Long-Term Treatment of Cuban Policosanol Attenuates Abnormal Oxidative Stress and Inflammatory Response via Amyloid Plaques Reduction in 5xFAD Mice

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder resulting in cognitive decline or dementia, the number of patients with AD is continuously increasing. Although a lot of great progress has been made in research and development of AD therapeutics, there is no fundamental cure for this disease yet. This study demonstrated the memory-improving effects of Cuban policosanol (PCO) in 5xFAD mice, which is an animal model of AD. Following 4-months of treatment with PCO in 5xFAD mice, we found that the number of amyloid plaques decreased in the brain compared to the vehicle-treated 5xFAD mice. Long-term PCO treatment in 5xFAD mice resulted in the reduction of gliosis and abnormal inflammatory cytokines level (interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF]-α) in the cortex and hippocampus. Levels of lipid peroxide (4-hydroxynonenal [4-HNE]) and superoxide dismutase (SOD1 and SOD2) levels were also recoverd in the brains of PCO-treated 5xFAD mice. Notably, PCO administration reduced memory deficits in the passive avoidance test, as well as synaptic loss (PSD-95, synaptophysin) in 5xFAD mice. Collectively, we identified the potential effects of PCO as a useful supplement to delay or prevent AD progression by inhibiting the formation of Aβ plaques in the brain.


Introduction
Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with cognitive decline. Recent investigations indicate that the number of dementia patients is projected to increase by more than 131 million worldwide by 2050, together with the cost of dementia [1]. AD has a neuropathological hallmark consisting of extracellular amyloid plaque deposition composed of amyloid beta (Aβ) peptide and intracellular neurofibrillary tangles (NFT) containing hyperphosphorylated tau [2][3][4]. Amyloid plaques and NFT are mainly deposited in the brain, such as the hippocampus, entorhinal cortex, and basal forebrain, which influence learning, memory, and emotional behavior [5]. Eventually, these neuropathologies result in damage and destruction of the synapses that mediate memory and cognition [6]. Although research on the treatment of AD is continuously progressing, currently no clear preventive or therapeutic drugs exist.

Animals
Two-month-old male transgenic 5xFAD (B6SJL) mice and wild-type litter-mate mice as controls were used in this study. The 5xFAD mice express APP [Swedish (K670N/M671L), Florida (I716V), and London (V717I) mutations] and PS1 (M146L/L286V mutations) transgenes. Mice were the progeny of male hemizygous 5xFAD mice selected from existing in-house animals and female wild-type F1 hybrid mice obtained from JAX Laboratories (Bar Harbor, ME, USA). The temperature and humidity of the breeding room were automatically maintained at 22 ± 2 • C and 50 ± 10%, respectively. A 12L:12D photoperiod was provided, and food and water were provided ad libitum during the acclimation period in cages. All the animal experiments were approved by the Institutional Animal Care and Use Committee of the Lee Gil Ya Cancer and Diabetes Institute, Gachon University (LCDI-2018-0145).

Passive Avoidance Test
The passive avoidance test was performed for three continuous days using a passive avoidance apparatus (Gemini Passive Avoidance System; San Diego Instruments, San Diego, CA, USA). It had two adjacent chambers that were bright and dark, and the chambers were connected by a remote operational gate. The bright chamber was illuminated by a 6 W LED light. During the test, the mice were placed in a bright room. On the first day, we allowed the mice to freely explore the chambers and trained them to enter the dark chamber when the gate was open. The next day, the mice were exposed to electric shock on their feet (2 mA for 2 s) after entering the dark chamber. By 24 h after the The scheme of the experiment procedure. After treatment with policosanol by oral injection (5 mg/kg) for 4 months and then performing the behavior test to assess the effect of cognitive behavior. (B) The bodyweight of mice was measured weekly. (C) Policosanol treatment for 4 months increased latency time in the 6 months aged 5xFAD mice. Values are expressed as the mean ± SEM (n = 5~6 per group). ** p < 0.01 vs. WT-V group, # p < 0.05 vs. WT-P, && p < 0.01 vs. 5xFAD-V group. Statistical analysis between the four groups was performed using the one-way ANOVA, followed by Turkey's post hoc test. WT-V: Vehicle-treated wild-type mice; WT-P: Policosanol-treated wild-type mice; 5xFAD-V: Vehicle-treated 5xFAD mice; 5xFAD-P: Policosanol-treated 5xFAD mice.

Passive Avoidance Test
The passive avoidance test was performed for three continuous days using a passive avoidance apparatus (Gemini Passive Avoidance System; San Diego Instruments, San Diego, CA, USA). It had two adjacent chambers that were bright and dark, and the chambers were connected by a remote operational gate. The bright chamber was illuminated by a 6 W LED light. During the test, the mice were placed in a bright room. On the first day, we allowed the mice to freely explore the chambers and trained them to enter the dark chamber when the gate was open. The next day, the mice were exposed to electric shock on their feet (2 mA for 2 s) after entering the dark chamber. By 24 h after the retention test, the time duration from the placement of the animal in the bright chamber until their entrance to the dark chamber was measured as the step-through latency time, which indicates their memory retrieval. All the daily procedures were performed between 9:00 and 14:00.

Tissue Preparation
Under anesthetizing with a mixture of Zoletil (16.7 mg/kg) and Rompun (15.5 mg/kg), the mouse's brain was extracted. The hemisphere of each mouse brain was fixed in 4% paraformaldehyde at 4 • C for 24 h and then was dehydrated in a 30% sucrose solution for 3 days. The dehydrated tissues were frozen in molds filled with optimal cutting temperature compounds (Sakura, Osaka, Japan). After frozen tissues were cut at a thickness of 22 µm using the cryomicrotome (Cryotome, Thermo Electron Corporation, Waltham, MA, USA), tissues were placed in a cryoprotectant solution (ethylene 30% and glycerol 30% in PBS), and stored at 4 • C.

Immunohistochemistry and Quantification
To determine the effects of policosanol on microglial and astrocyte activation in 5xFAD mice, immunohistochemical analysis was performed as previously described [30]. Briefly, cryostat brain sections were washed in PBS-T (0.3% Triton X-100 in PBS), and the slices were blocked in PBS-T containing 0.5% Bovine serum albumin (BSA) and 3% normal goat serum at room temperature for 30 min. Sections were then incubated with primary antibody overnight at 4 • C in PBS-T solution (glial fibrillary acidic protein [GFAP], 1:500, DAKO; Iba1, 1:500, Novus Biologicals, Centennial, CO, USA). The next day, the following incubation with fluorescent secondary antibody for 1 h at room temperature, and 4 ,6diamidino-2-phenylindole (DAPI) was counterstaining. Images were taken using a Zeiss AxioImager (White Plains, NY, USA) Z1 microscope at 100× magnification. To construct complete images of the hippocampus, separate images taken in the CA1, CA3, and dentate gyrus (DG) were combined according to their correlated parts of the images. Once the region of interests (ROIs) were defined, Alexa Fluro 488 (green) or Alexa Fluro 555 (red) signal was used to measure the fluorescence intensity or the percent of a red signal within each ROI (n = 3 per animal).

Amyloid Plaque Staining and Quantification
To stain amyloid plaques, thioflavin S stain was applied to the sections for 10 min at room temperature. Finally, the dyed tissues were mounted on glass slides and imaged for further analysis. Images were taken using a Zeiss AxioImager Z1 microscope at 100× magnification. To construct complete images of the hippocampus, separate images taken in CA1, CA3, and dentate gyrus were combined according to their correlated parts of the images. The number of amyloid plaques in the brain sections was counted manually using the fluorescent signal by thioflavin S in the entorhinal cortex, prefrontal cortex, and hippocampus. We used the Image J software (V1.4.3.67, National Institute of Health, Bethesda, MD, USA) for manual counting of the plaques.

Enzyme-Linked Immunosorbent Assay
Enzyme-linked immunosorbent assays (ELISAs) were used to quantify the levels of Aβ 1-42 , IL-1β, IL-6, and TNF-α. To create the total lysate, the cortical tissues of the mice were weighed and homogenized in RIPA buffer containing a cocktail of protease inhibitors (Roche Science, Mannheim, Germany) and a cocktail of phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Brain tissue homogenates were analyzed using the following ELISA kits: Aβ 1-42 (KHB3544, Invitrogen, Waltham, MA, USA), IL-1β (KET6013; Abbkine, Wuhan, China), IL-6 (KET7009; Abbkine, Wuhan, China), and TNF-α (KET7015; Abbkine), according to the manufacturer's instructions. All the experiments were analyzed in duplicate, and the mean value of the duplicate samples was calculated in all the assays. Quantification of Aβ 1-42 and cytokine levels was performed using a VICTOR X4 Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).

Statistical Analysis
All the data are presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 9.1.0 (221) software (GraphPad Software Inc., San Diego, CA, USA). One-way analysis of variance (ANOVA) was performed using the Turkey test for multiple comparisons among the groups. Statistical significance was set at p < 0.05.

Long-Term Administration of PCO Recovers the Memory Deficit in 5xFAD Mice
After the treatment of 2 months-old 5xFAD mice with PCO for 4 months, we examined the protective effect of PCO on memory deficits ( Figure 1A). The bodyweight of the mice was measured every week following injection; there were no significant differences among the groups ( Figure 1B).
The passive avoidance test was performed to confirm the protective effect on memory loss after the PCO treatment. The step-through latency time of their transition from the bright room to the darkroom was measured on the last day. The 5xFAD-V group (5xFAD-V, 57.00 ± 16.24 s, Figure 1C) showed significantly decreased latency time in the light compartment compared to the wild-type mice (WT-V, 255.20 ± 16.24 s, p < 0.01; WT-P, 216.20 ± 39.97 s, p < 0.05). In addition, the 5xFAD mice treated with PCO (5xFAD-P, 253.60 ± 39.77 s, p < 0.01) significantly increased the latency time compared with the 5xFAD-V group.

PCO Treatment Decreases the Formation of Amyloid Plaques in the Cortex and Hippocampus of 5xFAD Mice
To evaluate the amyloid pathological changes, we conducted two methodologies, Thioflavin-S staining for amyloid plaque load and ELISA analysis for Aβ 1-42 protein levels in the brains of 5xFAD mice. After 4 months of daily oral administration of PCO, we observed that amyloid plaques accumulation diminished in both the cortex and the hippocampus. Interestingly, the administration of PCO (99.40 ± 22.01, p < 0.05) decreased the number of amyloid plaques in the cortical regions of the 5xFAD mice (57.00 ± 16.24, Figure 2A). Quantification of Aβ 1-42 also showed that the amount of Aβ 1-42 of the 5xFAD-P (2.38 ± 0.41 ng/mg, p < 0.01) mice were significantly reduced compared to the 5xFAD-V group (4.81 ± 0.78, Figure 2B). hippocampus. Interestingly, the administration of PCO (99.40 ± 22.01, p < 0.05) decreased the number of amyloid plaques in the cortical regions of the 5xFAD mice (57.00 ± 16.24, Figure 2A). Quantification of Aβ1-42 also showed that the amount of Aβ1-42 of the 5xFAD-P (2.38 ± 0.41 ng/mg, p < 0.01) mice were significantly reduced compared to the 5xFAD-V group (4.81 ± 0.78, Figure 2B). The image and quantification of Thioflavin-S stain targeted the amyloid plaques in the cortex of the 5xFAD mice. The number of plaques significantly decreased in the 5xFAD-P (5 mg/kg) group compared to the 5xFAD-V group. (B) Amyloid beta1-42 in the cortex also decreased in the 5xFAD-P (5 mg/kg) group compared to the 5xFAD-V group. (C) The image and quantification of Thioflavin-S stain targeted the amyloid plaques in the cortex of the 5xFAD mice. The number of plaques was significantly decreased in the 5xFAD-P group compared to the 5xFAD-V group. (D) Amyloid beta1-42 in the hippocampus was also decreased in the 5xFAD-P group compared to the 5xFAD-V group. Values are expressed as the mean ± standard error of the mean (n = 5-6 per group). **** p < 0.0001, $$$ p < 0.001 vs. compared to WT-V or WT-P group, and & p < 0.05, && p < 0.01 vs. 5xFAD-V group. Statistical analysis between the four groups was performed using the one-way analysis of variance, followed by Turkey's post hoc test.
In addition to the cortex, the amount of neuritic plaque in the hippocampus of the 5xFAD-P group (21.40 ± 2.57, p < 0.05) was decreased compared to that in the 5xFAD-V group (38.67 ± 2.57, Figure 2C). In the hippocampus, the Aβ1-42 peptide in the 5xFAD-P group (1.95 ± 0.50, p < 0.01) significantly decreased compared to that in the 5xFAD-V group (3.64 ± 0.20, Figure 2D). These results indicate that the PCO treatment has the effect of not only reducing the number of amyloid plaques but also reducing the protein level of Aβ1-42 that composes the amyloid plaques.

Long-Term Administration of PCO Attenuated Gliosis in the Brain of 5xFAD Mice
Since markedly increased levels of reactive astrocytes and microglia were present near the amyloid plaques in AD, the effects of PCO treatment on gliosis in the brains of the 5xFAD mice were analyzed. First, we performed immunohistochemistry and Western blotting with the GFAP antibody, an astrocyte marker, in the brains of all the mice groups ( Figure 3). GFAP immunofluorescence staining and quantified results demonstrated that the PCO treatment reduced GFAP-reactive astrocytes in the 5xFAD-V mice ( Figure 3A). The intensity ratio of astrocyte was significantly increased in the cortex and hippocampus including the DG, CA1, and CA3 in the 5xFAD-V group (cortex, 6.25 ± 0.84; DG, 2.05 ± 0.17; CA1, 1.92 ± 0.10; CA3, 1.99 ± 0.40; Figure 3A) compared to the WT-V (cortex, 1.00 ± 0.15, p < 0.001; DG, 1.00 ± 0.06, p < 0.01; CA1, 1.00 ± 0.11, p < 0.01; CA3, 1.00 ± 0.02, p < 0.05) or WT-P group (cortex, 0.97 ± 0.21, p < 0.001; DG, 1.01 ± 0.17, p < 0.01; CA1, 0.78 ± 0.12, p <   in the cortex also decreased in the 5xFAD-P (5 mg/kg) group compared to the 5xFAD-V group. (C) The image and quantification of Thioflavin-S stain targeted the amyloid plaques in the cortex of the 5xFAD mice. The number of plaques was significantly decreased in the 5xFAD-P group compared to the 5xFAD-V group. (D) Amyloid beta  in the hippocampus was also decreased in the 5xFAD-P group compared to the 5xFAD-V group. Values are expressed as the mean ± standard error of the mean (n = 5-6 per group). **** p < 0.0001, $$$ p < 0.001 vs. compared to WT-V or WT-P group, and & p < 0.05, && p < 0.01 vs. 5xFAD-V group. Statistical analysis between the four groups was performed using the one-way analysis of variance, followed by Turkey's post hoc test.
In addition to the cortex, the amount of neuritic plaque in the hippocampus of the 5xFAD-P group (21.40 ± 2.57, p < 0.05) was decreased compared to that in the 5xFAD-V group (38.67 ± 2.57, Figure 2C). In the hippocampus, the Aβ 1-42 peptide in the 5xFAD-P group (1.95 ± 0.50, p < 0.01) significantly decreased compared to that in the 5xFAD-V group (3.64 ± 0.20, Figure 2D). These results indicate that the PCO treatment has the effect of not only reducing the number of amyloid plaques but also reducing the protein level of Aβ 1-42 that composes the amyloid plaques.

PCO Has the Antioxidant and Anti-Inflammatory Effect in the Brain of 5xFAD
Next, Western blots were used to determine the oxidative stress and inflammatory changes induced by altered Aβ and gliosis. As shown in Figure 5A, the protein level of 4-HNE in 5xFAD-V mice (1.65 ± 0.10) was significantly increased compared to that in the WT-V mice (1.00 ± 0.11, p < 0.01) or WT-P (1.18 ± 0.08, p < 0.05, Figure 5A) mice. The 5xFAD-P mice (0.93 ± 0.08, p < 0.001) showed a reduced 4-HNE level compared to the 5xFAD-V mice. SOD is the primary defense antioxidant that suppresses or prevents the formation of free radicals or reactive species in cells. Therefore, we investigated the level of SOD in the cortex of mice to confirm the effect of PCO on oxidative stress in the 5xFAD mice. The level of SOD1 in the 5xFAD-V mice (1.89 ± 0.17) increased compared to that in the WT Figure 4. Cuban policosanol (PCO) treatment attenuated induced microgliosis in 5xFAD mice. To observe microglia alteration, (A) image and quantification of Iba1 immunofluorescence stain in the cortex (CX) and hippocampus (DG, CA1, and CA3). The Iba1 expression level in the 5xFAD-P group was significantly decreased compared to the 5xFAD-V group. Western blot was performed in both (B) the cortex and (C) the hippocampus. The policosanol treatment in the 5xFAD attenuated overexpression level of Iba1, which is a marker of microglia, compared to the 5xFAD-V group. Values are expressed as the mean ± SEM (n = 4 per group). ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. WT-V group, ## p < 0.01, and ### p < 0.001 vs. WT-P group, & p < 0.05, and && p < 0.01 vs. 5xFAD-V group. Statistical analysis between four groups was performed using a one-way analysis of variance, followed by Turkey's post hoc test.

PCO Has the Antioxidant and Anti-Inflammatory Effect in the Brain of 5xFAD
Next, Western blots were used to determine the oxidative stress and inflammatory changes induced by altered Aβ and gliosis. As shown in Figure 5A, the protein level of 4-HNE in 5xFAD-V mice (1.65 ± 0.10) was significantly increased compared to that in the WT-V mice (1.00 ± 0.11, p < 0.01) or WT-P (1.18 ± 0.08, p < 0.05, Figure 5A) mice. The 5xFAD-P mice (0.93 ± 0.08, p < 0.001) showed a reduced 4-HNE level compared to the 5xFAD-V mice. SOD is the primary defense antioxidant that suppresses or prevents the formation of free radicals or reactive species in cells. Therefore, we investigated the level of SOD in the cortex of mice to confirm the effect of PCO on oxidative stress in the 5xFAD mice. The level of SOD1 in the 5xFAD-V mice (1.89 ± 0.17) increased compared to that in the WT mice (WT-V, 1.00 ± 0.18, p < 0.01; WT-P, 1.19 ± 0.11, p < 0.05; Figure 5B). The PCO treatment (1.21 ± 0.12, p < 0.05) significantly restored the protein level of SOD1 in the 5xFAD mice compared to that in the WT mice. However, the level of SOD2 protein in the 5xFAD-V (0.53 ± 0.03, Figure 5C) group significantly decreased compared to the WT groups (WT-V, 1.00 ± 0.08, p < 0.01; WT-P, 0.90 ± 0.09, p < 0.05). In the 5xFAD-P group, the protein level of SOD2 (1.21 ± 0.12, p < 0.05) recovered, similar to the WT group. mice (WT-V, 1.00 ± 0.18, p < 0.01; WT-P, 1.19 ± 0.11, p < 0.05; Figure 5B). The PCO treatment (1.21 ± 0.12, p < 0.05) significantly restored the protein level of SOD1 in the 5xFAD mice compared to that in the WT mice. However, the level of SOD2 protein in the 5xFAD-V (0.53 ± 0.03, Figure 5C) group significantly decreased compared to the WT groups (WT-V, 1.00 ± 0.08, p < 0.01; WT-P, 0.90 ± 0.09, p < 0.05). In the 5xFAD-P group, the protein level of SOD2 (1.21 ± 0.12, p < 0.05) recovered, similar to the WT group. Values are expressed as the mean ± SEM (n = 4-5 per group). ** p < 0.01, and *** p < 0.001 vs. WT-V group, # p < 0.05, and ### p < 0.001 vs. WT-P group, & p < 0.05, and &&& p < 0.001 vs. 5xFAD-V group. Statistical analysis between the four groups was performed using the one-way analysis of variance, followed by Turkey's post hoc test.
To investigate the effect of PCO treatment on the immune response, we measured the protein expression levels of pro-inflammatory cytokines such as IL-1β, IL-6, and TNFα. In the cortex of the 6-month aged 5xFAD-V mice, IL-1β was significantly increased compared to the control group (11.32 ± 3.574 pg/mL, p < 0.0001, WT-V; 23.13 ± 4.987 pg/mL, p < 0.001, WT-P; Figure 6A). However, the PCO treatment in the 5xFAD mice (32.55 ± 5.527 pg/mL, p < 0.01) decreased the expression level of IL-1β compared to the 5xFAD-V mice. , and iNOS (D) in the 5xFAD-P group recovered compared to the 5xFAD-V group. Values are expressed as the mean ± SEM (n = 4-5 per group). ** p < 0.01, and *** p < 0.001 vs. WT-V group, # p < 0.05, and ### p < 0.001 vs. WT-P group, & p < 0.05, and &&& p < 0.001 vs. 5xFAD-V group. Statistical analysis between the four groups was performed using the one-way analysis of variance, followed by Turkey's post hoc test.

PCO Treatment Attenuates Synaptic loss in 5xFAD Mice
In the 5xFAD mice, synaptic loss, including the presynapse and the post-synapse, was observed in the 6-month mice [31]. Therefore, we assessed the presynaptic marker synaptophysin, and postsynaptic marker PSD-95 in the cortex of the mice. The expression level of the synaptophysin in the brain of the 5xFAD-V mice (0.659 ± 0.035) was significantly decreased compared to that in the wild-type mice (WT-V, 1.00 ± 0.08, p < 0.05; WT-P, 0.97 ± 0.07, p < 0.05; Figure 7A). Interestingly, the protein expression level of synaptophysin in the 5xFAD-P group (1.01 ± 0.097, p < 0.05) recovered compared to that in the 5xFAD-V group. In addition, we also confirmed PSD-95, a postsynaptic marker, in the cortex of mice. The protein level of PSD-95 in the 5xFAD-V group (0.499 ± 0.057) was significantly lower than that of the control group (WT-V, 1.00 ± 0.049, p < 0.05; WT-P, 0.967 ± 0.117, p < 0.05; Figure 7B). In the PCO treatment of the 5xFAD mice (1.07 ± 0.14, p < 0.01), the PSD-95 expression level recovered similar to that in the wild-type group. Figure 6. The policosanol treatment appeared as an anti-inflammatory effect. In the 5xFAD-P group, the level of proinflammatory cytokines such as IL-1β (A), IL-6 (B), and TNF-α (C) was significantly decreased compared to the 5xFAD-V group. Values are expressed as the mean ± SEM (n = 5-6 per group). ** p < 0.01, and **** p < 0.0001 vs. WT-V group, ## p < 0.01, and ### p < 0.001 vs. WT-P group, & p < 0.05, && p < 0.01, and &&& p < 0.001 vs. 5xFAD-V group. Statistical analysis was performed using the one-way ANOVA, followed by Turkey's post hoc test.

PCO Treatment Attenuates Synaptic loss in 5xFAD Mice
In the 5xFAD mice, synaptic loss, including the presynapse and the post-synapse, was observed in the 6-month mice [31]. Therefore, we assessed the presynaptic marker synaptophysin, and postsynaptic marker PSD-95 in the cortex of the mice. The expression level of the synaptophysin in the brain of the 5xFAD-V mice (0.659 ± 0.035) was significantly decreased compared to that in the wild-type mice (WT-V, 1.00 ± 0.08, p < 0.05; WT-P, 0.97 ± 0.07, p < 0.05; Figure 7A). Interestingly, the protein expression level of synaptophysin in the 5xFAD-P group (1.01 ± 0.097, p < 0.05) recovered compared to that in the 5xFAD-V group. In addition, we also confirmed PSD-95, a postsynaptic marker, in the cortex of mice. The protein level of PSD-95 in the 5xFAD-V group (0.499 ± 0.057) was significantly lower than that of the control group (WT-V, 1.00 ± 0.049, p < 0.05; WT-P, 0.967 ± 0.117, p < 0.05; Figure 7B). In the PCO treatment of the 5xFAD mice (1.07 ± 0.14, p < 0.01), the PSD-95 expression level recovered similar to that in the wild-type group.

Discussion
This study demonstrates that long-term treatment with PCO has a protective effect on AD progression. In detail, we showed that the PCO treatment of 5xFAD mice for 4months attenuated memory impairment, amyloid plaque formation, and gliosis, Figure 7. The policosanol treatment inhibited the synaptic loss in the hippocampus of the 5xFAD mice. (A) Synaptophysin, which is a presynaptic marker, and (B) PSD-95, which is a postsynaptic marker were recovered by policosanol administration in the 5xFAD-P group. Values are expressed as the mean ± standard error of the mean (n = 4 per group). * p < 0.05 vs. WT-V group, # p < 0.05 vs. WT-P group, & p < 0.05, and && p < 0.01 vs. 5xFAD-V group. Statistical analysis between the four groups was performed using the one-way analysis of variance, followed by Turkey's post hoc test.

Discussion
This study demonstrates that long-term treatment with PCO has a protective effect on AD progression. In detail, we showed that the PCO treatment of 5xFAD mice for 4-months attenuated memory impairment, amyloid plaque formation, and gliosis, including the astrocytes and microglia, in the brain. Together with these results, we demonstrated that PCO consumption reduced lipid peroxidation and aberrant SOD proteins, thereby reducing the inflammatory response and eventually normalizing synaptic loss in the 5xFAD mice.
PCO derived from the wax component of plants is a long-chain aliphatic alcohol compound comprising eight kinds of sugar cane originating from aliphatic alcohols [26]. Previous studies have reported that PCO intake improves abnormal lipoprotein levels or cardiovascular disease [26,32]. It also showed enhancement of HDL-C functionality, reduction of total cholesterol [31], and LDL-cholesterol (LDL-C) in human blood samples [28,33]. These improvements imply that the PCO is a potent natural antioxidant [34]. In addition, PCO treatment has been reported to be effective in ameliorating neuronal damage or plasma oxidative stress in a stroke animal model [35]. Dyslipidemia is a common risk factor associated with degenerative brain diseases such as AD [36,37]. For example, several studies have suggested that a circulating high level of LDL-C is associated with cognitive decline or early-onset AD [38,39]. Furthermore, high HDL-C levels are associated with a decreased risk of AD in elderly individuals [40]. Although the specific mechanism has not been fully elucidated, apolipoprotein A-I (apoA-I), which is the most abundant lipoprotein in HDL, and discoidal HDL, have a protective effect in AD pathology, including amelioration of Aβ deposition and memory reduction as well as the antioxidant effect in preclinical studies [41][42][43][44]. ApoA-I and discoidal HDL was known to delay Aβ fibrillization in the brain through access via the blood-cerebrospinal fluid barrier at the choroid plexus [45]. It is well known that apoA-I and HDL can inhibit amyloid plaque aggregation and remove Aβ from the brain [45,46]. Moreover, in our study, PCO administration inhibited the formation of Aβ plaques, delaying AD pathology progression in 5xFAD mice.
Early-onset AD is mainly familial and is caused by mutations in the gene encoding APP, PS-1 (PSEN1), and PS-2 (PSEN2) [1]. Since 5xFAD mice include five familial AD (FAD) mutations consisting of amyloid protein precursor (APP) and presenilin-1 (PSEN1), they showed Aβ plaques and cognitive impairment in the early stage [47]. In our results, we confirmed the increased Aβ plaques and high protein levels of Aβ 1-42 in the 5xFAD mice of 6-months ( Figure 2). In the early stage of AD, amyloid plaques appear, and the reactivation or activation of glial cells, such as astrocytes and microglia, are located near the plaques [12]. Astrocytes and microglia play a major role in the Aβ clearance and degradation through the production of proteases capable of hydrolyzing Aβ at different cleavage sites or accelerating Aβ exit to the blood circulation [12,48]. Gliosis following Aβ plaque stimulation releases a variety of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α [49], resulting in a persistent inflammatory response [11]. However, Aβ exposure induces astrocytic cell death as well as neuronal cell death through upregulation of the inflammatory cytokines and nitric oxide release [50].
Activated microglia are also associated with the acceleration of neuronal dysfunction after the production of neurotoxic cytokines, chemokines, and reactive oxygen and nitrogen species [51,52]. The treatment agent which affects neuroinflammation has been focused as a new pharmacological tool for neurodegenerative disease [53]. In our results, the PCO treatment attenuated the abnormal astrocytes and microglia in the brain of the mice, as well as the inflammatory response (Figures 3, 4 and 6). Long-term PCO treatment considerably inhibited the formation of amyloid plaques composed of Aβ 1-42 , thereby suppressing gliosis and restoring pro-inflammatory cytokines.
The formation of amyloid plaques composed of amyloid-beta (Aβ) peptides is initiated and enhanced by oxidative stress and an imbalance between the oxidants and antioxidants [54]. Previous studies demonstrated that, in patients with AD, oxidative stress occurred in the brain region of abundant Aβ  , which led to lipid peroxidation [55]. Moreover, a higher concentration of iron (Fe) is found in the brains of patients with AD, such as the hippocampus and the parietal cortex [56], which results in damage to the neuronal cell membrane and cell death via lipid peroxidation [57]. Lipid peroxidation is a major target of neurodegenerative diseases since the brain, which consumes nearly 20% of inspired oxygen and comprises high levels of polyunsaturated fatty acids (PUFAs), is very susceptible to oxidative stress [58]. In particular, the attack of free radicals on the PUFAs in the membrane phospholipids results in the formation of reactive aldehydes such as 4-HNE and malondialdehyde [59]. Although the normal brain is protected from oxidative stress by antioxidant or free radical scavenging, impairment of these mechanisms results in excessive lipid peroxidation [60,61], and DNA oxidation [62], and protein oxidation [63] in the brain of a patient with AD. Numerous studies have demonstrated that the 4-HNE level, a lipid peroxidation marker, was increased in patients and animal models of AD [64][65][66]. The 4-HNE is highly neurotoxic, affects the histological alteration in the AD brain, and correlates with increased neuronal apoptosis [64,67]. The accumulation of free radical damage alters the activity or expression of antioxidants, including SOD or catalase, in the brains of patients with AD [68,69]. Although it is controversial for SOD expression levels in the AD brain, some research has shown that expression of SOD1 (CuZn-SOD), which is localized in the cytosol, was increased in clinical and preclinical studies. [23,70,71]. In contrast, SOD2 (Mn-SOD), which is the main superoxide scavenger in the mitochondria, exacerbates oxidative stress and the pathology of AD [72,73]. After 4-months of PCO treatment, excessive lipid peroxide (4-HNE) with abnormal SOD1 and SOD2 in the 5xFAD mice recovered ( Figure 5), PCO is considered to have inhibited the oxidative damage induced by amyloid plaques. Prolonged oxidative stress induced by Aβ plaques is known to result in synaptic loss associated with memory impairment [74]. However, long-term administration of PCO suppressed the formation of amyloid plaques, resulting in anti-inflammatory and anti-oxidative effects. Consequently, it was confirmed to restore damaged synaptic proteins such as synaptophysin and PSD-95 ( Figure 7) and memory impairment (Figure 1) in policosanol-treated 5xFAD mice.
This study had certain limitations. First, we evaluated the protective efficiency of PCO in the 5xFAD male mice. Since there is evidence of sex-specific patterns and sex differences in AD [75], the PCO effects in female mice need to be confirmed. Together with previous studies that examined the effect of PCO on ameliorating dyslipidemia, we confirmed the inhibition of amyloid plaque formation. Second, following the blood-brain barrier, studies on the clear mechanism of PCO action on the regulation between apoA-I and amyloid pathology should be performed. Finally, based on the report on the increase of HDL-C and improvement of HDL function with PCO treatment, we need to determine the protective efficacy of PCO against vascular dementia, the second most common cause of dementia, which could be prevented by upregulation of HDL-C level [40,45].
Taken together, there is a possibility that the treatment with PCO causes elevation of HDL-C and enhancement of HDL functionality to induce the reduction of oxidized species (4-HNE and iNOS), inflammatory cytokines (IL-1β, IL-6, and TNF-α), and amyloid plaque size. The current results could explain the relationship between higher serum HDL-C in middle age and the lower incidence of dementia in late age in a previous human study [76].
In summary, we demonstrated that PCO has antioxidant and anti-inflammatory effects by inhibiting the formation of Aβ plaques in the brain, and could be an effective supplement in delaying or preventing AD progression.