Effects of osteopontin combined with milk fat globule membrane proteins on scopolamine-induced learning and memory impairment in mice

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A dementia model was established through intraperitoneal injection of scopolamine, followed by random allocation into seven experimental groups: blank control, model control, OPN alone, MFGM alone, and three combination groups (low-, intermediate-, and high-dose OPN + MFGM). Cognitive performance was evaluated using Morris water maze testing, while biochemical analyses included assessments of hippocampal acetylcholinesterase (AChE) activity, antioxidant enzyme activities (superoxide dismutase [SOD] and glutathione peroxidase [GSH-Px]), and malondialdehyde (MDA) levels in both hippocampal tissue and serum. The intermediate- and high-dose combination groups demonstrated significant cognitive improvements compared to the model group, manifested by reduced escape latency, increased platform crossings, and prolonged target quadrant duration in water maze testing. Biochemical analyses revealed that these combination treatments significantly suppressed AChE activity in hippocampal tissue while enhancing antioxidant capacity through elevated SOD and GSH-Px activities, accompanied by reduced MDA levels in both brain and serum. This study demonstrated that the combination of OPN and MFGM administration improved learning and memory in mice with scopolamine-induced dementia through dose-dependent effects on the central cholinergic nervous and antioxidant systems. Osteopontin MFGM membrane proteins Morris water maze Cognition improvement Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Alzheimer’s disease (AD) is the most common form of dementia in older adults. Common pathological features of AD include amyloid plaques between neurons, intracellular neurofibrillary tangles, and impaired cholinergic signaling. AD is clinically manifested as cognitive and memory impairment, with subsequent behavioral dysfunction and loss of ability to live independently[ 1 , 2 ]. The prevalence of AD continues to increase due to the rapidly aging population. In the United States, there are currently 5.8 million adults aged 65 years or older with AD, and this number may grow to 13.8 million by 2050. Data from official death certificates indicated 122,019 deaths from AD in 2018, making AD the sixth-leading cause of death in the United States. Between 2000 and 2018, the number of deaths resulting from AD increased by 146.2%[ 3 ]. China has the highest number of individuals with dementia worldwide, including approximately 9.5 million patients with AD. Thus, AD has become a severe medical and social issue[ 4 ]. AD is a degenerative disease, in which the underlying symptoms and associated brain changes occur several years prior to the appearance of noticeable symptoms, such as memory and language impairment, thus AD is difficult to detect. Most existing clinical drugs for AD are given after the appearance of symptoms[ 5 – 10 ]. Therefore, the search for effective long-term treatment interventions without adverse effects to improve learning and memory impairment has become a main focus in the treatment of neurological diseases. Osteopontin (OPN) was initially discovered in bones and was later found to be present at a relatively high concentration in human milk. OPN is a highly glycosylated and phosphorylated acidic protein that contains the amino acid sequence arginine-glycine-aspartate. The importance of OPN in organisms has been demonstrated in many studies. In particular, OPN has been demonstrated as crucial in physiological processes of immune activation, bone repair, vascular regeneration, bone remodeling, and repair-promoting processes in the brain[ 11 ]. OPN contains an Arg-Gly-Asp (RGD) integrin-binding sequence[ 12 ]. The RGD integrinbinding site of mouse and human OPN is highly conserved. Therefore, the effects of OPN on mice may reflect its impact on human. Milk fat globular membrane (MFGM) is formed during the release of milk fat by mammary gland endothelial cells. It is a trilayer membrane structure composed of many proteins, including phospholipids and sphingolipids[ 13 ]. Although accounting for only 1–2% of total milk protein, MFGM contains a broad range of components with diverse functions, and thus can partially represent the proteins in lactating cells. Recent studies have revealed a variety of phospholipids and membrane proteins as the major components of MFGM. Further, MFGM has been shown to be a key player in neurodevelopment, metabolic regulation, and anti-infective activity in infants and young children[ 14 ]. Prior studies on MFGM supplementation in animal models have shown promising results in regards to improved cognition according to T-maze alternation and the Morris water maze tests[ 15 ]. The cooperation among OPN and MFGMs might boost their benefits in cognitive impairment. To date, the combined use of OPN and MFGM to promote intellect, specifically in promoting brain development and enhancing memory and learning ability, has not been reported. Therefore, this study aimed to investigate the effects of OPN and MFGM combined treatment on scopolamine-induced learning and memory impairment in mice. This study can support efforts in developing functional foods that promote brain development and enhance memory and learning ability by incorporating breast milk components, which may have significant implications for improving patient’s quality of life. 2 Materials and methods 2.1 Chemical OPN was purchased from Arla Foods (Basking Ridge, NJ, USA), and MFGM was purchased from Hilmar Ingredients (Hilmar, CA, USA). 2.2 Animal Model and Treatment Eight-four female Kunming mice (4-week-old, SPF-grade, weighed 18–20g) were purchased from Beijing Huafukang Bioscience Co., Inc. (license number: SCXK [Beijing] 2014-0004). The maintenance feed was produced by Beijing Keao Xieli Feed Co., Ltd. (license number: SCXK [Beijing] 2014-0010). The mice were kept in an environmentally controlled breeding room with temperature at 22 ± 1°C and relative humidity at 60 ± 5% for at least 3 days before experimentation. The mice were fasted overnight, but with free access to water prior to testing. Animal welfare and experimental procedures were strictly in accordance with the guide for the care and use of laboratory animals. After 3 days of adaptive feeding, the mice were randomly divided into seven groups of 12 mice each (Table 1 ). The mice in the experimental groups were gavaged different concentrations of the respective test substances at 9:00 ~ 9:15 a.m. for 14 days. Subsequently, the mice in the model and experimental groups received 5 mg/kg.bw scopolamine via intraperitoneal injection and were subjected to Morris water maze test, followed by oxidative stress evaluation. Table 1 Dosage volumes for each group of mice Group Dose group Dose (mg/kg.bw) 10 mL/kg.bw (distilled water) Blank control Blank control group - - Model Model group - - OPN High-dose OPN 200 20 mg/mL MFGM High-dose MFGM 7692 769 mg/mL Low Low-dose OPN + Low-dose MFGM 60 + 1923 6 + 192 mg/mL Intermediate Intermediate-dose OPN + Intermediate-dose MFGM 130 + 3846 13 + 385 mg/mL High High-dose OPN + High-dose MFGM 200 + 7692 20 + 769 mg/mL 2.3 Morris Water Maze Test The mice in each of the experimental groups were given different concentrations of the respective test substances for 14 days. Following, the pool used for the water maze test was filled with water (25 ± 1℃), and ink was added until the platform (quadrant 5) was invisible. The water level was set to 1 cm above the platform. Four 10 cm × 10 cm paper sheets of different shapes were fixed to the center of the wall of each quadrant (1–4). The positions of internal objects were fixed, and the light intensity was maintained constant throughout the experiment[ 16 ]. The blank group was not given any treatments. The mice in the model and experimental groups were given an intraperitoneal injection of 5 mg/kg.bw scopolamine. Thirty minutes after injection, the mice were placed in quadrant 4 to begin the experiment. The timer was stopped if the mice remained in quadrant 5 for more than 3 s or if latency reached 180 s. The corresponding data were recorded. All mice underwent three consecutive habituation trials at 15–17d after beginning of gavage (mice that did not spend time in quadrant 5 during the 180 s latency were manually placed in quadrant 5 and allowed to remain for 30 s). The mice continued to receive test substances during this period until the beginning of the acquisition trial on day 18. Probe trial was conducted 5 d after the acquisition trial. Scopolamine was not injected during the acqu isition (18 d) or probe (23 d) trials. 2.4 Biochemical Assays Following behavioral testing, retro-orbital bleeding was performed. The collected blood was centrifuged to obtain the serum and stored at − 80°C. After the mice were sacrificed, the brain tissue was collected and weighed. The tissue was quickly separated in an ice bath. The brain tissue was added with 9x normal saline and homogenized. Following, the homogenate was centrifuged at 3000 r/min for 15 min. The supernatant was collected, and the levels of AChE, SOD, GSH-PX, and MDA were measured according to the manufacturer’s protocol. 2.5 Statistical Analysis Data analysis was performed using GraphPad Prism (GraphPad Software 8.0.1), and results are presented as means ± SEM. Statistical differences between groups were analyzed using a one-way analysis of variance (ANOVA) and p < 0.05 was considered statistically significant. 3 Results 3.1 Effects of Test Substances on Morris Water Maze Test Perform h ance As shown in Fig. 1 A-C, the groups OPN and MFGM did not exibit significant improvements in escape latency, time spent in the platform quadrant, and number of platform crossings compared with the model group. However, these aspects were significantly improved in the different doses of OPN + MFGM groups, as compared with the OPN and MFGN groups. In particular, the intermediate and high groups achieved results that were close to or better than the OPN and MFGN groups. The intermediate and high groups spent significantly higher percentages of time and distance travelled in quadrant 2 compared with the OPN and MFGN groups, and the percentages were even higher than that in the blank group (Fig. 2 A-B). Next, the swim trajectories and strategies adopted by the mice to locate the platform during the Morris water maze test were analyzed (Fig. 3 ). After training, the swim trajectories of the mice in the model group were mainly random (Fig. 3 ). The mice in the blank group adopted an effective strategy with linear swim trajectories (Fig. 3 ). Moreover, the mice in the intermediate and high groups displayed a strategy that trended towards that of the OPN and MFGN groups (Fig. 3 ). These findings indicated that the OPN and MFGM combination exhibited a synergistic effect compared with either component alone. Further, treatment with this combination significantly improved memory and promoted brain development. 3.2 Effects of Test Substances on GSH-Px Activities in Mouse Brain Tissue and Serum GSH-P is an important enzyme found in many tissues that catalyzes the decomposition of hydrogen peroxide. Specifically, it catalyzes the reduction of hydrogen peroxide using reduced glutathione as the substrate, thereby protecting cell membrane structure integrity and function. Figure 4 A-B shows the results of biochemical assays on GSH-Px activities in the mouse hippocampus and serum. As shown in Fig. 4 A-B, compared with the blank group, GSH-Px activities in the hippocampus and serum of the model group was significantly reduced ( p < 0.01). By contrast, the test substances in the intermediate and high groups led to significant increase of GSH-Px activities in the mouse brain tissue and serum compared with the model group, and the activities were significantly higher than that in the OPN and MFGM groups. These results indicated that the OPN and MFGM combination significantly increased GSH-Px activity in the brain, as compared with either component alone, in a dose-dependent manner. 3.3 Effects of Test Substances on SOD Activities in Mouse Brain Tissue and Serum SOD is an important antioxidant enzyme in organisms that can eliminate oxygen free radicals produced in the body during metabolic processes. SOD protects cells by blocking oxygen free radical-induced cell damage and repairing damaged cells. SOD is also the primary free radical scavenger in the body, and its level can directly reflect aging and death in an organism. Figure 5 A-B shows the results of biochemical assays on SOD activities in the mouse hippocampus and serum. As shown in Fig. 5 A-B, compared with the blank group, SOD activities in the hippocampus and serum of the model group was significantly reduced ( p < 0.001). Further, the different doses of OPN + MFGM groups exhibited significant increases in SOD activity in the mouse brain tissue and serum compared with the model group ( p < 0.001), and the activities were significantly higher than that in the OPN and MFGM groups. Furthermore, antioxidant activity increased with increasing dose, displaying dose dependency. These results indicated that scopolamine could reduce SOD activity and alter the prooxidant-antioxidant balance in the mouse brain and serum, thereby increasing the oxidative stress level in the body. Compared with either component alone, the OPN and MFGM combination significantly increased SOD activities in brain tissue and serum to prevent cell damage. 3.4 Effects of Test Substances on MDA Levels in Mouse Brain Tissue and Serum MDA is a key product of lipid peroxidation, which when abnormally increased is linked to memory impairment. Its levels also indirectly reflect the degree of free radical-induced damage in the body. Biochemical assays were performed to measure MDA levels in the mouse hippocampus and serum (Fig. 6 A-B). Results showed significantly increased MDA levels in the hippocampus and serum of the model group compared with the blank group ( p < 0.001). By contrast, mice in all groups of OPN + MFGM showed significantly reduced MDA levels in the brain tissue and serum compared with the model group ( p < 0.001), and the levels were significantly higher than that in the OPN and MFGM groups. This indicated that scopolamine increased MDA levels in brain tissue and serum, thereby inducing memory impairment. However, the OPN and MFGM combination significantly reduced MDA levels in brain tissue compared with either component alone. 3.5 Effects of Test Substances on AChE Activities in Mouse Brain Tissue AChE is an enzyme that selectively hydrolyzes acetylcholine (ACh) into choline and acetic acid. It is primarily found in the synaptic cleft of cholinergic nerve terminals. Changes in AChE activity directly affect the level of ACh released by cholinergic neurons, as AChE regulates cholinergic signals in the synaptic cleft via ACh hydrolysis. Figure 7 shows the results of biochemical assays on AChE activities in the mouse hippocampus, which demonstrated significantly increased AChE activity following intraperitoneal injection of scopolamine in the model group. AChE expression and activation regulate the physiological concentration of ACh in the brain cholinergic synapses through increased ACh hydrolysis, thereby reducing ACh levels in the brain. This, in turn, affects signal transduction of the central cholinergic nervous system, leading to cognitive dysfunction in mice. However, intake of different doses of OPN and MFGM inhibited AChE activity in the mouse brain tissue, blocking ACh hydrolysis and enhancing cognitive function. In addition, the OPN and MFGM combination improved the learning and memory impairment induced by scopolamine injection compared with either component alone, which corroborated the results of the behavioral testing. 4 Discussion AD is a common degenerative central nervous system (CNS) disorder among older adults. The key clinical presentations include reduced intellect, communication difficulties, mobility issue, and affective disorders. More than 80% of patients exhibit cognitive dysfunction of varying degrees, which significantly impacts patients’ quality of life[ 17 ]. The pathogenesis of AD is not fully understood. Studies showed that oxidative damage constituted an important mechanism for early stages of AD[ 18 ]. Lipid peroxidation in the plasma and cerebrospinal fluid of patients resulted in decreased plasma antioxidant levels and increased oxidative markers. The primary goals of current AD treatments focus on inhibiting the progressive degeneration of brain function, reversing early pathological changes, and preserving the patient’s cognitive and memory functions. AD is mainly treated with antipsychotics, and classic antipsychotic drugs, such as perphenazine and haloperidol, have shown considerable efficacy. However, these drugs are also associated with significant cardiac and hepatic adverse reactions. These and other adverse reactions have limited their use. Therefore, the search for a safe and effective pharmacological treatment for AD is of great significance. Scopolamine is a muscarinic (M) receptor antagonist that readily penetrates the blood-brain barrier and enters the CNS. It can reversibly block M receptors in the CNS and reduce the binding of ACh to its receptor, causing dysregulation of the cholinergic nervous system. This induces memory loss and decline in learning ability, eventually leading to learning and memory impairment[ 19 ]. Scopolamine is commonly used for the construction of dementia models induced by damage to the central cholinergic nervous system and is widely used for memory impairment models for testing AD drugs[ 20 ]. In this study, scopolamine was used to investigate the possible effects of MFGM and OPN on the improvement of learning and memory impairment in a mouse model[ 21 , 22 ]. Morris water maze test was conducted and confirmed successful establishment of the model. Compared with the model and control groups, the mice that received the test substances exhibited significant increases in the number of platform crossings during the water maze test. This indicated that the OPN and MFGM combination significantly improved the impaired learning and memory of mice with scopolamine-induced dementia. ACh is a neurotransmitter of the central cholinergic nervous system that plays an crucial role in learning and memory processes of the hippocampus[ 23 ]. AChE is a key enzyme responsible for ACh metabolism and is a catabolic enzyme that regulates ACh levels in brain tissue. Thus, AChE activity can be used an as indicator of the effects of the test substances on improving cholinergic nerve functions[ 24 ]. Our study showed that the AChE activity in the hippocampus of mice in the high-dose group was significantly reduced following intervention with OPN plus MFGM, suggesting that the OPN and MFGM combination may regulate cholinergic nerve function by modulating AChE activity, thereby improving learning and memory function. Learning and memory impairment is a common symptom of many neurological diseases. Nerve tissues have a unique architecture that renders them more susceptible to oxygen free radical-mediated damages. Moreover, the large number of free radicals produced during cell metabolism can damage large molecules, such as DNA, which accelerates neuronal death and results in neurodegenerative diseases[ 25 ]. Studies have shown reduced free radical-scavenging ability or antioxidant capacity in patients with learning and memory impairment, which results in excessive amount of oxidative stress factors that causes neurotoxicity[ 26 , 27 ]. Under normal physiological state, free radicals are continuously produced, yet eliminated through the body’s free radical scavenging system, thus maintaining a dynamic prooxidant-antioxidant balance in the body. Under disease states, such as degeneration of central cholinergic neurons, the activities of antioxidant enzymes in the body continuously decline. The oxygen free radicals in the body cannot be eliminated in time, eventually leading to abnormal accumulation of free radicals[ 26 ]. SOD is an important enzyme that is widely present in organisms. It is involved in maintaining the dynamic balance between free radical production and scavenging in the body. It catalyzes the disproportionation of the free radical superoxide anion to yield hydrogen peroxide, which helps the body to combat against the cytotoxic effects of oxygen free radicals and other oxides. Thus, SOD is a crucial antioxidant enzyme for scavenging free radicals in the body[ 27 ]. GSH-Px is an important enzyme for peroxide decomposition that is widely present in the body. It catalyzes reduced glutathione into oxidized glutathione and reduces harmful peroxides to nontoxic hydroxyl compounds, thereby protecting the structure and function of cell membranes[ 28 ]. MDA is a key product of lipid peroxidation, and its abnormal increase in the body is linked to memory impairment[ 29 ]. This study found that high-dose OPN plus MFGM significantly increased SOD and GSH-Px activities and significantly reduced MDA levels in the hippocampus and serum of the model mice. Our findings suggested that the OPN and MFGM combination potentially improved learning and memory function through combating oxidative damage, which warrants further investigations. This research lays the foundation for the application of functional proteins. Conclusion This study revealed that osteopontin (OPN) combined with milk fat globule membrane (MFGM) proteins mitigated scopolamine-induced cognitive impairment in mice. Intermediate- and high-dose OPN + MFGM enhanced Morris water maze performance (reduced latency, increased platform crossings), suppressed acetylcholinesterase activity, elevated antioxidant enzymes (SOD, GSH-Px), and lowered lipid peroxidation (MDA). The synergy between OPN and MFGM highlights their dual-action mechanism targeting cholinergic and oxidative pathways, suggesting therapeutic potential for neurodegenerative diseases like Alzheimer’s. These findings advocate milk-derived components as promising candidates for cognitive-enhancing interventions. Declarations Author Contributions Xiaochen Liu contributed to the experimental design and writing of the manuscript, performed the experiments and analyzed the data. Bin Guo provided the experimental method. Da xi Ren funded the project. Bin Guo and Da xi Ren supervised the project. All authors read and approved the final manuscript. Funding This study was supported by Guangxi Science and Technology Base and Talent Program (2024AC38038). Data Availability No datasets were generated or analysed during the current study. Ethics Approval All studies received ethical approval from the Ethics Committee of Jilin University (Changchun, China). Competing Interests The authors declare no competing interests. References Liguori C, Pierantozzi M, Chiaravalloti A, Sancesario GM, Mercuri NB, Franchini F, Schillaci O, Sancesario G: When Cognitive Decline and Depression Coexist in the Elderly: CSF Biomarkers Analysis Can Differentiate Alzheimer's Disease from Late-Life Depression. Front Aging Neurosci 2018, 10:38. Scheltens P, Blennow K, Breteler MM, de Strooper B, Frisoni GB, Salloway S, Van der Flier WM: Alzheimer's disease. Lancet 2016, 388:505-517. Alzheimer's disease facts and figures. Alzheimers Dement 2020. Jia J, Wei C, Chen S, Li F, Tang Y, Qin W, Zhao L, Jin H, Xu H, Wang F, et al: The cost of Alzheimer's disease in China and re-estimation of costs worldwide. Alzheimers Dement 2018, 14:483-491. Villemagne VL, Burnham S, Bourgeat P, Brown B, Ellis KA, Salvado O, Szoeke C, Macaulay SL, Martins R, Maruff P, et al: Amyloid β deposition, neurodegeneration, and cognitive decline in sporadic Alzheimer's disease: a prospective cohort study. Lancet Neurol 2013, 12:357-367. Reiman EM, Quiroz YT, Fleisher AS, Chen K, Velez-Pardo C, Jimenez-Del-Rio M, Fagan AM, Shah AR, Alvarez S, Arbelaez A, et al: Brain imaging and fluid biomarker analysis in young adults at genetic risk for autosomal dominant Alzheimer's disease in the presenilin 1 E280A kindred: a case-control study. Lancet Neurol 2012, 11:1048-1056. Jack CR, Jr., Lowe VJ, Weigand SD, Wiste HJ, Senjem ML, Knopman DS, Shiung MM, Gunter JL, Boeve BF, Kemp BJ, et al: Serial PIB and MRI in normal, mild cognitive impairment and Alzheimer's disease: implications for sequence of pathological events in Alzheimer's disease. Brain 2009, 132:1355-1365. Bateman RJ, Xiong C, Benzinger TL, Fagan AM, Goate A, Fox NC, Marcus DS, Cairns NJ, Xie X, Blazey TM, et al: Clinical and biomarker changes in dominantly inherited Alzheimer's disease. N Engl J Med 2012, 367:795-804. Gordon BA, Blazey TM, Su Y, Hari-Raj A, Dincer A, Flores S, Christensen J, McDade E, Wang G, Xiong C, et al: Spatial patterns of neuroimaging biomarker change in individuals from families with autosomal dominant Alzheimer's disease: a longitudinal study. Lancet Neurol 2018, 17:241-250. Braak H, Thal DR, Ghebremedhin E, Del Tredici K: Stages of the pathologic process in Alzheimer disease: age categories from 1 to 100 years. J Neuropathol Exp Neurol 2011, 70:960-969. Sodek J, Ganss B, McKee MD: Osteopontin. Crit Rev Oral Biol Med 2000, 11:279-303. Jiang R, Lönnerdal B: Evaluation of Bioactivities of Bovine Milk Osteopontin Using a Knockout Mouse Model. J Pediatr Gastroenterol Nutr 2020, 71:125-131. Ali F, Wang ZX: Effect of pasteurization on the enzymatic cross-linking of milk proteins by microbial transglutaminase in view of milk fat globule membrane isolation. Food Bioscience 2021:43. Timby N, Domellöf M, Lönnerdal B, Hernell O: Supplementation of Infant Formula with Bovine Milk Fat Globule Membranes. Adv Nutr 2017, 8:351-355. Brink LR, Lönnerdal B: The role of milk fat globule membranes in behavior and cognitive function using a suckling rat pup supplementation model. J Nutr Biochem 2018, 58:131-137. Cahill JF, Baxter MG: Cholinergic and noncholinergic septal neurons modulate strategy selection in spatial learning. Eur J Neurosci 2001, 14:1856-1864. Ju Y, Tam KY: Pathological mechanisms and therapeutic strategies for Alzheimer's disease. Neural Regen Res 2022, 17:543-549. Youssef P, Chami B, Lim J, Middleton T, Sutherland GT, Witting PK: Evidence supporting oxidative stress in a moderately affected area of the brain in Alzheimer's disease. Sci Rep 2018, 8:11553. Jain NK, Patil CS, Kulkarni SK, Singh A: Modulatory role of cyclooxygenase inhibitors in aging- and scopolamine or lipopolysaccharide-induced cognitive dysfunction in mice. Behav Brain Res 2002, 133:369-376. Manral A, Meena P, Saini V, Siraj F, Shalini S, Tiwari M: DADS Analogues Ameliorated the Cognitive Impairments of Alzheimer-Like Rat Model Induced by Scopolamine. Neurotox Res 2016, 30:407-426. Gong Z, Huang J, Xu B, Ou Z, Zhang L, Lin X, Ye X, Kong X, Long D, Sun X, et al: Urolithin A attenuates memory impairment and neuroinflammation in APP/PS1 mice. J Neuroinflammation 2019, 16:62. Qi Y, Cheng X, Jing H, Yan T, Xiao F, Wu B, Bi K, Jia Y: Effect of Alpinia oxyphylla-Schisandra chinensis herb pair on inflammation and apoptosis in Alzheimer's disease mice model. J Ethnopharmacol 2019, 237:28-38. Weinberger NM, Miasnikov AA, Chen JC: Sensory memory consolidation observed: increased specificity of detail over days. Neurobiol Learn Mem 2009, 91:273-286. Dashniani MG, Beseliia GV, Maglakelidze GA, Burdzhanadze MA, Chkhikvishvili N: Effects of the selective lesions of cholinergic septohippocampal neurons on different forms of memory and learning process. Georgian Med News 2009:81-85. Markesbery WR, Lovell MA: Damage to lipids, proteins, DNA, and RNA in mild cognitive impairment. Arch Neurol 2007, 64:954-956. Fan Y, Hu J, Li J, Yang Z, Xin X, Wang J, Ding J, Geng M: Effect of acidic oligosaccharide sugar chain on scopolamine-induced memory impairment in rats and its related mechanisms. Neurosci Lett 2005, 374:222-226. Goverdhan P, Sravanthi A, Mamatha T: Neuroprotective effects of meloxicam and selegiline in scopolamine-induced cognitive impairment and oxidative stress. Int J Alzheimers Dis 2012, 2012:974013. Dringen R: Metabolism and functions of glutathione in brain. Prog Neurobiol 2000, 62:649-671. Hall ED, Bosken JM: Measurement of oxygen radicals and lipid peroxidation in neural tissues. Curr Protoc Neurosci 2009, Chapter 7:Unit 7.17.11-51. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7164889","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":489819873,"identity":"34afd810-9a3d-4119-ab52-0583f4dd5a90","order_by":0,"name":"Xiaochen Liu","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Xiaochen","middleName":"","lastName":"Liu","suffix":""},{"id":489819874,"identity":"bd40e87a-6735-4c52-89b7-f9b70adaee51","order_by":1,"name":"Bin Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYHACNoYEhv9y/AyHG4AcZqK1MBtLNhwkRQtQZeKGA4xEapGPSD724OEOtsTNBw+2STBUWCc2sJ89gFeL4Y20dIPEMzzG2w6AtJxJT2zgyUvAr2VGjplEYpuELFgLY9vhxAYJHgNitBgwbm4AaflHhBZ5CbCWBMUNDCAtDURoMeB5BvRL2wFjiQMHmy0SjqUbt/HkELClPfnYw59tB+T4Zxw+eONDjbVsP/sZArYcgLEkgKwEBkg04belAcbib8CtahSMglEwCkY2AADoNknvHCg8OgAAAABJRU5ErkJggg==","orcid":"","institution":"Jilin University","correspondingAuthor":true,"prefix":"","firstName":"Bin","middleName":"","lastName":"Guo","suffix":""},{"id":489819875,"identity":"36dcaed6-c293-45de-9e70-35b217dc5ce3","order_by":2,"name":"Daxi Ren","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Daxi","middleName":"","lastName":"Ren","suffix":""}],"badges":[],"createdAt":"2025-07-19 13:53:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7164889/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7164889/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11064-025-04612-7","type":"published","date":"2025-11-14T15:58:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87490049,"identity":"88d07693-43c7-498a-a9d4-6882355694c6","added_by":"auto","created_at":"2025-07-24 11:39:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":69829,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of test substances on Morris water maze test performance. (A) Escape latency (s); (B) Time spent in the platform quadrant(s); (C) Number of platform crossings. All data are expressed as means ± SEM. * \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.05; ** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.01; *** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164889/v1/5f272269c4617e86c6296f87.jpg"},{"id":87490021,"identity":"af3aca02-6d0f-49d0-b29e-76c0129fea05","added_by":"auto","created_at":"2025-07-24 11:38:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68321,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of test substances on Morris water maze test performance of mice. (A) Time spent in quadrant 2 percentage (%); (B) Distance travelled in quadrant 2 percentage (%); All data are expressed as means ± SEM. ** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.05; ** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.01; *** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164889/v1/8175359aacd73a0caf4aa20e.jpg"},{"id":87490457,"identity":"b0bb385b-8429-49e6-934f-1614bdfd418b","added_by":"auto","created_at":"2025-07-24 11:46:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":125048,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe swim trajectories and strategies adopted by the mice in the Morris water maze test.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164889/v1/420e2c486df5af4ca599237c.jpg"},{"id":87490032,"identity":"b448d457-e342-46f8-b621-072d92e5cf4f","added_by":"auto","created_at":"2025-07-24 11:38:59","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68910,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of test substances on GSH-Px activities in mouse brain tissue and serum. (A) GSH-Px activitiy in the mouse hippocampus; (B) GSH-Px activitiy in the mouse serum; All data are expressed as means ± SEM. * \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.05; ** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.01; *** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164889/v1/eec0c78573dc1fd2da91721e.jpg"},{"id":87491306,"identity":"65572856-e3db-4401-b3cc-8aa3b4f01ba8","added_by":"auto","created_at":"2025-07-24 11:55:00","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":64066,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of test substances on SOD activities in mouse brain tissue and serum. (A) SOD activitiey in the mouse hippocampus; (B) SOD activitiey in the mouse serum; All data are expressed as means ± SEM. * \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.05; ** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.01; *** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164889/v1/2054818160ef72bb53e67277.jpg"},{"id":87490056,"identity":"6c1d705e-164d-495e-93eb-10efc618d82d","added_by":"auto","created_at":"2025-07-24 11:39:00","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59346,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of test substances on MDA level in mouse brain tissue and serum. (A) MDA level in the hippocampus; (B) MDA level in the serum; All data are expressed as means ± SEM.* \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.05; ** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.01; *** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164889/v1/a1fc2d0d26ca11f36ce659b1.jpg"},{"id":87490063,"identity":"da31000d-be77-43ba-8d16-5133ed017f89","added_by":"auto","created_at":"2025-07-24 11:39:00","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":145389,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of test substances on AChE activities in mouse brain tissue. All data are expressed as means ± SEM. * \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.05; ** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.01; *** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt; 0.001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164889/v1/445abe64f95e95f8b4562e32.jpg"},{"id":96105958,"identity":"df06cd32-98bf-4a22-ad3c-ca38e527e186","added_by":"auto","created_at":"2025-11-17 16:12:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1834580,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7164889/v1/2750fa16-1c5d-4089-8b0a-29219c7da080.pdf"},{"id":87490034,"identity":"0fa29f60-17b7-4ea8-a404-3dafb05a5011","added_by":"auto","created_at":"2025-07-24 11:38:59","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":201842,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164889/v1/48afdc6d41da9d3a130959f0.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of osteopontin combined with milk fat globule membrane proteins on scopolamine-induced learning and memory impairment in mice","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD) is the most common form of dementia in older adults. Common pathological features of AD include amyloid plaques between neurons, intracellular neurofibrillary tangles, and impaired cholinergic signaling. AD is clinically manifested as cognitive and memory impairment, with subsequent behavioral dysfunction and loss of ability to live independently[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The prevalence of AD continues to increase due to the rapidly aging population. In the United States, there are currently 5.8\u0026nbsp;million adults aged 65 years or older with AD, and this number may grow to 13.8\u0026nbsp;million by 2050. Data from official death certificates indicated 122,019 deaths from AD in 2018, making AD the sixth-leading cause of death in the United States. Between 2000 and 2018, the number of deaths resulting from AD increased by 146.2%[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. China has the highest number of individuals with dementia worldwide, including approximately 9.5\u0026nbsp;million patients with AD. Thus, AD has become a severe medical and social issue[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. AD is a degenerative disease, in which the underlying symptoms and associated brain changes occur several years prior to the appearance of noticeable symptoms, such as memory and language impairment, thus AD is difficult to detect. Most existing clinical drugs for AD are given after the appearance of symptoms[\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, the search for effective long-term treatment interventions without adverse effects to improve learning and memory impairment has become a main focus in the treatment of neurological diseases.\u003c/p\u003e\u003cp\u003eOsteopontin (OPN) was initially discovered in bones and was later found to be present at a relatively high concentration in human milk. OPN is a highly glycosylated and phosphorylated acidic protein that contains the amino acid sequence arginine-glycine-aspartate. The importance of OPN in organisms has been demonstrated in many studies. In particular, OPN has been demonstrated as crucial in physiological processes of immune activation, bone repair, vascular regeneration, bone remodeling, and repair-promoting processes in the brain[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. OPN contains an Arg-Gly-Asp (RGD) integrin-binding sequence[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The RGD integrinbinding site of mouse and human OPN is highly conserved. Therefore, the effects of OPN on mice may reflect its impact on human.\u003c/p\u003e\u003cp\u003eMilk fat globular membrane (MFGM) is formed during the release of milk fat by mammary gland endothelial cells. It is a trilayer membrane structure composed of many proteins, including phospholipids and sphingolipids[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Although accounting for only 1\u0026ndash;2% of total milk protein, MFGM contains a broad range of components with diverse functions, and thus can partially represent the proteins in lactating cells. Recent studies have revealed a variety of phospholipids and membrane proteins as the major components of MFGM. Further, MFGM has been shown to be a key player in neurodevelopment, metabolic regulation, and anti-infective activity in infants and young children[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Prior studies on MFGM supplementation in animal models have shown promising results in regards to improved cognition according to T-maze alternation and the Morris water maze tests[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe cooperation among OPN and MFGMs might boost their benefits in cognitive impairment. To date, the combined use of OPN and MFGM to promote intellect, specifically in promoting brain development and enhancing memory and learning ability, has not been reported. Therefore, this study aimed to investigate the effects of OPN and MFGM combined treatment on scopolamine-induced learning and memory impairment in mice. This study can support efforts in developing functional foods that promote brain development and enhance memory and learning ability by incorporating breast milk components, which may have significant implications for improving patient\u0026rsquo;s quality of life.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Chemical\u003c/h2\u003e\u003cp\u003eOPN was purchased from Arla Foods (Basking Ridge, NJ, USA), and MFGM was purchased from Hilmar Ingredients (Hilmar, CA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Animal Model and Treatment\u003c/h2\u003e\u003cp\u003eEight-four female Kunming mice (4-week-old, SPF-grade, weighed 18\u0026ndash;20g) were purchased from Beijing Huafukang Bioscience Co., Inc. (license number: SCXK [Beijing] 2014-0004). The maintenance feed was produced by Beijing Keao Xieli Feed Co., Ltd. (license number: SCXK [Beijing] 2014-0010). The mice were kept in an environmentally controlled breeding room with temperature at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and relative humidity at 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% for at least 3 days before experimentation. The mice were fasted overnight, but with free access to water prior to testing. Animal welfare and experimental procedures were strictly in accordance with the guide for the care and use of laboratory animals.\u003c/p\u003e\u003cp\u003eAfter 3 days of adaptive feeding, the mice were randomly divided into seven groups of 12 mice each (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The mice in the experimental groups were gavaged different concentrations of the respective test substances at 9:00\u0026thinsp;~\u0026thinsp;9:15 a.m. for 14 days. Subsequently, the mice in the model and experimental groups received 5 mg/kg.bw scopolamine via intraperitoneal injection and were subjected to Morris water maze test, followed by oxidative stress evaluation.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDosage volumes for each group of mice\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDose group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDose (mg/kg.bw)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10 mL/kg.bw (distilled water)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlank control\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBlank control group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eModel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eModel group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOPN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHigh-dose OPN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20 mg/mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMFGM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHigh-dose MFGM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7692\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e769 mg/mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLow-dose OPN\u0026thinsp;+\u0026thinsp;Low-dose\u0026nbsp;MFGM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e60\u0026thinsp;+\u0026thinsp;1923\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6\u0026thinsp;+\u0026thinsp;192 mg/mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIntermediate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIntermediate-dose OPN\u0026thinsp;+\u0026thinsp;Intermediate-dose MFGM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e130\u0026thinsp;+\u0026thinsp;3846\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13\u0026thinsp;+\u0026thinsp;385 mg/mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHigh-dose OPN +\u003c/p\u003e\u003cp\u003eHigh-dose MFGM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e200\u0026thinsp;+\u0026thinsp;7692\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20\u0026thinsp;+\u0026thinsp;769 mg/mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Morris Water Maze Test\u003c/h2\u003e\u003cp\u003eThe mice in each of the experimental groups were given different concentrations of the respective test substances for 14 days. Following, the pool used for the water maze test was filled with water (25\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃), and ink was added until the platform (quadrant 5) was invisible. The water level was set to 1 cm above the platform. Four 10 cm \u0026times; 10 cm paper sheets of different shapes were fixed to the center of the wall of each quadrant (1\u0026ndash;4). The positions of internal objects were fixed, and the light intensity was maintained constant throughout the experiment[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe blank group was not given any treatments. The mice in the model and experimental groups were given an intraperitoneal injection of 5 mg/kg.bw scopolamine. Thirty minutes after injection, the mice were placed in quadrant 4 to begin the experiment. The timer was stopped if the mice remained in quadrant 5 for more than 3 s or if latency reached 180 s. The corresponding data were recorded. All mice underwent three consecutive habituation trials at 15\u0026ndash;17d after beginning of gavage (mice that did not spend time in quadrant 5 during the 180 s latency were manually placed in quadrant 5 and allowed to remain for 30 s). The mice continued to receive test substances during this period until the beginning of the acquisition trial on day 18. Probe trial was conducted 5 d after the acquisition trial. Scopolamine was not injected during the acqu isition (18 d) or probe (23 d) trials.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Biochemical Assays\u003c/h2\u003e\u003cp\u003eFollowing behavioral testing, retro-orbital bleeding was performed. The collected blood was centrifuged to obtain the serum and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. After the mice were sacrificed, the brain tissue was collected and weighed. The tissue was quickly separated in an ice bath. The brain tissue was added with 9x normal saline and homogenized. Following, the homogenate was centrifuged at 3000 r/min for 15 min. The supernatant was collected, and the levels of AChE, SOD, GSH-PX, and MDA were measured according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Statistical Analysis\u003c/h2\u003e\u003cp\u003eData analysis was performed using GraphPad Prism (GraphPad Software 8.0.1), and results are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical differences between groups were analyzed using a one-way analysis of variance (ANOVA) and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.1 Effects of Test Substances on Morris Water Maze Test Perform\u003c/b\u003eh\u003cb\u003eance\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C, the groups OPN and MFGM did not exibit significant improvements in escape latency, time spent in the platform quadrant, and number of platform crossings compared with the model group. However, these aspects were significantly improved in the different doses of OPN\u0026thinsp;+\u0026thinsp;MFGM groups, as compared with the OPN and MFGN groups. In particular, the intermediate and high groups achieved results that were close to or better than the OPN and MFGN groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe intermediate and high groups spent significantly higher percentages of time and distance travelled in quadrant 2 compared with the OPN and MFGN groups, and the percentages were even higher than that in the blank group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, the swim trajectories and strategies adopted by the mice to locate the platform during the Morris water maze test were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). After training, the swim trajectories of the mice in the model group were mainly random (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The mice in the blank group adopted an effective strategy with linear swim trajectories (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Moreover, the mice in the intermediate and high groups displayed a strategy that trended towards that of the OPN and MFGN groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese findings indicated that the OPN and MFGM combination exhibited a synergistic effect compared with either component alone. Further, treatment with this combination significantly improved memory and promoted brain development.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Effects of Test Substances on GSH-Px Activities in Mouse Brain Tissue and Serum\u003c/h2\u003e\u003cp\u003eGSH-P is an important enzyme found in many tissues that catalyzes the decomposition of hydrogen peroxide. Specifically, it catalyzes the reduction of hydrogen peroxide using reduced glutathione as the substrate, thereby protecting cell membrane structure integrity and function. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B shows the results of biochemical assays on GSH-Px activities in the mouse hippocampus and serum.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B, compared with the blank group, GSH-Px activities in the hippocampus and serum of the model group was significantly reduced (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). By contrast, the test substances in the intermediate and high groups led to significant increase of GSH-Px activities in the mouse brain tissue and serum compared with the model group, and the activities were significantly higher than that in the OPN and MFGM groups. These results indicated that the OPN and MFGM combination significantly increased GSH-Px activity in the brain, as compared with either component alone, in a dose-dependent manner.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Effects of Test Substances on SOD Activities in Mouse Brain Tissue and Serum\u003c/h2\u003e\u003cp\u003eSOD is an important antioxidant enzyme in organisms that can eliminate oxygen free radicals produced in the body during metabolic processes. SOD protects cells by blocking oxygen free radical-induced cell damage and repairing damaged cells. SOD is also the primary free radical scavenger in the body, and its level can directly reflect aging and death in an organism. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B shows the results of biochemical assays on SOD activities in the mouse hippocampus and serum.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B, compared with the blank group, SOD activities in the hippocampus and serum of the model group was significantly reduced (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Further, the different doses of OPN\u0026thinsp;+\u0026thinsp;MFGM groups exhibited significant increases in SOD activity in the mouse brain tissue and serum compared with the model group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the activities were significantly higher than that in the OPN and MFGM groups. Furthermore, antioxidant activity increased with increasing dose, displaying dose dependency. These results indicated that scopolamine could reduce SOD activity and alter the prooxidant-antioxidant balance in the mouse brain and serum, thereby increasing the oxidative stress level in the body. Compared with either component alone, the OPN and MFGM combination significantly increased SOD activities in brain tissue and serum to prevent cell damage.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Effects of Test Substances on MDA Levels in Mouse Brain Tissue and Serum\u003c/h2\u003e\u003cp\u003eMDA is a key product of lipid peroxidation, which when abnormally increased is linked to memory impairment. Its levels also indirectly reflect the degree of free radical-induced damage in the body. Biochemical assays were performed to measure MDA levels in the mouse hippocampus and serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). Results showed significantly increased MDA levels in the hippocampus and serum of the model group compared with the blank group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). By contrast, mice in all groups of OPN\u0026thinsp;+\u0026thinsp;MFGM showed significantly reduced MDA levels in the brain tissue and serum compared with the model group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the levels were significantly higher than that in the OPN and MFGM groups. This indicated that scopolamine increased MDA levels in brain tissue and serum, thereby inducing memory impairment. However, the OPN and MFGM combination significantly reduced MDA levels in brain tissue compared with either component alone.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Effects of Test Substances on AChE Activities in Mouse Brain Tissue\u003c/h2\u003e\u003cp\u003eAChE is an enzyme that selectively hydrolyzes acetylcholine (ACh) into choline and acetic acid. It is primarily found in the synaptic cleft of cholinergic nerve terminals. Changes in AChE activity directly affect the level of ACh released by cholinergic neurons, as AChE regulates cholinergic signals in the synaptic cleft via ACh hydrolysis. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the results of biochemical assays on AChE activities in the mouse hippocampus, which demonstrated significantly increased AChE activity following intraperitoneal injection of scopolamine in the model group. AChE expression and activation regulate the physiological concentration of ACh in the brain cholinergic synapses through increased ACh hydrolysis, thereby reducing ACh levels in the brain. This, in turn, affects signal transduction of the central cholinergic nervous system, leading to cognitive dysfunction in mice. However, intake of different doses of OPN and MFGM inhibited AChE activity in the mouse brain tissue, blocking ACh hydrolysis and enhancing cognitive function. In addition, the OPN and MFGM combination improved the learning and memory impairment induced by scopolamine injection compared with either component alone, which corroborated the results of the behavioral testing.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eAD is a common degenerative central nervous system (CNS) disorder among older adults. The key clinical presentations include reduced intellect, communication difficulties, mobility issue, and affective disorders. More than 80% of patients exhibit cognitive dysfunction of varying degrees, which significantly impacts patients\u0026rsquo; quality of life[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The pathogenesis of AD is not fully understood. Studies showed that oxidative damage constituted an important mechanism for early stages of AD[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Lipid peroxidation in the plasma and cerebrospinal fluid of patients resulted in decreased plasma antioxidant levels and increased oxidative markers. The primary goals of current AD treatments focus on inhibiting the progressive degeneration of brain function, reversing early pathological changes, and preserving the patient\u0026rsquo;s cognitive and memory functions. AD is mainly treated with antipsychotics, and classic antipsychotic drugs, such as perphenazine and haloperidol, have shown considerable efficacy. However, these drugs are also associated with significant cardiac and hepatic adverse reactions. These and other adverse reactions have limited their use. Therefore, the search for a safe and effective pharmacological treatment for AD is of great significance.\u003c/p\u003e\u003cp\u003eScopolamine is a muscarinic (M) receptor antagonist that readily penetrates the blood-brain barrier and enters the CNS. It can reversibly block M receptors in the CNS and reduce the binding of ACh to its receptor, causing dysregulation of the cholinergic nervous system. This induces memory loss and decline in learning ability, eventually leading to learning and memory impairment[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Scopolamine is commonly used for the construction of dementia models induced by damage to the central cholinergic nervous system and is widely used for memory impairment models for testing AD drugs[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, scopolamine was used to investigate the possible effects of MFGM and OPN on the improvement of learning and memory impairment in a mouse model[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Morris water maze test was conducted and confirmed successful establishment of the model. Compared with the model and control groups, the mice that received the test substances exhibited significant increases in the number of platform crossings during the water maze test. This indicated that the OPN and MFGM combination significantly improved the impaired learning and memory of mice with scopolamine-induced dementia.\u003c/p\u003e\u003cp\u003eACh is a neurotransmitter of the central cholinergic nervous system that plays an crucial role in learning and memory processes of the hippocampus[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. AChE is a key enzyme responsible for ACh metabolism and is a catabolic enzyme that regulates ACh levels in brain tissue. Thus, AChE activity can be used an as indicator of the effects of the test substances on improving cholinergic nerve functions[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Our study showed that the AChE activity in the hippocampus of mice in the high-dose group was significantly reduced following intervention with OPN plus MFGM, suggesting that the OPN and MFGM combination may regulate cholinergic nerve function by modulating AChE activity, thereby improving learning and memory function.\u003c/p\u003e\u003cp\u003eLearning and memory impairment is a common symptom of many neurological diseases. Nerve tissues have a unique architecture that renders them more susceptible to oxygen free radical-mediated damages. Moreover, the large number of free radicals produced during cell metabolism can damage large molecules, such as DNA, which accelerates neuronal death and results in neurodegenerative diseases[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Studies have shown reduced free radical-scavenging ability or antioxidant capacity in patients with learning and memory impairment, which results in excessive amount of oxidative stress factors that causes neurotoxicity[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Under normal physiological state, free radicals are continuously produced, yet eliminated through the body\u0026rsquo;s free radical scavenging system, thus maintaining a dynamic prooxidant-antioxidant balance in the body. Under disease states, such as degeneration of central cholinergic neurons, the activities of antioxidant enzymes in the body continuously decline. The oxygen free radicals in the body cannot be eliminated in time, eventually leading to abnormal accumulation of free radicals[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSOD is an important enzyme that is widely present in organisms. It is involved in maintaining the dynamic balance between free radical production and scavenging in the body. It catalyzes the disproportionation of the free radical superoxide anion to yield hydrogen peroxide, which helps the body to combat against the cytotoxic effects of oxygen free radicals and other oxides. Thus, SOD is a crucial antioxidant enzyme for scavenging free radicals in the body[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. GSH-Px is an important enzyme for peroxide decomposition that is widely present in the body. It catalyzes reduced glutathione into oxidized glutathione and reduces harmful peroxides to nontoxic hydroxyl compounds, thereby protecting the structure and function of cell membranes[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. MDA is a key product of lipid peroxidation, and its abnormal increase in the body is linked to memory impairment[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This study found that high-dose OPN plus MFGM significantly increased SOD and GSH-Px activities and significantly reduced MDA levels in the hippocampus and serum of the model mice. Our findings suggested that the OPN and MFGM combination potentially improved learning and memory function through combating oxidative damage, which warrants further investigations. This research lays the foundation for the application of functional proteins.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study revealed that osteopontin (OPN) combined with milk fat globule membrane (MFGM) proteins mitigated scopolamine-induced cognitive impairment in mice. Intermediate- and high-dose OPN\u0026thinsp;+\u0026thinsp;MFGM enhanced Morris water maze performance (reduced latency, increased platform crossings), suppressed acetylcholinesterase activity, elevated antioxidant enzymes (SOD, GSH-Px), and lowered lipid peroxidation (MDA). The synergy between OPN and MFGM highlights their dual-action mechanism targeting cholinergic and oxidative pathways, suggesting therapeutic potential for neurodegenerative diseases like Alzheimer\u0026rsquo;s. These findings advocate milk-derived components as promising candidates for cognitive-enhancing interventions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eXiaochen Liu contributed to the experimental design and writing of the manuscript, performed the experiments and analyzed the data. Bin Guo provided the experimental method. Da xi Ren\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efunded the project. Bin Guo and Da xi Ren supervised the project. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study was supported by Guangxi Science and Technology Base and Talent Program (2024AC38038).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability \u0026nbsp;\u003c/strong\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u0026nbsp;\u003c/strong\u003eAll studies received ethical approval from the Ethics Committee of Jilin University (Changchun, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLiguori C, Pierantozzi M, Chiaravalloti A, Sancesario GM, Mercuri NB, Franchini F, Schillaci O, Sancesario G: When Cognitive Decline and Depression Coexist in the Elderly: CSF Biomarkers Analysis Can Differentiate Alzheimer\u0026apos;s Disease from Late-Life Depression. \u003cem\u003eFront Aging Neurosci \u003c/em\u003e2018, 10:38.\u003c/li\u003e\n\u003cli\u003eScheltens P, Blennow K, Breteler MM, de Strooper B, Frisoni GB, Salloway S, Van der Flier WM: Alzheimer\u0026apos;s disease. \u003cem\u003eLancet \u003c/em\u003e2016, 388:505-517.\u003c/li\u003e\n\u003cli\u003eAlzheimer\u0026apos;s disease facts and figures. \u003cem\u003eAlzheimers Dement \u003c/em\u003e2020.\u003c/li\u003e\n\u003cli\u003eJia J, Wei C, Chen S, Li F, Tang Y, Qin W, Zhao L, Jin H, Xu H, Wang F, et al: The cost of Alzheimer\u0026apos;s disease in China and re-estimation of costs worldwide. \u003cem\u003eAlzheimers Dement \u003c/em\u003e2018, 14:483-491.\u003c/li\u003e\n\u003cli\u003eVillemagne VL, Burnham S, Bourgeat P, Brown B, Ellis KA, Salvado O, Szoeke C, Macaulay SL, Martins R, Maruff P, et al: Amyloid \u0026beta; deposition, neurodegeneration, and cognitive decline in sporadic Alzheimer\u0026apos;s disease: a prospective cohort study. \u003cem\u003eLancet Neurol \u003c/em\u003e2013, 12:357-367.\u003c/li\u003e\n\u003cli\u003eReiman EM, Quiroz YT, Fleisher AS, Chen K, Velez-Pardo C, Jimenez-Del-Rio M, Fagan AM, Shah AR, Alvarez S, Arbelaez A, et al: Brain imaging and fluid biomarker analysis in young adults at genetic risk for autosomal dominant Alzheimer\u0026apos;s disease in the presenilin 1 E280A kindred: a case-control study. \u003cem\u003eLancet Neurol \u003c/em\u003e2012, 11:1048-1056.\u003c/li\u003e\n\u003cli\u003eJack CR, Jr., Lowe VJ, Weigand SD, Wiste HJ, Senjem ML, Knopman DS, Shiung MM, Gunter JL, Boeve BF, Kemp BJ, et al: Serial PIB and MRI in normal, mild cognitive impairment and Alzheimer\u0026apos;s disease: implications for sequence of pathological events in Alzheimer\u0026apos;s disease. \u003cem\u003eBrain \u003c/em\u003e2009, 132:1355-1365.\u003c/li\u003e\n\u003cli\u003eBateman RJ, Xiong C, Benzinger TL, Fagan AM, Goate A, Fox NC, Marcus DS, Cairns NJ, Xie X, Blazey TM, et al: Clinical and biomarker changes in dominantly inherited Alzheimer\u0026apos;s disease. \u003cem\u003eN Engl J Med \u003c/em\u003e2012, 367:795-804.\u003c/li\u003e\n\u003cli\u003eGordon BA, Blazey TM, Su Y, Hari-Raj A, Dincer A, Flores S, Christensen J, McDade E, Wang G, Xiong C, et al: Spatial patterns of neuroimaging biomarker change in individuals from families with autosomal dominant Alzheimer\u0026apos;s disease: a longitudinal study. \u003cem\u003eLancet Neurol \u003c/em\u003e2018, 17:241-250.\u003c/li\u003e\n\u003cli\u003eBraak H, Thal DR, Ghebremedhin E, Del Tredici K: Stages of the pathologic process in Alzheimer disease: age categories from 1 to 100 years. \u003cem\u003eJ Neuropathol Exp Neurol \u003c/em\u003e2011, 70:960-969.\u003c/li\u003e\n\u003cli\u003eSodek J, Ganss B, McKee MD: Osteopontin. \u003cem\u003eCrit Rev Oral Biol Med \u003c/em\u003e2000, 11:279-303.\u003c/li\u003e\n\u003cli\u003eJiang R, L\u0026ouml;nnerdal B: Evaluation of Bioactivities of Bovine Milk Osteopontin Using a Knockout Mouse Model. \u003cem\u003eJ Pediatr Gastroenterol Nutr \u003c/em\u003e2020, 71:125-131.\u003c/li\u003e\n\u003cli\u003eAli F, Wang ZX: Effect of pasteurization on the enzymatic cross-linking of milk proteins by microbial transglutaminase in view of milk fat globule membrane isolation. \u003cem\u003eFood Bioscience \u003c/em\u003e2021:43.\u003c/li\u003e\n\u003cli\u003eTimby N, Domell\u0026ouml;f M, L\u0026ouml;nnerdal B, Hernell O: Supplementation of Infant Formula with Bovine Milk Fat Globule Membranes. \u003cem\u003eAdv Nutr \u003c/em\u003e2017, 8:351-355.\u003c/li\u003e\n\u003cli\u003eBrink LR, L\u0026ouml;nnerdal B: The role of milk fat globule membranes in behavior and cognitive function using a suckling rat pup supplementation model. \u003cem\u003eJ Nutr Biochem \u003c/em\u003e2018, 58:131-137.\u003c/li\u003e\n\u003cli\u003eCahill JF, Baxter MG: Cholinergic and noncholinergic septal neurons modulate strategy selection in spatial learning. \u003cem\u003eEur J Neurosci \u003c/em\u003e2001, 14:1856-1864.\u003c/li\u003e\n\u003cli\u003eJu Y, Tam KY: Pathological mechanisms and therapeutic strategies for Alzheimer\u0026apos;s disease. \u003cem\u003eNeural Regen Res \u003c/em\u003e2022, 17:543-549.\u003c/li\u003e\n\u003cli\u003eYoussef P, Chami B, Lim J, Middleton T, Sutherland GT, Witting PK: Evidence supporting oxidative stress in a moderately affected area of the brain in Alzheimer\u0026apos;s disease. \u003cem\u003eSci Rep \u003c/em\u003e2018, 8:11553.\u003c/li\u003e\n\u003cli\u003eJain NK, Patil CS, Kulkarni SK, Singh A: Modulatory role of cyclooxygenase inhibitors in aging- and scopolamine or lipopolysaccharide-induced cognitive dysfunction in mice. \u003cem\u003eBehav Brain Res \u003c/em\u003e2002, 133:369-376.\u003c/li\u003e\n\u003cli\u003eManral A, Meena P, Saini V, Siraj F, Shalini S, Tiwari M: DADS Analogues Ameliorated the Cognitive Impairments of Alzheimer-Like Rat Model Induced by Scopolamine. \u003cem\u003eNeurotox Res \u003c/em\u003e2016, 30:407-426.\u003c/li\u003e\n\u003cli\u003eGong Z, Huang J, Xu B, Ou Z, Zhang L, Lin X, Ye X, Kong X, Long D, Sun X, et al: Urolithin A attenuates memory impairment and neuroinflammation in APP/PS1 mice. \u003cem\u003eJ Neuroinflammation \u003c/em\u003e2019, 16:62.\u003c/li\u003e\n\u003cli\u003eQi Y, Cheng X, Jing H, Yan T, Xiao F, Wu B, Bi K, Jia Y: Effect of Alpinia oxyphylla-Schisandra chinensis herb pair on inflammation and apoptosis in Alzheimer\u0026apos;s disease mice model. \u003cem\u003eJ Ethnopharmacol \u003c/em\u003e2019, 237:28-38.\u003c/li\u003e\n\u003cli\u003eWeinberger NM, Miasnikov AA, Chen JC: Sensory memory consolidation observed: increased specificity of detail over days. \u003cem\u003eNeurobiol Learn Mem \u003c/em\u003e2009, 91:273-286.\u003c/li\u003e\n\u003cli\u003eDashniani MG, Beseliia GV, Maglakelidze GA, Burdzhanadze MA, Chkhikvishvili N: Effects of the selective lesions of cholinergic septohippocampal neurons on different forms of memory and learning process. \u003cem\u003eGeorgian Med News \u003c/em\u003e2009:81-85.\u003c/li\u003e\n\u003cli\u003eMarkesbery WR, Lovell MA: Damage to lipids, proteins, DNA, and RNA in mild cognitive impairment. \u003cem\u003eArch Neurol \u003c/em\u003e2007, 64:954-956.\u003c/li\u003e\n\u003cli\u003eFan Y, Hu J, Li J, Yang Z, Xin X, Wang J, Ding J, Geng M: Effect of acidic oligosaccharide sugar chain on scopolamine-induced memory impairment in rats and its related mechanisms. \u003cem\u003eNeurosci Lett \u003c/em\u003e2005, 374:222-226.\u003c/li\u003e\n\u003cli\u003eGoverdhan P, Sravanthi A, Mamatha T: Neuroprotective effects of meloxicam and selegiline in scopolamine-induced cognitive impairment and oxidative stress. \u003cem\u003eInt J Alzheimers Dis \u003c/em\u003e2012, 2012:974013.\u003c/li\u003e\n\u003cli\u003eDringen R: Metabolism and functions of glutathione in brain. \u003cem\u003eProg Neurobiol \u003c/em\u003e2000, 62:649-671.\u003c/li\u003e\n\u003cli\u003eHall ED, Bosken JM: Measurement of oxygen radicals and lipid peroxidation in neural tissues. \u003cem\u003eCurr Protoc Neurosci \u003c/em\u003e2009, Chapter 7:Unit 7.17.11-51.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Osteopontin, MFGM membrane proteins, Morris water maze, Cognition improvement","lastPublishedDoi":"10.21203/rs.3.rs-7164889/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7164889/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the effects of osteopontin (OPN) combined with milk fat globule membrane (MFGM) proteins on scopolamine-induced learning and memory impairment in mice. A dementia model was established through intraperitoneal injection of scopolamine, followed by random allocation into seven experimental groups: blank control, model control, OPN alone, MFGM alone, and three combination groups (low-, intermediate-, and high-dose OPN\u0026thinsp;+\u0026thinsp;MFGM). Cognitive performance was evaluated using Morris water maze testing, while biochemical analyses included assessments of hippocampal acetylcholinesterase (AChE) activity, antioxidant enzyme activities (superoxide dismutase [SOD] and glutathione peroxidase [GSH-Px]), and malondialdehyde (MDA) levels in both hippocampal tissue and serum. The intermediate- and high-dose combination groups demonstrated significant cognitive improvements compared to the model group, manifested by reduced escape latency, increased platform crossings, and prolonged target quadrant duration in water maze testing. Biochemical analyses revealed that these combination treatments significantly suppressed AChE activity in hippocampal tissue while enhancing antioxidant capacity through elevated SOD and GSH-Px activities, accompanied by reduced MDA levels in both brain and serum. This study demonstrated that the combination of OPN and MFGM administration improved learning and memory in mice with scopolamine-induced dementia through dose-dependent effects on the central cholinergic nervous and antioxidant systems.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"Effects of osteopontin combined with milk fat globule membrane proteins on scopolamine-induced learning and memory impairment in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-24 11:38:52","doi":"10.21203/rs.3.rs-7164889/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-04T09:23:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-04T02:16:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-29T12:04:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21463248795980385247727804049395780846","date":"2025-07-25T09:56:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83817809331851636140764226134554637622","date":"2025-07-25T09:31:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"203846247367485545664782735034911189097","date":"2025-07-24T12:31:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"176390184275604338208261925686372444783","date":"2025-07-23T14:10:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292192491964219464871357507519031240611","date":"2025-07-22T14:41:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259180855056642480421571296793332123026","date":"2025-07-22T12:47:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-22T12:01:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-21T15:22:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-21T00:58:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Neurochemical Research","date":"2025-07-19T13:46:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7bb99020-4148-4a6f-9e38-bc11ddfeea6b","owner":[],"postedDate":"July 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:10:25+00:00","versionOfRecord":{"articleIdentity":"rs-7164889","link":"https://doi.org/10.1007/s11064-025-04612-7","journal":{"identity":"neurochemical-research","isVorOnly":false,"title":"Neurochemical Research"},"publishedOn":"2025-11-14 15:58:13","publishedOnDateReadable":"November 14th, 2025"},"versionCreatedAt":"2025-07-24 11:38:52","video":"","vorDoi":"10.1007/s11064-025-04612-7","vorDoiUrl":"https://doi.org/10.1007/s11064-025-04612-7","workflowStages":[]},"version":"v1","identity":"rs-7164889","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7164889","identity":"rs-7164889","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}