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D-12 Long-Term Treatment of Cuban Policosanol Attenuates Abnormal Oxidative Stress

and Inflammatory Response via Amyloid Plaques Reduction in 5xFAD Mice

Jin-Ho Kim , Dong-Kyun Lim , Yoo-Hun Suh 2,3 and Keun-A Chang 1,2,4,*
2
1

1 Department of Health Sciences and Technology, Gachon Advanced Insiue for Healh Sciences & Technology, Gachon University, Incheon 21999, Korea

2 Neuroscience Research Institute, Gachon University, Incheon 21565, Korea

3 Department of Pharmacology, College of Medicine, Seoul National University, Seoul 03080, Korea

4 Department of Pharmacology, College of Medicine, Gachon University, Incheon 21999, Korea

Antioxidants, volume 10, Issue 8, 2021

Background & Purpose Methods

Alzheimer’s disease (AD) is a progressive neurodegenerative disease associated with cognitive decline. Recent investigations indicate that the • Animals : 5xFAD • Policosanol

number of dementia patients is projected to increase by more than 131 million worldwide by 2050, together with the cost of dementia. AD has a 1mo 3mo 5mo 7mo 9mo 12mo 15mo 18mo+ Absent Unknown

neuropathological hallmark consisting of extracellular amyloid plaque deposition composed of amyloid beta (Aβ) peptide and intracellular

neurofibrillary tangles (NFT) containing hyperphosphorylated tau. Amyloid plaques and NFT are mainly deposited in the brain, such as the 1.5mo 2mo

hippocampus, entorhinal cortex, and basal forebrain, which influence learning, memory, and emotional behavior. Eventually, these

neuropathologies result in damage and destruction of the synapses that mediate memory and cognition. Although research on the treatment of AD Changes in Neuronal Synaptic
Cognitive
is continuously progressing, currently no clear preventive or therapeutic drugs exist. Plaque Gliosis impairment LTP/LTD Loss Loss Tangle

Cuban policosanol (PCO) is a mixture of alcohols separated and purified from sugar cane (Saccharum officinarum L). Especially, PCO reported Two-month-old male transgenic 5xFAD (B6SJL) mice and wild-type litter-mate mice as

safety as well as the effect on dyslipidemia in the meta-analysis. A previous report suggested that PCO consumption by patients with type II controls were used in this study. The 5xFAD mice express APP [Swedish (K670N/M671L),

hypercholesterolemia, type 2 diabetics with hypercholesterolemia, and combined hypercholesterolemia improved the lipid-related risk factors Florida (I716V), and London (V717I) mutations] and PS1 (M146L/L286V mutations)

such as low-density lipoprotein (LDL) oxidation in cardiovascular disease. Moreover, in a human study in Korea, short-term (12 weeks) and transgenes.

long-term (24 weeks) consumption of PCO resulted in decreased blood pressure and serum total cholesterol with the elevation of high-density • Behavior test : passive avoidance test 1-tetracosanol (C24H49OH, 0.1–20 mg/g), 1-nonacosanol
(C29H59OH, 1.0–20.0 mg/g), 1-heptacosanol (C27H55OH, 1.0–30.0
lipoprotein-cholesterol (HDL-C) in a dose dependent manner. In addition to its lipid-lowering effect, PCO showed antioxidant and anti-glycation mg/g), 1-tetratriacontanol (C34H69OH, 1.0–50.0 mg/g), 1-

effects to enhance HDL functionality. Since abnormalities in lipid metabolism are important risk factors in AD pathology, PCO treatment may be hexacosanol (C26H53OH, 30.0–100.0 mg/g), 1-dotriacontanol

an effective substrate in the management of AD. In the current study, we aimed to analyze the effects of long-term policosanol consumption on (C32H65OH, 50.0–100.0 mg/g), 1-triacontanol (C30H61OH, 100.0–

memory impairment in 5xFAD mice, which is an animal model of AD. 150.0), and 1-octacosanol (C28H57OH, 600.0–700.0 mg/g).
nd
1 day (Adaptation) 2 nd day (Acquisition Trial) 3 day (Retention Trial)
st
Results

Figure 1. The policosanol effect on cognitive dysfunction and pathophysiology of Alzheimer’s Figure 5. The policosanol showed an antioxidant effect on oxidative stress. In the Western

disease in 5xFAD mice. (A) The scheme of the experiment procedure. After treatment with blot, (A) the overexpression of 4-hydroxynonenal (4-HNE), which is a marker of oxidative

policosanol by oral injection (5 mg/kg) for 4 months and then performing the behavior test to stress, in the 5xFAD group was significantly attenuated by policosanol treatment. (B–D)

assess the effect of cognitive behavior. (B) The bodyweight of mice was measured weekly. (C) Abnormal expressions of SOD1 (B), SOD2 (C), and iNOS (D) in the 5xFAD-P group

Policosanol treatment for 4 months increased latency time in the 6 months aged 5xFAD mice. recovered compared to the 5xFAD-V group. Values are expressed as the mean ± SEM (n

Values are expressed as the mean ± SEM (n = 5~6 per group). ** p < 0.01 vs. WT-V group, # p Figure 3. Cuban policosanol (PCO) treatment attenuated induced astrogliosis in the 5xFAD = 4–5 per group). ** p < 0.01, and *** p < 0.001 vs. WT-V group, # p < 0.05, and ### p

< 0.05 vs. WT-P, && p < 0.01 vs. 5xFAD-V group. Statistical analysis between the four groups mice. (A) Image and quantification of glial fibrillary acidic protein (GFAP) immunofluorescence < 0.001 vs. WT-P group, & p < 0.05, and &&& p < 0.001 vs. 5xFAD-V group. Statistical

was performed using the one-way ANOVA, followed by Turkey’s post hoc test. WT-V: Vehicle- stain in the cortex and hippocampus (DG, CA1,and CA3). The GFAP expression level in the analysis between the four groups was performed using the one-way analysis of variance,

treated wild-type mice; WT-P: Policosanol-treated wild-type mice; 5xFAD-V: Vehicle-treated 5xFAD-P group significantly decreased compared to the 5xFAD-V group. After brain collection followed by Turkey’s post hoc test.

5xFAD mice; 5xFAD-P: Policosanol-treated 5xFAD mice. in the 6-months aged mice, Western blot was performed to observe the changes in the astrocytes.
In both (B) the cortex and (C) hippocampus, the expression level of GFAP, which is a marker of
astrocyte, in the 5xFAD treated policosanol group was reduced compared to the 5xFAD-V group.

Values are expressed as the mean ± standard error of the mean (n = 3 per group). * p < 0.05, **
p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. WT-V group, # p < 0.05, ## p <0.01, and ### p

< 0.001 vs. WT-P group, & p < 0.05, && p < 0.01, and &&&& p < 0.0001 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.

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.

Figure 2. Policosanol treatment attenuated the accumulation of amyloid plaques. (A) 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 Figure 4. Cuban policosanol (PCO) treatment attenuated induced microgliosis in 5xFAD mice. To Figure 7. The policosanol treatment inhibited the synaptic loss in the hippocampus of the

of the 5xFAD mice. The number of plaques was significantly decreased in the 5xFAD-P observe microglia alteration, (A) image and quantification of Iba1 immunofluorescence stain in the 5xFAD mice. (A) Synaptophysin, which is a presynaptic marker, and (B) PSD-95, which is a

group compared to the 5xFAD-V group. (D) Amyloid beta1–42 in the hippocampus was cortex (CX) and hippocampus (DG, CA1, and CA3). The Iba1 expression level in the 5xFAD-P postsynaptic marker were recovered by policosanol administration in the 5xFAD-P group.

also decreased in the 5xFAD-P group compared to the 5xFAD-V group. Values are group was significantly decreased compared to the 5xFAD-V group. Western blot was performed in Values are expressed as the mean ± standard error of the mean (n = 4 per group). * p < 0.05

expressed as the mean ± standard error of the mean (n = 5–6 per group). **** p < 0.0001, both (B) the cortex and (C) the hippocampus. The policosanol treatment in the 5xFAD attenuated vs. WT-V group, # p < 0.05 vs. WT-P group, & p < 0.05, and && p < 0.01 vs. 5xFAD-V

$$$ p < 0.001 vs. compared to WT-V or WT-P group, and & p < 0.05, && p < 0.01 vs. overexpression level of Iba1, which is a marker of microglia, compared to the 5xFAD-V group. group. Statistical analysis between the four groups was performed using the one-way analysis

5xFAD-V group. Statistical analysis between the four groups was performed using the Values are expressed as the mean ± SEM (n = 4 per group). ** p < 0.01, *** p < 0.001, and **** p of variance, followed by Turkey’s post hoc test.

one-way analysis of variance, followed by Turkey’s post hoc test. < 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.

Conclusion References

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This study demonstrates that long-term treatment with PCO has a protective effect on AD progression. In detail, we showed that the PCO doi:10.1101/cshperspect.a006189.

treatment of 5xFAD mice for 4-months attenuated memory impairment, amyloid plaque formation, and gliosis, including the astrocytes and 2. Haque, R.U.; Levey, A.I. Alzheimer’s disease: A clinical perspective and future nonhuman primate research opportunities. Proc. Natl. Acad. Sci. USA 2019, 116, 26224–26229,

microglia, in the brain. Together with these results, we demonstrated that PCO consumption reduced lipid peroxidation and aberrant SOD doi:10.1073/pnas.1912954116.

proteins, thereby reducing the inflammatory response and eventually normalizing synaptic loss in the 5xFAD mice. 3. Campora, M.; Francesconi, V.; Schenone, S.; Tasso, B.; Tonelli, M. Journey on Naphthoquinone and Anthraquinone Derivatives: New Insights in Alzheimer’s Disease.
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Acknowledgments

Funding: This study was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) & Institutional Review Board Statement: All the animal experiments were approved by the Institutional Animal Care and Use Committee of

funded by the Korean government (MSIT) (2020M3A9E4104384). The funders had no role in study design, data collection, and analysis, the Lee Gil Ya Cancer and Diabetes Institute, Gachon University (LCDI-2018-0145: 2019-01-03).
decision to publish, or preparation of the manuscript.