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Introduction

Alzheimer's disease (AD), the most common form of dementia, which currently has no effective available treatment, is commonly diagnosed in individuals over 65 years old (1). There are numerous common symptoms including confusion, irritability, aggression, mood swings, trouble with language and long-term memory loss, which ultimately leads to mortality. On average, the life expectancy following diagnosis is 3.2–6.6 years (2). β-amyloid (Aβ) can aggregate to form flexible soluble oligomers, which may exist in several forms. Senile plaques are extracellular deposits of Aβ in the brain associated with degenerative neurons and an abundance of microglia and astrocytes (3). As a neurodegenerative disorder, AD is associated with the presence of these neurotoxic senile plaques in the brain (4). Oxidative stress is another important independent adverse factor in the development of AD (5).

Obesity may increase the risk of various diseases such as heart disease, type 2 diabetes, obstructive sleep apnea, certain types of cancer and osteoarthritis. Given that obesity is associated with vascular and metabolic diseases, it is suggested that it is associated with AD (6). As a novel determinant of body weight and obesity, iroquois homeobox protein 3 (IRX3) serves as a functional long-range target of obesity-associated variants within fat mass and obesity-associated (FTO) genes, influencing obesity (7). It is a member of the iroquois homeobox gene family and serves a role in the early stages of neural development (8). Obesity-associated single nucleotide polymorphisms have been demonstrated to be associated with IRX3 instead of fat mass and obesity-associated protein in the human brain (7).

Catalpol is an iridoid glucoside, and large quantities can be isolated from the genus Rehmannia (Orobanchaceae). Previous studies have indicated that catalpol serves a role in neuroprotection, however, the precise mechanisms remain unclear (9,10). The current study predominantly focuses on elucidating whether catalpol preserves neural function and reduces the formation of senile plaques in AD using an AD mice model. In addition, the effects of catalpol on IRX3 and obesity-associated genes were investigated.

Materials and methods

Generation of AD mice

A total of 18 male 8-month-old Kunming mice provided by the Animal Center of The Third Affiliated Hospital of Soochow University (Changzhou, China). Mice were maintained under 50±5% relative humidity at 22±2°C with a 12-h light/dark cycle and free access to food and water. The mice were randomly divided into 3 groups with 6 mice/group: The AD group; AD mice treated with catalpol group (AD+C); and the control group (CON). For generation of the AD mouse model, 12 mice (6 each from the AD and AD+C groups) were injected intraperitoneally with D-(+)-galactose (120 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) and sodium nitrite (90 mg/kg; Sigma-Aldrich), once daily continuously for 60 days. In order to enhance the progression of senile dementia, a one-off injection of 2 µl Aβ1–42 (amino acid sequence: DAEFGHDSGFEVRHQKLVFFAEDVGSNKGAIIGLMVGGVVIA; synthesized by Sangon Biotech Co., Ltd., Shanghai, China) into the left lateral ventricle of the 12 mice was conducted when the mice were 9-months old. At 10 months, the mice were considered to have AD. Catalpol (20 mg/kg; Sigma-Aldrich) was administered at 10 months for 30 days by intragastric administration in the AD+C group. No further treatment was administered in the AD group. The CON group mice were 11-months old without any chemical treatment. All mice used in the experiments of the current study were 11 months old. All experiments were approved by the Ethics Committee of Soochow University (Suzhou, China).

Detection of reactive oxygen species (ROS)-associated enzymes

Following sacrification of the mice by CO2 inhalation, the cerebral cortex of the mouse brain was dissected and centrifuged at a speed of 12,000 × g for 15 min in Tris-buffered saline with Protease Inhibitor Cocktail (1:1,000; Sigma-Aldrich) for 30 min at 4°C. The supernatant of the cerebral cortex was extracted and transferred to a tube. ROS-associated enzymes from homogenates were detected using the following kits purchased from Beyotime Institute of Biotechnology (Haimen, China): Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px3), catalase (CAT) and malondialdehyde (MDH).

Detection of soluble Aβ40 and Aβ42

The above-mentioned supernatant containing soluble Aβ was homogenized in 5 M guanidine HCl and 50 mM Tris-HCl (pH 8.0) buffer, and aliquots of the homogenates were diluted 40 fold. The supernatant was used for the determination of the concentrations of Aβ40 and Aβ42 peptides using Mouse ELISA kits (Thermo Fisher Scientific, Waltham, MA, USA). The optical density value was detected at λ=495 nm using the PR-310 ELISA reader (Shenzhen Procan Electronics Inc., Shenzhen, China).

Thioflavin-S staining

Mouse brains in the three groups were dissected and embedded in optimum cutting temperature compound (Sakura Finetek USA Inc., Torrance, CA, USA) and rapidly frozen on dry ice. Coronal sections (10 µm) were cut and deparaffinized in 4% paraformaldehyde for 20 min. Slides were placed in 1% thioflavin-S solution (catalog no. T1892; Sigma-Aldrich) for 12 min, then were differentiated twice in 70% fresh alcohol for 10 min. Subsequent to rinsing in distilled water, the slides were mounted in glycerin jelly. Images were captured using a confocal microscope (A1R+; Nikon Corporation, Tokyo, Japan) and analyzed by the relative area (%) of senile plaques (white spot as indicated by the arrows) using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Detection of IRX3 and obesity-associated genes

The expression of obesity-associated genes and IRX3 at the gene level was conducted using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using the ABI 7500 cycler (Applied Biosystems; Thermo Fisher Scientific). Total RNA was isolated from three groups using TRIzol® reagent (Ambion; Thermo Fisher Scientific) according to the manufacturer's instructions. For each group, 2.5 µg RNA in a 20-µl reaction mixture was reverse-transcribed into cDNA using the SuperScript® VILO™ cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, tube contents were mixed and incubated at 25°C for 10 min, then incubated at 42°C for 1 h and terminated at 85°C for 5 min. The products of cDNA were used as templates in the following qPCR. The following cycling conditions were used: Stage 1, 1 cycle of 95°C for 30 sec; stage 2, 40 cycles of 95°C for 5 sec and 60°C for 20 sec; and stage 3, 1 cycle of 65°C for 15 sec. The Obesity PCR Array was performed using a 96-well plate (SABiosciences; Qiagen Inc., Valencia, CA, USA) to detect the obesity-associated genes, which predominantly include orexigenic genes, anorectic genes and genes associated with energy expenditure. The PCR array was a set of optimized primer assays in a 96-well plate and was used for gene expression analysis. cDNA template was mixed with the PCR master mix, and then run the PCR program The 102 bp primer sequence for IRX3 (Sangon Biotech Co., Ltd.) was as follows: Forward, 5′-GAAAACTTAGACAGCGCGGCAG-3′ and reverse, 5′-AGTTTTGCAGTCCGAAATGGGT-3′. cDNA in these groups was reverse transcribed and used as templates with the SYBR® Premix Ex Taq™ II (Perfect Real Time) kit (Takara Biotechnology Co., Ltd., Dalian, China). Gene expression values were determined relative to that of the control group, which was set as 1. 2−ΔΔCT was used for analysis (11)

Western blot analysis

A total of 30 µg protein isolated from the mouse cortex was boiled for 5 min then cooled on ice and injected into 10% SDS-PAGE for electrophoresis. The proteins were then transferred onto a Immobilon-PFL polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) and blocked in 5% fat-free milk for 1 h. Membranes were incubated overnight at 4°C with rabbit polyclonal immunoglobulin (Ig)G anti-BACE1 (70 kDa; 1:1,000; cat. no. ab2077; Abcam, Cambridge, UK), rabbit polyclonal IgG anti-IDE (118 kDa; 1:1,000; catalog no. ab32216; Abcam), rabbit polyclonal IgG anti-NEP (86 kDa; catalog no. AB5458; Chemicon; EMD Millipore) and rabbit monoclonal IgG anti-IRX3 (52 kDa; 1:1,500; cat. no. ab174307; Abcam) primary antibodies, and for 1 h at room temperature followed by three washes with phosphate-buffered saline for 10 min each. Subsequently, the membranes were incubated with the alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibody (1:2,000; cat. no. 65-6122; Thermo Fisher Scientific). A polyclonal rabbit anti-glyceraldehyde 3-phosphate dehydrogenase antibody (cat. no. G9545; Sigma-Aldrich) was used as an internal control. Immunoreactive bands were visualized using Pierce™ ECL Substrate (Thermo Fisher Scientific) and then scanned (Fujifilm LAS 4000; Fujifilm, Tokyo, Japan).

Morris water maze test

The Morris water maze was used to detect the abilities of learning and memory of the spatial location via training mice to find a hidden platform. The swimming pool (120 cm in diameter, 50 cm in height, 30 cm depth of water made opaque with milk) was divided into 4 equal quadrants. A submerged platform (10 cm in diameter, 29 cm in height) was placed inside the target quadrant and 1 cm below the surface of the water while other three quadrants were entry points of the mice. Mice were trained to swim and find the hidden platform during the Morris water maze test for five consecutive days, including four days of place navigation with three consecutive trials per day, and the probe trial performed on the last day. On the testing day (day 5), the platform was removed and mice were allowed to explore the pool for 90 sec. The time (sec) spent in the target quadrant and the number of crossings of the former location of the hidden platform were recorded and considered as an index of spatial learning and memory.

Statistical analysis

Data are presented as the mean ± standard deviation. Calculations were performed using SPSS software, version 16.0 (SPSS, Inc., Chicago, IL, USA). The results were evaluated by one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

Results

Effects of catalpol on ROS-associated enzymes

The activities of the ROS-associated enzymes, SOD, GSH-Px and CAT, and the concentration of MDA were detected separately using the kits mentioned above. As shown in Fig. 1, the activity of SOD, GSH-Px and CAT was significantly reduced in the AD mice (P<0.01), which suggested the suppressive capacities of ROS clearance in the AD group. Subsequent to treatment with catalpol, the activity of these enzymes was increased significantly although were still lower than those of the CON group. The concentration of MDA was increased significantly in the AD group, suggesting the enhanced induction of ROS, however, no clear effect on MDA levels was detected following treatment with catalpol.

Figure 1 Detection of reactive oxygen species-associated enzymes in the supernatant of the cerebral cortex of mouse brains in 3 groups. (A) SOD; (B) GSH-Px; (C) CAT; and (D) MDA. Data are presented as the mean ± standard deviation. ##P<0.01, vs. CON; *P<0.05, vs. AD; **P<0.01, vs. AD. SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; CAT, catalase; MDA, malondialdehyde; AD, Alzheimer's disease; CON, control; C, catalpol.

Effects of catalpol on reduction of Aβ40 and Aβ42

As presented in Fig. 2, the concentration of Aβ40 (Fig. 2A) and Aβ42 (Fig. 2B) in the cortex of the brain was increased in AD mice as expected. Subsequent to treatment with catalpol, the level of Aβ40 and Aβ42, particularly Aβ40, in the AD+C group reduced significantly but remained higher than the level in the CON group.

Figure 2 Concentrations of soluble Aβ40 and Aβ42 in the cerebral cortex of mouse brains in the 3 groups. (A) Aβ40; and (B) Aβ42. Data are presented as the mean ± standard deviation. ##P<0.01, vs. CON; *P<0.05, vs. AD; **P<0.01, vs. AD. Aβ, β-amyloid; AD, Alzheimer's disease; CON, control; C, catalpol.

Effects of catalpol on the formation of senile plaques

Senile plaques in the hippocampus and cortex of mice were detected using thioflavin-S staining. As presented in Fig. 3A, the senile plaques (white spots indicated by arrows) were seldom detected in the hippocampus and cortex of mice in the CON group. Senile plaques were observed to be significantly increased in the AD group (Fig. 3B). Subsequent to treatment with catalpol, the number of senile plaques in the hippocampus and cortex were observed to be significantly reduced in the AD+C group (Fig. 3C), but remained higher than that in the CON group. The result indicated that catalpol reduced the formation of senile plaques in the hippocampus and cortex. The quantification of the plaque area is presented in Fig. 3D.

Figure 3 Senile plaques (white spots indicated by arrows; scale bar =100 µm) in the hippocampus and cortex of mouse brains in the 3 groups. (A) CON group; (B) AD group; (C) AD+C group. (D) Relative area of senile plaques (% of total area). Data are presented as the mean ± standard deviation. ##P<0.01, vs. CON; *P<0.05, vs. AD. AD, Alzheimer's disease; CON, control; C, catalpol.

Effects of catalpol on the expression levels of IRX3 and obesity-associated genes

As presented in Fig. 4A, no significant alterations in the expression levels of IRX3 were detected at the gene level. Additionally, no significant alterations in the expression levels of obesity-associated genes were observed. In addition, no alterations in the protein expression levels of IRX3 were observed (Fig. 4B). These results indicated that catalpol was not able to regulate the levels of IRX3. The alterations were considered significant if the fold change was >2 fold (increased) or <0.5 fold (reduced).

Figure 4 (A) The expression of IRX3 at the gene level in the mouse brain in 3 groups. Data are presented as the mean ± standard deviation. (B) The expression of IRX3, BACE-1, IDE and NEP at the protein level in the mouse brains in 3 groups as observed by western blotting. GAPDH was used as an internal control. Lane 1, CON group; lane 2, AD group; lane 3, AD+C group. IRX3, iroquois homeobox protein 3; BACE-1, β-secretase 1; NEP, neprilysin; IDE, insulin-degrading enzyme; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CON, control; AD, Alzheimer's disease; C, catalpol.

Effects of catalpol on enzyme regulation

BACE-1, IDE and NEP are three key enzymes regulating the formation of senile plaques during the progression of AD (12). As presented in Fig. 4B, compared with the CON group, the expression of IDE was downregulated in the AD group. Subsequent to treatment with catalpol, the expression of IDE was observed to be significantly upregulated in the AD+C group. However, no alterations to the expression of BACE-1 and NEP were detected among these groups. These results indicated that catalpol protected the brain by upregulating the expression of IDE.

Effects of catalpol on learning and memory promotion

The target quadrant time and the average number of hidden platform crossings in the CON, AD and AD+C groups were recorded. As presented in Fig. 5, the time spent in the target quadrant determined by the representative swim paths in the pool was longer in the CON group, however was significantly reduced in the AD group (P<0.01). The number of hidden platform crossings in the AD mice was significantly reduced when compared with that of the CON group (P<0.01). Following catalpol treatment, these data were increased compared with the AD group (P<0.05), but remained lower than that in the CON group. These results indicate the effects on learning and spatial memory in the AD mice, which may be ameliorated and by catalpol.

Figure 5 Morris water maze test in the 3 groups of mice. (A) The target quadrant time. (B) Number of hidden platform crossings. Data are presented as the mean ± standard deviation. ##P<0.01, vs. CON; *P<0.05, vs. AD. AD, Alzheimer's disease; CON, control; C, catalpol.

Discussion

Catalpol is an iridoid glucoside isolated from the genus Rehmannia (Orobanchaceae). Previous experiments by our group suggested that catalpol can stimulate adrenocortical hormones, increasing the production of androgens (unpublished data). Catalpol has additionally been identified to produce anti-inflammatory (13,14), anticancer (15,16), diuretic and hypoglycemic effects (17,18). Previously, catalpol was reported to exhibit neuroprotective effects, including protection against neurodegeneration (19). However, the mechanisms remain unclear.

Oxidative stress serves a key role in AD, which is the most common form of dementia and currently has no cure. It reflects an imbalance in ROS, which is regulated by numerous enzymes, and damages the neurons in the brain. ROS are regulated by many enzymes. The activity of ROS-associated enzymes, SOD, GSH-Px and CAT, and the concentration of MDA are important in the process of AD (20). SOD catalyzes the dismutation of the toxic superoxide (O2−) radical into O2 or H2O2. It is an important antioxidant in the majority of living cells exposed to oxygen. GSH-Px reduces lipid hydroperoxides to their corresponding alcohols and reduces free hydrogen peroxide to H2O. CAT catalyzes the decomposition of hydrogen peroxide to water and oxygen, and is an important enzyme in protecting the cell from oxidative damage. MDA is a marker of oxidative stress as ROS degrades polyunsaturated lipids, forming MDA. The activities of these enzymes were observed to be significantly reduced in AD mice following the progression of disease. Subsequent to treatment with catalpol, the activities of these enzymes were increased significantly but remained lower than those of the CON group. These results suggested that catalpol may partially ameliorate the effects on the levels of these enzymes. Notably, there was no clear inhibition on MDA detected following treatment with catalpol, however this requires further investigation in order to be verified.

Previous studies suggest that Aβ, which is a peptide of 36–43 amino acids in length, serves a central role in the pathological development of AD; increases in Aβ40 and Aβ42 have been implicated in the pathogenesis of AD (21,22). Levels of Aβ40 and Aβ42, which aggregate to form flexible soluble oligomers that are toxic to neurons, were observed to be significantly elevated in the brains of patients with AD (23). Senile plaques, the presence of which are an important criterion of the neurohistopathological verification of AD, contain Aβ40 and Aβ42 (24). In the current study, the concentrations of Aβ40 and Aβ42 in the cortex of the brain were increased in AD mice as hypothesized. Following treatment with catalpol, the levels of Aβ40 and Aβ42, particularly Aβ40, in the AD+C group reduced significantly but remained higher than the levels in the CON group. Thus, catalpol was shown to reduce the formation of senile plaques in the hippocampus and cortex, which are increased in AD. Aβ is removed and cleaved by several amyloid-degrading enzymes, including BACE-1, IDE and NEP (12). The results of the current study indicated that catalpol protected the brain by upregulating IDE, rather than the other two enzymes.

IRX3, a member of the iroquois homeobox gene family, serves a key role in early stages of neural development. A previous study demonstrated tha IRX3 is involved in neural formation and growth. IRX3 was previously identified to influence obesity (7). However, no alterations on the expression of IRX3 were detected at the gene and protein levels, thus suggesting that catalpol was not able to regulate IRX3. In addition, no significant alterations in the expression of obesity-associated genes were observed following treatment with catalpol. Although obesity has an important role in AD due to vascular defects, impaired insulin metabolism and defects in glucose transport in the brain, the results indicated that catalpol is not capable of regulating obesity.

The Morris water maze test is widely used to investigate spatial learning and memory, whilst it has been additionally used to measure the effect of drugs on neurocognitive disorders (25). The current study identified that catalpol was able to increase the time spent in the target quadrant and the average number of hidden platform crossings, suggesting that the effects on learning and spatial memory in the AD mice were partially ameliorated by catalpol.

In conclusion, the present study indicates that catalpol reduces the levels of oxidative stress in the cerebral cortex of the hippocampus by regulating SOD, GSH-Px and CAT. Catalpol is additionally suggested to reduce the levels of soluble Aβ40 and Aβ42 in the cerebral cortex of the hippocampus and thus inhibit the formation of senile plaques, which are regulated by IDE. Catalpol is not able to directly regulate the expression of IRX3 and obesity-associated genes. The learning and memory impairments were additionally identified to be relieved following treatment with catalpol. Thus, catalpol may be a potential drug for the treatment of neurodegenerative diseases such as AD.

Acknowledgments

The current study was supported by the Third Affiliated Hospital of Soochow University.

References