptosis is obviously different from apoptosis, pyrodeath and autophagy, mainly showing that the volume of mitochondria decreases, the density of bilayer membrane increases, and the matrix decreases or disappears (Liu et al., 2020).Our experiments also confirmed that ferroptosis occurred after TBI, the expression levels of key ferroptosis proteins GPX4 and TfR1 changed, and the corresponding membrane shrinkage and ridge disappearance occurred in mitochondria.
Previous studies have shown that AA has a variety of pharmacological effects such as anti-inflammatory, anticancer, anti-convulsant and anti-oxidation, and can improve body damage by inhibiting inflammation and oxidative stress (Yang et al., 2018;Gao et al., 2022;Gomes Junior et al., 2020;Luiz Gomes et al., 2018).However, the improvement of persistent cognitive deficits caused by TBI by AA has not been clearly studied.In this experiment, we first confirmed that AA has an improving effect on nerve function deficit caused by TBI, and further verified whether AA has an anti-ferroptosis effect.The results showed that AA had anti-ferroptosis and anti-inflammatory effects, alleviating brain tissue defects, protecting BBB and reducing nerve function defects.At the same time, we also noticed that AA and curcumin have some of the same structure.Curcumin is a relatively widely studied drug, which has anti-inflammatory, antioxidant and brain tissue protective effects in TBI (Qian et al., 2021).Moreover, curcumin plays an important role in the treatment of other diseases such as acute kidney injury and liver injury (Wei et al., 2020;Zheng et al., 2022).As AA with similar structure to curcumin, its therapeutic effect in TBI may be the same as that of curcumin.We will analyze drugs with similar structure in subsequent studies, focusing on clarifying the structure that plays the main pharmacological effect.
Our experiment found that AA has anti-ferroptosis effect in TBI, which can affect the expression level of ferroptosis key proteins.
Moreover, both AA and Fer-1 can reduce iron deposition in tissues and reduce ferroptosis.By examining the degree of tissue defect and BBB, we found that both AA and Fer-1 could reduce the damage caused by TBI.However, in behavioral experiments, we found that AA had a better effect than Fer-1 in improving the neurological impairment and behavioral cognitive ability of TBI mice, which indicates that AA not only has an anti-ferroptosis effect, but also has other therapeutic effects on TBI.In further experiments, we found that AA also has antiinflammatory effects in TBI, which is why AA has a greater effect on improving neurological function (Fig. 8).At the same time, by measuring the expression level of IL-6, we found that Fer-1 could inhibit TBI-mediated inflammation to a certain extent, but its effect was not as good as that of AA at the selected time node.In addition, the effect of AA on TfR1 and GPX4 protein expression and iron deposition can only partially explain the anti-ferroptosis effect of AA, and further evidence will be added if oxidative lipidomics studies are carried out.Due to the various forms of TBI secondary injury, its treatment requires comprehensive treatment (Meyfroidt et al., 2022).AA can be anti-ferroptosis, anti-inflammatory and improve BBB permeability.More pharmacological effects of AA in TBI need to be further discovered and used in the clinical treatment of TBI.
In conclusion, AA can inhibit ferroptosis caused by TBI.Secondly, AA can reduce brain tissue defects and reduce BBB damage.AA therapy can reduce TBI-induced neuronal loss neurodegeneration.The reason why AA is better than Fer-1 in improving nerve function deficit after TBI is that AA not only has anti-ferroptosis effect, but also can reduce inflammation after TBI.

Declaration of Competing Interest
We are hereby submitting our manuscript entitled "Anacardic acid improves neurological deficits in traumatic brain injury by antiferroptosis and anti-inflammation" online.The authors declare no competing financial interest.

Fig. 2 .
Fig. 2. Verification of ferroptosis characteristics after TBI.(A) The ultrastructure of the cortex on day 7 after TBI in mice was captured by transmission electron microscopy.The red arrow indicates mitochondria.(B) Western blot was performed to observe the time course of changes in the expression of iron metabolism-related proteins (Tfr1 and GPX4).(C) The relative densities of each protein were normalized against GAPDH.ImageJ software was used for western blot band quantification.*P < 0.05, **P < 0.01, and ***P < 0.001 vs sham.

Fig. 3 .
Fig. 3. AA inhibits TBI-induced ferroptosis.(A-C) Representative western blots indicating the expression changes of TfR1 and GPX4 in the injured cortex in different groups on 24 h after TBI, respectively, with the right bar graphs.GAPDH was used as a loading control.Western blot bands were quantified using ImageJ software.(D-G) Representative images of immunohistochemical staining of TfR1 and GPX4 in the injured cortex.Quantification of TfR1 and GPX4 levels for different models.Scale bar is 100 μm.(H, I) Representative pictures showing the results of iron accumulation in these above groups.Scale bar is 50 μm.*P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4 .
Fig. 4. Protection of tissue defect and BBB by AA. (A, B) Brain tissue sections were stained with HE staining and quantitatively analyzed with ImageJ software.Representative histogram showing the volume ratio of ipsilateral to contralateral brains.Scale bar is 50 μm, n = 6 animals per group.(C) Representative pictures of EB extravasation in the right brains of various groups at day 3 after TBI (n = 6).(D) Quantitative analysis of EB leakage (n = 6).*P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5 .
Fig. 5. AA reduces TBI-induced neuronal loss and neurodegeneration.(A, B) Representative pictures showing the results of Nissl and FJB staining on 12 h post TBI in the Sham, TBI, Fer-1 and AA treatment groups.(C, D) The results of Nissl staining were shown by the number of normal nerve cells in each visual field.The degree of neurodegeneration is presented as the number of FJB + cells per vision field.Scale bar is 100 μm.*P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 6 .
Fig. 6.Determination of the therapeutic effect and concentration of Anacardic acid.(A) The NOR test was tested on day 28 post TBI.(B) mNSS scores were examined at day 1, 3, 7, 14 after TBI.(C) Computer printouts of the swimming trajectories of mice from each group on day 8 of the Morris water maze test.(D) At day 8, mice from the Sham and TBI + AA groups had more times of crossing.(E) At day 8, mice from the Sham and TBI + AA groups spent a greater percentage of time in the target quadrant.*P < 0.05, **P < 0.01, and ***P < 0.001.