Efficiency of Biobran/MGN-3, an Arabinoxylan Rice Bran, in Attenuating Diabetes-Induced Cognitive Impairment of the Hippocampus via Oxidative Stress and IR/Akt/NF-κB in Rats

Type 2 diabetes mellitus (T2DM) is a common metabolic disease accompanied by cognitive impairment, hippocampal malfunctioning, and inflammation. Biobran/MGN-3, an arabinoxylan rice bran, has been shown to have an antidiabetic effect in streptozotocin-induced diabetic rats. The present study investigates Biobran's effect against diabetes-induced cognitive impairment and synaptotoxicity in the hippocampus via oxidative stress and the IR/A/NF-κB signaling pathway in rats. Diabetes was induced via i.p. injection of streptozotocin (STZ) (40 mg/kg BW); STZ-treated rats were then administered Biobran (100 mg/kg BW) for 4 wks. Biobran supplementation improved motor coordination and muscular strength, as assessed by Kondziella's inverted screen test. Biobran also improved concentration levels of glutathione (GSH), antioxidant enzymes, acetylcholine (ACh), dopamine, serotonin, insulin receptor (IR), and alpha serine-threonine protein kinase (Akt); it protected against elevated levels of glucose, total cholesterol, triglycerides, oxidative stress markers, TBARS, NO, AChE, and MAO; and it significantly decreased inflammatory cytokines levels of IL-1β, NF-κB, TNF-α, and amyloid β1-42. Moreover, Biobran ameliorated hippocampal histological alterations. Immunohistochemical observations showed that Biobran reduced overexpression of hippocampal synaptophysin and Ki67 relative to untreated diabetic rats. Biobran may ameliorate hippocampal alterations in diabetic rats via its antidiabetic, antiproliferative, anti-inflammatory, antiapoptotic, and antioxidant effects.


Introduction
Diabetes mellitus (DM) is one of the most common chronic endocrine metabolic disorders. It is characterized by hyperglycemia [1] and its most common form, type 2 diabetes mellitus (T2DM), is characterized by relative insulin insufciency and insulin resistance [2]. T2DM is associated with long-term complications afecting the brain, heart, blood vessels, kidneys, and eyes [3]. Moreover, chronic hyperglycemia through the polyol pathway and the oxidative stress state induced by the increased formation of reactive oxygen species have been proposed as major pathophysiological links between T2DM progression and the onset of diabetic complications [4]. Similarly, the pathogenesis of diabetes and diabetic complications is associated with increased infammation [5]. Diabetes and insulin-related effects are associated with changes in serotonin (5-HT) and dopamine (DA) neurotransmitters, and the insulin signaling mechanism alpha serine-threonine protein kinase (Akt) has been implicated in hippocampal neurogenesis [5]. Akt's signal transduction pathway is comprised of Akt, the Bcl-xL/ Bcl-2-associated death promoter (Bad), and nuclear factor kappa B (NF-κB), and this pathway is involved in cell metabolism, diferentiation, proliferation, and apoptosis [6].
Recently, evidence has grown indicating that chronic T2DM patients can have neurobehavioral changes and various intracranial neuropathies [7]. Several reports have found a strong relationship between DM and cognitive defcits, neuronal impairment, and dementia [7,8]. Te primary clinical features associated with cognitive impairment induced by diabetes have included psychomotor retardation, learning and memory impairment, and decreased mental fexibility [8]. Te hippocampus is particularly sensitive to DM's chronic hyperglycemia which can lead to cognitive impairment, to delayed responses to surroundings, and to hippocampal neuron number reduction and abnormal morphology [6].
Many antidiabetic drugs are being explored in clinical practice, but unfortunately those that are available are impotent against DM complications, including cognitive impairment, and they are associated with disagreeable side efects [8]. Given this situation, it is immensely important to investigate novel and multitargeted natural products that can mitigate DM pathogenesis. Biobran/MGN-3, a rice bran arabinoxylan, is a polysaccharide containing beta-1,4 xylopyronase hemicellulose [9] that has been proven to be an efective antioxidant that prevents free radical formation and enhances the antioxidant defense system [10]. We have also recently reported Biobran's benefcial efects on aging [11,12] and neurodegenerative diseases [10], and recent studies have furthermore reported the antidiabetic efect of Biobran in streptozotocininduced diabetic rats [13]. Te current study investigates Biobran's efect against diabetes-induced cognitive impairment and hippocampal synaptotoxicity via oxidative stress and the IR/Akt/NF-κB signaling pathway in rats. We hypothesized that Biobran's distinct antioxidant properties would relieve diabetes-induced cognitive impairment. Tis hypothesis was tested by systematically investigating Biobran's therapeutic efects against diabetes-induced cognitive impairment and hippocampal synaptotoxicity via oxidative stress and the IR/ Akt/NF-κB pathway in rats.

Biobran.
Biobran is a processed hemicellulose that is obtained by reacting rice bran hemicelluloses with multiple carbohydrate hydrolyzing enzymes from shiitake mushrooms. Te main chemical structure of Biobran is an arabinoxylan, with a xylose in its main chain and arabinose in its side chain [9]. Biobran is water soluble and solutions of Biobran were freshly prepared every day by mixing Biobran with saline (0.9% w/v). Te dose used in the present study (100 mg/kg body weight every day for 4 wks) was chosen in accordance with our previous work [14].

Diabetic
Model. Te diabetic model in this experiment used STZ and a high-fat diet (HFD) consisting of 66.5% commercial feed (Labina), 13.5% cholesterol, and 20% sugar. HFD feeding was used to induce insulin resistance and STZ at low dose was used to cause initial dysfunction in beta cells, which closely mimics the natural metabolic events of human T2DM.

Animals and Treatment
. 24 male Wistar albino rats were obtained from the Animal Care Unit, Experimental Animal Center, Medical Research Institute, Alexandria University (Alexandria, Egypt). Rats weighed 170-180 g. Rats were separated into 4 groups of 6 and housed in a well-ventilated animal house in stainless steel wire cages. Rats were fed a basal diet, supplied with tap water ad libitum, and kept under constant environmental conditions at 22 ± 3°C temperature and a 12/12-h-light/dark cycle. Te control group and Biobran group were fed commercial feed. Te diabetic untreated group and diabetic plus Biobran group were fed a HFD. All animals were given their respective diets for two weeks, and on the 15th day, the animals in the diabetic groups were fasted for 12 h and intraperitoneally administered a single dose of STZ (40 mg/kg body weight) [15]. Control animals were given only vehicle (0.01 M citrate bufer, pH 4.5). Tree days after STZ induction (on the 18th day of treatment), measurements of blood glucose were taken to confrm diabetes' establishment. Animals with postprandial glycaemia values ≥288 mg/dL were considered diabetic. Animals treated with Biobran were orally administered 100 mg Biobran per kg body weight every day for 4 wks [10]. No mortality was observed in any groups during the experiment. All animal handling and experimental procedures were performed according to the guidelines approved by Alexandria University Institutional Animal Care and Use Committee (ALEXU-IACUC), a member of the International Council for Laboratory Animal Science (ICLAS) (approval number: AU 04 22 06 27 3 01).

Behavioral Analysis.
Kondziella's inverted screen test was used to measure animals' muscular strength in all limbs [16]. Te inverted screen is made of wire 1 mm in diameter shaped into 12 mm squares over a total square mesh size of 43 cm. Te screen border consists of wood beading 4 cm deep, preventing the rats from moving to the opposite side. Rats are placed in the screen center, following which the screen is rotated (declining the rat's head) for 120 seconds to a fully inverted position. Measurements were made of the time it took for the rat to fall of the screen.

Histological and Immunohistological Assessments.
After fxation of hippocampal tissues in 10% bufered neutral formalin solution, the tissues were embedded in parafn, sectioned, stained with hematoxylin and eosin (H&E), and examined microscopically for the evaluation of histopathological changes [21].
For immunohistochemical staining, xylene was used to deparafnize parafn sections (5 μm thick) over 1-2 min. Tese were then rehydrated with ethanol of decreasing grades (100%, 95%, and 70% ethanol), using two changes each of 5 mins, with a fnal 5 min stage with distilled water. PBS was then used to rinse sections and 0.1% H 2 O 2 was used to block for 30 min as an activity inhibitor of endogenous peroxidase. Following the PBS rinse, incubation of sections was conducted at room temperature (21°C) for 1 h in blocking solution (10% normal goat serum), followed by incubation (21°C) for 1 h with the primary antibody (synaptophysin and Ki67). PBS was again used to rinse sections, after which sections were incubated (21°C) with secondary biotinylated antibody for 20 min. PBS was used to rinse sections, and then enzyme conjugate "streptavidinhorseradish peroxidase" solution was applied over 10 min. Visualization of secondary antibody binding was carried out using 3,3′-diaminobenzoic acid (DAB) dissolved in PBS to which H 2 O 2 was added for a concentration of 0.03% immediately before use. Finally, PBS was used to rinse sections and hemotoxylin (100 μl, 2 drops) was used to counterstain the slides. Distilled water was used to wash the slides until sections became blue. Slides were dehydrated with increasing grades (70%, 95%, and 100%) of ethanol for 5 min each, cleared in xylene, and cover-slipped with histomount mounting solution.
Immunohistochemical assessments were carried out using fve nonoverlapping felds (400x) of each section photographed randomly with an Olympus digital camera in the hippocampus. Every brain section and every marker were used to analyze total dentate gyral area.

Statistical Analysis.
Results reported are mean-± standard error (SE); six rats were used per group (n � 6). Te data were subjected to statistical analyses using a oneway analysis of variance (ANOVA) (PC-STAT, University of Georgia, 1985); groups were compared against each other using the LSD test. p < 0.05 was used as the standard for statistical signifcance. Analyses were calculated with assistance from the Statistical Package for the Social Sciences software (SPSS, version 16.0).

Behavioral Assessment.
Following the induction of diabetes, the muscular strength and motor coordination of all rat paws were assessed via Kondziella's inverted screen test ( Figure 1). Te time of falling decreased signifcantly (p < 0.05) for the untreated diabetic group in comparison with controls. Diabetes afected muscular strength so that rats quickly fell down because they were unable to hold onto the inverted screen. Diabetic rats treated with Biobran had signifcant increases (p < 0.05) in their time of falling in comparison with untreated diabetic rats.

Glucose, Insulin, and Lipid Profle Levels.
Serum measurements indicated that there were signifcantly increased glucose levels (p < 0.05) and signifcantly decreased insulin Evidence-Based Complementary and Alternative Medicine levels (p < 0.05) for the untreated diabetic group relative to control (Table 1). Biobran signifcantly reversed these efects (p < 0.05) for diabetic rats' glucose and insulin levels in comparison with the untreated diabetic rats. Te serum measurements also showed signifcantly increased TC and TG concentrations (p < 0.05) for diabetic rats in comparison with control, but Biobran again signifcantly reversed the efect on serum TC and TG concentrations (p < 0.05) relative to the untreated diabetic group.

Hippocampal Oxidative Stress Markers and Antioxidant
Enzymes. Brain levels of oxidative stress markers TBARS and NO showed signifcant elevation (p < 0.05) for untreated diabetic rats in comparison with control (Table 2), along with signifcant reduction (p < 0.05) for reduced glutathione (GSH) and the antioxidant enzyme activities of GPX and SOD. Biobran administration was efective at reversing all of these efects. Biobran treatment signifcantly reduced (p < 0.05) elevated TBARS and NO levels and signifcantly boosted (p < 0.05) concentrations of GSH, GPX, and SOD relative to the untreated diabetic group. Table 3, the untreated diabetic group displayed signifcant decreases in brain levels of acetylcholine (ACh), serotonin, and dopamine relative to control. However, oral administration of Biobran signifcantly (p < 0.05) augmented brain levels of ACh, serotonin, and dopamine relative to untreated diabetic rats. In addition, activities of brain AChE and MAO signifcantly increased (p < 0.05) for the untreated diabetic group in comparison with control, while Biobran supplementation to diabetic rats led to signifcant decreases (p < 0.05) in the activities of brain AChE and MAO compared to untreated diabetic group.

Histological and Immunohistochemical Assessments.
For control and Biobran-treated rats, histological examination of H&E-stained sections revealed normal hippocampal structure. Untreated diabetic rats showed markedly degenerated neurons with darkly stained pyknotic nuclei enveloped by large perinuclear spaces, dilated blood vessels in the molecular layer, and red neurons in polymorphic and granular layers. Treatment with Biobran to diabetic rats caused improvement with few cells present with darkly stained nuclei and perinuclear space (Figure 2). Synaptophysin, the major synaptic vesicle protein, and the cell proliferation marker Ki67 were used to measure the number of proliferating cells. Control and Biobran-treated rats reacted positively for both synaptophysin and Ki67, while there was a decline in reaction for synaptophysin and Ki67, respectively, in the diabetic group compared to control. Biobran treatment to diabetic rats resulted in signifcantly increased positive reactions for synaptophysin and Ki67 relative to the untreated diabetic group (Table 5, Figures 3 and 4).

Discussion
Streptozocin (STZ) treatment was used here to induce T2DM in rats, as manifested in the serum by signifcantly decreased insulin and signifcantly increased glucose. Te protective efect of the arabinoxylan rice bran product Biobran/MGN-3 against this STZ-induced diabetes and attendant brain damage in rats was then assessed. Biobran is a nutraceutical with health-promoting properties that include potent antioxidant, antiangiogenic, antiinfammatory, and immunomodulatory properties [9-12, 22, 23]. Supplementation with Biobran to diabetic rats protected against elevations in the levels of glucose, total cholesterol, triglycerides, oxidative stress markers, TBARS, NO, AChE, and MAO, and it led to signifcant decreases in infammatory cytokine levels for TNF-α, NF-κB, IL-1β, and amyloid β 1-42 . Furthermore, Biobran ameliorated hippocampal histological alterations and helped restore neuromuscular strength.
Biobran supplementation has been shown to improve blood glucose spikes as well as metabolism of proteins and lipids in diabetic rats [13,14,23]. Moreover, recent work has demonstrated that rice bran is efective at lowering blood glucose and improving insulin resistance because it results in increased levels of adiponectin which is linked to wholebody insulin sensitivity [24]. Rice bran diet has the ability to reduce total blood cholesterol, mainly by increasing fecal lipid excretion and regulating lipogenic enzyme activities [24]. Rats fed with Biobran have shown signifcantly lower triglycerides, total cholesterol, and total protein [13], and Biobran supplementation of diabetic rats has led to Evidence-Based Complementary and Alternative Medicine signifcantly lower glucose levels as well as lower total cholesterol levels in comparisons with control rats [14].
Te increased blood glucose levels found in diabetes mellitus cause oxidative stress. Several studies showed that STZ-induced diabetes is associated with signifcantly changed oxidative stress biomarkers [2,25]. Tese fndings are consistent with the present study, where diabetes was shown to signifcantly change oxidative stress biomarkers in the hippocampus as indicated by decreased GSH levels and antioxidant enzymes activities of SOD and GPx and by increases in the level of TBARS and NO. However, it is very interesting that Biobran supplementation for STZ-induced diabetic rats caused restorative efects on the functioning of the antioxidant defense system and benefcial decreasing of oxidative stress biomarkers, TBARS, and NO. Te antioxidant potential of Biobran noted in this study post i.p. injection of STZ in diabetic rats was further confrmed in our recent model using intracerebroventricular injection of STZ in mice [10]. Tose Biobran-fed mice were found to be protected against sporadic Alzheimer's disease and showed  Values are mean ± S.E.; each group has n � 6. a Signifcantly diferent relative to the control group, p < 0.05. b Signifcantly diferent relative to the diabetic group, p < 0.05. Values are mean ± S.E.; each group has n � 6. a Signifcantly diferent relative to the control group, p < 0.05. b Signifcantly diferent relative to the diabetic group, p < 0.05. Values are mean ± S.E.; each group has n � 6. a Signifcantly diferent relative to control group, p < 0.05. b Signifcantly diferent relative to diabetic group, p < 0.05.
Evidence-Based Complementary and Alternative Medicine signifcantly increased GSH content and signifcantly decreased MDA and IL-6 [10]. Reduced infammatory response indicators and oxidative stress levels have also been found in the study of Biobran's positive efects on mice for alleviating intestinal barrier dysfunction induced by radiation [26].
Neurotransmitters are essential to the health of the mind and body. Low levels for any kind of neurotransmitter can lead to disease development. Te current study examines the levels of several important neurotransmitters in rats with STZ-induced diabetes, and they had lower levels of ACh, serotonin, and dopamine as compared to control, while hippocampal activities of AChE and MAO were increased compared to the control, in agreement with previous reports [27,28]. Biobran treatment of diabetic animals led to signifcantly elevated (p < 0.05) monoamine neurotransmitter levels relative to the diabetic group while decreasing hippocampal AChE and MAO activities.
We noted that the brains of STZ-treated rats have reduced concentrations of IR and Akt and increased concentrations of IL-1β, NF-κB, TNF-α, and amyloid β  . In diabetic rat studies, STZ has been reported to disrupt hypothalamic signaling either down-stream (PKB) or upstream (IRS-2) of PI3K and/or to block signal transduction through Akt [29]. Our results show that Biobran can enhance the insulin signaling pathway through activation of IR and Akt. Biobran acted as an immunomodulator by reducing the brain's levels of IL-1β, NF-κB, TNF-α, and amyloid β  . Tis agrees with our recent study which showed that Biobran supplementation led to signifcantly declined levels of proinfammatory cytokines and amyloid β 1-42 in mice brains with sporadic Alzheimer's disease [10]. Amyloid plaque's  Values are mean ± S.E.; each group has n � 6. a Signifcantly diferent relative to the control group, p < 0.05. b Signifcantly diferent relative to the diabetic group, p < 0.05. major component, amyloid β, exists predominantly as the Aβ 1-42 form, a form that is more likely than Aβ 1-40 to aggregate because it is less soluble. Aβ 1-42 has become a pathological biomarker for neuro-degenerative diseases, and its increased concentration in diabetes strongly suggests it is more likely associated with hyperglycemia [30]. Moreover, rice bran exhibits anti-infammatory activity, as evidenced by reductions of TNF-α and IL-6 (proinfammatory cytokines) and increased IL-10 (antiinfammatory cytokine) in Raw264.7 macrophage cells [31]. It has been appreciated for a long time that hippocampal formation is responsible for memory and learning. Diseases like diabetes could afect the groups of eferent systems involved with these processes, including sensory processing and integration, afective and social learning, and episodic memory [32]. In the current study, we carried out histological and immunohistochemical analyses of diabetic rats' hippocampi in the presence and absence of Biobran treatment. In H&E-stained hippocampal sections of untreated diabetic rats, there were signifcant increases in degenerated neurons that had stained pyknotic nuclei enveloped by large perinuclear spaces. In the polymorphic and granular layers, there were red neurons, and in the molecular layer, there were dilated blood vessels. In contrast, histological examination showed Biobran's protective efects. Diabetic rats treated with Biobran showed marked improvement in the abovementioned symptoms. We noted only few cells with darkly stained nuclei and perinuclear space. Tis could be explained by the ability of Biobran to improve the levels of blood glucose and augment the function of the antioxidant defense system [13,14,23].
Further study also showed that diabetes induced a signifcant decrease of synaptophysin and Ki67-positive cells. Synaptophysin is a major synaptic vesicle protein and is known as a marker of synapses that is afected by several neurodegenerative diseases; several studies have confrmed that diabetes results in the degradation of synaptophysin protein [33,34]. Spine plasticity and synaptic integrity are key to learning and memory. Diabetes and the resulting insulin signaling disruption have been found to potentiate or exaggerate synaptic damage linked to learning defcits [33]. Earlier studies also reported that hyperglycemia impaired neurogenesis via reduced neuronal proliferation, migration, and maturation and caused cognitive impairments [35]. Our fndings show that Biobran can antagonize the suppression of neurogenesis induced by diabetes, indicating that oxidative stress may reasonably be assumed to be indirectly or directly implicated in suppressing neurogenesis in diabetes. Biobran also enhances health-related quality of life for healthy old adults [36]. Our previous results showed that Biobran supplementation of aged subjects caused enhancement in physical and mental component levels in comparison with placebo-treated subjects and baseline values. Taken together, our fndings indicate that Biobran is a potent psychoneuroimmune modulatory agent [36]. Te neuromuscular impairment caused by diabetic neuropathy is also a well-established phenomenon in type 2 diabetic patients [37,38] and rodents [39,40]. In our study, we noted that STZ-induced diabetic rats showed a decline in neuromuscular strength as compared with normal rats as shown by Kondziella's inverted screen test. Te neuromuscular impairment caused by diabetic neuropathy is consistent with the results by others [39][40][41]. Treatment with Biobran helped to restore the neuromuscular strength as demonstrated in increased skeletal muscle strength for Biobran-treated diabetic rats.

Conclusions
Biobran/MGN-3 was found to have curative efects against STZ-induced cognitive impairment, neurotoxicity, neuroinfammation, and neuronal death. All neurotoxic and oxidative parameters induced by STZ were attenuated. Biobran/MGN-3 improved the levels of the neurochemical transmitters ACh, dopamine, serotonin, AChE, and MAO in the hippocampal tissue. Tus, Biobran administration to diabetic rats attenuates cognitive impairment and synaptotoxicity in the hippocampus by acting on oxidative stress and the IR/Akt/NF-κB pathway. Biobran/MGN-3 could potentially act as a novel therapeutic agent for treating neuro-degenerative disorders.

Data Availability
Te data that support the fndings of this study are available from the corresponding author upon request.