Protective Effect of Biobran/MGN-3 against Sporadic Alzheimer’s Disease Mouse Model: Possible Role of Oxidative Stress and Apoptotic Pathways

Abstract

Alzheimer’s disease (AD) is a debilitating and irreversible brain disease that affects an increasing number of aged individuals, mandating the development of protective nutraceuticals. Biobran/MGN-3, an arabinoxylan from rice bran, has potent antioxidant, antiaging, and immunomodulatory effects. The aim of the present study was to investigate the protective effect of Biobran against sporadic Alzheimer’s disease (SAD). SAD was induced in mice via intracerebroventricular injection of streptozotocin (STZ) (3 mg/kg). STZ-treated mice were administered with Biobran for 21 days. The effects of Biobran on memory and learning were measured via the Morris water maze, novel object recognition, and Y-maze tests. Biomarkers for apoptosis, oxidative stress, and amyloidogenesis were measured using ELISA and western blot analysis. Histopathological examination was performed to confirm neuronal damage and amyloid-beta deposition. Biobran reversed the spatial memory deficit in SAD-induced mice, and it increased the expression of glutathione, reduced malondialdehyde, decreased IL-6, decreased intercellular adhesion molecule-1 (ICAM-1), and significantly increased nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant response element (ARE). Moreover, Biobran exerted a protective effect against amyloid-beta-induced apoptosis via the suppression of both cleaved caspase-3 and the proapoptotic protein Bax and via the upregulation of the antiapoptotic protein Bcl-2. Furthermore, it reduced the expression of forkhead box class O proteins. It could be concluded from this study that Biobran may be a useful nutritional antioxidant agent for protection against SAD through its activation of the gene expression of Nrf2/ARE, which in turn modulates the apoptotic and amyloidogenic pathways.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Alzheimer’s disease (AD) is the most common neurodegenerative disorder characterized by progressive loss of memory and cognition. The appearance of oxidative stress markers is one of the hallmarks of AD; it leads to the build-up of amyloid deposits and neurofibrillary tangles and to the progression of the disease [1]. Oxidative stress is involved in many disorders, including Parkinson’s disease, chronic inflammation, and AD [2]. Neurons produce energy at a high rate and show high oxygen consumption; they are at extremely high risk to oxidative damage from reactive oxygen species (ROS) [3]. Currently, the process by which amyloid-beta (Aβ) accumulation occurs in the central nervous system is uncertain, but the generation of ROS during Aβ self-aggregation is a potential mechanism by which Aβ may cause neuronal damage and death. This effect ultimately leads to synaptic membrane depolarization, excessive calcium influx, and mitochondrial impairment [4, 5]. One of the common regulators of the oxidative stress pathway in AD is the expression of the nuclear factor erythroid 2-related factor 2 (Nrf2). It is present mainly in the cytoplasm of the hippocampal neurons [6], and in AD animal models, the pathology of Aβ is linked with altered expression of Nrf2 target genes [7]. Nrf2 can act as a molecular switch in neurons that mediates the antioxidant system [8]. Active Nrf2 protects cells against oxidative injury by binding to the antioxidant response element (ARE) under oxidative stimuli and promoting antioxidative genes [9]. Therefore, the restoration of Nrf2 expression could alleviate cognitive impairment by protecting neurons against oxidative injury and decreasing Aβ accumulation [10].

Neuroinflammation plays an important role in AD pathogenesis through the production of inflammatory cytokines like IL-6, which is highly prevalent in AD [11, 12], and the intercellular adhesion molecule-1 (ICAM-1), which is highly expressed in the neuritic plaques in AD brains. ICAM-1 has been implicated in neurodegeneration through its role as an important mediator of immune cell activation and inflammatory response in AD [13]. ICAM-1 plays a key role in cell survival in the brain and induced the upregulation of proapoptotic proteins, Bax and cleaved caspase-3, and downregulation of the antiapoptotic proteins, Bcl-2 [14, 15]. This pathway has many downstream targets like the transcriptional factor forkhead box proteins of class O3a (FOXO3a), a factor that, when translocated to the nucleus, can trigger cell apoptosis. Evidence is growing that apoptotic markers can directly target FOXO3a and lead to cell apoptosis [16]. The family of FOXO proteins is extensively involved in the cell signal transduction of apoptosis and in oxidative stress. This effect is important in the survival of cerebral endothelial vascular cells [17], oxidative stress injury in mouse cerebellar granule neurons [18], and hippocampal neuronal injury [19]; and it can also lead to caspase 3-induced apoptotic death [20, 21].

STZ injection in the brain is linked to brain insulin resistance, neuroinflammation, oxidative stress, and deposition of Aβ, as well as tau protein aggregation leading to impairment in memory and learning functions that mimic sporadic Alzheimer’s disease (SAD) in humans [22]. ICV-STZ injection induces the activation of microglial cells, which produce massive amounts of inflammatory mediators and free radicals that provoke neuronal damage [23].

Currently, there are no dietary supplements or prescribed medications for decreasing the risk of AD [24], and current FDA-approved treatments for AD are only symptomatic [25]. Biobran/MGN-3 is a denatured hemicellulose from rice bran that has shown promising effect as a natural adjuvant to existing immunotherapies for cancer [26, 27] through its antioxidant properties [28]. Biobran was shown previously to enhance natural killer cell activity in aged mice [29] as well as healthy elderly human subjects [30], with improvement in their health-related quality of life [31]. Few studies have been done on the beneficial effects of Biobran on aging and neurodegenerative diseases. So, the present study is aimed at investigating the possible protective effect of Biobran in a SAD model through the modulation of oxidative stress, amyloidogenesis, inflammation, and apoptotic pathways. Behavioral and biochemical experiments were performed to illuminate the mechanisms underlying its potential neuroprotective effect in the STZ model of SAD.

2. Material and Methods

2.1. Animals

Adult male Swiss albino mice, weighing 25-30 g, were used in the present study. Mice were obtained from the animal facility of the National Research Center, Cairo, Egypt, and they were housed 6 mice per cage. Mice were allowed to acclimate to their environment for a period of one week prior to the study. Animals were maintained in a controlled environment with constant temperatures ($25\pm 2^{°}\text{C}$), light/dark cycles (12/12 h), and relative humidity ($60\pm 10\%$). Animals were provided with a standard chow diet and allowed water ad libitum. This study complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health [32] and was approved by the Institutional Animal Care and Use Committee, Cairo University (CU-IACUC), approval number: CU-III-F-26-20. Animal discomfort and suffering was minimized as much as possible.

2.2. Drug Treatments

STZ was purchased from Sigma-Aldrich Co. (St Louis, MO, USA) and dissolved in saline solution (0.9% NaCl). Biobran is a denatured hemicellulose that is extracted from rice bran by reacting rice bran hemicellulose with carbohydrate-hydrolyzing enzymes obtained from Shiitake mushrooms. Biobran’s main chemical structure is arabinoxylan with an arabinose polymer in its side chain and a xylose in its main chain [26]. Daiwa Pharmaceutical Co. Ltd. (Tokyo, Japan) kindly provided Biobran. Biobran was prepared in saline (0.9% $w/v$) and was freshly prepared each day (Figure 1).

[figure omitted; refer to PDF]

The number of amyloid plaques was investigated in different experimental groups through visualization with Congo red stain. Normal mice showed no amyloid deposition in the brain sections. Meanwhile, ICV injection of STZ showed multifocal deposition of the amyloid deposition in the brain tissue especially in the inflammatory lesion that showed focal gliosis. The administration of Biobran (50 mg/kg) resulted in a marked reduction of the number of amyloid plaques in the brain tissue. Moreover, mice that received Biobran (100 mg/kg) showed few amyloid plaques, and mice that received Biobran (200 mg/kg) showed an absence of amyloid plaques in most examined brain tissue (Figure 12).

[figure omitted; refer to PDF]

4. Discussion

The current study evaluated the protective effect of Biobran/MGN-3 against STZ-induced SAD in mice. Biobran, a natural biological response modifier, has been shown to possess antiaging [29–31] and antioxidant [37] properties. Biobran is proved previously to exhibit potent immunomodulatory functions [26, 27, 46–48] and exert beneficial effects against cancer, viruses, and microbes [49–52].

In the present study, STZ-treated mice were unable to discriminate between novel and familiar objects, as demonstrated by the NOR task. The ICV-STZ group revealed marked deterioration in memory and learning functions as observed in the Morris water maze and manifested by a significant decrease in the time spent in the target quadrant as well as in the Y-maze tests demonstrated by a significant decline in the spontaneous alternation behavior. These findings are in agreement with previous studies reporting that ICV-STZ injection was implicated in decreased spontaneous alternation behavior in the Y-maze test and a decline in spatial learning and reference memory in Morris water maze trials as well as the test day [53, 54]. This indicates obvious memory and learning deficits in these mice. The ICV injection of STZ is a well-known model of sporadic Alzheimer’s disease in rodents with similar progressive pathology of AD as in the human brain [55–57]. However, it was of great interest to note that Biobran supplementation prevented the STZ-induced impairments of short-term and spatial memory. In a dose-dependent manner, Biobran reduced the MEL time, extended the time spent in the target quadrant, and reversed the discrimination and preference indices as well as decreasing the spontaneous alternation behavior in the Y-maze task.

Coherent to the aforementioned findings, it was found that the cognitive dysfunction exerted a positive impact on the amyloidogenic and oxidative stress pathways. Aβ peptide is one of the hallmarks of AD causing neuronal loss in the brain and resulting into deficits in memory and learning. Previous studies revealed that antioxidant compounds could be promising therapeutic or preventive interventions for AD patients because they inhibit Aβ fibril formation and protect the brain from Aβ neurotoxicity [58]. In the current study, Biobran exerted a significant antioxidant effect in the model of SAD, which is in agreement with previous studies that revealed Biobran’s antioxidant activity against murine solid Ehrlich carcinoma [28], as well as its ability to significantly alleviate the increase in MDA content and prevent the irradiation-induced depletion of GSH in mice spleens [59].

ROS generation caused by mitochondrial oxidative phosphorylation can have profound effects on cellular functions and result in the initiation of many diseases, including aging [60] and AD [2, 61]. During oxidative stress, ROS can lead to neuronal synaptic dysfunction [62, 63] and may cause neuronal damage and death during Aβ self-aggregation [64]. Biobran has been shown previously to upregulate the oxidative stress in the liver and to inhibit the levels of these biomarkers including MDA, total free radicals, and nitric oxide in murine Ehrlich carcinoma [28]. This suggests that Biobran induces oncostatic activity by providing protection against oxidative stress, modulating lipid peroxidation, and enhancing the antioxidant defense system. Moreover, it was reported previously that Nrf2 is suppressed in AD patients’ neurons [65], which is in harmony with the results of the present study. For AD animals, there is a decrease in Nrf2 expression, as well as in the expression of the Nrf2/ARE pathway’s target genes [66]. Altered expression in Nrf2 is associated with cognitive deficits and impaired spatial memory in mouse models of AD [6] and a deficiency in Nrf2 results in vulnerability to oxidative stress [67], phosphorylated-Tau [68], and enhanced autophagic dysfunction [7]. On the other hand, it was revealed previously that neurons can be protected against Aβ pathology and oxidative proteotoxic stress by the upregulation of the Nrf2/ARE pathway [69, 70]. Several studies have shown that neuropathological changes such as AD and Parkinson’s are also associated with faulty inflammatory processes such as increased expression of the proinflammatory cytokine IL-6 in the brain [11, 12]. In the current study, IL-6 and ICAM-1 were significantly increased after STZ administration, but Biobran supplementation caused a significant decrease in the levels of these biomarkers. Interestingly, a recent clinical study revealed the increased concentrations of IL-6 and ICAM-1 in AD patients’ cerebrospinal fluid (CSF) [71, 72]. These effects were more evident in patients with abnormal CSF Aβ levels, indicating that, in the presence of Aβ pathology, associations between cerebrovascular, neurodegenerative, and neuroinflammatory processes may be aggravated and contribute to tau aggregation, leading to cognitive impairment and disease progression [71]. Therefore, focusing on these biomarkers offers potential targets for novel therapeutic interventions. In the present work, the administration of Biobran significantly inhibited the levels of ICAM-1 and IL-6 in the hippocampi of SAD-induced mice with significant accumulation of amyloid plaques.

In the present study, Biobran exerted an antiapoptotic effect against STZ in a dose-dependent manner. This effect could be due to suppression of cleaved caspase-3 as well as the proapoptotic protein Bax and through the downregulation of the antiapoptotic protein Bcl-2. It has been suggested previously that Aβ activates the neuronal apoptotic pathway via its accumulation in the mitochondrial membrane and impairment of mitochondrial function [73]. The membrane of mitochondria becomes permeable during mitochondrial apoptosis, and ROS gets released [74]. Apoptogenic proteins such as cytochrome c can thereby be produced, and proapoptotic factors can be introduced into the cytosol from the mitochondria, ultimately activating procaspases and inducing apoptosis [75]. Neuronal loss can be caused by mitochondrial dysfunction via the regulation of proapoptotic proteins like caspase-3 and Bax and antiapoptotic proteins like Bcl-2 [14, 15, 20]. The ability of Biobran to exert a protective effect against STZ-induced apoptosis is in accordance with our earlier studies showing that Biobran treatment upregulated Bax expression, activated caspase-3, and downregulated Bcl-2 expression; these well-established molecular events in apoptosis have shown that Biobran can protect against glandular stomach carcinogenesis in rats [49], inhibit hepatocarcinogenesis in rats [50], and enhance fractionated X-ray irradiation’s anticancer effects for Ehrlich solid tumor-bearing mice [59].

The effect of Biobran on FOXO protein expression in STZ-injected mice hippocampi was also examined. FOXO proteins have a range of biological functions. They are present throughout the body and are selectively expressed in the nervous system. The complex interaction between signal transduction pathways and FOXO proteins in the presence of oxidative stress can significantly impact apoptosis and autophagy [18, 20, 76]. Under oxidative stress conditions, autophagy can be induced by FOXO proteins along with the promotion of cell survival [64]. STZ-injected mice showed significantly higher levels of the FOXO protein expression. Biobran decreased significantly these values in a dose-dependent manner. This demonstrates Biobran’s protective effect against FOXO-mediated apoptosis in STZ-treated mice. Recently, it was reported that Biobran/MGN-3 is a promising psychoneuroimmune modulatory agent that could improve the quality of life in healthy old adults [31].

Histopathological analysis using H & E and Congo red stainings further revealed the protective effect of Biobran against STZ-induced neuronal damage in the cerebral cortex and hippocampal sections of mice. Treatment with Biobran reduced the neuronal toxicity observed in STZ-injected mice, with fewer eosinophilic-stained neurons and more healthy neurons with prominent nuclei. This indicates that Biobran could act as a potential candidate to attenuate neurodegeneration and preserve cognitive functions. Hippocampal sections of Biobran-treated groups also exhibited a dose-dependent protective effect against Aβ plaque formation. These positive histological effects of Biobran are in agreement with our behavioral and biochemical assessments. The highest dose of Biobran exerted better protection compared to the low and moderate doses.

5. Conclusions

Biobran exerts a dose-dependent protective effect against sporadic AD. This effect is achieved through the targeting of the Nrf2/ARE antioxidant signaling that modulates amyloidogenesis as well as the Bcl2/Bax/caspase-3 pathway. To our knowledge, the present study is the first to investigate the protective effect of Biobran against SAD. Our findings suggest that Biobran’s activity is able to reduce Aβ generation and promotes cognitive function recovery. They may suggest the possible applicability of Biobran in clinical trials of human subjects in the management of SAD.

Authors’ Contributions

M Ghoneum and N El Sayed planned the study and wrote the manuscript. N El Sayed designed and performed the experiments. Both authors approved the manuscript.

Acknowledgments

Biobran/MGN-3 was provided by Daiwa Pharm. Co., Ltd, Japan. This work was funded by Diawa Pharmaceutical Co., Ltd., Tokyo, Japan; Grant #T0099108.

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