Disorders of Hippocampus Facilitated by Hypertension in Purine Metabolism Deficiency is Repressed by Naringin, a Bi-flavonoid in a Rat Model via NOS/cAMP/PKA and DARPP-32, BDNF/TrkB Pathways

Individuals who are hypertensive have a higher tendency of predisposition to other genetic diseases including purine metabolism deficiency. Therefore, the search for nontoxic and effective chemo protective agents to abrogate hypertension-mediated genetic disease is vital. This study therefore investigated the repressive effect of naringin (NAR) against disorder of hippocampus facilitated by hypertension in purine metabolism deficiency. Male albino rats randomly assigned into nine groups (n = 7) were treated for 35 days. Group I: control animals, Group II was treated with 100 mg/kg KBrO3, Group III was treated with 250 mg/kg caffeine, and Group IV was treated with 100 mg/kg KBrO3 + 250 mg/kg caffeine. Group V was administered with 100 mg/kg KBrO3 + 100 mg/kg haloperidol. Group VI was administered with 100 mg/kg KBrO3 + 50 mg/kg NAR. Group VII was administered with 250 mg/kg caffeine + 50 mg/kg NAR, and Group VIII was administered with 100 mg/kg KBrO3 + 250 mg/kg caffeine + 50 mg/kg NAR. Finally, group IX was treated with 50 mg/kg NAR. The sub-acute exposure to KBrO3 and CAF induced hypertension and mediated impairment in the hippocampus cells. This was apparent by the increase in PDE-51, arginase, and enzymes of ATP hydrolysis (ATPase and AMPase) with a simultaneous increase in cholinergic (AChE and BuChE) and adenosinergic (ADA) enzymes. The hypertensive-mediated hippocampal impairment was associated to alteration of NO and AC signaling coupled with lower expression of brain-derived neurotrophic factor and its receptor (BDNF-TrkB), down regulation of Bcl11b and DARPP-32 which are neurodevelopmental proteins, and hypoxanthine accumulation. However, these features of CAF-mediated hippocampal damage in KBrO3-induced hypertensive rats were repressed by post-treatment with NAR via production of neuro-inflammatory mediators, attenuation of biochemical alterations, stabilizing neurotransmitter enzymes, regulating NOS/cAMP/PKA and DARPP-32, BDNF/TrkB signaling, and restoring hippocampal tissues.


Introduction
Hippocampus is a cognition-centric brain region rooted within the temporal lobe (Gilbert and Brushfield 2009). It is also responsible for learning and memory (Maria et al. 2019). The high level of susceptibility of the hippocampus to pathological insults has been intricately linked with neurologic disorders including cerebrovascular disease, stroke, and vascular cognitive damage (Hainsworth and Markus 2008;Van der Flier et al. 2018). Correspondingly, Kohler et al. (2014) established that the level of worsening in memory function is less prominent in normotensive individuals compared to hypertensive ones. In the same vein, deterioration in learning, a function of the hippocampus was observed in experimentally induced hypertensive mice, which is consistent with the phenotypic signs of aging Csiszar et al. 2013;Toth et al. 2014). Thus, chronic hypertension may affect brain regions involved in learning and memory. The brain-derived neurotrophic factor (BDNF) is a signaling protein expressed in the brain and spinal cord (Gadad et al. 2021). There are two neurotrophin receptors: tropomyosin-related kinase (Trk) receptor family and tumor necrosis factor (TNF) receptor family (Barbacid 1994;Bothwell 1995). TrkB, a receptor of Trk (Ohira and Hayashi 2009), is expressed in the central nervous system (Choo et al. 2017;Liao et al. 2019). Previous findings established that the control of BDNF-TrkB signaling pathway may ameliorate psychiatric and neurological diseases (Sheldrick et al. 2017;Park and Lee 2018) via stimulation of several efficient downstream cascades resulting in the expression of antioxidant enzymes which guard the neurons from cell death due to reactive oxygen species (ROS) and the activation of several kinases ranging from the cAMP-dependent protein kinase A (PKA) and calcium/calmodulin-regulated protein kinases CaMKII (Bruna et al. 2018;Nitti et al. 2018) which have regulatory functions on hippocampus and other regions of the brain (Park et al. 2021).
Research has shown that extracellular nucleotides which are enzymes of the purine metabolic pathway may contribute to the regulation of hypertension and blood homeostasis in mammals (Jinnah et al. 2013). In order to initiate pathological response, these nucleotides break down adenosine triphosphate (ATP), giving rise to AMP (adenosine-5-monophosphate) and diphosphate (Gardani et al. 2019). Ecto-5 1 -nucleotidase catalyzes the formation of adenosine from AMP, while adenosine amino hydrolase, otherwise regarded as adenosine deaminase (ADA), irreversibly catalyzes the deamination of adenosine yielding inosine and hypoxanthine (Cortés et al. 2015). Consequently, the regulation of adenine nucleotides and nucleoside by these ectoenzymes contribute to the maintenance of hypertension (Sajjan andMakandar 2016: Kutryb-Zajac et al. 2020).
Furthermore, the connection between alteration in the action of nitric oxide (NO) and hypertension or other vascular diseases cannot be over emphasized (Petrie et al. 2018). The reaction which leads to the formation of NO from L-arginine is catalyzed by nitric oxide synthase (NOS). The mechanism of action of NO, a vasodilator, may involve the positive modulation of guanylate cyclase (GC) and production of cyclic guanosine monophosphate (cGMP) which eventually triggers protein kinase G (PKG), thus implicating lowered level of NO in the pathogenesis of hypertension and neuroinflammation (Miguel et al. 2015). Additionally, signaling pathways which activate AC and upregulate intracellular PKA would in sequence of events and potentiate the phosphorylation of dopamine and cAMP-regulated phosphoprotein of relative molecular mass 32,000 Daltons (DARPP-32) at Thr 34 residue: an activation required for improved brain function (Qian et al. 2015).
Individuals with hypertension may concurrently have hippocampal impairment due to continuous exposure to toxicants. Caffeine (CAF) which is widely consumed with coffee and other items could also influence neurotoxicity (Jee et al. 2020). Equally, KBrO 3 a food additive, which is present in adulterated bread, has been established to be a promoter of neurodisorder and other diseases (Saad et al. 2017). Thus, ardent consumers of adulterated bread and coffee may be at higher risk of hypertension and neurodisorder. Hence, this study suggests the ability of KBrO 3 to induce hypertension and co-promote neurotoxicity with CAF. Based on copious evidences available on the grape fruit naringin and its aptitudes to attenuate neurotoxicity (Akintunde et al. 2020a, b) and alleviate the deleterious effects of hypertension via several signaling pathways (Akintunde et al. 2020a, b), we therefore propose that naringin (iso-biflavonoid) might induce the activation of cAMP/PKA/DARPP-32 cascade and trigger BDNF-TrkB signaling which altogether may act as a vital mechanistic therapy for patients with hippocampal impairment mediated by hypertension in purine metabolism deficiency.
Naringin, a flavanone glycoside found in grapes and citrus fruits (Ufuk 2007), have been proven to show numerous pharmacological activities among which are free radicals scavenging ability, anti-oxidative, anti-inflammatory, anti-mutagenic, anti-cancer, anti-microbial, and reduction of cholesterol levels (Supriy et al. 2017;Mu et al. 2019;Akintunde et al. 2020a, b). According to our recent studies, naringin was found to replenish NO and inhibit cAMPphosphodiesterase (cAMP-PDE), oculopathy in hypertensive rats on exposure to bisphenol-A (Akintunde et al. 2020a, b). Given these potentials of naringin, this study, therefore, was designed to assess whether iso-flavonoid naringin would repress disorders of hippocampus facilitated by hypertension in purine metabolism deficiency in a rat model via NOS/ cAMP/PKA and DARPP-32, BDNF/TrkB pathways.

Animal Management
Sixty-three adult male Wistar rats (100-120 g) were procured from the Department of Physiology, University of Ibadan, Nigeria. They were moved to the animal house, Department of Biochemistry, Federal University of Agriculture, Abeokuta, Nigeria. They were kept in well-aired cages at 25 °C and maintained on standard rodent feed and water ad libitum under a 12-h light/dark cycle. The animals were acclimatized for 2 weeks before the study. All the animals received gentle care following the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science and published by the National Institute of Health Public Health Service (PHS 1996). The guidelines were followed to ensure the protection of the animals' well-being during the experiment. The experimental approved number of the researcher is 20160901.

Treatment Procedure
The male rats were grouped (n = 7) as follows: • Group I (control) served as the untreated control and was given distilled water for 35 days. • Group II (KBrO 3 ONLY) served as KBrO 3 -induced rats with 100 mg/kg KBrO 3 for 21 days and post-administered with distilled water for 14 days. • Group III (CAF ONLY) served as exposure to 250 mg/kg caffeine for 21 days and post-administered with distilled water for 14 days. • Group IV (CAF + KBrO 3 ) served as co-exposure to 100 mg/kg KBrO 3 and 250 mg/kg CAF for 21 days and post-administered with distilled water for 14 days. • Group V (KBrO 3 + HAL) served as exposure to 100 mg/ kg KBrO 3 for 21 days and post-treated with 100 mg/kg haloperidol for 14 days. • Group VI (KBrO 3 + NAR) served as exposure to 100 mg/ kg KBrO 3 for 21 days and post-treated with 50 mg/kg naringin for 14 days. • Group VII (CAF + NAR) served as exposure to 250 mg/ kg caffeine for 21 days and post-treated with 50 mg/kg naringin for 14 days. • Group VIII (KBrO 3 + CAF + NAR) served as posttreatment with 50 mg/kg Naringin for 14 days against mixture of 100 mg/kg KBrO 3 and 250 mg/kg Caffeine for 21 days. • Group IX (NAR ONLY) served as the post-treated control with 50 mg/kg naringin for 14 days and pre-administered with distilled water for 21 days.

Selection of Dose
The KBrO 3 was given orally by gavage for 21 days at a dose of 100 mg/kg body weight because of its ability to induce neurotoxicity and nephrotoxicity in rats (Oseni et al. 2015;Saad et al. 2017). The nephrotoxic ability of 100 mg/kg KBrO 3 suggests its hypertensive potential which was in line with the previous study (Oseni et al. 2015). The doses of 50 mg/kg NAR and 250 mg/kg CAF were selected following the studies by Akintunde et al. (2020a, b) and Willson (2018), respectively. Naringin shows no toxicity in different diseased models. Olive oil was used as vehicle due to its ability to dissolve organic solvent. Post-administration technique was adopted in this study because individuals that have hippocampal disorders due to hypertension may have been susceptible to purine metabolism deficiency as a result of co-exposure to KBrO 3 from consumption of adulterated bread and CAF from drinking of caffeine laden tea and drinks.

Determination of Systolic and Diastolic Blood Pressure
Blood pressure was measured in conscious rats in line with Tata et al. (2019), using non-invasive tail-cuff plethysmography (CODA™ 8 Non-Invasive Blood Pressure System, Kent Scientific Corporation, USA) as per manufacturer's instructions. Systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg or both indicates hypertension in humans (Williams et al. 2018). Rats were conditioned with the apparatus before measurements were taken, and systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured and recorded at the end of the experiment (35th day). The measurements were carried out blindly by the same set of personnel in all experimental animals, otherwise stated.

Hippocampal Tissue Preparation
The hippocampus was carefully removed from the cranial cavity and placed in 10 volumes of 0.1 M of phosphate buffer, pH 7.4, on ice and gently homogenized in a glass potter. After, the homogenates were centrifuged at 10,000 rpm for 15 min at 4 °C. Aliquot part of supernatants was stored in a refrigerated condition until the subsequent experimental analysis. Protein was determined by the Coomassie blue method described by Bradford (1976), using bovine serum albumin as standard solution.

Lipid peroxidation Assay
Lipid peroxidation was quantified as malondialdehyde (MDA) according to the method of Ohkawa et al. (1979) and expressed as nmoles MDA/mg protein.

Determination of Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) Activities
The activities of AChE and BuChE were determined in the hippocampus by the modified method of Ellman (Perry et al. 2000) and expressed as AChE activity/min/mg protein and BuChE activity/min/mg/protein, respectively.

Monoamine oxidase-A activity assay
Monoamine oxidase (MAO-A) activity was estimated using benzylamine as the MAO substrate according to the method described by Kettler et al. (1990), and the result was expressed as MAO-A activity/mg protein.

Determination of Arginase Activity
The hippocampal arginase activity was evaluated using the method of Zhang et al. (2001) and expressed as arginase activity/mg protein.

Assay of NO as a Marker of NO Synthesis
Nitric oxide level was determined by method described by Miranda et al. (2001) and was expressed as μM/mg protein.

NTPDase (Determination of ATPase and AMPase) Activities Assay
The NTPDase enzymatic assay of the hippocampus was carried out as described by the method of Schetinger et al. (2007). The released inorganic phosphate (Pi) was measured and enzyme activities are reported as nmol Pi released/mg of protein.

Adenosine Deaminase Activity Determination (ADA)
ADA activity estimation was performed using the method described by Guisti and Galanti (1984).

Isolation of RNA
Total RNA was isolated from tissue after homogenization with 100 mg/1 ml hippocampal tissue TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA), according the manufacturer's instructions. The hippocampus collected in TRIzol was taken out, and about 100 mg of tissue was homogenized in 1 ml Ribozol ™ reagent (USA) with Polytron homogenizer. Chloroform (100 µl) was mixed with the homogenate. Small portions of the samples (hippocampus) from the different groups were homogenized separately using a hand auto-homogenizer with plastic Teflon under sterile, nuclease-free conditions. To the homogenates, 100 µL of chloroform, the separating medium, was added for partitioning. The mixtures was allowed to stand for 10 min and centrifuged at 11, 300 rpm to give three distinct phases. The upper phase was aspirated into new sterile 1.5 mL Eppendorf tubes (appropriately labeled), and isopropyl alcohol (250 µL) was added. The content of each tube was mixed by inversion and allowed to stand for 10 min, to precipitate the total mRNA. The tubes were centrifuged at 12,000 rpm for 10 min, and the supernatants were decanted, leaving the pellets (mRNA). The pellets were washed with 500 µL of 75% ethanol, and the solution was spun in a centrifuge at 9,100 rpm for 5 min. The ethanol in each tube was decanted, and the pellets were dried in a DNA concentrator. The contents of each tube were then reconstituted using 50 µL of nuclease-free water and the concentration of mRNA in each tube was estimated using a NanoDrop at 260 nm. The mRNA in each tube was then diluted with RNase free water appropriately until equal concentration was achieved in each tube.

Real-Time PCR analysis of Hippocampal HGPRTI, DARPP, TrKB BCI11b, and BDNF
The isolated RNA was subjected to RT-PCR analysis according to the manufacturer's instructions. Total RNA was reversely transcribed using RevertAid™ First strand cDNA synthesis kit (Thermo Fisher Scientific, USA) as per specific primers (Table 1) for HGPRT1, DARPP, TrKB, BCl11b, BDNF and β-actin (as a reference gene).

Synthesis of Complementary DNA (cDNA)
Into clean, dry, well labeled Eppendorf tubes, 25 µL of the purified mRNA was measured for the synthesis of cDNA, using reverse transcriptase super mix (EasyScript ® One-Step RT -PCR SuperMix Cat. No. AE411).

Amplification of cDNA
The cDNAs obtained from 3.9.2 above were amplified, using the polymerase chain reaction (PCR) technique. Briefly, equal volumes (2 µL) of the DNA templates were dispensed into PCR tubes, and 10 µL of the PCR stock (containing nuclease-free water (2.4 µL), forward and backward primers for the gene of interest (0.2 µL each), and 5 µL of the 2X ES one-step reaction mix, EasyScript ® One-Step enzyme mix (0.2 µL)) was added. The tubes were then transferred to the PCR thermocycler for 30 cycles of amplification.

Gel Preparation and Electrophoresis of the Amplicon
Agarose (1 g) was dissolved in 100 mL of 1 × Tris-boric acid-ethylenediamine-tetra acetic acid (TBE) buffer and gently dissolved by heating in a microwave for 2 min. At 30-s intervals, the flask was removed and swirled to ensure proper dissolution. The gel was allowed to cool for easy handling, and ethidium bromide (4 µL) was added to stain the nucleic acids and allow visualization under ultraviolet light. The gel was gently poured into an already prepared electrophoretic tank and allowed to solidify after which 1 × TBE buffer was poured into the tank, followed by the removal of the combs and stoppers. DNA amplicons were loaded into the wells created by the combs and properly covered. Electrophoresis was allowed to run at a voltage of 90 mV and a current of 250 mA for 10 min. The gel was transferred to a Fotophoresis U/V transilluminator (Thermo Fischer, Germany) machine for viewing. The snapshot of the migrated bands was subjected to densitometry scanning, and the band intensity of the cDNA fragment of each gene was normalized against the band intensity of the cDNA fragment of the house keeping control gene (β-actin), using Image-J software as described by Schneider et al. (2012).

Examination of Histopathology
After the treatment, the hippocampus in each group was assessed via a cut in the sagittal plane and placed in 4% para-formaldehyde at 4 °C for 2 days. After dehydration, transparency, paraffin immersion, and paraffin embedding, the hippocampus was sliced along the median anteroposterior axes at a thickness of 6 μm. The section was stained with hematoxylin and eosin for morphological observation and defining positions. Sections were read and images were captured using microscope.

Statistical Analysis
No statistical method was used to determine sample size. Sample size was arbitrarily set to 63 (9 groups with 7 animals each). Results are presented as means ± SEM of the number of experiments indicated. Statistical significance was assessed by one-way analysis of variance followed by Turkey's test. p < 0.05 was considered to represent a significant difference in all experiments. The statistical analyses were performed using the software package GraphPad Prism 5. Figure 1A reveals that SBP was obviously activated (p < 0.05) by the administration of 100 mg/kg/day KBrO 3 compared to control. Similar pattern was observed in rats exposed to 250 mg/kg CAF intoxication when compared with control rats. Also, mixed exposure of 100 mg/kg/ day KBrO 3 and 250 mg/kg/day CAF remarkably hiked the systolic blood pressure to the tune of about 200 mmHg in compared to control rats. Conversely, Fig. 1A reveals that the post-administration of NAR with KBrO 3 only and CAF only caused a significant reduction (p < 0.05) in SBP similar to HAL treated group. In the same vein, post-administration of NAR against the mixture of KBrO 3 + CAF showed a significant decrease (p < 0.05) in SBP compared to the exposed groups. Furthermore, Fig. 1B shows a diastolic blood pressure greater than 90 mmHg following the administration of single exposure of KBrO 3 and CAF compared to control. Exposure to mixture of 100 mg/kg/day KBrO 3 and 250 mg/ kg/day CAF for 21 days significantly (p < 0.05) increased DBP compared to control. However, post-administration of NAR with KBrO 3 only, CAF only significantly (p < 0.05) reduced DBP compared to the exposed group. Similar effects were observed in KBrO 3 and CAF treated rats with NAR. Put together, NAR led to a significant reduction (p < 0.05) of SBP and DBP, predicting the restoration of normotension in rats on exposure to KBrO 3 and CAF.

Effect of Bi-flavonoid on Hippocampal MDA in Purine Metabolism Deficiency of Hippocampal Rats Exposed to KBrO 3 and CAF Intoxication
As observed in Fig. 2, the level of hippocampal MDA (hMDA) was elevated remarkably (p < 0.05) when compared to the control after treatment with 100 mg/kg/day KBrO 3 . The level of hippocampal MDA (hMDA) was also raised significantly (p < 0.05) when compared with the control after exposure to 250 mg/kg/day CAF intoxication (Fig. 2). Similarly, exposure to mixture of 100 mg/kg/day KBrO 3 and 250 mg/ kg/day CAF for 21 days significantly (p < 0.05) triggered the hMDA level (Fig. 2). The post-administration of NAR with KBrO 3 only, CAF only, and mixture of KBrO 3 + CAF caused a significant (p < 0.05) restoration of hippocampal membrane integrity similar to HAL treated group when compared to control (Fig. 2). Figure 3 shows the effect of 50 mg/kg/day of NAR on hippocampal tissue in rats exposed to 100 mg/kg/day KBrO 3 and 250 mg/kg/day CAF on the activity of ectonucleotidases; NTPDase (ATP as substrate, graph A and AMP as substrate, graph B) 5 1 -nucleotidase, and adenosine deaminase (adenosine as substrate, graph C). The inhibition of nitric oxide synthesis by KBrO 3 only, CAF only, and their mixture significantly (p < 0.05) increased the activity of NTPDase (ATP and AMP hydrolysis) and adenosine deaminase in relation to their corresponding controls. The post-treatment with NAR (50 mg/kg/day) remarkably (p < 0.05) down-regulated the activity of NTPDase (ATP and AMP hydrolysis) and ADA in rats exposed to KBrO 3 only, CAF only, and their mixture in comparison to the exposed groups ( Fig. 3A−C).

Effect of Bi-flavonoid on Neurotransmitter Enzymes (AChE, BuChE, and MAO-A) in Purine Metabolism Deficiency of Hippocampal Rats Exposed to KBrO 3 and CAF Intoxication
Figure 4A−C shows the effect of NAR on the activity of AChE, BuChE, and MAO-A in purine metabolism deficiency of hippocampal rats on exposure to CAF and KBrO 3 intoxication. As shown in Fig. 4A, the activity of hippocampal MAO-A was remarkably (p < 0.05) diminished upon administration of KBrO 3 only when compared to control. Equally, an obvious decrease (p < 0.05) was observed in rats due to 250 mg/kg/day CAF intoxication when compared to the control. In the same vein, rats exposed to both 100 mg/kg/day KBrO 3 and 250 mg/kg/day CAF showed significant (p < 0.05) reduction in the activity of hippocampal MAO-A in relation to the control rats (Fig. 4A). However, post-treatment with NAR significantly (p < 0.05) elevated the activity of hippocampal MAO-A in rats exposed to KBrO 3 only similar to HAL post-treated group in relation to the exposed group. In the NAR and CAF co-treated group, a significantly (p < 0.05) elevated activity of hippocampal MAO-A was resulted when compared to the toxicant group. Similar effects were observed in KBrO 3 and CAF treated rats post-treated with NAR (Fig. 4A). Conversely, Fig. 4B shows the effect of NAR on the activity of AChE in KBrO 3 -induced hippocampal impaired rats on exposure to CAF intoxication. Hippocampal AChE activity was obviously (p < 0.05) activated by the administration of 100 mg/ kg/day KBrO 3 in relation to the control. The same trend was observed in rats exposed to 250 mg/kg/day CAF intoxication when compared to the control rats, while mixed exposure of 100 mg/kg/day KBrO 3 and 250 mg/kg/day CAF significantly (p < 0.05) elevated the hippocampal AChE activity in relation to the control rats (Fig. 4B). The post-treatment with NAR (50 mg/kg/day) remarkably (p < 0.05) decreased hippocampal AChE activity in rats exposed to KBrO 3 only, CAF only and their mixture relative to the exposed groups (Fig. 4B). Furthermore, Fig. 4C shows that the activity of BuChE in hippocampus was obviously (p < 0.05) activated by the administration of 100 mg/kg/day KBrO 3 in relation to the control. Same trend was observed in rats exposed to 250 mg/kg/day CAF intoxication when compared to the control rats. Similarly, mixed exposure of 100 mg/kg/day KBrO 3 and 250 mg/kg/day CAF significantly (p < 0.05) elevated the hippocampal BuChE activity in relation to the control rats (Fig. 4C). Conversely, the post-treatment with NAR (50 mg/kg/day) remarkably (p < 0.05) decreased hippocampal BuChE activity in purine metabolism deficiency of rats exposed to KBrO 3 only, CAF only, and their mixture in comparison to the toxicant groups (Fig. 4C).

Effect of Bi-flavonoid on the Activity of Arginase in Purine Metabolism Deficiency of Hippocampal Rats Exposed to KBrO 3 and CAF Intoxication
Figure 5 reveals the effect of naringin, a bi-flavonoid on arginase activity in purine metabolism deficiency of rats exposed to KBrO 3 and CAF intoxication. Exposure of rats to 100 mg/kg/day KBrO 3 only, 250 mg/kg/day CAF only, and mixture of KBrO 3 and CAF outstandingly (p < 0.05) triggered the activity of hippocampal arginase in relation to control rats. The increase in arginase activity was evidently (p < 0.05) restored by naringin in the post-treated groups as shown in Fig. 5.
Naringin, a Bi-flavonoid Reduces the Activity of PDE-5 1 Activity in Purine Metabolism Deficiency of Rats Exposed to KBrO 3 and CAF Intoxication Figure 6 provides the account of PDE-5 1 activity in purine metabolism deficiency rats on exposure to KBrO 3 and CAF intoxication. Exposure of rats to 100 mg/kg/day KBrO 3 only substantially (p < 0.05) increased hippocampal PDE-5 1 activity in relation the control. A similar pattern was observed in animals exposed to 250/mg/kg/day CAF in comparison to control rats. Pointedly, exposure to mixture of 100 mg/kg/day KBrO 3 and 250 mg/kg/day CAF significantly (p < 0.05) enhanced PDE-5 1 activity in comparison to the control. Contrastly, a significant (p < 0.05) restoration to normalcy following post-treatment with NAR was observed in PDE-5 1 activity (as depicted in Fig. 6) in rats exposed to KBrO 3 only, CAF only, and KBrO 3 + CAF.  Fig. 3 A, B, and C Effect of naringin on ATP hydrolytic enzymes (ATPase and AMPase) and adenosine deaminase (ADA) in purine metabolism deficiency of hippocampal rats on exposure to KBrO 3 and CAF intoxication. Bars which are mean ± SEM of seven (n = 7) rats, bearing different letters are significantly different at p < 0.05 Fig. 4 A, B, and C Effect of naringin on neurotransmitter enzymes (MAO-A, AChE, and BuChE) in purine metabolism deficiency of hippocampal rats on exposure to KBrO 3 and CAF intoxication. Bars which are mean ± SEM of seven (n = 7) rats, bearing different letters are significantly different at p < 0.05   Figure 7 depicts the NO level in purine metabolism deficiency of hippocampus rats treated with 100 mg/kg/day KBrO 3 and 250/mg/kg/day CAF for 21 days. Owing to the involvement of NOS/AC/PKA/PKG signaling pathway in the regulation of enzymes, we studied the nitric oxide (NO) synthesis metabolites. It was observed that purine metabolism deficiency rats on exposure to KBrO 3 showed a significant (p < 0.05) reduction level of hippocampal NO content relative to the control. Correspondingly, exposure to 250/mg/kg/day CAF only and mixture of KBrO 3 and CAF considerably (p < 0.05) decreased the hippocampal NO content relative to the control group. Post-administration of NAR however significantly (p < 0.05) reconciled the effect of NO level in the hippocampus (Fig. 7) in relation to the exposed groups. This is suggestive of the fact that NAR may serve as NO donor, capable of acting on NO-dependent or NO-independent pathway.

Effect of Bi-flavonoid on BCI11b Transcript in Purine Metabolism Deficiency of Rats Exposed to KBrO 3 and CAF Intoxication
The effect of NAR on B-cell lymphoma/leukemia 11b (BCI11b) protein in purine metabolism deficiency rats on exposure to KBrO 3 and CAF intoxication is presented in Fig. 8. Exposure to KBrO 3 only significantly (p < 0.05) decreased the level of BCI11b compared to control, while rats' exposure to CAF only equally indicated (p < 0.05) low level of BCI11b compared to control. Combined exposure (KBrO 3 + CAF) also showed low significant (p < 0.05) low level of BCI11b proteins in relation to control. Conversely, post-treatment of NAR significantly (p < 0.05) improved the up-regulation of BCI11b proteins in relation to their corresponding exposed groups. Group of rats administered with NAR only significantly (p < 0.05) showed highest level of BCI11b protein when compared with other groups (Fig. 8).

Effect of Bi-flavonoid on DARPP-32, BDNF, and TrkB in Purine Metabolism Deficiency Rats on Exposure to KBrO 3 and CAF Intoxication
Since the production of DARPP-32 and BDNF-TrkB signaling pathway play regulatory functions on hippocampus and other regions of the brain to ameliorate psychiatric and neurological diseases, we therefore examined the effect of NAR on the level of dopamine and cAMP-regulated phosphoprotein of molecular weight 32 kDa (DARPP-32), brain-derived neurotrophic factor (BDNF), and tropomyosin-related kinase B (TrkB) in rat model (Figs. 9, 10, and 11). Exposure to KBrO 3 only, CAF only and KBrO 3 + CAF significantly (p < 0.05) lowered the hippocampal DARPP-32, BDNF, and TrkB proteins in relation to their corresponding control groups. Inversely, post-administration of NAR significantly (p < 0.05) heightened the levels of DARPP-32, BDNF and TrkB proteins in relation to the exposed groups.

Effect of Bi-flavonoid on the Expression of a Key Purinergic Enzyme, HGPRT1, in Hippocampal Rats Exposed to KBrO 3 and CAF Intoxication
The effect of NAR on HGPRT1 protein in rats exposed to KBrO 3 and CAF intoxication is presented in Fig. 12. Exposure to KBrO 3 only, CAF only and KBrO 3 + CAF significantly (p < 0.05) down-regulated the expression of HGPRT1 protein in relation to their corresponding control groups. Contrariwise, post-administration of NAR significantly  Fig. 7 Effect of naringin on NO level in purine metabolism deficiency of hippocampal rats on exposure to KBrO 3 and CAF intoxication. Bars which are mean ± SEM of seven (n = 7) rats, bearing different letters are significantly different at p < 0.05 (p < 0.05) enhanced the expression of HGPRT1 when compared to the exposed groups. However, a significant (p < 0.05) higher regulation of HGPRT1 was indicated in the groups of NAR + KBrO 3 and NAR only, when compared to others.

Effect of Bi-flavonoid on Histology of Purine Metabolism Deficiency of Hippocampal Rats upon Exposure to KBrO 3 and CAF Intoxication
Effect of bi-flavonoid on histology of purine metabolism deficiency of hippocampal rats upon exposure to KBrO 3 and CAF intoxication is presented in Fig. 13. In the control rats, the hippocampus shows normal hippocampal formation, there is normal structural organization of neuronal cells in CA2-CA4, the layers appear compact without scattering of neuronal cells, and the pyramidal cells seen are normal with mild scattering of pyramidal layers in CA 1 (black arrow) (13A). The hippocampus of rats exposed to KBrO 3 only and CAF only for 21 days shows depletion of pyramidal layers in CA4 (black arrow) (13B) and depletion and shrunken pyramidal layers in CA4 (black arrow) (13C), respectively. The hippocampus of rats exposed to mixture of KBrO 3 and CAF for 21 days (13D) shows depletion of pyramidal layers in CA4 (black arrow). Hence, post-treatment of NAR for 14 days shows normal structural organization of neuronal cells (white arrow) in CA1-CA4, the layers appear compact without scattering of neuronal cells, and the pyramidal cells seen are normal, with mild depletion of pyramidal layers in CA4 and CA1 (black Fig. 8 Effect of naringin on B-cell lymphoma/leukemia 11b normalized against housekeeping gene, beta-actin (β-actin) in purine metabolism deficiency of hippocampal rats on exposure to KBrO 3 and CAF intoxication. Bars which are mean ± SEM of seven (n = 7) rats, bearing different letters are significantly different at p < 0.05 Fig. 9 Effect of naringin on brain-derived neurotrophic factor (BDNF) normalized against housekeeping gene, beta-actin (β-actin) in purine metabolism deficiency of hippocampal rats on exposure to KBrO 3 and CAF intoxication. Bars which are mean ± SEM of seven (n = 7) rats, bearing different letters are significantly different at p < 0.05 arrows) (Fig. 13F−H). While hippocampus of rats treated with therapeutic drug shows normal structural organization of neuronal cells (white arrow) in CA1-CA4, the layers appear compact without scattering of neuronal cells, and the pyramidal cells seen are normal. However, there is mild vascular congestion (slender arrow) and thrombosis (black arrow) (Fig. 13E). The hippocampus of rats treated with NAR only shows normal structural organization of neuronal cells (white arrow) in CA2-CA4, the layers appear compact without scattering of neuronal cells, and the pyramidal cells seen are normal with mild scattering of pyramidal layers in CA1 (black arrow).

Discussion
The outcome of our study showed that post-treatment with NAR, a bi-flavonoid from grape fruit ameliorated hippocampal impairment mediated by hypertension in rats. This is evidenced by the attenuation of biochemical indices, including depletion of PDE-5 1 activities and restoration of histological architecture of the hippocampal tissues via NOS/cAMP/PKA and DARPP-32, BDNF/TrkB signaling pathways after exposure to KBrO 3 and CAF for 21 days. NAR was applied in the management of reactive oxygen species (ROS), inflammation, and cancer (Salehi Fig. 10 Effect of naringin on the expression of dopamine-and cAMP-regulated phosphoprotein 32 (DARPP-32) normalized against housekeeping gene beta-actin (β-actin) in purine metabolism deficiency of hippocampal rats on exposure to KBrO 3 and CAF intoxication. Bars which are mean ± SEM of seven (n = 7) rats, bearing different letters are significantly different at p < 0.05

Fig. 11
Effect of naringin on the expression of tropomyosinrelated kinase B (TrkB) normalized against housekeeping gene beta-actin (β-actin) in purine metabolism deficiency of hippocampal rats on exposure to KBrO 3 and CAF intoxication. Bars which are mean ± SEM of seven (n = 7) rats, bearing different letters are significantly different at p < 0.05 et al. 2019). Being an antioxidant, whose source is natural, it has a robust free radical scavenging ability due to its hydroxyl group which is capable of interacting with several biomolecules, thus leading to the stability of numerous physiological processes (Cavia-Saiz et al. 2010;Deenonpoe et al. 2019). We observed that KBrO 3 and CAF increased the systolic and diastolic blood pressure relative to control. Also, the combined exposure of rats to KBrO 3 and CAF significantly (p < 0.05) hiked both SBP and DBP. This asserts that a combined or single intoxication of these toxicants may be a potent cause of hypertension in mammals. This finding is similar to that of Williams et al. (2018) which places the diagnostic threshold of hypertension at Systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg or both. Interestingly, post-treatment with NAR significantly attenuated the increase in blood pressure and restored normotension in the animals.
Oxidative stress: the balance between oxidative and antioxidative processes is essential in many pathological conditions. Our study revealed that exposure to KBrO 3 caused an increase in MDA in the hippocampus of rats, suggesting its role in generating free radical and consequently high blood pressure triggered by oxidative stress. Our study also agrees with the previous studies that mouse exposure to KBrO 3 can lead to generation of free radicals which can attack polyunsaturated fatty acids (PUFAs) in the bio membrane, leading to its dysfunction (Saad et al. 2017). CAF intoxication significantly increased the MDA level compared to KBrO 3 intoxication. Although, previous findings on coffee consumption and its association with the incidence of hypertension are not homogeneous and still inconsistent (Nadia et al. 2021). Nevertheless, mixed exposure of KBrO 3 and CAF caused an up-surge in the MDA level which may be connected to the ability of these toxicants to obstruct the formation of nitric oxide, a potent vasodilator molecule that is capable of blocking hippocampal nitric oxide synthase (NOS) activity (Yurach et al. 2011;Sultan et al. 2015). In a remarkable manner, post-treatment with fruit active NAR caused an outstanding reduction of hippocampal MDA level, thus affirming the ability of plants flavonoids to restore normotension (Akintunde et al. 2020a, b).
It is noteworthy to report that the decline of AChE and BuChE activities may increase the bioavailability of neurotransmitters which could stimulate the production of NO in the hippocampal endothelial cell. From our findings, both single and combined exposure to KBrO 3 and CAF intoxication caused a noticeable increase in AChE and BuChE activities. However, post-treatment with NAR prevented this effect. Expectedly, NAR only showed the strong protection when compared with the exposed groups. The metabolic reduction of AChE and BuChE activities caused the bioavailability of acetylcholine and butyrylcholine, respectively (Akintunde and Oboh 2015), thus stimulating the production of NO in the hippocampal endothelial cell. This effect can be attributed to the intake of NAR against disorders of hippocampus facilitated by hypertension in rats. However, MAO-A activity did not follow similar pattern compared with AChE and BuChE. Studies have shown that caffeine shows positive effects in the management of some neurological disorders in animal models, in part by modulating neurotransmitters and dopaminergic signaling in the mesocorticolimbic brain regions (Alasmari 2020). Corroborating the pattern shown by MAO-A, Diogo (2010) reported that high doses of caffeine may lead to psychosis and anxiety, while reasonable consumption of caffeine (less than 6cups/day) has the ability to attenuate cognitive failure, suicide, and symptoms of major depressive disorder, thus suggesting the need for further dose regimen elucidation in MAO-A activity in KBrO 3 and CAF intoxicated rats. From our study, it is therefore safe to conclude that the fall in the level of AChE Fig. 12 Effect of naringin on the expression of hypoxanthineguanine phosphoribosyl transferase 1 (HGPRT1) normalized against housekeeping gene, beta-actin (β-actin) in the hippocampus of experimental rats. Bars which are mean ± SEM of seven (n = 7) rats, bearing different letters are significantly different at p < 0.05 and BuChE after exposure to KBrO 3 and CAF intoxication in a single and mixed manner may be the basis for neurodisorder while the decreasing pattern shown by MAO-A activity suggest irrational behaviors, an indicator of Lesch Nyhan syndrome (LNS), as all monoamine (neurotransmitter) has been completely depleted. Our study is in tune with previous findings which indicates that a surge in these enzymes may interplay at the center of several pathophysiology (Tarr . 2013) and that that the inhibition of these enzymes may confer disorderliness in cognitive function (Marucci et al. 2020). Inversely, post-treatment with 50 mg/kg NAR prevented this effect by reducing the activities of acetylcholinesterase, butyrylcholinesterase, thus up-regulating the level of NO to promote its therapeutic effect. Low NO with a concomitant increased PDE-5 1 activity could lead to severe damages in vascular muscles (Loeb et al. 2012). The intoxication of KBrO 3 and CAF in purine metabolism deficiency in rats was heightened in the activity of PDE-5 1 with a concurrent reduction of NO bioavailability in rats' hippocampus. The fall in the level of NO enhanced the binding of cGMP to the allosteric site of PDE-5 1 (i.e., phosphorylation of PDE-5), exerting its effect. It is of essence to report here that exposure of hippocampal impaired hypertensive patients to KBrO 3 and CAF may increase the phosphorylation of PDE-5 1 by binding to cGMP to initiate disorder. Conversely, NAR impeded the degradative action of cGMP-specific phosphodiesterase type 5 (PDE-5 1 ) on cyclic GMP in the hippocampus. For this reason, NAR is capable of causing vasodilation in the cells of the hippocampus, enabling unhindered blood flow to the hippocampus. An increase in NO level has been further elucidated to result in the positive modulation of guanylate cyclase (GC) and production of cyclic guanosine monophosphate (cGMP) which eventually triggers protein kinase G (PKG), thus implicating decreased level of NO in the pathogenesis of hypertension and neuroinflammation (Miguel et al. 2015). In the same vein, disturbance in the activity of arginase has been intricately connected to further deterioration of hypertensive patients (Johnson et al. 2015). Our study appears to be the first which establishes an up-surge in the activity of arginase after CAF intoxication in purine metabolism deficiency of hypertensive rats. This may depict a drop in the level of L-arginine needed to create NO by nitric oxide synthase (NOS), thus implicating CAF intoxication in neurological damage. The connection between increased arginase and lowered cyclic adenosine monophosphate (cAMP) which should eventually trigger protein kinase A (PKA) (Mason et al. 2015), may also be responsible for hippocampal impairment. Thus, exposure to CAF and KBrO 3 intoxication might have contributed to hippocampal impairment mediated by hypertension in rats by increasing arginase activity, thereby lowering cAMP and simultaneously lowering NO, thus down-regulating cGMP. Similar to the findings of Johnson et al. (2015), decreased expression of NO with a concurrent increase in arginase activity is connected to hypertensive-induced impairment. Consequently, the oral administration of fruit active NAR caused a significant reduction in the hippocampal arginase activity to increase endothelium-dependent relaxation of hippocampal cells (Xu et al. 2015) by conferring stability on NO/cGMP pathway (Anwar et al. 2018).
Nitric oxide (NO), a strong vasodilator, plays multifunctional roles in disorders by activating the soluble guanylate cyclase, leading to increased production of cyclic adenosine monophosphate (cGMP). In the same vein, NO has been established to provide a relationship between neurological and vascular diseases and the enzymes that cause ATP breakdown. In order to activate a pathological response, these nucleotides hydrolyze adenosine triphosphate (ATP), resulting to AMP (adenosine-5-monophosphate) and diphosphate (Gardani et al. 2019). We therefore examined the possibility of alterations in the activity of NO yielding an increase in adenosine deaminase and enzymes of ATP hydrolysis thus acting as the basis for hippocampal impairment mediated by hypertension in rats exposed to CAF intoxication. In this study, we observed that single administration or mixed exposure of rats to KBrO 3 and CAF remarkably increased the ATP hydrolytic enzymes (ATPase and AMPase) and adenosine deaminase activity (ADA). In fact, the combined exposure showed a greater increase in the NTPDase compared to the single administration of KBrO 3 and CAF. Altogether, these increases signify pathophysiology in prostatic homeostasis and some other organs (Gardani et al. 2019). The mechanism of action of adenosine is similar to NO, cGMP, and neurotransmitters (Layland et al. 2014). It mediates its action by regulating adenylyl cyclase (AC) and the production of cAMP (Raker et al. 2016). Adenosine-mediated cAMP further induces protein kinase A (Pleili et al. 2018), thus enhancing regulatory functions on hippocampus and other regions of the brain (Park et al. 2021). We therefore report that NAR might occasion the hydrolysis of ATP to adenosine, thus activating AC which promotes the breakdown of ATP to form cAMP (a secondary messenger).
Hypoxanthine-guanine phosphoribosyl transferase (HGPRT1) is a transferase coded for in humans by the HPRT1 gene (Finette et al. 2002). Its function is to catalyze the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. Thus, it is required for efficient recycling of purine nucleotides. The deamination of adenosine is catalyzed by adenosine deaminase (ADA) into inosine which can then be converted into hypoxanthine and then retrieved as IMP or exported out of the cell as hypoxanthine or uric acid (Camici et al. 2018). From our findings, KBrO 3 and CAF intoxication caused a decrease in HGPRT1 expression which led to the accumulation of hypoxanthine, a basis for neurological disorders (Agrahari et al. 2019), known as Lesch-Nyhan syndrome. Also, exposure to KBrO 3 and CAF facilitates the accumulation of hypoxanthine, which is a consequence of HPRT1 deficiency that triggers neurological abnormalities (Torres et al. 2016), an indication of LNS in mammals. It equally caused the dysregulation of cAMP-PKA signaling and phosphodiesterase expression due to HPRT1-deficiency (Guibinga et al. 2013). Interestingly, post-NAR-administration caused a significant increase in the expression of HGPRT1 in the rats signifying amelioration of CAF and KBrO 3 -induced hippocampal perturbations associated with LNS. Furthermore, the control of BDNF-TrkB signaling pathway has been established to ascertain the ameliorative capacity in combating neurological diseases (Sheldrick et al. 2017;Park and Lee 2018). Exposure of rats to KBrO 3 and CAF has reduced these signaling pathways and consequently lowered the expression of antioxidant enzymes which possess safeguarding functions on the neurons and up-regulation of cAMP dependent protein kinase A (PKA) and calcium/calmodulin-regulated protein kinases CaMKII (Bruna et al. 2018;Nitti et al. 2018). Also, the expression of BDNF-TrkB may be down-regulated in CAF and KBrO 3 groups due to low production of HGPRT1. On the contrary, post oral administration of NAR upregulated the expression of BDNF-TrkB, signifying protection against purine metabolism deficiency associated with hypertension and LNS. This study synchronizes well enough with the findings of Zhang et al. (2016) which emphasizes the therapeutic roles of BDNF-TrkB signaling in psychiatric disorders. Also, activation of adenyl cyclase (AC) with an attendant upregulation of intracellular PKA due to the down regulation of adenosine in the cascade may cause the phosphorylation of Dopamine and cAMP-regulated phosphoprotein of relative molecular mass 32,000 Daltons (DARPP-32) at Thr 34 residue on post-treatment with NAR. This interconnected activation is required for improved brain function (Qian et al. 2015) as corroborated by (Pleili et al. 2018;Park et al. 2021). Consequently upon the administration of CAF and KBrO 3 in purine metabolism deficiency rats, we observed a significant reduction in the expression of DARPP-32 which could be suggestive of neurological disorders like schizophrenia (Wang et al. 2017). This further establishes the neurodegenerative ability of these toxicants (Saad et al. 2017;Alasmari 2020). However, the study hypothesized that bi-flavonoid known as naringin may be a pharmacological supplement against neurological hypertension connected to LNS via DARPP-32/BDNF-TrkB signaling cascades in rats.
Lastly, B cell leukemia 11b (Bcl11b) is a transcription factor belonging to zinc finger protein family (Lennon et al. 2017). It is essential for the development of the nervous and the immune system. Its deficiency in mice causes brain defects, abridged learning ability, and compromised immunity (Lessel et al. 2018). In our study, Bcl11b transcript levels were down-regulated on exposure to CAF and/or KBrO 3 in relation to the control group. Molecular examination revealed that Bcl11b expression was reduced in the hippocampus of diseased rats, which further supports the decrease in its level due to CAF and KBrO 3 exposure. However, post-administration of naringin markedly upregulated its expression in the hippocampus to provide protection against CAF and KBrO 3 exposure in purine metabolism deficiency rats. Several studies have demonstrated the role of caffeine in LNS, and there have been diverse views till date. However, this study corroborates the findings of Lin et al. (2021) which established the interference of habitual daily consumption of caffeine with sleep-wake regulation and could disturb neural homeostasis.

Conclusion
Post-treatment with NAR for 14 days on exposure to KBrO 3 and CAF for 21 days repressed disorders of hippocampus facilitated by hypertension in rats via depletion of arginase, reduction of elevated malondialdehyde level, enzymes of ATP hydrolysis, and alteration of biochemical indices in the hippocampal tissue. Also, exposure to KBrO 3 and CAF for 21 days down-regulated the expression of HGPRTI, DARPP, TrKB BCI11b, and BDNF molecules. However, naringin, a bi-flavonoid, may serve as a fruit therapy against hippocampal hypertension in purine metabolism deficiency via NOS/ cAMP/PKA and DARPP-32, BDNF/TrkB signaling pathways. We therefore recommended naringin for further studies especially on clinical trials.