Molecular Targeting of Nrf2 and NFkB Signaling by 3-acetyl-11-keto- β-boswellic Acid and Piperine in Fluid Percussion Rat Model of Traumatic Brain Injury

Background & purpose: Traumatic brain injury in rats through lateral uid percussion injury (LFPI) causes elevation in intracranial pressure which leads to impairments in motor and cognitive behavior. 3-acetyl-11-keto-β-boswellic acid (AKBA) is a well-known anti-inammatory agent but it has very low bioavailability. The current study was established to investigate the neuroprotective effect of AKBA in combination with bioenhancer piperine in LFPI induced TBI experimental rats. Experimental approach: Fluid percussion injury was created by delivering 50 mmHg of pressure for 3 minutes to the exposed brain. AKBA 25 mg/kg and 50 mg/kg orally and AKBA ((25 mg/kg, p.o.) in combination with piperine (2.5 mg/kg, p.o.) was administered from day 1 to day 14. On 1 st , 7 th and 14 th day, the behavioral parameters were checked. On 15 th day, animals were euthanized. Then the cortex was isolated for the estimation of biochemical levels (MDA, nitrite, reduced GSH, catalase), neuroinammatory markers (TNF-α, IL-1β, IL-6), and neurotransmitters (norepinephrine, dopamine, 5-HT, GABA, glutamate). From some animals, hippocampus and cortex were isolated for histopathological analysis and expressions of Nrf2 and NFkB was measured by immunohistological study. Key results: Treatment with AKBA signicantly attenuated LFPI induced abnormalities, biochemical and neurotransmitter changes in experimental rats. Further nding AKBA in combination with piperine signicantly prevented histopathological changes, increased Nrf2 positive cells and reduced NFkB expression in the cortical region. Conclusion & implication: The present study concluded that AKBA along with piperine achieved antioxidant, anti-inammatory, neuromodulatory effects as well as prevented neuronal injury via targeting Nrf2 and NFkB.


Introduction
Worldwide, traumatic brain injury (TBI) causes disability followed by death in between teenager and adults [1]. The rate of injuries is increasing yearly. TBI can be demonstrated as an alteration of normal brain physiology that results from sudden external mechanical injury to the skull [2]. Road accidents are the major cause of head injury; however, falls are the leading cause of post-TBI morbidity [3]. TBI signi cantly impairs healthcare system of body and also alters socio-economical status of patients, and is often tagged as a silent epidemic. The long-term sequelae of diffuse TBI impair cognition, emotional and sensory functions [4]. Post-TBI neurovascular outcomes require experimentation that involves circulatory, immune alteration, nervous systems in vivo rather than in vitro and in silico modeling.
Various well-established animal models of TBI are available for neurotrauma investigations and each model deals with speci c clinical etiology. The uid percussion injury (FPI) model is a commonly used and well-characterized preclinical TBI model [5]. FPI model was established for the establishment of midline injury, later it was modi ed to lateral injury in adult rats. Post-TBI observable effects including transient apnoea, seizure activities are very common [6]. The outcome that was investigated following the FPI is neuronal damage which results in neuralgia after 7 days of injury.
Nuclear factor-erythroid 2-related factor 2 (Nrf2), a nuclear factor which grabs researchers attention in recent years [10]. Various reports showed that Nrf2 has involvement in different pathological consequences including reperfusion injury, nervous systems [11,12]. Mainly, Nrf2 present in the cytoplasm in normal conditions in association with Kelch-like ECH-associated protein 1 (Keap1), but under pathological conditions, the cysteine residue of Keap1 promotes the release of Nrf2. However, the Nrf2 is then translocate to the nucleus and attached to the antioxidant response element (ARE) and subsequently triggers various downstream protective factors (HO-1, NADPH: quinine oxidoreductase 1), which further regulates intracellular redox balance [13,14]. The goals of the current study are to investigate the role of AKBA treatment following LFPI, check neurological insu ciencies in rats, and for the determination of the neuroprotective effect of Nrf2/HO-1 activator AKBA.

Animals
For current research protocol, male wistar rats (200-230 g) were procured from ISF College of Pharmacy, Moga, Punjab, India. The animals were kept in a 12-hour light/dark cycle and maintained the room temperature of 22 ± 1° C. Before conducting the experiment, the protocol was checked and approved by Institutional Animal Ethics Committee (IAEC). The procedures of handling and maintaining were followed according to the guidelines given by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India.

AKBA and other chemicals
AKBA and piperine were procured from Sigma Aldrich, India. ELISA kits (TNF-α, IL-1β, IL-6) were purchased from Abcam, USA. Nrf2 antibody and NF-kB antibody kits were purchased from ELK Biotechnology, Wuhan. All the chemicals were freshly prepared before starting the experimental procedures.

Craniotomy
Before starting the experiment, the rats were anaesthetized with intraperitoneal injection of ketamine (65 mg/kg) and xylazine (10 mg/kg). Anaesthetized rats were placed in the stereotaxic surgery frame in a prone position. A midline incision (2-cm) was made from the eyes towards neck by using a surgical blade, and the skin was apped for exposing the right parietal bone. The location of the craniotomy was selected in the right hemisphere midpoint of bregma and lambda. The craniotomy was performed using drill machine. The bone dust was cleaning continuously to prevent from any further damage to the dura. After the craniotomy, a micropipette tip was attached to the surgery area of the skull and sealed with dental cement. The micropipette tip (1 ml) was lled with 5% dextrose solution, and then attached with a sphygmomanometer connecting tube for delivering uid pressure.

Fluid percussion injury (FPI)
The lateral uid percussion injury model was used to create mild traumatic brain injury. This model causes injury by inducing uid pressure into the skull through sphygmomanometer. The sphygmomanometer rubber tube is connected with a Y-tube connector. One arm of this tube connector was linked with the cuff and other one was connected with a micro-pipette tip. After the surgical procedure was done, the micro-pipette tip was xed onto the skull at the craniotomy region. After gaining normal respiration, the uid pressure of 50 mmHg was delivered through sphygmomanometer for 3 minutes to the brain. Following the LFPI, the tip was removed very deliberately. The skin was then sutured and the rats were placed in sterilized cages and then housed.

Experimental design
After the habituation period was over, the rats were trained in grip strength test, rotarod apparatus, beam crossing task, open eld test and Morris water maze for 5 days. After the completion of training, rats were distributed randomly into the following groups-Group I: Normal, Group II: TBI control, Group III: TBI+ piperine (2.5 mg/kg), Group IV: TBI+AKBA (25 mg/kg), Group V: TBI+AKBA (50 mg/kg), and Group VI: TBI+ piperine (2.5 mg/kg) + AKBA (25 mg/kg). Each group consisted of 7 animals except the normal group (n=6). The drug administration was started from day 1 of injury to till day 14. On the 1 st , 7 th , and 14 th day of post-injury, the behavioral study was performed. On the 15 th day, cervical dislocation method was proceeded for animals sacri ce and then brains were taken out and kept it at -80° C. On the day of analysis, the cortex part was isolated for analysing biochemicals (MDA, nitrite, glutathione, catalase), neuroin ammatory markers (TNF-α, IL-1β, IL-6), and neurotransmitters (norepinephrine, dopamine, 5-HT, GABA, glutamate). Some isolated brains were kept in 10% formalin at room temperature for histological and immunohistological studies. The gripping ability of the forelimbs of experimental rats was analyzed with grip strength apparatus (Make: Chatillon). Before initiating of protocol, the rats were trained carefully. During the experimental session, rats were individually placed on apparatus and free to grab the mesh with forelimbs. Then rats were pulled back by the tail to record the grip strength reading in KgF [15].

Rotarod test
The rotarod apparatus (Model: Medicraft 519/E-40, INCO) was used to evaluate the motor coordination of rats [16]. The rats were pre-trained for 5 days (three sessions per day) to acclimatize the rotarod activity. On the rotating rod (25 rpm speed), rats were placed and the cut-off time was 3 minutes. On the rotating rod, the average retention time was recorded for evaluation.

Open eld test
The open eld test apparatus was used to evaluate the locomotion and exploration using wooden, square, greyish white colored apparatus (100 cm x 100 cm, wall: 35 cm high). The apparatus was designed with 25 sub-square (20 cm x 20 cm) in the oor. The rat was introduced at the middle of the apparatus and the primary free exploration time was for 5 minutes. The exploratory behavior alteration for each group was observed. After conducting each test, the oor of the apparatus was cleaned with a wet cloth. During the observation, line crossing counts (crossing of one square with forelimbs), rearing (stands with the hind legs), and grooming (mouth or head rubbing with paws) were recorded and the scoring was done by a blind observer [17].

Narrow beam task
This test was performed to evaluate the motor coordination and function by checking the ability of animals to cross the narrow beam. The beam was made up of two platforms (start point and end point) and connected by a wooden beam (length:100 cm, width: 5.5 cm, and thickness: 1 cm). The beam was 100 cm above from the ground. A sponge pad was placed at the ground below the beam to prevent from another injury during the performance of rats. For the acclimatization to the wooden beam, the rats were allowed for 5 minutes of exploration before processing of training phase. The training session was started by placing the rats on the starting point. During travelling onto the beam, the slipping of foot occurred. Time taken to travel the beam and the numbers of foot slip during travelling were recorded [18].

Morris water maze
To check the spatial and learning memory impairment after TBI, the Morris water maze was used [19].
This apparatus was made up of an iron-coated round pool (diameter 6 feet x height 3 feet). Tap water was used for lling up of the maze and the controlled temperature of water was at 26 ± 1 °C. The cotton thread was used for dividing the maze into four equal quadrants. During the acquisition phase, one quadrant was selected for keeping a round platform (5 cm diameter) just 1 cm above the water level, but during the retention phase, the pool water volume was increased so as to maintained the platform 1.5 cm below the water level. The animals were placed in pool one by one for four times and the gap between two training session was 5 minutes. The animals were placed at the rst quadrant and recorded the time taken by each animal to reach the platform. Then the rats were permitted to sit on the round platform for 20 seconds. If the rat was able to nd the platform within 2 minutes, represents good training. But if the rat was unable to nd the platform within the given time, the same was guided to nd the platform and stay on the same position for next 20 seconds. After 24 hours of last training session, a probe trial was also conducted. The time spent in the target quadrant (TSTQ) was recorded and it showed learning as well as spatial memory function after training. After the training period was over, the escape latency was checked, and then milk was poured to water to make the pool opaque. For checking the nal spatial memory, TSTQ and escape latency was measured. 2.7. Biochemical parameters studies 2.7.1. Dissection and homogenization: On the 15 th day, animals were sacri ced and immediately removed the brain samples and placed on ice for isolating the cortex, and also for different brain morphological evaluation. The isolated brains were rinsed with ice-cold saline (0.9 %) for biochemical estimation, and further processing was conducted in cold condition (-4 °C). After weighing each isolated brain samples, they were homogenized in phosphate buffer (0.1 M) with a of pH 7.4, and subsequently centrifuged for 15 minutes at 10,000g and the supernatant were separated for the estimation of biochemical and molecular markers.

Measurement of MDA:
The concentration of the malondialdehyde (MDA) in the cortical region was measured and the procedures was completed according to the method of wills et al [20]. The MDA concentration (measurement of lipid peroxidation) was analyzed via reaction with thiobarbituric acid at a wavelength of 532 nm using a spectrophotometer (UV-1700, Shimadzu, Japan). The estimated values were expressed in nmol/ mg tissue protein.

Estimation of nitrite:
The nitrite accumulation in cortical supernatant was determined by colorimetric assay using Griess reagent (0.1% N-(1-naphthyl) ethylene diamine dihydrochloride, 1%sulfanilamide and 2.5% phosphoric acid) according to the method described by Green et al [21]. Both the supernatant and Griess reagent were mixed in equal volume and incubation was done for 10 minutes at 37° C. Absorbance of the nal solution was measured at 540 nm in a spectrophotometer (UV-1700, Shimadzu, Japan) and expressed as µg/ml. 2.7.4. Measurement of reduced GSH: The concentration of glutathione in the brain cortex was measured according to the method demonstrated by Ellman [22]. In the tissue supernatant, 1 ml of 4% salicylic acid was added at 4 °C till precipitation. Then the mixture was then centrifuged at 12000 g. After the separation of supernatant, it was mixed with 0.1 M phosphate buffer until yellow color appears. The absorbance is measured at 412 nm using Shimadzu spectrophotometer and the estimated values expressed as µmol/mg protein.

Estimation of catalase:
The accumulation of catalase in the cortical zone was estimated by the method demonstrated by Sinha [23] and Aebi [24]. To proceed with the reaction, 2.9 ml of 10 mM H 2 O 2 was mixed in 50 µM potassium phosphate buffer and maintained the pH 7.4. Then 0.1 ml of tissue homogenate was mixed into that mixture. The decreased rate of absorbance at 240 nm was recorded for 3 min. The calculated data was expressed as µg /mg tissue protein.

Neurochemical analysis:
2.8.1. Estimation of Brain catecholamines by HPLC-ECD: Catecholamines (Nor-epinephrin, dopamine and serotonin) levels were estimated by using high performance liquid chromatography connected with electrochemical detector. Sodium citrate buffer (pH 4.5) and acetonitrile with a ratio of 87:13 (v/v) were used for preparing the mobile phase. Sodium citrate buffer was prepared with 10 mM citric acid, 25 mM NaH 2 HPO 4 , 25 mM EDTA, and 2 mM of 1-heptane sulfonic acid. For the uninterrupted separation, the separation rate was maintained with a ow rate of 0.8 ml/min. At the injector point, total 20 µl of samples was injected after being ltrated through 0.2 nm nylon lter [26]. On the experimental day, the brain samples were cut and 0.2 M perchloric acid was used for homogenization of brain samples. Then the samples were centrifuged for 5 minutes at 12000 g. The supernatant was ltered and injected. Breeze software was used for recording of plotted graph and the concentrations of neurotransmitters level were calculated from standard curve [27].
2.8.2. GABA and Glutamate estimation by HPLC-ECD: The levels of GABA and Glutamate was estimated according to the method described by Donzanti and Yamamoto et al [28]. 100 mM Na 2 HPO 4 , 25 mM EDTA and 22 % methanol (pH 6.5) were used for the preparation of the mobile phase. For uninterrupted separation, the ow rate was maintained at 1.2 ml/min. Total 20 µl of samples was injected through rheodyne valve injector. 0.2 M perchloric acid was used for the homogenization of the brain tissues. Then the centrifugation of brain samples was done for 15 minutes at 12,000 g. Derivatization of supernatant was done using OPA/-ME and before injecting the sample, the samples were being ltered through nylon lters (0.22 mm). Recorded data was analyzed with the help of breeze software [29].
2.8.3. Pre-column derivatization procedure: Before injecting in the HPLC, the solution was derivatized with OPA/β-ME according to the method demonstrated by Donzanti and Yamamoto [28]. The derivatization stock solution was made by dissolving 27 mg of OPA in 1 ml of methanol. Then in the solution, 5 ml of β -ME was mixed with 9 ml of tetraborate buffer (0.1 M sodium tetraborate) at a pH of 10.3. The solution was kept stable in dark condition upto 5 days. OPA/ β-ME stock solution (5 ml) was mixed with tetraborate buffer (7.5 ml) for the preparation of the working solution of OPA/ β-ME. Before experiments, the solution must be prepared freshly. The brain tissue samples and the derivatized reagents was mixed with each other with a ratio of 1:1.5 [30,31].

Histological Examination:
After isolating the brain, the brain samples were individually cut with a microtome (Leica RM 2025; Nassloch, Germany) with a thickness of 5 µm. The sections were xed by immersing in 10% buffered formalin, then dehydrated in alcohol and embedded in para n. Two different sections were examined for individual brain samples (cortex and hippocampus). The samples were then stained with hematoxylin and eosin (H&E) for assessing histopathological changes under a blindfold condition with standard microscopy.
2.9.1. Immunohistochemical procedures: For the collection of lesioned cortices, coronal sections with a thickness of 4 μm were cut from the bregma region (1.0-3.0 mm). For IHC staining, formalin-xed para n-embedded sections (4 mm) were prepared. The sections were then transferred through xylene for half an hour for the depara nization and then slides were rehydrated with absolute alcohol 95%, 70%, and 50% in decreasing order. The endogenous peroxide activity was blocked by adding 3% H 2 O 2 in distilled water for 30 minutes, followed by tap water washing for 30 minutes. The sections were then incubated with blocking buffer (5% goat serum in PBS) for 30 minutes for inhibiting the non-speci c binding of antibodies. Then tris buffer solution (pH 7.4) was used for rinsing coronal sections for 10 minutes. Then sections were incubated with Nrf2 (1:100, ELK Biotechnology) and NFkB (1:100, ELK Biotechnology) for 1 hour. Then slides were washed with PBS for 5 min, followed by incubation with secondary antibodies (1:500, ELK Biotechnology) for 1 hour. Again, tris buffer solution (pH 7.4) used for rinsing coronal sections for 10 minutes. The sections were then incubated with substrate (3,3′-diaminobenzidine, DAB) and examined for colour change to brown, appeared within 10 min and then counterstained with hematoxylin. In immunochemical staining, the numbers of immune-positive stained cells were counted under light microscopy with a magni cation of x 400 under blindfold conditions.

Statistical analysis
The data obtained of all results were represented as mean ± standard deviation (SD) and analysed using GraphPad Prism 5.0. Two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test for multiple comparison was used for analysing behavioral parameters. Whereas, one-way analysis of variance (ANOVA) followed by Tukey post-hoc test for comparison was used for analysing biochemical, neurotransmitter, and neuroin ammatory markers. P<0.05 was considered as statistically signi cant. The grip strength of rats was signi cantly reduced in the TBI control group as compared with the normal group. AKBA (25 mg/kg) and AKBA (50 mg/kg) signi cantly increased grip strength as compared with the TBI control group. Furthermore, AKBA (50 mg/kg) in combination with piperine (2.5 mg/kg), signi cantly enhanced grip strength as compared with the TBI control group, piperine per se and AKBA treatment group and restored toward normal (Fig 1). 3.1.2. Effect of combination treatment with AKBA and piperine on rotarod test in TBI rat model TBI control group showed signi cant decrease in motor co-ordination (rotarod activity) as compared with normal group. The rotarod activity in AKBA (25 mg/kg) and AKBA (50 mg/kg) group was signi cantly increased as compared with TBI control group. AKBA (25 mg/kg) in combination with piperine (2.5 mg/kg) signi cantly increased the rotarod activity as compared with TBI control, piperine per se and AKBA treatment group (Fig 2).

Effect of combination treatment with AKBA and piperine on open eld task in TBI induced experimental rats
In the open elds task, line crossing, grooming and rearing behavior was measured. In the TBI induced rat group, the task activity was gradually decreased as compared with normal group. The line crossing activity was signi cantly increased in AKBA (25 mg/kg) and AKBA (50 mg/kg) treated group as compared with TBI control group. However, combination of AKBA (25 mg/kg) with piperine (2.5 mg/kg) signi cantly increased the line crossing as compared with TBI control group, piperine per se and AKBA treatment group (Fig 3a).
After TBI, the numbers of grooming (Fig 3b) and rearing (Fig 3c) was signi cantly decreased in TBI induced rat group as compared with the normal group. On the day 7, AKBA (25 mg/kg and 50 mg/kg) and combination group showed signi cant improvement in rearing while on day 14, all treatment groups restored altered counts as compared with TBI induced rat group. In piperine treated animals, the rearing and grooming activity on day 7 as well as day 14 were signi cantly improved (p<0.05 and 0.01, respectively).
3.1.4. Effect of combination treatment with AKBA and piperine on narrow beam task performance (time taken to cross the beam and no. of foot slip) in TBI induced experimental rats TBI induced rats showed increase in the time taken to cross the beam pathway and numbers of foot slip as compared with normal group. Treatment with AKBA (25 mg/kg) and AKBA (50 mg/kg) both signi cantly and dose dependently decreased reach time and foot slips as compared to TBI control group. Among all treatment groups, AKBA (25 mg/kg) and piperine (2.5 mg/kg) combination group at day 14 was more effective in improving narrow beam walk activity (Fig 4a and 4b).
3.1.5. Effect of combination treatment with AKBA and piperine on ELT and TSTQ in MWM TBI induced experimental rats The TBI control group had taken more time in ELT to nd out the hidden platform as compared with the normal group which clearly indicates deterioration in learning performance (p<0.001). The both doses of AKBA (25 mg/kg and 50 mg/kg) signi cantly improved in learning performance at day 7 whereas at day 14, both doses of AKBA more signi cantly improved than day 7. However, combination with AKBA (25 mg/kg) and piperine (2.5 mg/kg) signi cantly decreased the escape latency and intensi ed the TSTQ than TBI control group, piperine per se and AKBA (25 & 50 mg/kg), which indicating memory performance retention (Fig 5a and 5b).

Biochemical parameters estimation
3.2.1. Effect of combination treatment with AKBA and piperine on brain oxidative stress parameters (LPO, nitrite, reduced GSH, catalase) in TBI induced experimental rats The levels of LPO and nitrite were elevated signi cantly (p<0.001), whereas GSH and catalase levels were signi cantly reduced (p<0.001) in TBI induced rats as compared with normal group. However, AKBA (25 mg/kg and 50 mg/kg) treatment group signi cantly alleviated (p<0.001) the level of LPO and nitrite and the levels of GSH and catalase were signi cantly increased as compared with TBI induced rat group. Whereas, the combination treatment group (AKBA + piperine) signi cantly decreased (p<0.001) LPO and nitrite level and increased GSH and catalase level signi cantly (p<0.001) as compared to TBI control group and AKBA high dose (50 mg/kg) group (Table 1).

Neuroin ammatory markers
3.3.1. Effect of combination treatment with AKBA and piperine on neuroin ammatory cytokines levels (TNF-α, IL-1β, IL-6)) in TBI induced experimental rats The biochemical result revealed that neuroin ammatory level has changed post induction of TBI presented by an elevation of IL-1β (p<0.001), TNF-α (p<0.001) and IL-6 (p<0.001) as compared with normal group. The elevated levels of IL-1β, TNF-α and IL-6 was decreased signi cantly (p<0.001) after administration of AKBA (25 mg/kg and 50 mg/kg). However, combination group of AKBA (25 mg/kg) and piperine (2.5 mg/kg) signi cantly alleviated (p<0.001) the level of IL-1β, TNF-α, and IL-6 as compared with TBI induced group, piperine per se and 50 mg/kg of AKBA treated group (Fig 6). In the brain, dopamine is metabolized by monoamine oxidase with two intermediates, 3,4dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). In comparison to normal group, the level of catecholamines were signi cantly reduced (p<0.001) in TBI control group. The treatment with AKBA 25 mg/kg and AKBA 50 mg/kg, signi cantly improved (p<0.001) the level of dopamine, norepinephrine, and serotonin. Also, the combination group of AKBA (25 mg/kg) and piperine (2.5 mg/kg) signi cantly restored (p<0.001) the level of catecholamines as compared to TBI group and AKBA 50 mg/kg treatment group (Table 1). Further TBI induced rats results signi cant increase in the level of DOPAC and HVA. Rats treated with AKBA (25 mg/kg and 50 mg/kg) showed signi cant decreased levels of DOPAC and HVA in comparison to TBI induced rats. However, combination of AKBA (25 mg/kg) and piperine (2.5 mg/kg) signi cantly inhibited the increased levels of catecholamine metabolites as compared to TBI induced rats ( Table 2).

3.4.2.
Effect of combination treatment with AKBA and piperine on GABA and glutamate levels in TBI induced experimental rats TBI control group revealed that the level of GABA (cortical) was signi cantly decreased (p<0.001) as compared with normal group. The treatment groups with AKBA (25 mg/kg and 50 mg/kg) prevented the GABA level alteration signi cantly (p<0.001) as compared with TBI induced rats. Whereas, the combination group with AKBA (25 mg/kg) and piperine (2.5 mg/kg) more signi cantly improved the altered level of GABA as compared to TBI group and AKBA high dose.
In the TBI control group, there was signi cant elevation (p<0.001) in the level of glutamate as compared with normal group. Treatment with AKBA (25 mg/kg and 50 mg/kg) signi cantly decreased (p<0.001) the altered level of glutamate as compared with TBI induced group. Moreover, AKBA (25 mg/kg) and piperine (2.5 mg/kg) combination group signi cantly decreased (p<0.001) the level of glutamate as compared to high dose of AKBA and TBI control group (Table 3).

Effect of combination treatment with AKBA and piperine on neuronal loss in the cerebral cortex and hippocampal CA1 regions
Based on the results shown in gure 17a (hippocampal CA1 regions) and gure 17b (cerebral cortex (CC), the morphological status after TBI was examined by hematoxylin and eosin (H&E) staining. TBI control group was found to have smaller number of intact neurons in cerebral cortex and hippocampal CA1 regions as compared to normal group rats indicating neuronal loss. 25 mg/kg and 50 mg/kg of AKBA treatments on traumatic rats signi cantly protected the CC and CA1 regions from neuronal loss as compared with TBI induced rats. Moreover, the combination treatment of AKBA with piperine markedly showed protection from decline neuron numbers as compared with TBI induced group and High dose of AKBA group (Fig 7a and 7b).

Effect of combination treatment with AKBA and piperine in increasing Nrf2 and NFkB positive cells in the cortical regions
As shown in the gure 19, the normal rats did not have expression of Nrf2 positive cells in the nucleus. The TBI control group enhanced Nrf2 expression in the nucleus as compared with normal rats. AKBA (25 mg/kg and 50 mg/kg) treatment signi cantly enhanced Nrf2 positive cell expressions. Moreover, the combination group with AKBA and piperine more signi cantly increased the Nrf2 positive cell expressions which indicate the ability of AKBA to promote Nrf2 translocation from cytoplasm to nucleus and thereby elevate the biding to the downstream genes (Fig 8).
Elevation of NFkB positive cell expressions was found signi cantly more in the TBI control group as compared to normal group. AKBA (25 mg/kg and 50 mg/kg) treatment to the TBI induced group, signi cantly lowered the NFkB positive cells as compared with TBI control group. Moreover, simultaneous administration of AKBA and piperine signi cantly reduced the generation of NFkB positive cell expressions as compared with TBI control and AKBA high dose treatment group (Fig 9).

Discussion
The present study result showed that the potential protective effect in the combination group of AKBA and piperine against lateral uid percussion induced TBI in experimental rats. Lateral uid percussion injury (LFPI) model is the most utilized model because it shows construct validity whereas it successfully recreates etiological outcomes of human TBI. The basic principle of LFPI is to induce uid pressure over the exposed skull. The traumatic brain injury causes motor coordination impairment in animals as well as in human beings [32]. Several available data represent the behavioral alteration in rats and humans after TBI [33,34]. The current investigation on gripping activity, open eld task, beam crossing task, Morris water maze and rotarod activity have shown a signi cant impairment in motor function and cognition post-TBI. The AKBA (25 mg/kg), AKBA (50 mg/kg) and combination of AKBA (25 mg/kg) and piperine (2.5 mg/kg) have also shown signi cant restoration in altered motor function. Wei et al. shown that there was an improvement in cognition after treatment with AKBA [35].
There are some impairments in cognition as well as in motor functions that are observed in the early stage post -TBI and which may persist up to several years after TBI [36,37]. Draper et al. showed in their study that learning behavior is altered both in animals and humans with TBI [38]. Cortical as well as the hippocampal area is important for learning and memory processing [39] and which is disrupted post-TBI.
The current study showed that AKBA along with piperine signi cantly improved the memory de cits or cognitive impairments in traumatic rats.
Post TBI brain causes activation of microglial and astroglia cells, also activation of in ammatory mediators within the brain. Microglia has a role in increasing proin ammatory cytokines.
Neuroin ammation-induced secondary injury in post TBI patients has been associated with serious neurodegenerative diseases. Neuroin ammation may have some bene ts as well as detrimental role after TBI. Various reports have been published for the con rmation of enhancement of oxidative stress post brain injury [40][41][42]. In the current experimental study, we have also noticed that the elevation of oxidative stress markers in TBI induced rats as compared with the normal group. An increase in oxidative stress is also linked with increase in neuroin ammatory markers including IL-6, TNF-α, IL-1β. Also, upregulation of in ammatory responses increased NADPH oxidase enzyme. Ahmed et al. observed that in a study of oxygen-glucose deprivation/reperfusion model of bEND.3 cells, the treatment with AKBA signi cantly reduced the level of oxidative stress in the endothelial cells [43]. In this current study, we have investigated that there was increased in ammation at cortical region (increased LPO and decreased GSH and CAT). 25 mg/kg and 50 mg/kg of AKBA groups and combination group (AKBA and piperine) revealed that there was a reduction in the oxidative stress and neuroin ammation in TBI control group rats as compared with normal group of animals which consider the bene cial effect of AKBA. Roy et al. have shown a signi cant reduction of malondialdehyde level and elevation of GSH level after the administration of AKBA treatment [44].
The increased level of in ammatory cytokines is linked with increased production of quinolinic acid and an elevated level of quinolinic acid is also linked to increased excitatory neurotransmitter (glutamate). In the synaptic cleft, the excess level of glutamate also causes neurodegeneration. After TBI, in the hippocampal region, there is an activation of extra-synaptic NR2B-containing NMDARs. The imbalance in the excitatory and inhibitory neurotransmitters shows neuronal dysfunction because of increased glutamate release, faulty reuptakes. So, the level of GABA and glutamate is altered following TBI [43]. In our current investigation on the neurotransmitter level, we have analysed that after administration of AKBA and also in combination with piperine, there was signi cant improvement in the altered level of GABA and glutamate level. Catecholamines such as dopamine, nor-epinephrin and serotonin also have a role to maintain normal brain physiology. So, we have checked the level of these catecholamines. In the present study, after induction of TBI in the experimental rats, there was a signi cant decrease level of DA, NE and 5-HT in the TBI control rats as compared to normal rats. Piperine which is a bio enhancer in combination with AKBA signi cantly restored the levels of catecholamines. Kawa et al. reported that the level of catecholamines was also impaired after the induction of TBI in rats [45]. Several reports have demonstrated that there is an altered level of catecholamines in the neurodegenerative diseases [25,46].
Post-TBI brain shows major histopathological changes which lead to behavioral alteration, cognitive de cits [47]. In the current study, we have performed the histopathological analysis to nd the structural and molecular changes after TBI. In the TBI control groups, there were decreased numbers of intact neurons in both hippocampal and cortical regions after 2 weeks of TBI-induction. Piperine along with AKBA more signi cantly increased the counts of intact neurons and causes neurogenesis more profoundly.
AKBA is known to be an Nrf2 activator that shows a protective defence mechanism through HO-1 neuroprotective pathway. Nrf2 is an antioxidant defence mechanism regulator [48]. In a basal condition, Nrf2 resides in the cytoplasm along with Kelch-like ECH associated protein 1 (Keap1). Nuclear gathering of Nrf2 upregulates endogenous antioxidant mechanism [49]. One study suggested that AKBA has a role for the translocation of cytoplasmic Nrf2 to the nucleus [50]. Another useful study demonstrated that stimulation of Nrf2 expression by triterpenoid could alleviate MPTP-induced oxidative stress in the mice model whereas Nrf2 knockout mice were unable to show protection in MPTP induced neurotoxicity [51].
In our study, we have also observed the translocations of cytoplasmic Nrf2 to the nuclear region of cortex under oxidative stress. The expression of Nrf2 positive cells (binding of AKBA to Nrf2) was markedly increased in the combination group of AKBA and piperine as compared to TBI control group, AKBA alone groups. On the other side, in normal condition, nuclear factor kappa B (NFkB) remains in the cytoplasm but upon activation, translocate to nucleus. NFkB is linked with various human diseases like Parkinson's, Alzheimer's disease, atherosclerosis [52]. The activated level of NFkB is increased following TBI and resides for the prolonged time [53]. One study revealed that upon AKBA administration, the elevated level of NFkB was prevented in experimental mice [54]. The cells showed a high level of NFkB expression in the cortical region following TBI. Piperine alone with AKBA treatment showed signi cant decreased in the counts of NFkB positive cells in the cortical regions as compared with the TBI induced group. In addition, more experiments using TUNNEL assay and western blotting are to be proposed in the future. Conclusions on concluding remarks, the current research demonstrates that treatment with 25 mg/kg and 50 mg/kg of AKBA orally and its combination with piperine (2.5 mg/kg, po) has signi cantly improved the LFPI induced behavioral, biochemical, neurotransmitter level alteration. The neuroprotective effect of AKBA in combination with piperine revitalize its inhibitory nature towards oxidation, anti-in ammatory effect and modulatory effect in catecholamines level in the cortical regions. However, it can be concluded that AKBA in combination with piperine could be a promising therapeutic intervention in the traumatic brain insult. Format for Availability of Data and Materials: As per Journal policy we will share on request.

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Format for Consent for Publication: All the authors agreed to submit paper.
Funding: The authors received no nancial support for the research, authorship, and/or publication of this article.
Con ict of Interest: The authors declare no con ict of interest, nancial or otherwise.       Effect of AKBA in combination with piperine on narrow beam task performance in TBI model of rats -a: Time to cross the beam (sec), b: No of foot slips. Results are expressed as the mean ± SD and analysed by two-way analysis of variance followed by Bonferroni's post hoc test. *P<0.001 vs. normal, &P<0.001 vs TBI normal, #p<0.001 vs TBI + AKBA 50 mg/kg.  Effect of AKBA in combination with piperine on pro-in ammatory cytokines levels (TNF-α, IL-1β, IL-6) in TBI induced experimental rats. Results are expressed as the mean ± SD and analysed by one-way analysis of variance followed by Tukey's post hoc test. *P<0.001 vs. normal, $P<0.05 vs TBI control, &P<0.001 vs TBI control, #p<0.001 vs TBI + AKBA 50 mg/kg.  Effect of AKBA in combination with piperine on immuno-histochemical study (Nrf2). As compared with the normal group, TBI control group signi cantly presented a morphology with more Nrf2 nucleus concentration. AKBA 25 mg/kg and 50 mg/kg signi cantly increased the numbers of Nrf2 positive cell (*p<0.001 vs TBI control). After treating with AKBA and piperine combination, the concentration morphology was more apparent (#p<0.001 vs TBI+ AKBA 50 mg/kg). Effect of AKBA in combination with piperine on immuno-histochemical study (NFkB). As compared with the normal group, TBI control group signi cantly presented a morphology with more NFkB nucleus concentration (*P<0.001). AKBA 25 mg/kg treated group signi cantly reduced the level of nuclear NFkB (#P<0.05). AKBA 50 mg/kg treated group signi cantly decreased the NFkB level (@P<0.01). After administering AKBA (25 mg/kg) and piperine (2.5 mg/kg) combination, the concentration morphology