Protective effect of chlorogenic acid on cognitive impairment in rats with early Alzheimer's disease via Wnt signaling pathway

Abstract

Background:

Chlorogenic acid (CGA) has neuroprotective properties associated with Alzheimer's disease (AD). However, the exact mechanism by which CGA prevents cognitive impairment in AD remains unclear. The purpose of this study was to investigate the protective effect of CGA on cognitive impairment in rats with early AD via the Wnt signaling pathway.

Objective:

To investigate the protective effect of CGA on cognitive impairment in an early AD rat model via the Wnt signaling pathway.

Methods:

Forty male rats were randomly divided into the control group (CON), AD group (AD), CGA 100 groups and CGA 150 groups with 10 rats in each group. In addition to CON group, the other three groups of rats were injected with 10 μL Aβ_25-35 in the unilateral ventricle to create the model. After 3 days of molding, CGA100 group (gavage of CGA at a dose of 150 mg/kg/d) and CGA150 group (gavage of CGA at a dose of 150 mg/kg/d) were given CGA for 6 weeks. Morris water maze test, Nissl staining test, and western blot test were used.

Results:

CGA reduced the escape latency of Aβ_25-35-induced early AD rats, shortened the swimming distance, and extended the activity time of the target quadrant. CGA increased the number of Nissl, decreased the expression of inflammatory factors, decreased the expression levels of GSK-3β, GFAP, and tau, and increased the expression levels of DVL2 and β-catenin.

Conclusions:

CGA can protect the cognitive impairment of early AD rats via Wnt signaling pathway.

Keywords

Alzheimer's disease chlorogenic acid memory impairment Wnt signaling pathway

Introduction

Alzheimer's disease (AD) is a neurodegenerative disease occurring in middle and old age. The main clinical manifestations are progressive memory loss, cognitive impairment, visuospatial and executive dysfunction.¹ The two main pathological changes of AD are amyloid plaques and neurofibrillary tangles, which are mainly caused by amyloid and tau proteins, but their pathogenesis has not been fully clarified,^2,3 With the development of medicine, the survival time of patients with AD has been significantly prolonged, and the prevalence of cognitive impairment in AD has risen rapidly. Therefore, it is practically significant to study the pathogenesis of cognitive impairment in AD and find an effective treatment plan.

Chlorogenic acid (CGA), chemical formula C16H18O9, is a phenolic acid formed by caffeic acid and quinic acid, which is a phenylpropanoid compound produced by the shikimic acid pathway in the process of aerobic respiration in plants. CGA is an effective phenolic antioxidant, which belongs to the extract of honeysuckle and other Chinese medicinal materials. CGA has a many biological activities, including anti-inflammatory, antioxidant, mutation inhibition, and anti-tumor,^4,5 and can reduce the risk of diabetes and cardiovascular diseases. Among them, the anti-inflammatory effect and neural protection of CGA have been extensively studied.⁶ Studies have shown that CGA can inhibit the activation of NF-κB and its signaling pathway, thereby inhibiting neurotoxicity and slowing down the development of AD.⁷ CGA has a neuroprotective effect on transient forebrain ischemia and can reduce cognitive impairment.⁸ Research shows that preventive effect of CGA on cognitive dysfunction of AD in APP/PS2 transgenic mice.⁹ However, the protective effect of CGA on cognitive impairment and its molecular mechanism remains unclear.

The Wnt signaling pathway exists widely invertebrates, among which the classic Wnt/β-catenin signaling pathway not only activates the expression of nuclear target genes but also is closely associated with the occurrence of a variety of neurological diseases.¹⁰ Early studies have shown that Wnt signaling is involved in brain formation, and knockout of the Wnt3a can damage the hippocampal gyrus of mouse embryos, thus affecting embryonic development.¹¹ Studies have proved that when people show symptoms of AD such as memory loss, the Wnt signaling pathway in the body is in a closed state. When drugs are used to inhibit neuroinflammation and oxidative stress, the Wnt signaling pathway may be activated, to achieve the goal of treating AD.¹² Studies have shown that the dysfunction of the Wnt/β-catenin signaling pathway plays an important role in the pathogenesis of AD, and the abnormality of the Wnt pathway is the main cause of abnormal tau phosphorylation and synaptic loss in AD patients.^13,14 However, the protective effect of CGA on cognitive impairment in AD via Wnt signaling is far from fully elucidated.

Therefore, we hypothesized that activation of the Wnt signaling pathway can improve cognitive dysfunction in AD rats. The main purpose of this study is to verify the protective effect of CGA on cognitive impairment in rats with early AD via Wnt signaling pathway, which provides a new idea and new direction for clinical prevention and treatment of AD.

Methods

Animals

Male SD rats were purchased from the Jinzhou Medical University Animal Center weighing 220 g to 240 g, aged 4–5 weeks. The feeding conditions were standard with room temperature 23 ± 2°C, humidity 50 ± 5%, indoor ventilation, and natural lighting. The mice could eat and drink independently. All animal studies were carried out in strict accordance with the laws of the People's Republic of China on the use and care of laboratory animals¹⁵ and the guidelines formulated by the Institute of Laboratory Animals of Jinzhou Medical University.

Materials and experimental design

CGA (content > 99%) (Changsha Xintian Biotechnology Co., China.) Aβ_25-35 (Sigma, USA), antibodies of GSK-3β, β-catenin and DVL2 (Proteintech, USA); GFAP and Tau (Abcam, USA). The water maze system (Zhenghua Biological Corporation, China), and the electrophoresis apparatus (Bio-rad Corporation, USA.) were used in this study.

A total of 40 male SD rats were randomly fed for 7 days and were divided into four groups: Control group (CON group), AD group (AD group), CGA100 group (gavage of CGA at a dose of 100 mg/kg/d) and CGA150 group (gavage of CGA at a dose of 150 mg/kg/d) for with 10 rats in each group. After the rats were anesthetized by intraperitoneal injection of 0.3% pentobarbital sodium (1 mL/100 g), animals were injected with 10 μL Aβ_25-35 in the unilateral ventricle. In addition to CON group, the other three groups of rats were injected with 10 μL Aβ_25-35 in the unilateral ventricle to create the model. One week before modeling, the CGA100 group and CGA150 group CGA gavage were given, except for the control group, 3 days after the completion of molding, the remaining groups continued to administer for 6 weeks. The stereotaxic coordinates for the injection were according to the brain atlas (0.8 mm AP, 1.2 mm ML and 3.7 mm DV, according to Bregma), and the needle was slowly injected, pushed 2 μL Aβ every 2 min, and left for 5 min. The needle was removed slowly, and the wound was sutured with penicillin powder after surgery.

Morris water maze

Morris water maze (MWM) are classical tasks widely used to assess memory parameters and deficits in rodents.¹⁶ The MWM was composed of a circular pool, a computer and a MWM video analysis system, which was divided into four quadrants The MWM used by rats was around a reservoir with a diameter of about 150 cm and a height of 60 cm, which could be filled with water (25 ± 1°C) about 30 cm deep. A colorless transparent platform with a diameter of 10 cm was placed in the pool, which was 1 cm below the water surface. The experiment process is divided into two parts: positioning voyage (days 1 to 5) and space exploration (day 6). The escape latency, stay time in the target quadrant and times of crossing the platform were recorded within 60 s.

Field experiments

Field experiments, also known as open-box experiments, test the autonomous behavior, exploration behavior and tension of experimental animals in an unfamiliar environment.¹⁷ The experiment was carried out in four black experimental boxes divided into 40 cm × 40 cm × 35 cm. Half an hour before the experiment, the rats were put into the experimental environment. Three experimenters cooperated with each other. One experimenter controlled the computer, and two of them simultaneously put four rats into squares facing the box wall and let them explore the environment freely for 10 min. The experimental environment is kept absolutely quiet to avoid disturbing the rats. After the experiment, the experimental box is cleaned with disinfection.

Western blotting

Mice were decapitated after they were anesthetized by 0.3% sodium pentobarbital (1 ml/100 g, intraperitoneally). The hippocampal tissue was separated the protein of the hippocampus was extracted and the sample was fully denaturated. The protein samples were separated by SDS-PAGE and electrically transferred to a polyvinylidene difluoride membrane (PVDF). The non-specific binding sites on the membrane were sealed with 1% bovine serum albumin (BSA) in Tris-buffered saline Tween-20 (TBST) buffer at the concentration for 2 h. They were then incubated with specific primary antibodies: GSK-3β, β-catenin, DVL2 (1:1000; Proteintech), and GFAP, Tau (1:1000; Abcam), at 4°C in a shaking bed overnight. TBST was washed and placed into an antibody diluent containing a secondary antibody (HRP labeled Goat anti-rabbit IgG, 1:5000) for 2 h at room temperature. ImageJ software analyzed the banding results.

Nissl staining

Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (1 ml/100 g, intraperitoneally), perfused with 0.9% normal saline and subsequently with 4% paraformaldehyde. The brain tissue was removed and fixed in 4% paraformaldehyde solution for 48 h. The fixed brain tissue was repaired and the paraffin section was carried out. The 5 μm slices were roasted at 60°C for 1 h, dewaxed to water, soaked in xylene I and xylene II solution for 15 min, soaked in 100% I and 100% II alcohol for 10 min, soaked in 95% I and 95% II alcohol for 5 min, soaked in 80% and 70% alcohol for 5 min, and washed with double steaming for 5 min, 3 times in total. The method of methyl violet staining was adopted. First, methyl violet staining solution was used for 10 min, and then the differentiation solution was washed with double steam water. The differentiation solution was differentiated for 4～8 s, dehydrated and transparent, and neutral gum was sealed for microscopic observation.

Immunohistochemistry

After soaking the whole brain tissue in PFA solution for 72 h, the whole brain tissue was pruned and put into 30% sucrose solution for precipitating sugar treatment. Frozen sections were made with a thickness of 20 μm. The sections were washed three times with PBS, and incubated with 50 μL endogenous peroxidase in each tissue for 10 min at room temperature. The sections were washed three times with PBS, added 50 μL serum to each tissue and incubated for 1 h at room temperature. The tissue sections were directly added with primary antibody (GFAP 1:200) and put in the refrigerator at 4°C overnight. The sections were rewarmed for 1 h the next day, washed three times with PBS, dropped secondary antibody, and then incubated at room temperature for 1 h .The sections were washed three times with PBS, then horseradish peroxidase was dropped and incubated at room temperature for 10 min. The sections were washed three times with PBS and carried out DAB color rendering. The reaction was terminated immediately after the chromogenic reaction was completed. After the completion of color development, the reaction was terminated in time for ∼5–20 min of hematoxylin staining, and the reverse blue was washed with running water and dehydrated in gradient alcohol, soaked in 80% alcohol, 90% alcohol and 95% alcohol for 5 min, soaked in 100% alcohol I and alcohol II for 10 min, and soaked in xylene I and xylene II for 10 min. The neutral gum was sealed and observed under the microscope.

Statistical analysis

All data were analyzed by Graph Pad Prism 8.0 (Graph Pad Software Inc., San Diego, USA) was used for performing all statistical analysis. Data were expressed as mean ± SD, and p < 0.05 was considered statistically significant. Data were analyzed by One-way or Two-way ANOVA. The Student's t-test was used to compare the two groups. The experimental results agree with the normal distribution.

Results

The movement of rats in the open field

Two weeks after modeling, an open field test was conducted to verify the autonomous behavior and exploration behavior of the rats after modeling in a strange environment. The results showed that the movement track of normal rats in the open field was mostly carried out at the edge of the test chamber. Due to the deposition of Aβ, the rats were anxious and nervous about the unfamiliar environment, and the activity track began to move to the center of the activity box and the activity frequency increased, suggesting that the rats had nervous and anxious emotions. Compared with the AD group, tension was improved and central activity locus decreased in the CGA100 group after treatment. After treatment with CGA150, their tension also decreased. The therapeutic effect of CHA100 was better than the CHA150 group. The results showed that CGA could improve learning memory and cognitive impairment in AD rats Figure 1).

Figure 1.

The movement trajectory of rats in each group was changed in the open field. Control group (CON group), Alzheimer's disease group (AD group), Chlorogenic acid 100 group (CGA100 group; gavage of chlorogenic acid at a dose of 100 mg/kg/d), and Chlorogenic acid 150 group (CGA150 group; gavage of chlorogenic acid at a dose of 150 mg/kg/d).

Effects of CGA on learning and memory ability and cognitive ability of Ad rats

MWM test was used to detect the changes in learning memory ability and cognitive ability of rats. The results showed that compared with the CON group, the swimming path of the AD group was chaotic and complicated, and even the platform could not be found within the specified time. The escape latency was significantly prolonged, and the number of platform crossings was reduced. Compared with the AD group, the swimming path and the escape latency of the CGA group were all shortened significantly (p < 0.05), and the times of platform crossing were increased (p < 0.05). After the CHA100 treatment, the escape latency and swimming path of rats were significantly shortened. After treatment, the time to find the platform in CHA150 group is shorter than that in the AD group, but longer than that in CHA100 group, indicating that CHA can improve the learning memory and cognitive impairment of AD rats, and the therapeutic effect of CHA100 group is better than that in CHA150 group. The result indicated that the CGA group could effectively improve the learning, memory and cognitive impairment of AD rats (Tables 1 and 2, Figure 2).

Figure 2.

Swimming trajectory of rats. Control group (CON group), Alzheimer's disease group (AD group), Chlorogenic acid 100 group (CGA100 group; gavage of chlorogenic acid at a dose of 100 mg/kg/d) and Chlorogenic acid 150 group (CGA150 group; gavage of chlorogenic acid at a dose of 150 mg/kg/d).

Table 1.

Comparison of escape latency time (s) ( $\bar{X}$ ±S n = 10).

Group/day	One day	Two days	Three days	Four days	Five days
CON	54.5 ± 5.0	43.2 ± 8.3	35.2 ± 8.3	27.5 ± 15.0	21.2 ± 8.8
AD	57.1 ± 3.9	55.4 ± 3.6*	50.4 ± 4.0*	44.2 ± 7.9*	35.6 ± 14.8*
CGA100	55.9 ± 3.6	37.1 ± 4.0*	37.1 ± 11.1^#	31.6 ± 9.9^#	23.1 ± 11.6^#
CGA150	56.9 ± 2.5	49.1 ± 4.2*	42.7 ± 6.1^#	33.6 ± 6.3^#	24.2 ± 9.9^#

*p < 0.05 compared with the CON group; ^#p < 0.05 compared with the AD group.

Table 2.

Comparison of times of crossing platform of SD rats ( $\bar{X}$ ±S n = 10).

Group	Sample size	Number of platform crossings
CON	10	4.5 ± 0.3
AD	10	1.45 ± 0.55*
CGA100	10	3.1 ± 0.27^#
CGA150	10	2.0 ± 0.5^#

*p < 0.05 compared with the CON group; ^#p < 0.05 compared with the AD group.

Effects of CGA on hippocampal neurons in Ad rats

To verify the changes in the number of Nissl positive neurons in the hippocampal tissues of rats after modeling and the effects of different doses of CGA on the number of Nissl and neurons, the hippocampal tissues of rats in each group were stained with Nissl. The results showed that in the hippocampal CA1 region of the rats, compared with the CON group, the AD group had fewer Nissl. Compared with the AD group, Nissl increased in the CGA100 group. Compared with the AD group, Nissl also increased in the CGA150 group. However, the increase was not as large as that in the CHA100 group, and the treatment effect in the CHA100 group was better than that in the CHA150 group. CGA can alleviate the learning and memory impairment of rats to a certain extent (Figure 3).

Figure 3.

(Top) Effects of CGA on hippocampal neurons in AD rats. (A) CON group; (B) AD group; (C) CGA100 group; (D) CGA150 group. (Bottom) The cell number of hippocampal neurons in the CA1 area of Nissl staining.

Effect of CGA on GFAP content in the hippocampus of Sd rats

To explore whether CGA can inhibit the inflammatory expression in the hippocampus of AD rats, immunohistochemical analysis was performed on brain sections of rats, and the expression of GFAP in the hippocampus of different groups of rats was detected. Glial scar formation of astrocytes in the area of CNS injury by exogenous chemicals or trauma is determined by the expression of GFAP. In the hippocampal CA1 region of AD rats, the expression of GFAP increased, indicating the excessive deposition of Aβ and tau proteins in the hippocampus of AD rats, resulting in inflammatory expression. The expression of the CGA100 group was lower than that of the AD group. The expression of the CGA150 group was lower than that of the AD group. However, the expression of CHA150 group was higher than that of the CHA100 group, indicating that the therapeutic effect of the CHA100 group was better than that of the CHA150 group. Therefore, it can be verified that CGA can inhibit inflammatory expression and excessive deposition of Aβ and tau proteins in the hippocampus of AD rats (Figure 4).

Figure 4.

Effect of CGA on GFAP content in the hippocampus of SD rats. (A) CON group; (B) AD group; (C) CGA100 group; (D) CGA150 group.

Effects of CGA on GSK-3β, β-catenin, DVL2, GFAP, and tau proteins in the hippocampus of Sd rats

To explore whether CGA can improve learning, memory and cognitive impairment in AD rats via Wnt signaling pathway, brain tissues of each group were extracted for detection. When Aβ was over deposited, the Wnt pathway was inhibited, and the protein content in the pathway was changed. The results showed that GSK-3β protein content was increased in the AD group compared with the CON group. After CGA treatment, the GSK-3β expression level of the CGA100 group was decreased. Compared with the CON group, the contents of β-catenin and DVL2 were decreased in the AD group (p < 0.05). Compared with the AD group, β-catenin and DVL2 contents were increased in the CGA100 group (p < 0.05). Compared with the AD group, the contents of β-catenin and DVL2 in the CGA150 group also increased (p < 0.05).The results showed that CGA could improve the learning and memory ability and cognitive impairment of AD rats via Wnt signaling pathway (Figure 5).

Figure 5.

The effects of CGA on GSK-3β, β-catenin in the hippocampus of SD rats. (A) The result of GSK-3β, β-catenin, and DVL2. (a) CON group; (b) AD group; (c) CGA100 group; (d) CGA150 group. (B) The relative expression of GSK-3β protein. (C) The relative expression of β-catenin protein; (D) The relative expression of DVL2 protein. *p < 0.05 compared with the CON group; ^#p < 0.05 compared with the AD group, All results were represented as mean ± SD (n = 10). The significant difference from the respective values was determined by one-way analysis of the variance test. n = 3 for western blotting.

Western Blot was used to detect the content of GFAP and tau proteins, and verify the therapeutic effect of CGA. Compared with the CON group, GFAP andtTau protein expression increased in the AD group, and GFAP and tau protein expression decreased in the CGA100 and the CGA150 groups compared with the AD group. The results showed that CGA could improve learning, memory and cognitive impairment in AD rats (p < 0.05) (Figure 6).

Figure 6.

The effects of CGA on GFAP and tau proteins in the hippocampus of SD rats. (A) The result of GFAP and tau. (a) CON group; (b) AD group; (c) CGA100 group; (d) CGA150 group. (B) The relative expression of GFAP protein. (C) The relative expression of Tau protein. *p < 0.05 compared with the CON group; ^#p < 0.05 compared with the AD group, All results were represented as mean ± SD (n = 10). The significant difference from the respective values was determined by one-way analysis of the variance test.

Discussion

AD is an inflammatory neurodegenerative disease with insidious onset and slow symptom process.¹⁸ AD is a growing global health problem that has a huge impact on individuals and societies, which is becoming a major challenge for health and social care worldwide.^19,20 Research shows that patients with AD are at risk of cognitive impairment due to decreased cognitive activity and understanding.^21–23 In our present study, we found that the Aβ_25-35 injection into the lateral ventricle leads to impaired learning and memory and spatial impairment in AD rats, which is consistent with the findings of Xiao et al.²⁴ Studies have also shown that AD is closely associated with learning memory and cognitive impairment.^25,26

As Aβ in the body mainly comes from the cleavage of amyloid-β protein precursor (AβPP), and the β-secretase and γ-secretase generated in the cleavage process will promote the formation of Aβ. Studies have confirmed that injecting Aβ fragments into the hippocampus of experimental SD rats will lead to excessive deposition of Aβ in the hippocampus of rats, so as to prepare the AD model.²⁷ Studies have found that when Aβ is over-deposited in the brain of SD rats, the expression level of tau protein increases accordingly. Therefore, some scholars have proposed that tau protein may not be a single pathogenic factor, but the occurrence of AD may be caused by the simultaneous over deposition of Aβ and tau protein, resulting in senile plaques in the hippocampus, leading to memory impairment and cognitive impairment.^28,29 In this study, the expression of tau protein was detected after injection of Aβ, and it was found that the expression level of tau protein in the AD model group increased significantly, and decreased after CGA treatment. Therefore, the mechanism of AD in this study is that the combined action of Aβ and tau protein leads to memory impairment and cognitive impairment in AD rats.

Currently, the drugs used to treat AD do not completely prevent the progression of the disease. CGA is a polyphenolic substance extracted from traditional Chinese medicine Honeysuckle, etc. Studies have shown that CGA has anti-inflammatory, antioxidant, antibacterial and antiviral effects and is widely used in tumor and cardiovascular fields.^30,31 Studies have shown that CGA can regulate a variety of metabolism to achieve anti-inflammatory and antioxidant effects, protect nerves and slow down apoptosis.³² At the level of the signal pathway, studies have shown that CGA has been proven to have good neuroprotective properties for AD-related neurodegeneration, and has been applied to conduct potential molecular studies in mice with nerve injury and APP/PS1 common defect.³³ CGA had a neuroprotective effect on the CI/R rats by regulating the oxidative stress-related Nrf2 pathway.³⁴ Other studies have shown that CGA increases the MMP intensity of hippocampal neurons induced by Aβ_25-35, reverses the expression effect of apoptosis-related proteins, and provides direction for AD.³⁵ We found that CGA shortens the escape latency, improves learning and memory, and improves cognitive impairment in AD rats. To further study the protective effect of CGA on cognitive impairment in early AD, we explored the mechanism of action and better understand the positive significance of this effect mechanism.

In the brain, the classical Wnt/β-catenin signaling pathway not only plays a critical role in neuronal survival and neurogenesis, but it also plays an important role in regulating synaptic plasticity and the integrity and function of the blood-brain barrier.³⁶ The brain of AD patients is regulated by the Wnt/β-catenin signaling pathway, and the Wnt/β-catenin signaling pathway in the brain is inhibited, the level of β-catenin decreases, and the level of GSK-3β increases, resulting in Aβ accumulation, leading to the occurrence of AD.^37,38 Studies have proved that the Wnt signaling pathway can regulate hippocampal plasticity, participate in the formation of neuronal dendrites and axons, and regulate the formation of neuronal circuits in the hippocampus of adults.³⁹ Wnt loss signaling promoted cognitive impairment, tau phosphorylation, and Aβ₁₋₄₂ production in the hippocampus of wild-type mice, thereby contributing to AD-like neuropathology.⁴⁰ In this experiment, western blot detection results showed that in the AD model group, the β-catenin level decreased, the GSK-3β level increased, and the DVL2 level decreased, suggesting that the Wnt signal was inhibited. After CGA treatment, the changes of catenin, GSK-3β and DVL2 levels were all reversal suggesting that the CGA can improve the AD rat model via the Wnt signal pathway.

Conclusion

In conclusion, our study provides evidence that CGA may improve learning and memory by activating the Wnt signaling pathway, which may be a new therapeutic target mechanism for AD. To better guide the clinical application of CGA, many experiments are needed to study the mechanism of CGA.

Footnotes

Acknowledgments

We acknowledge and appreciate our teachers and classmates for their valuable efforts and comments on this paper.

ORCID iDs

Liang Hou

Deyu Zheng

Author contributions

Deyu Zheng (Resources); Lei Wang (Investigation); Xuehua Wang (Investigation); Lei Wang (Investigation); Xuehua Wang (Writing – original draft); Liang Hou (Data curation); Yingxue Liu (Methodology); Jiangsheng Liu (Investigation); Deqiang Zhang (Formal analysis; Software); Suyan Yao (Resources); Deyu Zheng (Conceptualization; Resources).

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported Natural Science Foundation of Liaoning Province.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability

All data from this research can be made available upon request from the corresponding author.

References

Vicente-Zurdo

Romero-Sanchez

Rosales-Conrado

, et al. Ability of selenium species to inhibit metal-induced Abeta aggregation involved in the development of Alzheimer's disease. Anal Bioanal Chem 2020; 412: 6485–6497.

Naseri

Wang

Guo

, et al. The complexity of tau in Alzheimer's disease. Neurosci Lett 2019; 705: 183–194.

Scheltens

De Strooper

Kivipelto

, et al. Alzheimer's disease. Lancet 2021; 397: 1577–1590.

Heitman

Ingram

. Cognitive and neuroprotective effects of chlorogenic acid. Nutr Neurosci 2017; 20: 32–39.

Miao

Xiang

. Pharmacological action and potential targets of chlorogenic acid. Adv Pharmacol 2020; 87: 71–88.

García-Morales

Huerta-Reyes

González-Cortazar

, et al. Anti-inflammatory, antioxidant and anti-acetylcholinesterase activities of Bouvardia ternifolia: potential implications in Alzheimer's disease. Arch Pharm Res 2015; 38: 1369–1379.

Hwang

Kim

Park

, et al. Anti-inflammatory effects of chlorogenic acid in lipopolysaccharide-stimulated RAW 264.7 cells. Inflamm Res 2014; 63: 81–90.

Lee

Kang

Kim

, et al. Experimental pretreatment with chlorogenic acid prevents transient ischemia-induced cognitive decline and neuronal damage in the hippocampus through anti-oxidative and anti-inflammatory effects. Molecules 2020; 25: 3578.

Ishida

Yamamoto

Misawa

, et al. Coffee polyphenols prevent cognitive dysfunction and suppress amyloid β plaques in APP/PS2 transgenic mouse. Neurosci Res 2020; 154: 35–44.

10.

Gaesser

Fyffe-Maricich

. Intracellular signaling pathway regulation of myelination and remyelination in the CNS. Exp Neurol 2016; 283: 501–511.

11.

Parr

Mirzaei

Christian

, et al. Activation of the Wnt/β-catenin pathway represses the transcription of the β-amyloid precursor protein cleaving enzyme (BACE1) via binding of T-cell factor-4 to BACE1 promoter. FASEB J 2015; 29: 623–635.

12.

Folke

Pakkenberg

Brudek

. Impaired Wnt signaling in the prefrontal cortex of Alzheimer's disease. Mol Neurobiol 2019; 56: 873–891.

13.

Inestrosa

Tapia-Rojas

Cerpa

, et al. WNT Signaling is a key player in Alzheimer's disease. Handb Exp Pharmacol 2021; 269: 357–382.

14.

Yang

Zhang

. Microglia and Wnt pathways: prospects for inflammation in Alzheimer's disease. Front Aging Neurosci 2020; 12: 110.

15.

Zhao

Huang

, et al. Research on biosafety risk management of laboratory animals in universities. Exp Technol Manag 2024; 41: 242–249.

16.

Lissner

Wartchow

Toniazzo

, et al. Object recognition and Morris water maze to detect cognitive impairment from mild hippocampal damage in rats: a reflection based on the literature and experience. Pharmacol Biochem Behav 2021; 210: 173273.

17.

Wang

Liu

, et al. Application of open field test in mouse behavior analysis. Chin J Cell Biol 2011; 33: 6.

18.

Shieh

Huang

Lin

. Alzheimer's disease and diabetes: insulin signaling as the bridge linking two pathologies. Mol Neurobiol 2020; 57: 1966–1977.

19.

Lane

Hardy

Schott

. Alzheimer's disease. Eur J Neurol 2018; 25: 59–70.

20.

Sun

Zhu

, et al. Clinical research on Alzheimer's disease: progress and perspectives. Neurosci Bull 2018; 34: 1111–1118.

21.

Denny

Bartley

Edwards

, et al. AD8 patient-informant discrepancy predicts insight and cognitive impairment in Alzheimer's disease. Geriatr Nurs 2021; 42: 262–267.

22.

Margolis

Kelly

Daiello

, et al. Anticholinergic/sedative drug burden and subjective cognitive decline in older adults at risk of Alzheimer's disease. J Gerontol A Biol Sci Med Sci 2021; 76: 1037–1043.

23.

Wilson

Wang

, et al. Cognitive activity and onset age of incident Alzheimer disease dementia. Neurology 2021; 97: e922–e929.

24.

Xiao

Wang

, et al. Celastrol attenuates learning and memory deficits in an Alzheimer's disease rat model. Biomed Res Int 2021; 2021: 5574207.

25.

Shen

Chen

, et al. Cognitive decline, dementia, Alzheimer's disease and presbycusis: examination of the possible molecular mechanism. Front Neurosci 2018; 12: 394.

26.

Yao

Sweeney

Nagorski

, et al. Quantifying cognitive resilience in Alzheimer's disease: the Alzheimer's disease cognitive resilience score. PLoS One 2020; 15: e0241707.

27.

Hemonnot-Girard

Valverde

Hua

, et al. Analysis of CX3CR1 haplodeficiency in male and female APP/PSEN1 mice along Alzheimer disease progression. Brain Behav Immun 2021; 91: 404–417.

28.

Alexander

Kalev

Mehrabian

, et al. Familial early-onset dementia with complex neuropathologic phenotype and genomic background. Neurobiol Aging 2016; 42: 199–204.

29.

Paudel

Angelopoulou

Jones

, et al. Tau related pathways as a connecting link between epilepsy and Alzheimer's disease. ACS Chem Neurosci 2019; 10: 4199–4212.

30.

Ajiboye

Ojo

Okesola

, et al. In vitro antioxidant activities and inhibitory effects of phenolic extract of Senecio biafrae (Oliv and Hiern) against key enzymes linked with type II diabetes mellitus and Alzheimer's disease. Food Sci Nutr 2018; 6: 1803–1810.

31.

Luo

, et al. Anti-acne vulgaris effects of chlorogenic acid by anti-inflammatory activity and lipogenesis inhibition. Exp Dermatol 2021; 30: 865–871.

32.

Fang

Wang

Wei

, et al. Beneficial effects of chlorogenic acid on alcohol-induced damage in PC12 cells. Biomed Pharmacother 2016; 79: 254–262.

33.

Gao

Meng

, et al. Chlorogenic acid alleviates Aβ-induced autophagy and cognitive impairment via the mTOR/TFEB signaling pathway. Drug Design Dev Ther 2020; 14: 1705–1716.

34.

Liu

Wang

Zhang

, et al. Protective effects of chlorogenic acid on cerebral ischemia/reperfusion injury rats by regulating oxidative stress-related Nrf2 pathway. Drug Des Devel Ther 2020; 14: 51–60.

35.

Shi

Sun

Wang

, et al. CGA Restrains the apoptosis of Aβ-induced hippocampal neurons. Int J Neurosci 2020; 130: 700–707.

36.

Jia

Piña-Crespo

. Restoring Wnt/β-catenin signaling is a promising therapeutic strategy for Alzheimer's disease. Mol Brain 2019; 12: 104.

37.

Huang

Liu

. Dickkopf-1: current knowledge and related diseases. Life Sci 2018; 209: 249–254.

38.

Zhang

. The correlation between LncRNA-17A expression in peripheral blood mononuclear cells and Wnt/β-catenin signaling pathway and cognitive function in patients with Alzheimer disease. Am J Transl Res 2021; 13: 11981–11986.

39.

Inestrosa

Varela-Nallar

. Wnt signalling in neuronal differentiation and development. Cell Tissue Res 2015; 359: 215–223.

40.

Tapia-Rojas

Inestrosa

. Loss of canonical Wnt signaling is involved in the pathogenesis of Alzheimer's disease. Neural Regen Res 2018; 13: 1705–1710.