Anti‑aging trait of whey protein against brain damage of senile rats

Aging mammalian results in impaired bio-functions and neurological disorders. The current study investigated whether whey protein (WP) syrup supplementation may improve age-related changes in diseased brain indicators like tau protein, β-amyloid and α-amylase. The study was carried out in conjunction with immunohistochemistry, histology, and flow cytometry of apoptosis. At the ages of 8 and 30 months, Wistar albino rats ( Rattus novergicus ) were divided into four groups ( n = 8; G1; 8 months old rats; G2, 8 months old rats supplemented WP; G3, 30 months old rats; G4, 30 months old rats supplemented WP), with or without whey syrup administration. For 2 months, oral whey supplementation in 2 mL/ kg doses is given twice a day every 12 h. Rats were sacrificed, and their brains were subjected to biochemical, histological, immunohistochemistry, and flow cytometric investigations. Aged rats had lower levels of superoxide dismutase (SOD), adenosine triphosphate (ATP), serotonin (5-HT), and dopamine (DA). These observations were parallel with increased inflammatory markers [tumor necrosis factor α- and 5-lipoxygenase (5-LO)], lipid peroxidation products (MDA), as well as apoptotic marker caspase-3, annexin-v, tau protein, β-amyloid, and α-amylase. Whey administration to aged rats reduced inflammatory and oxidative stress markers as well as improved neurotransmitters, tau protein, β-amyloid, and α-amylase. The advantages of supplementation were validated by improved histology and immunohistochemistry in aged rats’ cerebrum, cerebellum, and hippocampus. In addition, apoptosis was reduced, according to flow cytometry analysis of annexin-v. In conclusion, WP contains amino acids and bioactive compounds that could decrease brain oxidative stress and restore normal metabolic function. Furthermore, increased antioxidant defense and DA and 5-HT neurotransmitters, while decreasing brain tau protein and β-amyloid, were associated with better histology in aged rats’ cerebrum, cerebellum, and hippocampus.


Introduction
Aging is characterized by arterial stiffness, abnormal autonomic dysregulation, and damage to the blood-brain barriers. Cortical and subcortical micro-infarcts, scattered white matter disorders have all been connected to these processes. These injuries resulted in demyelination and axonal damage [1][2][3][4]. In addition, the ischemic damaging of the neuronal cells and the development of cerebral micro-hemorrhages of venous origin facilitate neurodegenerative disorders and dementia [5,6]. Aging also increased transactive response DNA-binding protein (TDP-43) [7], which increases glial and neuronal cell inflammation. The inflammation resulted in the accumulation of pro-inflammatory microRNA cytokines targeting genes involved in neuronal apoptosis [8]. In addition, old age showed irregular glycolytic enzyme activity that impede synaptic function and trigger neuronal cell loss [9]. Besides, old age might lead to heme degradation because of heme-oxygenase-1 regulation, which causes injury to the mitochondrial membrane in neuronal cell [10]. Parkinson's disease, Alzheimer's disease, and glaucoma are common disorders connected with aging. Glaucoma manifests oxidative stress assessed by mitochondrial and endoplasmic reticulum dysfunction and endothelial cell damage. The glaucomatous patient exhibited an abnormal aggregation of β-amyloid or tau protein in the retinal ganglion cell [11].
Whey protein (WP) is a bio-waste from cheese processing. Whey is a rich source of amino acids and plays a vital role in treating type 2 diabetes and glucose homeostasis [12][13][14][15]. Mainly bovine serum albumin and lactoferrin in WP are the main components of milk [16,17]. These nutrients are critical constituents of the human diet [18] and found commonly in infant formula [19]. It was shown that animals fed on a diet containing milk fat globule, lactoferrin, and a polydextrose/galactooligosaccharide probiotic led to a high increase in dendritic spine density in hippocampal dentate gyrus neurons [20].
Supplementation (1 g/kg b.w./day) of WP improved antioxidant potential and decreased free radicals and protein carbonyl [21]. Administration of WP to animals fed a diet containing a high phytoestrogen for ten weeks improved estradiol levels, T4, and glucose homeostasis [22]. Female mice (C57BL/6 J) fed (100 g WP/L water) with WP for 12 weeks showed activated brain function with increased levels of cytochromes [23].
Diabetes mellitus (DM) could damage the brain through oxidative stress [24]. The WP treatment alleviated DM progression by increasing blood flow, insulin secretion, cerebral oxygen, and decreased blood sugar levels [25]. The consumption of fermented dairy products alleviated cognitive function and enhanced symptoms of Alzheimer's in mouse models. Ano et al. [26] reported that tryptophan-tyrosine (Trp-Tyr)-dipeptide (WY)-containing peptides increased dopamine level, while decreasing monoamine oxidase-β activities in brain tissues. The oxidative stress implicated in injured neurons was reduced in CD1 mice administered Immunocal ® (WP). The reduction alleviated axonal demyelination and brain-derived neurotrophic factor [27]. Patients with Parkinson's disease who used WP supplements had lower plasma homocysteine and higher plasma glutathione level, upregulation of branched and essential amino acids [28]. Sarcopenic older adults given WP, essential amino acids (containing leucine), and vitamin D exhibited increased muscle strength with a handgrip dynamometer, as well as enhanced health conditions based on blood biochemical indicators [29].
The goal of the current research was to investigate the aid of whey supplementation effects on neurodegenerative markers and brain redox state, apoptosis and increase the antioxidant defense parallel with improving histology and immunohistochemistry of brain regions during the aging process.

Characterization of whey syrup
Fresh bovine whey was obtained from the Dairy Product Laboratory (Faculty of Agriculture, Mansoura University, Egypt). WP was analyzed according to Parris and Baginskla [30]. The method used reversed-phase HPLC for denaturation of WP (Whey from bovine milk, Sigma-Aldrich Chemie, Taufkirchen, Germany), which is precipitated at pH 4.6. Un-denatured WP's absorbance and nitrogen concentration were compared to known WP standard to quantify WP nitrogen. Lactose was quantified according to Essig and Kleyn [31]. Lactose is hydrolyzed to β-galactose and glucose in the presence of β-galactosidase. In the presence of β-galactose dehydrogenase, β-galactose is oxidized by NAD to galactonic acid. Reduced nicotinamide-adenine dinucleotide resulted in stoichiometric with lactose amount determined at 340 nm.
The antioxidant content was determined according to Lim and Quah [32]. One mL of a methanol solution (100 µM) of 2,2-diphenyl-1-picrylhydrazyl (DPPH·) and the different concentrations of WP samples (0-30 mg/ mL) were mixed. The mixtures were incubated for 20 min, and the absorbance was detected at 517 nm. The data expressed as radical scavenging activity (%).
According to Karami et al. [33], the number of lactobacilli colonies in WP was estimated. This was performed by incubating 2 mL of whey in a flask containing MRS broth media containing 100 mL distilled water at 37 °C. After 24 h, 100 μL enriched sample was spread over MRS agar and cultured in an anaerobic environment for 48 h at 37 °C. The number of bacterial colonies was counted.

Animal grouping and investigation
Male adult Wistar albino rats (Rattus novergicus) (8 months old, n = 16, weight 200 ± 10 g) and senile rats (30 months old, n = 16, weight 400 ± 10 g) were obtained from the Breeding Lab (Ministry of Health and Population, Egypt). Adult (8 months old) and old rats (30 months old) were divided into four groups (n = 8; G1; 8 months old rats; G2, 8 months old rats supplemented whey; G3, 30 months old rats; G4, 30 months old rats supplemented whey), with or without whey syrup administration. For 2 months, oral whey supplementation was administered twice a day in 2 mL/kg of whey syrup (every 12 h) [34,35]. The animals were housed in an aerated environment with a 12 h light/dark cycle and 180-200 lx light intensity [34,35]. Free access to diet and water are ad libitum. At the end of treatment, the animals were starved overnight and euthanized with halothane (2-bromo-2-chloro-1,1,1-trifluoroethane), followed by cervical dislocation and dissection. The blood was collected from the heart, coagulated, centrifuged, and serum separated and stored in the refrigerator. The brain was dissected and divided into two halves, one of which was preserved in the refrigerator, and the other was fixed in phosphate-buffered formalin (10%, pH 7.4) for immunohistochemical and histological studies.
The Experimental Animal Ethical Committee (Faculty of Science, Mansoura University, Egypt) approved the experiment (Approved Statement No. RZ19004).

Body and brain weight
The whole absolute body and brain weights (g) were recorded. The relative weights are calculated by dividing the brain weight on the body weight and multiplying by 100, followed by statistical analysis. With or without whey supplementation, absolute and relative brain weights were determined in the animals.

Brain dopamine (DA) and serotonin (5-HT)
According to the manufacturer's instructions, the assayed neurotransmitters were determined using the ELISA Kit of CUSABIO TECHNOLOGY (Houston, USA). The ELISA Kit was used to assay dopamine (DA). Serotonin (5-HT) was determined using Kit-Cat Nu. E-El-0033.

TNF-α, 5-lipoxygenase and caspase 3
Caspase-3 (Catalog No. CSB-E08857r) and TNF-α (Catalog No. CSB-e11987r) were measured according to the instruction of the ELISA kit from CUSABIO TECHNOLOGY (Houston, USA). In addition, rat My BioSource assayed 5-lipoxygenase (5-LO) using ELSA kit (Catalog. No. MBS722629). The competitive inhibitory response method involved labeling biotin and either TNF-α or 5-LO, then incubating with horseradish peroxidase-conjugated with avidin. The absorbance was calculated at 540 nm with the aid of the standard curve.
To visualize the cerebellum, cerebrum, and hippocampus changes, sections were examined under light microscopy.

Immunohistochemistry of caspase-3 and synaptophysin
Serial 5-μm thick histological paraffin sections were cut and mounted onto super frost t plus glass slides (Fisher Thermo Scientific, Nepean, Canada). Tissue sections were processed for antigen retrieval by digestion in trypsin (0.05%, pH 7.8) at 37 °C for 15 min, then incubated against either mouse anti-synaptophysin (Thermo Fisher Scientific, Catalog MA5-14532) or caspase-3 (Catalog MA5-11516) overnight at 4 °C. These followed by treatment with a horseradish peroxidase streptavidin, then DAB plus Chromagen to detect the immunoactivity, and counterstained with Mayer hematoxylin. The negative control sections were incubated with non-immune rabbit serum (1%) instead of the caspase-3 and synaptophysin antibody. The brain regions were examined and photographed with a Leica BM5000 microscope (Leica Microsystems, Wetzlar, Germany). Image processing was carried out at 40 X objective and an Olympus digital camera fixed on an Olympus microscope with 1/2 X frame adaptor. Video Test Morphology Software analyzed the resulting images, and the percentage area was calculated and reported.

Flow cytometry assessments of annexin-v
The flow cytometric analysis of annexin-v was performed using V-FITC/PI double staining method. First, brain tissue was lysed with tris-EDTA solution (pH 7.4) and fixed in 70% ethyl alcohol. After that, cells were washed with PBS, suspended at 0.1-0.3 × 10 6 /mL, and stained with fluorescein isothiocyanate-conjugated annexin-v (annexin V-FITC). Specimens were incubated at room temperature for 15 min and determined by Becton Dickinson Fac Scan Fluorescence-Activated Cell Analyzer (Becton Dickinson, CA, USA).

Statistical analysis
The results were presented as means ± standard deviations (SD). Statistical analysis was conducted using SPSS (Version 13) one-way ANOVA post hoc analysis for windows, comparing the adult and aged groups and WP supplementation. The significance is at p < 0.05.

Whey characterization
The nutritional content of WP, including total protein, lactose content, total antioxidant activity, and lactobacilli content, is given in Table 1.

Absolute and relative brain weights
When compared to the non-supplemented group, whey syrup supplementation resulted in a non-significant increase in absolute brain weight and decreased relative brain weight in the 8 months old animals. Thus, whey syrup supplementation decreased the absolute brain weight of old age while the nonsignificant increase was in the relative brain weight (Fig. 1A-C).

Serum neurodegenerative markers
The serum amounts of xanthine oxidoreductase (XOR), creatine kinase (CK), and acetylcholinesterase increased significantly in aged rats, with a percent of +

Brain superoxide dismutase and lipid peroxidation product (malondialdehyde)
The brain SOD of the adult group (G1) was 15.4, while it was decreased in the aged group (G3), which was 9.81 with a percentage of reduction of 36.5%. When compared to G1, G2 supplemented whey revealed a non-significant increase in SOD activity. However, it increased significantly in G4, reaching 12.7 to the percentage increase of + 30.2 compared to G3. There were no variations in brain MDA concentrations between G1 and G2 on the other side. Compared to G1 (4.66), a significant increase of MDA was detected in G3 (6.97) with a percentage increase of + 49.5%. On the other hand, whey supplementation decreased MDA content (5.78) in G4 with a percent reduction of − 17.0%. When comparing G4 to G1, the percent of increased MDA during treatment reached + 24.0%, which is half-folded of the G3 value (Table 3).

Brain neurotransmitters dopamine and serotonin
In G1 and G2, there were no differences in serotonin (5-HT), dopamine (DA), and adenosine triphosphate (ATP). However, DA, 5-HT, and ATP levels in the brain were considerably decreased in G3 during aging, reaching 6.78, 103.5, and 82.6, respectively, compared to 13.05, 147.3, and 117.6 in G1. The decrease percentages were − 48.2, − 29.5, and − 29.6%, respectively. Whey administration to the aged group (G4) enhanced the assayed neurotransmitters and ATP levels, and their percentages of reduction were still higher in DA at − 37.0. Meanwhile, 5-HT and ATP exhibited less decreased percent at − 10.3 and − 18.8%, respectively, compared to G1 (Table 3).

Histopathological observations
In adult groups with or without supplementation (G1 & G2), the cerebral external granular (OGL) outer ganglionic layer showed a dense distribution of pyramidal and stellate cells with centrally located nuclei ( Fig. 2A, A1, B, B1). In the aged rats (G3), many neuroglial cells exhibited either chromatolysis (karyolysis) or clumping nuclear chromatin (pyknosis). Angiogenesis of the blood vessels seems to be widely spread throughout the cerebral tissue clarifying the diseased pathological feature. The glial cells seem to be grouped, manifesting inflammation of the brain tissues. Necrotic, edematous, and spongiform degenerated foci were comparatively increased (Fig. 2C, C1). Whey syrup supplementation enhanced these aged-related alterations (G4) (Fig. 2D, D1). The cerebellar cortex of 8 months old and whey supplemented groups (G1 & G2) exhibited normal structures of the molecular (MCL), Purkinje cell layer (PCL), and the granular cell layer (GCL) (Fig. 2A2, B2). In the aged rat (G3), the Purkinje cells had either pyknotic or karyolysed nuclei embedded in necrotic spaces. In addition, the damaged granular cells were invaded by a large glomerular space (Fig. 2C2). Whey syrup supplementation to the aged group (G4) enhanced the cerebellar structure, particularly Purkinje cells (Fig. 2D2).
Histological investigations of the hippocampus of 8 months old rats with or without whey syrup supplementation (G1 & G2) revealed a well-defined pyramidal, polymorphic, and molecular layer. The pyramidal layer comprises small pyramidal cells that are densely packed together. Each cell had a large polygonal shape with rounded nuclei, prominent nucleoli, and scanty cytoplasm. The dentate gyrus makes up of small granule cells. The molecular layer has regular neuronal axons and dendrites distribution (Fig. 2A3, B3). The aged group (G3) had chromatolysis or grouping nuclear chromatin that resulted from apoptosis in pyramidal cells (Fig. 2C3). Whey syrup treatment impaired the progress of angiogenesis in cerebral tissues of aging rats and vanishing most of them. However, there were just a few spots of primitive angiogenesis (Fig. 2D3).

Flow cytometry of annexin-v
The average number of apoptotic cells significantly increased in the aging brain compared to the aged group (G4) that fed whey. The percentages of apoptotic neuronal cells reached 54.1% compared to 92.4% in the experimental aged group (G4). Adult animals with or without whey syrup supplementation showed 14.3% and 17.3%, respectively (Fig. 5A, B).

Discussion
Rats with advanced age had a significant increase in body weight, an increase in absolute brain weight, and a decrease in relative brain weight. The increase in body weights and decreased relative brain weight reflected the decline of metabolic function of body organs [38,39] and the loss in body fat oxidation [40]. Thus, WP supplementation improved body weight gain and brain weight. In addition, whey supplements promoted protein synthesis and improved brain function during aging [41]. These resulted from its high content of amino acids [27] and its antioxidant activity to eliminate free radicals [42]. In elderly rats, the activity of SOD was significantly depleted, resulting in increasing MDA. The generation of free radicals caused damage to neuronal cells in the cerebrum, cerebellar cortex, and hippocampus. Damaged cerebral neurons led to the development of cerebral ischemia assessed by increased cerebral vasculogenesis. The hippocampus's pyramidal layer becomes atrophied, and nearly all of its neurons become pleomorphic. Increased brain caspase-3, UR + LR of annexin-v flow cytometry, and caspase-3 immunohistochemistry confirmed apoptotic cell death.
The current findings supported those of Li et al. [43], who reported that aged rats had higher levels of β-galactosidase, and MDA as well as decreased SOD activity. In addition, aging decreased catalase activity, glutathione/oxidized glutathione ratio, and MDA increase in an aging group compared to adult and aging group supplemented whey syrup. Each result represent the mean ± SD (n = 5); asterisk means significant at p < 0.05. G1; 8 months old rats; G2, 8 months old rats supplemented whey; G3, 30 months old rats; G4, 30 months old rats supplemented whey in the animal brain cortex [44,45]. These facilitated the release of an active form of oxygen, a cell death promoter [46,47].
Whey supplementation also improved the histologic structure by increasing brain SOD activity and decreasing MDA levels, reducing caspase-3 immune reaction, and synaptophysin proliferation in neurons. These seemed to be connected to an increase in cysteine, an amino acid that serves as a precursor to glutathione, the machine of antioxidative activity [48].
The increase of inflammatory markers TNF-α and 5-lipoxygenase during the aging process activated the angiogenesis of blood capillaries and the dense grouping of glia cells in the cerebrum. It also caused a comparative increase of Purkinje and granular cells and the widespread occurrence of pleomorphic neuronal cells in the necrotic zones of the hippocampus.
These results were consistent with studies carried out by Garg et al. [41], who highlighted increased inflammatory markers (TNF-α, IL-1β, and IL-6) and reduced acetylcholinesterase activity in aged rats. These resulted in impaired learning and memory function, particularly in the subgranular region of the dentate gyrus, considering the hippocampus more susceptible to aging-related damage [49].
TNF-α and 5-LO were downregulated in aged rats therapeutically treated with whey. These findings matched Hashemilar et al. [50], who supplemented WP in critically ill patients with ischemic stroke. In addition, Banerjee and Poddar [51] mentioned that neurodegeneration enhanced 5-LO coincides with increased synthesis of leukotriene and inflammatory eicosanoids.
The observed neuronal damage of the aged brain reflected the significant depletion of DA, 5-HT, and ATP. In addition, the depletion of ATP content expressed the loss of mitochondrial function and failure of neuronal cellular energy [52] and a reduction of energy requirements for brain function [53]. The data revealed that the impaired brain function of elderly rats resulted in a significant increase in brain β-amyloid, tau protein, and α-amylase levels. Tau is a highly regulated microtubule-associated protein in neurons. The abnormal aggregation of insoluble tau has been linked to neuronal cell loss and synapse degeneration in pathological conditions [54]. Tau phosphorylation is a secondary effect of β-amyloid accumulation related to aging [55]. Disrupted brain metabolism has been associated with extracellular β-amyloid plaques and intracellular tau phosphorylated protein [56]. The hippocampus has been linked to memory development [57]. In aged rats, decreasing synaptophysin expression in neuronal axons predicted neurodegeneration and the onset of cognitive failure [58].
In addition, when compared to the adult rat, the aged brain had a higher level of α-amylase. Periodic acid-Schiff showed positive polyglucosan bodies in the brain of AD patients, correlated with the increased α-amylase activity [59]. Furthermore, aged rats exhibited a significant increase in serum levels of xanthine oxidoreductase (XOR), creatine kinase (CK), and acetylcholinesterase while having a lower decrease in NGF levels and BNP. In vitro antidiabetic effect exhibited inhibition of α-amylase activity by 60%, and in vivo activities tested in albino rats showed that the final product helped regulate the blood glucose [60].
The elevated serum acetylcholinesterase seen in the elderly brain reflected the neurodegenerative condition described by García-Ayllón et al. [61] in Alzheimer's patients. Additionally, an XOR-generated ROS enhanced catabolism of purine bases [62] by liberating electrons to oxygen and generating O 2 − and H 2 O 2 within the cell [63], results in an imbalance in the antioxidant systems. Furthermore, increased serum creatine kinase reflected damage in the aged rats that reflected mitochondrial damage [64]. Finally, reducing the serum nerve growth factor [65] and natriuretic peptides mirrored the neurodegenerative condition [66].
According to the current findings, orally supplemented whey improves MDA, caspase-3, inflammatory markers, β-amyloid, tau protein, and α-amylase is, reflecting increased SOD activity and ATP contents of the brain in aged rats. These changes increased brain function and necrotic cells. UR + LR illustrating apoptosis. B Chart illustrating the apoptosis in the brain of aged rats and percent of improvement post whey supplementation. G1 8 months old rats, G2 8 months old rats supplemented whey, G3 30 months old rats, G4 30 months old rats supplemented whey. Data represent as the mean ± SD (n = 5); UR + LR in G2 is significant at p < 0.05 improved histology and immunohistochemistry in the cerebrum, hippocampus, and cerebellum.
The current findings are inconsistent with previous brain research. WP has been shown to improve rats with aging-related galactosaemic disease associated with SOD depletion and MDA. Adult (8 months) and old Wistar rats (30 months) consumed WP (300 mg/kg b.w.) for 28 days showed a reduction in inflammatory markers (TNF-α, interleukin (IL)-1β, IL-6) associated with oxidative stress in aged animals [43]. Whey treatment also reduced MDA and improved brain coordination in diabetic animals [67,68], increased mitochondrial activity [19], and managed brain structure and function [69,70]. Sixteen weeks of leucineenriched WP supplementation and combined with resistance-based exercise enhanced cardiometabolic health markers in old adults [16]. The inclusion of WP increased levels of liver antioxidant enzymes and decreased MDA and alanine aminotransferase (ALT) activities. Thus, WP could be recommended to enhance the growth performance and liver antioxidant enzymes in broiler chickens challenged with ethyl alcohol [12]. Reversal of the WP/Cas ratio in milk enhanced the insulin response, an effect possibly mediated by amino acids and/or incretins [15].

Conclusion
The current work investigated whether WP supplementation may improve age-related changes in diseased brain indicators like tau protein, β-amyloid and α-amylase. In conclusion, WP contains bioactive compounds and amino acids, which decrease brain oxidative stress and restore normal cognitive function. The increased antioxidant defense and DA and 5-HT neurotransmitters, while decreased brain tau and β-amyloid, were associated with better histology in aged rats' cerebrum, cerebellum, and hippocampus.
Author contributions MEE and HIHE collected and analyzed the samples. HIHE, MEE, AAE, AHA, SHQ, and WMF carried out experimental analyses. MFR reviewed and edited the article. All authors read and approved the manuscript.
Availability of data and materials Not applicable. Our manuscript has no associated data

Declarations
Conflict of interest The authors declare that they have no conflict of interest.

Ethics approval and consent to participate The Experimental Animal
Ethical Committee (Faculty of Science, Mansoura University, Egypt) approved the experiment (Approved Statement No. RZ19004).

Consent for publication Not applicable.
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