Neuroprotection of Tropical Fruit Juice Mixture via the Reduction of iNOS Expression and CRH Level in β-Amyloid-Induced Rats Model of Alzheimer's Disease

This study aimed to determine the effects of tropical fruit juice mixture (pomegranate, white guava, and Roselle) on biochemical, behavioral, and histopathological changes of β-amyloid- (Aβ-) induced rats. Formulation 8 (F8) of tropical fruit juice mixture was chosen for this present study due to its high phenolic content and antioxidant capacity. Forty Wistar male rats were divided into five groups: dPBS (sham-operated control), dAβ (Aβ control), JPBS (F8 and PBS), JAβ (F8 and Aβ), and IBFAβ (ibuprofen and Aβ). F8 (5 ml/kg BW), and ibuprofen (10 ml/kg BW) was given orally daily for four weeks before the intracerebroventricular infusion of Aβ for two weeks. Histological analysis and neuronal count of hippocampus tissue in the Cornu Ammonis (CA1) region showed that supplementation with F8 was able to prevent Aβ-induced tissue damage and neuronal shrinkage. However, no significant difference in locomotor activity and novel object recognition (NOR) percentage was detected among different groups at day 7 and day 14 following Aβ infusion. Only effect of time differences (main effect of day) was observed at day 7 as compared to day 14, where reduction in locomotor activity and NOR percentage was observed in all groups, with F (1, 7) = 6.940, p < 0.05 and F (1, 7) = 7.152, p < 0.05, respectively. Besides, the MDA level of the JAβ group was significantly lower (p < 0.01) than that of the dPBS group. However, no significant changes in SOD activity were detected among different groups. Significant reduction in plasma CRH level (p < 0.05) and iNOS expression (p < 0.01) in the brain was detected in the JAβ group as compared to the dAβ group. Hence, our current findings suggest that the tropical fruit juice mixture (F8) has the potential to protect the rats from Aβ-induced neurotoxicity in brain hippocampus tissue possibly via its antioxidant properties and the suppression of iNOS expression and CRH production.


Introduction
Alzheimer's disease (AD) is one of the most common neurodegenerative diseases in the elderly [1]. e clinical manifestations of AD include progressive loss of memory (dementia) and cognitive function, which are always accompanied by behavioral disorders [2]. e neuropathological hallmarks of AD include "positive" lesions such as the presence of amyloid plaques and cerebral amyloid angiopathy, neurofibrillary tangles (NFT), and glial responses and "negative" lesions such as loss of neurons and synapses [3]. While the senile amyloid plaques and NFT are the classical neuropathological lesions of AD, they likely only represent the surface of the pathological alterations that cause the cognitive decline associated with AD. Despite their cross-sectional nature, postmortem studies have enabled the staging on the progression of both amyloid and tangles which consequently lead to the development of diagnostic criterias that are being used worldwide currently [3]. Natural polyphenols have been reported to exert beneficial effects in preventing cardiovascular-related conditions but studies on its neuroprotective mechanisms are still lacking. Many of the biological effects of polyphenols have been attributed to their antioxidant properties, either through their reducing capacities per second or through their possible influences on intracellular redox status [4]. As such, polyphenols may protect cell constituents against oxidative damage and have been reported to limit the risk of various oxidative related degenerative diseases. e brain is particularly vulnerable to oxidative damage because it requires considerable amounts of energy and consumes a lot of oxygen in normal physiological conditions. Besides, the brain is also more susceptible to oxidative stress as compared to other organs due to the lower antioxidant defense capacity in the brain. e occurrence of oxidative stress in the brain is a key feature of sporadic Alzheimer's disease (AD) and manifests predominantly as lipid peroxidation [5]. e neuroprotective effects of many polyphenols rely on its ability to permeate the blood-brain barrier (BBB) and directly scavenge pathological concentration of reactive oxygen and nitrogen species and chelate transition metal ions [6]. Distinctive polyphenols were shown to have different scavenging activities and abilities to activate key antioxidant enzymes in the brain, thus breaking the vicious cycle of oxidative stress and tissue damage [7,8]. Red-wine polyphenols with distinct structures have been documented to increase endothelial nitric oxide synthase (eNOS) while downregulating inducible nitric oxide synthase (iNOS) activity [9,10]. Besides, tea polyphenols have been shown to increase neurological scores and reduce brain injury in oxyhemoglobininduced subarachnoid hemorrhage mice [11]. Furthermore, the neuroprotective effects of polyphenols were also achievable by reducing neurological injury, which subsequently leads to the improvement in behavioral outcomes [12,13].
Recently, accumulating evidence suggested that the classical hydrogen-donating antioxidant activity of polyphenols is unlikely to be the sole explanation for its cellular effects in vivo [4]. Polyphenols have exhibited several additional properties in a complex biological system, yet the mechanisms remain poorly understood. It is evident that polyphenols are potent bioactive molecules; therefore, a clear understanding of their precise mechanisms of action as either antioxidants or modulators of cell signaling is crucial to evaluate their potential as inhibitors of neurodegenerative diseases [4]. Besides, there are still limited studies on the neuroprotective effects of polyphenol-rich tropical fruits. Hence, this study aimed to determine the effects of tropical fruit juice mixture (pomegranate, white guava, and Roselle) on biochemical, behavioral, and histopathological changes of the Aβ-induced rat model of Alzheimer's disease. is is to explore the potential of the tropical fruit juice mixture to be developed as part of the therapeutic intervention in the prevention of chronic neurodegenerative diseases.

Analysis of Tropical Fruit Juice Mixture.
e antioxidant capacity of the tropical fruit juice mixture was analyzed using FRAP assay, DPPH assay, and TPC analysis, with slight modification of the method proposed by Aziz et al. [15]. ese assays were used to determine the best formulation in terms of antioxidant capacity and TPC content in the tropical fruit juice mixture. e best formulation was selected for further testing in the in vivo experiment. . Rats were kept at constant room temperature (24°C) under a 12-hour light/dark cycle (lights on from 7 : 00 AM to 7 : 00 PM) and were acclimatized at least seven days before the experiment and grouped into five groups with eight rats per group. Group one was the sham-operated control group (dPBS) supplemented with distilled water. Group two was the Aβ control group (dAβ) supplemented with distilled water. Group three (JPBS) was the sham-operated control group supplemented with tropical fruit juice mixture. As for group four (JAβ) and five (IBFAβ), they were Aβ-induced groups supplemented with tropical fruit juice mixture and ibuprofen (IBF), respectively.

Administration of Tropical Fruit Juice Mixture and
Ibuprofen. Tropical fruit juice mixture and IBF (Sigma-Aldrich Chemical, Saint Louis, USA) were given daily via oral gavage at a modified dosage of 5 ml/kg body weight [16] and 1 mg/kg/10 ml [17], respectively, for four weeks before Aβ infusion. For the dPBS group and dAβ group, distilled water (5 ml/kg body weight) was given orally to the rats instead of tropical fruit juice mixture. e experimental schedule of the study is summarized in Figure 1.

Intracerebroventricular Surgery of Beta-Amyloid.
Synthetic Aβ 1-42 (0.1 mg/ml) was dissolved in phosphatebuffered saline (PBS) and incubated at 37°C for three days to allow the formation of Aβ 1-42 aggregation. e rats were anesthetized using a mixture of ketamine, tiletamine, and xylazine (KTX) (0.1 ml/200 g) by intraperitoneal (i.p.) injection. Aβ was injected intracerebroventricularly (i.c.v.) using a bone microdrill, as described previously [18,19]. A small incision was made on the head of the anesthetized rats to expose the skull. en, one hole was drilled on the exposed skull (anteroposterior +1.2 mm from Bregma, mediolateral +2.0 mm, dorsoventral +4.0 mm) by using a stereotaxic apparatus. e cannula was affixed to the skull by using cyanoacrylate loctite glue (Loctite 454, USA). A subcutaneous pocket was prepared in the midscapular region of the back of the rats to receive the mini osmotic pump (ALZET, USA). e pump was then implanted in the subcutaneous pocket and was attached via polyvinylchloride tubing to the brain cannula. Aβ 1-42 solution and vehicle (PBS) were spontaneously infused into the left lateral cerebral ventricle of the brain through the cannula by a mini osmotic pump for two weeks at a constant rate (0.5 μl/hour), and the wound was stapled closed with wound clips (Reflex 7, USA). When the pump is properly placed, the tubing should have a generous amount of slack to permit free motion of the rats' head and neck. e rats require no restraint or handling during the delivery period.
2.6. Open Field Test. Rats were trained two days before drug and juice administration. e open field test (OFT) was performed before the novel object recognition (NOR) task. In this study, OFT was performed to measure locomotor activity in rats and exploration time in the central square of the arena [20]. e experiment was conducted in a soundproofed room illuminated with red light (20 watts) for easier recording of rats' activity. Each rat was placed in an open field arena (72 cm × 72 cm × 38 cm) and its exploration activity was recorded for five minutes. Recorded videos were analyzed, and the behaviors of rats were scored based on the frequency of line crossing, rearing, freezing, and time spent in the central square zone [21]. Locomotor activity was measured by the total frequency of line crossing and rearing.

Novel Object Recognition.
e test was carried out with a slight modification of the method proposed by Broadbent et al. [22]. Each rat was acclimatized for 45 minutes in a room inside the cage and one minute in an empty arena before the experiment. NOR was divided into three phases: habituation, familiarization, and novel with a duration of seven days. e first phase was habituation for five minutes (day 1 st and 2 nd ), followed by the familiarization phase for 15 minutes from the 3 rd day until the 6 th day and lastly the novel phase for 10 minutes on day 7 th . Rats were given two objects with similar shape and size during the familiarization phase but one of the familiar objects was replaced with a novel object during the novel phase. e object exploration was scored based on time spent to explore each object. Exploration was scored when the distance between the rat's noses was one cm from the placed object and there was movement of vibrissae [23]. Rats' preference for a novel object was expressed as the percentage of NOR exploration time compared to a familiar object.

Western Blot.
Rats from each group were decapitated, and brains were rapidly removed using the aid of dried ice to maintain the necessary temperature. Following dissection, each hippocampus was weighed and homogenized with lysis buffer. Brain lysates containing protein were used for the preparation of loading samples. Western blot was performed with slight modification of the method by Mahmood and Yang [24]. Samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. By using a conventional method, the membrane was blocked with 4% bovine serum albumin (BSA) solution before washing with tris-buffered saline with 0.1% Tween 20 (TBST) solution 5 times (5 minutes for each wash). en, the membrane was incubated with rabbit polyclonal anti-iNOS primary antibody (Abcam, USA; 1 : 1000 dilution) or rabbit monoclonal anti-β actin primary antibody (Abcam, USA; 1 : 1000 dilution) for 16 hours at 4°C and followed by 2 hours of incubation with HRP-conjugated anti-rabbit secondary antibody (Abcam, USA; 1 : 1000 dilution) at room temperature. e membrane was washed with TBST solution 5 times after every cycle of antibody incubation. Protein detection was conducted on the membrane by using Amersham enhanced chemiluminescence (GE Health Care, UK)

Histological Analysis of Hippocampus and Neuronal
Count. e hippocampus of the brain was first sectioned and isolated by using brain matrices (Tedpella, USA). After fixing with 10% formaldehyde, the hippocampus tissue was dehydrated, embedded in paraffin, and sliced into 5 μmthick section before staining with Nissl stain [25]. Prepared slides were examined under a light microscope, and the neuronal count was performed within the selected area (100 μm 2 ) of each hippocampus tissue.
2.11. Statistical Analysis. Statistical analysis was performed using SPSS version 22 (IBM, USA). e data were expressed as mean ± standard error of the mean (S.E.M) while p value less than 0.05 was considered as statistically significant. For the behavioral test, repeated-measures ANOVA was carried out to determine the significant differences between different groups and days of β-amyloid infusion. On the other hand, the biochemical tests and the neuronal count data were analyzed by using one way ANOVA and followed by Tukey HSD post hoc test to determine the significant differences between different groups. For nonparametric data, the Kruskal-Wallis analysis was performed and followed by the Mann-Whitney U test. Figure 2(a), F9 (4725.25 ± 158.70 μg/ml LAA) has the highest FRAP value and followed by F8 (4310.30 ± 185.40 μg/ml LAA). On the other hand, F10 has the highest (78.51 ± 5.67%) DPPH radical removal activity among all the fruit juice formulations, as shown in Figure 2(b). For TPC (Figure 2(c)), the highest phenolic content was reported in F9 (690.27 ± 21.18 μg GAE/ml). e TPC value of F8 (686.32 ± 7.32 μg GAE/ml) was slightly lower than F9. However, no significant difference in the TPC value can be detected between these two formulations. Both F8 and F9 were reported to have higher TPC and FRAP values as compared to the other formulations. However, the DPPH radical removal activity of F8 was higher than F9. Hence, the best formulation chosen for this study was F8 due to its high phenolic content, FRAP value, and DPPH radical removal activity in overall. . Tables 1 and 2 showed the locomotor activity in OFT and NOR percentage, respectively, between groups at day 7 and day 14 following Aβ infusion. Only effect of time differences (main effect of day) was observed at day 7 as compared to day 14, where reduction in locomotor activity and NOR percentage was observed in all groups (dPBS, dAβ, JPBS, JAβ, IBFAβ), with F (1, 7) � 6.940, p < 0.05 and F (1, 7) � 7.152, p < 0.05, respectively. No significant difference in locomotor activity and NOR percentage was detected among different rat groups at these two time points.

Effects of Tropical Fruit Juice Mixture on Aβ-Induced
Histological Changes

Histological Analysis of the Cornu Ammonis Region in
Hippocampus. Figure 3 shows the cross-sectional histology of hippocampus tissue in the Cornu Ammonis (CA1) region under 400x magnification. In the sham-operated control group (dPBS), neuron cells were normal in shape and arranged in an orderly and compact manner (Figure 3(a)). Similar results were also seen in the JPBS group (Figure 3(c)). For the dAβ group (Figure 3(b)), prominent tissue damage and shrinkage of neuronal cells were observed following the infusion of Aβ via i.c.v. Besides, neuron cells were not orderly arranged with the presence of gaps or spaces in between the cells. However, normal-shaped neuron cells were observed in the CA1 region of the hippocampus (Figure 3(d)) of the JAβ group after Aβ infusion, indicating that supplementation with tropical fruit juice mixture was able to prevent Aβ-induced neuronal shrinkage and damage.
On the other hand, ibuprofen-treated groups (IBFAβ group) showed an order and compact arrangement of neuron cells (Figure 3(e)) following Aβ infusion.

Neuronal Count in the Cornus Ammonis Region of
Hippocampus. Figure 4 shows the results of the neuronal count of the CA1 region of the hippocampus. In normal conditions, supplementation with F8 did not cause any significant increment in neuronal count as compared to the dPBS control group. Infusion of Aβ by i.c.v. into the brain hippocampus of the dAβ group (38.00 ± 2.00) caused a significant reduction (p < 0.05) in neuronal count as compared to the sham-operated control group (dPBS; 77.00 ± 1.00). However, supplementation with tropical fruit juice mixture or treatment with ibuprofen was able to attenuate the neurotoxic effects of Aβ infusion. Significant increment in neuronal count was observed in the JAβ group (p < 0.05; 71.50 ± 6.50) and IBFAβ group (p < 0.01; 93.00 ± 4.00) as compared to the dAβ group (38.00 ± 2.00).

MDA Concentration and SOD Activity in Brain
Homogenate. Infusion with Aβ did not cause any significant changes in MDA concentration in the brain homogenate of the dAβ group as compared to the dPBS group ( Figure 5(a)).
Only rats supplemented with F8 (JAβ group) showed a significant reduction (p < 0.01) in MDA concentration following Aβ infusion as compared to the dPBS group. On the other hand, no significant difference in SOD activity was detected in the brain homogenate of different rat groups, as shown in Figure 5(b).

iNOS Expression in Brain
Homogenate. Figure 6 shows the expression level of iNOS in brain homogenates among different rat groups. Infusion of Aβ (dAβ group) caused an increment in iNOS expression. Supplementation with F8 (JAβ group) was able to suppress the induction of iNOS expression significantly (p < 0.01), as compared to the dAβ group.

Concentration of CRH in Blood
Plasma. e plasma CRH concentration of different rat groups is shown in Figure 7. CRH concentration was significantly lower in the JAβ group (p < 0.01; 0.244 ± 0.089 μg/ml) and IBFAβ group (p < 0.05; 0.336 ± 0.088 μg/ml) as compared to the dPBS group

Discussion
is study aimed to explore the neuroprotective effects of the tropical fruit juice mixture from three different polyphenolrich fruits (pomegranate, white guava, and Roselle). e formulation F8 contains high phenolic content and antioxidant capacity as determined via FRAP, DPPH, and TPC analysis. ese fruits have been documented to be rich in flavonoid, tannin, and phenolic acid [26][27][28][29][30] that help in increasing and sustaining antioxidant levels more effectively.
is formulation was particularly rich in pomegranate which possesses high levels of antioxidant than other fruit juices and beverages [31,32]. Some in vivo studies also suggested that pomegranate extract may be helpful in the prevention of AD [33]. On the other hand, ibuprofen, which is a nonsteroidal anti-inflammatory drug (NSAID), was used as a positive control for Aβ-induced AD-like conditions in this present study. Previously, ibuprofen has been shown to suppress senile/amyloid plaque pathology and inflammation in a transgenic AD mouse model [34].
Although the cause of AD remains unclear, Aβ accumulation in the brain was proposed to play a major role in the onset of this disease [35,36]. Partial reproduction of AD neuropathological conditions and cognitive deficits can be achieved with pharmacological drug-induced approaches  Evidence-Based Complementary and Alternative Medicine and Aβ overexpression [37]. In this study, Aβ-induced neurotoxicity (AD model) was chosen due to the ability of the Aβ protein-peptide to produce the symptoms and pathophysiology that mimics the slow evolution of AD in humans [19,38]. e induction of neurotoxicity had successfully been achieved in this study since prominent tissue damage, shrinkage, and loss of neuronal cells were observed following the infusion of Aβ for 14 days consecutively. e previous study also demonstrated similar histological changes in the hippocampus region of rats following Aβ administration [26]. Histological analysis of the CA1 region in the hippocampus demonstrated that supplementation with the tropical fruit juice mixture can attenuate the neurotoxic effects of Aβ. Increase in the number of neuronal cells and the presence of neuronal cells with normal morphology following infusion of Aβ suggested that tropical fruit juice mixture possesses neuroprotective effects and has the potential to prevent the development and progression of neuronal diseases such as AD. Our current findings were further supported by the previous study where the Aβ-induced rat group supplemented with resveratrol was able to increase the neuronal count and reduced the neuronal damage caused by the neurotoxic effects of Aβ [26]. Central infusion of different types of Aβ such as Aβ 1-40 , Aβ 1-42 , or Aβ 25-35 into the intracerebral ventricle or hippocampus produced memory impairment by forming oligomer assemblies in rodents, as confirmed by data from various behavioral tests [38][39][40][41][42]. However, our present study showed that Aβ alone did not cause any significant behavioral changes, as demonstrated via OFT and NOR tests after 7 days and 14 days of infusion. is could be due to the concentration of Aβ (0.1 mg/ml) being used in this current study to induce the neurotoxic conditions in rats was relatively lower as compared to others previous studies, which is possibly inadequate to cause any detectable changes in OFT and NOR tests within the study time frame [38,42]. Hence, we were unable to investigate the protective effects of tropical fruit juice mixture against Aβ-induced behavioral changes in this present study under such circumstances.
In normal physiological conditions, redox status in cells is maintained by the antioxidant defense system [43].
However, excessive production of reactive oxygen species (ROS) may overwhelm the capacity of the antioxidant defense system, hence leading to the occurrence of oxidative stress. Oxidative stress is known to play an important role in the development of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease [44]. It has been reported that the presence of Aβ can increase ROS production in neuronal cells, hence causing oxidative stress in the brain [45]. A previous study has demonstrated that high level of MDA was found in the brain tissue of AD [26]. On the other hand, lower SOD activity was observed in the Aβ-induced group as compared to the control group following Aβ infusion into hippocampal tissues [46,47]. However, our current findings showed contrasting results where no significant changes in MDA concentration and SOD activity were detected between the dAβ group and the dPBS group following the infusion of Aβ. It is possible that the rats might have developed compensatory mechanisms against Aβ-induced oxidative stress during the study period since we infused the rats with a relatively lower dose of Aβ for 14 days.
Our current findings showed that supplementation with tropical fruit juice mixture can reduce the MDA concentration in the brain of rats following Aβ infusion. However, it does not have any effects on SOD activity. Hence, we suggest that the neuroprotective effects of tropical fruit juice mixture against Aβ-induced neurotoxicity were solely due to its high phenolic content and antioxidant capacity present in the tropical fruit juice mixture. It has been suggested that antioxidant therapy may help in the prevention and treatment of neurodegenerative diseases [48,49]. e presence of antioxidants derived from food sources has been suggested as a promoter for the endogenous antioxidant activity to reduce oxidative stress-aided process [50]. Food antioxidants derived from tropical fruit juice mixture of three different fruits (pomegranate, guava, and Roselle) contain different types of antioxidants that are beneficial to health, as proven previously [26][27][28][29][30]. us, we believed the composition of different fruits helps in increasing antioxidant levels more effectively.
Previous reports also demonstrated that Aβ protein is a potent neurotoxin that leads to the occurrence of neuroinflammation in the brain [51,52]. e induction of neuroinflammation by Aβ infusion may induce the expression of iNOS in neuronal cells. iNOS, which is the inducible isoform of NOS catalyzes the conversion of L-arginine into one of the reactive nitrogen species (RNS) known as nitric oxide (NO) [53]. NO is a short-lived free radical that plays an essential role in various physiological functions, including inflammation. Overproduction of NO can be deleterious since it is cytotoxic, and it may cause tissue destruction [54]. Our current findings demonstrated that Aβ infussion can induce the expression of iNOS, and supplementation with the tropical fruit juice mixture can attenuate the induction of iNOS expression by Aβ. is observation was supported by previous findings where supplementation with polyphenols derived from lychee seed and resveratrol was able to reduce the iNOS expression in rats following Aβ induction [26,55].
is is probably because the infusion of Aβ which is neurotoxic and can cause neuronal cell death, as demonstrated via histological analysis of the CA1 region of the hippocampus and neuronal count. e loss of CRH neurons may lead to the overall reduction in CRH production and secretion even in the presence of Aβ that has been shown to activate the HPA axis. Furthermore, inconsistent data with regards to the CRH level was reported previously, where the CRH level in the cerebrospinal fluid of AD subjects was found to increased, decreased, or remain unchanged in separate studies [59][60][61].
Disruption of the HPA axis has been consistently demonstrated in AD, usually preceding the cognitive decline [62]. It has been proved that hypothalamic dysfunction may drive the progression of AD. In a transgenic mouse model of AD, chronic stress can induce Tau pathology, neurodegeneration, and learning impairment, which can be blocked by CRH receptor 1 antagonists and enhanced by CRH overexpression [56]. Previous studies have demonstrated that Crf mutant animals showed a reduction in both Aβ-and Tau-related pathologies [63,64]. Moreover, mutations in CRH receptor 1 have been proved to reduce Tau hyperphosphorylation and Aβ deposition in response to stress [65][66][67]. ese data suggest that the activation of the HPA axis and subsequent increment in CRH concentration could contribute to brain dysfunction in AD. Results from this present study demonstrated that supplementation with the tropical fruit juice mixture was able to reduce the plasma CRH level in rats following Aβ infusion, suggesting that tropical fruit juice mixture has the potential to prevent the progression of AD by suppressing the activation of HPA axis. Besides that, the increase in the concentration of CRH was positively correlated to depression [68].
us, we suggest that tropical fruit juice may help in reducing depression and stress.

Conclusion
In conclusion, supplementation with formulation F8 of tropical fruit juice mixture (5 ml/kg body weight/day) can protect the CA1 region of the hippocampus of rats from prominent tissue damage, shrinkage, and neuronal loss following Aβ infusion for 14 days, possibly via its antioxidant properties and the suppression of iNOS expression and CRH production. us, the tropical fruit juice mixture (F8) has the potential to be developed as a neuroprotective agent in improving neuroinflammation and other neurodegenerative-related conditions. Data Availability e data sets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.