Magnolol (analytical standard, purity ≥98%) was provided by Shanghai Yuanye Biotechnology Co., Ltd. Congo red dye and Aβ_1-42 were acquired from MilliporeSigma, while the hematoxylin dye was supplied by Beyotime Institute of Biotechnology. Goat serum was obtained from Gibco; Thermo Fisher Scientific, Inc. RIPA lysis buffer, bovine serum albumin, ECL Kit and BCA Protein Assay Kit were obtained from Thermo Fisher Scientific, Inc. The QIAwave RNA Mini Kit, First Strand Kit and QuantiFast SYBR^® Green PCR Kit were obtained from Qiagen GmbH. The primary antibodies used in the present study were IL-6 (Abcam; cat. no. ab290735), IL-1β (Abcam; cat. no. ab315084), tau (Abcam; cat. no. ab254256), phosphorylated (p-)tau (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MN1020), GSK3β (Abcam; cat. no. ab32391), p-GSK3β (Abcam; cat. no. ab75814), synaptophysin (Proteintech Group, Inc.; cat. no. 17785-1-AP), BDNF (Proteintech; cat. no. 28205-1-AP), glial fibrillary acidic protein (GFAP; Proteintech Group, Inc.; cat. no. 16825-1-AP), ionized calcium binding adaptor molecule 1 (Iba1; Proteintech Group, Inc.; cat. no. 10904-1-AP) and GAPDH (Proteintech Group, Inc.; cat. no. 60004-1-Ig). The HRP-conjugated anti-rabbit (cat. no. A21020) and HRP-conjugated anti-mouse (cat. no. A21010) secondary antibodies were obtained from Abbkine Scientific Co., Ltd. FITC-labeled goat anti-mouse secondary antibody (cat. no. ab6785) was obtained from Abcam.

Healthy male C57BL/6 mice (age, 10-12 weeks; weight, 25-30 g) with normal locomotor and cognitive functions were purchased from Charles River Laboratories, Inc. Before grouping the mice, visible-platform training was carried out to determine the baseline differences in vision and motivation. Mice with abnormal locomotor abilities were excluded. Subsequently, mice were housed in specific pathogen-free cages under standard laboratory conditions including 22-25˚C temperature, 40-55% relative humidity and 12-h light/dark cycle, with free access to water and food. The Aβ_1-42 stock solution was prepared at a concentration of 1 mg/ml in sterile PBS and then incubated at 37˚C for 5 days to facilitate oligomerization.

A total of 30 C57BL/6 mice were randomly divided into five groups (n=6 mice/group): i) Control; ii) AD model; iii) 5 mg/kg magnolol + AD model; iv) 10 mg/kg magnolol + AD model; and v) 20 mg/kg magnolol + AD model. All surgical procedures were performed under aseptic conditions. The AD model was established as follows: The mice were anesthetized by intraperitoneal (i.p.) injection of pentobarbital sodium (50 mg/kg), and then subsequently secured onto a brain stereotaxic device in a prone position. As previously described (30,31), the hippocampal CA1 region serves a critical role in cognitive functions such as learning and memory, and damage or lesions in this region are closely related to cognitive impairment in AD. Additionally, bilateral injection can simulate the pathological changes of diseases more comprehensively, improving the stability and reliability of the model (32). Therefore, the bilateral hippocampal CA1 region was selected as the targeted injection area. The specific coordinate position was 2.4 mm dorsoventral, 0.2 mm anteroposterior and 1 mm mediolateral to the bregma. The aggregated form of Aβ_1-42 was administered via intracerebroventricular injection at a volume of 3 µl and a speed of 1 µl/min. The injector was left in place for 5 min. Mice in the control group received an equivalent amount of normal saline. To consider animal welfare, animal distress was alleviated as much as possible. A 0.2-ml dose of gentamicin was injected to the suture site for anti-inflammatory treatment when the incision was sutured. Meanwhile, the animals were housed individually and permitted to recuperate from the anesthesia on a heated mat in order to regulate body temperature at 37.5±0.5˚C. After 7 days, the treatment groups were administered varying doses of magnolol (5, 10 and 20 mg/kg) by gavage every day, while mice in the control and AD model groups received an equal volume of normal saline. During the experiments, changes in body weight were monitored once a week, and changes in food and water intake were observed. Although it was not expected, a rapid decrease in normal body weight >20% was defined as a humane endpoint for the present study. In this study, none of the mice reached the humane endpoint.

A Morris water maze (MWM) test was conducted after 2 months of magnolol treatment. No animals died naturally during the experiments, and all mice were euthanized by overdose with pentobarbital sodium (200 mg/kg; i.p.). The death was confirmed by cardiac and respiratory arrest, along with the absence of response to tail clamping. A total of 3 mice were randomly selected from each group, and the hippocampal tissues were collected for Congo red and immunofluorescence staining. The hippocampal tissues of the remaining mice were stored at -80˚C until use. The whole experiment lasted 73 days and a schematic diagram of the experimental protocol is shown in Fig. 1A. The animal experiments were conducted in compliance with the guidelines outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (33) and received approval from The Ethics Committee of The School of Medicine of Jinhua Polytechnic (Jinhua, China; approval no. AL-JSYJ202334).

As previously described (25), the MWM test was applied to evaluate the spatial memory of the mice. A circular pool with a height of 50 cm and a diameter of 150 cm was used. The water was filled to a depth of 30 cm, while maintaining a temperature of 24˚C. The swimming pool was artificially partitioned into four quadrants, designated as quadrant I, II, III and IV. During the 5-day spatial navigation test, a round platform with a diameter of 10 cm was positioned at the center of quadrant IV and submerged 1 cm below the water surface, followed by removing it when the 1-day probe task was conducted. The mice were each subjected to two tests per day, with a minimum interval of 15 min between tests. Each mouse was introduced into the pool and given 60 sec to seek the platform. The time taken to locate the concealed platform was recorded as escape latency (sec). On day 6, the hidden platform was removed to evaluate the memory ability. Subsequently, the mice were permitted to swim freely for 60 sec to seek the removed platform. A video tracking system (version 3.0; SMART, Panlab) was employed to record the distance to target, escape latency and swimming speed.

The hippocampal tissues were collected and promptly fixed in 4% paraformaldehyde (PFA) at 4˚C overnight. Subsequently, the specimens were immersed in a solution of 30% sucrose-PFA until they sank to the bottom. The samples were then embedded in paraffin and flash-frozen using liquid nitrogen. Slices with a thickness of 4 µm were obtained using a cryostat (Leica Microsystems GmbH), stored at -80˚C, and used for Congo red and immunofluorescence staining.

Congo red staining was used for Aβ plaque visualization in the CA1 region of the hippocampus, as previously reported (34,35). The sections underwent deparaffinization and hydration with distilled water, followed by staining in a solution of 0.5% Congo red for 20 min at room temperature. Subsequently, the sections were rinsed in distilled water and rapidly differentiated in an alkaline alcohol solution. The sections were then washed in tap water for 1 min, followed by counterstaining with hematoxylin for 30 sec at room temperature and subsequent rinsing again in tap water for 2 min. Finally, the sections were dehydrated and mounted using resinous mounting medium. For semi-quantification of Congo red staining, images were captured from three hippocampal sections per sample, and Congo red-positive cells were counted manually from the CA1 region of the hippocampus using five random regions, under an optical microscope (scale bar, 50 µm; Olympus Corporation). The number of Aβ plaques was represented by the number of Congo red-positive cells.

The tissue slices underwent deparaffinization in xylene for 10 min at room temperature and rehydration with descending concentrations of ethanol (100, 95 and 70% for 3-5 min each), followed by antigen retrieval in heated sodium citrate at 80˚C for 25 min. Subsequently, the slices were blocked with 4% goat serum for 15 min at room temperature, after which, the slices were incubated overnight at 4˚C with the following primary antibodies: IL-1β, IL-6, p-tau, Iba1 and GFAP (all diluted to 1:50). After three washes with PBS, the slices were incubated with FITC-labeled goat anti-mouse secondary antibody (1:200) for 1 h in the dark at room temperature. DAPI was used for nuclear staining at room temperature for 15 min. For the expression determination, images were captured from three hippocampal sections per sample. The IL-1β-, IL-6-, p-tau-, Iba1- and GFAP-positive cells were evaluated under a fluorescence microscope (Olympus Corporation; scale bar, 100 µm) from five different CA1 regions of hippocampus. Image-Pro-Plus (version 6.0; Media Cybernetics) was adopted to analyze the fluorescence intensity.

Proteins were extracted from hippocampal tissues using RIPA lysis buffer with protease inhibitors, and their concentration was determined using the BCA Protein Assay Kit. A total of ~25 µg proteins were separated by SDS-PAGE on a 10% gel and subsequently transferred to a PVDF membrane. The membrane was then blocked with 5% bovine serum albumin for 2 h at room temperature before being incubated overnight at 4˚C with primary antibodies against tau (1:1,000), p-tau (1:1,000), GSK3β (1:5,000), p-GSK3β (1:5,000), synaptophysin (1:20,000), BDNF (1:1,000), GFAP (1:5,000), Iba1 (1:1,000) and GAPDH (1:50,000). After washing the membranes three times with Tris-buffered saline-Tween-20 (0.05%), the HRP-conjugated secondary antibodies (1:10,000) were added and incubated at room temperature for 1 h. The internal reference used was GAPDH. Finally, the immunoreactive protein bands were visualized using an ECL Kit under a Gel-Proanalyzer (version 4.0; MilliporeSigma).

The QIAwave RNA Mini Kit was employed for RNA isolation. Subsequently, cDNA was synthesized using the First Strand Kit according to the manufacturer's protocol, and subjected to qPCR analysis using a QuantiFast SYBR Green PCR Kit on the Real-time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 95˚C for 10 min, followed by 40 cycles at 94˚C for 10 sec, 60˚C for 20 sec and 72˚C for 34 sec. Gene expression levels were determined using the 2^-ΔΔCq method (36) with GAPDH used as the internal control. The gene primers utilized in this study are detailed in Table I.

The data analysis was conducted using SPSS software version 20.0 (IBM Corp.) and the results are presented as the mean ± standard deviation. Statistical differences among the groups were determined using one-way ANOVA, followed by Tukey's multiple comparisons post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The body weight of mice in different groups was monitored. As shown in Fig. 1B, compared with that in the control group, the body weight of mice with AD was significantly decreased; however, compared with in the AD model group, administration of different doses of magnolol, particularly 20 mg/kg, significantly restored the body weight. The track plots of mice in the MWM test are shown in Fig. 1C. First, the speed of mice swimming in the water maze was assessed to exclude false-positive results. The results revealed that there was no significant difference in the swimming speed of each group of mice (Fig. 1D). Next, it was revealed that the AD model group spent more time to reach the platform compared with the control group in the spatial navigation test (Fig. 1E). However, administration of different doses of magnolol (5, 10 and 20 mg/kg) significantly ameliorated the effects of Aβ_1-42 on escape latency. In the spatial probe test, the memory retention of the platform location performed on the day following the spatial navigation test was assessed. It was shown that compared with the mice in the AD model group, magnolol-treated AD mice showed improved spatial learning and memory ability. This was reflected by significant progressive reductions in distance traveled to the target platform (Fig. 1F).

The accumulation of Aβ plaques is a crucial characteristic feature of AD. Therefore, the hippocampal tissues were subjected to Congo red staining for Aβ plaque deposition detection. Sections with Aβ plaque accumulation displayed a red-colored Congo stain in the CA1 region of the hippocampus. As shown in Fig. 2A, almost no Congo red-positive cells were observed in the control group, indicating that there was almost no accumulation of plaques in the hippocampal CA1 region of the control mice. Hippocampal sections from Aβ_1-42-induced mice in the model group showed more dense Congo red stains, indicating that Aβ plaques had accumulated. Administration of magnolol, specifically at a dosage of 20 mg/kg, notably reduced the number of Congo red-positive cells in hippocampal tissues and indicated that treatment with magnolol significantly inhibited the accumulation of Aβ plaques (Fig. 2B). After activation with plaque accumulation, astrocytes and microglia release a diverse array of pro-inflammatory cytokines that lead to impaired neuronal function in the hippocampus, which is crucial for learning and memory formation (37). Immunofluorescence staining was conducted to evaluate the expression of IL-1β and IL-6 in the hippocampus. It was indicated that relative expression levels of IL-1β and IL-6 were notably enhanced in the model group, which was in contrast to the control group (Fig. 2C and D). However, treatment with varying doses of magnolol significantly decreased IL-1β and IL-6 levels in the hippocampus of Aβ_1-42-induced mice.

The occurrence of AD involves numerous crucial pathological mechanisms, such as the formation of intracellular neurofibrillary tangles, disrupted synaptic plasticity, and the activation of astrocytes and microglia (38-40). Therefore, an investigation was conducted into the expression of pertinent factors. As illustrated in Fig. 3A, in comparison with the control group, the protein levels of tau, p-tau, GFAP and Iba1 were increased in the model group, whereas GSK3β, p-GSK3β, synaptophysin and BDNF levels were decreased. However, treatment with magnolol, specifically at a dosage of 10 or 20 mg/kg, significantly decreased the protein levels of tau, p-tau, GFAP and Iba1, and restored the levels of GSK3β, p-GSK3β, synaptophysin and BDNF in Aβ_1-42-induced mice. RT-qPCR analysis detected the mRNA expression levels of synaptophysin, BDNF, GFAP and Iba1 (Fig. 3B), while immunofluorescence staining assessed p-tau, Iba1 and GFAP expression (Fig. 3C and D), and the results provided further validation for the aforementioned findings.