Ganoderma lucidum Triterpenoids (GLTs) Reduce Neuronal Apoptosis via Inhibition of ROCK Signal Pathway in APP/PS1 Transgenic Alzheimer’s Disease Mice



Alzheimer’s disease (AD) is the most common cause of dementia among senior citizen. Ganoderma lucidum triterpenoids (GLTs) have nutritional health benefits and has been shown to promote health and longevity, but a protective effect of GLTs on AD damage has not yet been reported. The objective of this research was to elucidate the phylactic effect of GLTs on AD model mice and cells and to explore its underlying mechanisms. Morris water maze (MWM) test was conducted to detect changes in the cognitive function of mice. Hematoxylin-eosin (HE) staining was applied to observe pathological changes in the hippocampus. Silver nitrate staining was applied to observe the hippocampal neuronal tangles (NFTs). Apoptosis of the hippocampal neurons in mouse brain tissue was determined by TUNEL staining. The expression levels of apoptosis-related protein Bcl2, Bax, and caspase 3/cleaved caspase 3; antioxidative protein Nrf2, NQO1, and HO1; and ROCK signaling pathway-associated proteins ROCK2 and ROCK1 were measured by western blot. In vivo experiments show that 5-month-old APP/PS1 mice appeared to have impaired acquisition of spatial learning and GLTs could reduce cognitive impairment in AD mice. Compared to normal mice, the hippocampus of APP/PS1 mouse’s brains was severely damaged, while GLTs could alleviate this symptom by inhibiting apoptosis, relieving oxidative damage, and inactivating the ROCK signaling pathway. In in vitro cell experiments, Aβ25-35 was applied to induce hippocampal neurons into AD model cells. GLTs promoted cell proliferation, facilitated

1. Introduction

Alzheimer’s disease (AD), a widespread, progressive, nonreversible, and devastating set of neurodegenerative disorders, is characterized by progressive impairment of memory, motion disorders, and judging and reasoning abilities that eventually results in aphronesia [12]. The pathological features mainly include senile plaques (SP) formed by extracellular amyloid β-protein (Aβ) deposition and endocellular neurofibrillary tangles (NFTs) formed by hyperphosphorylation of tau protein in neurons, along with the increasingly attenuate number and capacity of synapses and neurons [3]. The NFTs are composed of amyloid fibrils, which are associated with synapse loss and neurodegeneration, and eventually lead to memory impairment and other cognitive problems [4]. The pathogenic causes linked with the incidence of AD include poor mental performance, traumatic brain injury, cerebral stroke, low social activity, age, social exclusion, and physical inactivity, and low education level [5]. There is an estimated 46.8 million people worldwide who suffered from AD or interrelated dementia disease in 2015, and the morbidity of AD throughout the world is anticipated to exceed 1.315 billion by year 2050 [67]. The disease is clinically basically characterized by a severe dysfunction of cognition and ascensive degradation of memory, resulting in loss of self-care ability and eventually needing all-time medical solicitude [8]. Nevertheless, up to now, there appears no efficient cure or prevention for AD, and the treatments simply moderate symptoms without affecting the disease development, laying a tremendous millstone on public health and society.

Ganoderma lucidum (Glucidum) is a Basidiomycetes fungus from the order Polyporales and acclaimed officinal agaric used as a folk remedy in Asia since ancient times as a result of its multitudinous health-promoting capabilities [910]. It has been manifested that this eumycete is beneficial in preventing and treating high blood pressure, hyperglycemia, chronic bronchitis, hepatitis, asthma, cancer, heart diseases, and HIV [1113]. Ganoderma lucidum triterpenoids (GLTs) is the major variety of bioactive and medicative components in Ganoderma lucidum (Glucidum) [14]. The mother nucleus made of isoprene is an important chemical structure of GLTs [15]. But the chemical structures of GLTs are more sophisticated as a result of the highly enriched oxidized states of these compounds. Most GLTs exhibit a large scale of bioactivity, including anticancer [16], antihypertensive, anti-HIV-1, antiangiogenic, immunomodulatory [17], antiandrogenic, antioxidant, antihepatitis B, antimicrobial activities, and anticomplement [18]. GLTs are efficient as adjuvant therapies and enhance health when united with other pharmaceuticals to cure fatigue syndrome, hepatitis, and prostate cancer [19]. However, research investigating the mechanism and application of Glucidum or GLTs in the treatment of diseases remains preliminary in terms of both the utilization efficacy and product type. Moreover, the therapeutic effect and molecular mechanism of GLTs on AD needs further research.

The present research was designed to evaluate the possible neuroprotective impact of GLTs on antiapoptosis in AD course. Firstly, we determined the effect of GLTs on cognitive disorder in APP/PS1 transgenic AD model mice compared to the control normal mice by place navigation test and spatial probe test. Furthermore, in consideration of the neurons, apoptosis is an important pathological process in AD; the roles of GLTs in the hippocampus’ apoptosis of mice were investigated. What’s more, we also probed into the function of GLTs on Aβ25-35-induced hippocampal neuron cell AD model. In addition, it was found that ROCK signal pathway may participate in the regulation process of GLTs on AD. We hope that improved mechanistic understanding of these phenomena may lay the foundation for selecting new drugs and screening targets to treat this devastating disease.

2. Materials and Methods

2.1. Animal

Male APPswe/PS1dE9 (APP/PS1) transgenic mice with a C57BL/6J background and nontransgenic littermates (C57BL/6 mice) were purchased from the Jiangsu ALF Biotechnology Co., Ltd (Nanjing, China). All mice were housed under controlled room temperature (20–24°C) and humidity (60–80%) and received food and water ad libitum. Three-month-old APP/PS1 mice and C57BL/6 mice were used for this study, and the experimental protocol was approved by the Medicine Animal Welfare Committee of the First Affiliated Hospital of Hunan Normal University (Hunan Provincial People’s Hospital).

2.2. Drug Treatment

GLTs were provided by the Supercritical Fluid Technology Research Center, Institute of Geochemistry, Chinese Academy of Sciences (Guiyang, China). Donepezil was purchased from Apharm Co., Ltd. (Daegu, South Korea). A total of 50 APP/PS1 mice were separated into five experimental groups: AD group (10 mL kg-1 normal saline), low-dose GLT group (0.35 g kg-1 GLTs), high-dose GLT group (1.40 g kg-1 GLTs), positive control group (0.38 g kg-1 donepezil), and solvent group (10 mL kg-1 edible oil). The above dosage was given by gavage once a day for 60 days. The other 10 C57BL/6 mice were in the normal control group (10 mL kg-1 normal saline).

2.3. Morris Water Maze (MWM) Test

An MWM test (recording system produced by Techman, Chengdu, China) was conducted after the intragastric gavage of GLTs and donepezil to determine changes in cognitive ability. The MWM experiments were divided into place navigation test and spatial probe test. The mice in place navigation test were trained once a day in the morning lasting for 9 days. The mice were put into the water from any of the three quadrants outside the safe platform to the wall of the pool, and the time of finding the safe platform within 120 s (escape latency) and the length of the swimming path (search distance) were recorded, and the mice were allowed to stay on the platform for 10 s. If the security platform cannot be found after 120 s, it is recorded as 120 s, and the mice were guided to the security platform. The place navigation test’s results, including the escape latency and search distance, were represented as the average of the results obtained in 1-9 days. The safety platform was removed on the 10th day to conduct spatial probe test. The mice were put into the water from the same entry point, and the search time (exploration time) of the mice in the quadrant of the original safety platform within 120 s was recorded, as well as the percentage of the swimming distance of the rats in the quadrant of the original safety platform in the total distance (exploration distance percentage). The experiments were carried out at 8 am and 3 pm to preclude the influence of circadian rhythm, and the laboratory was kept quiet, and the temperature and light intensity were as consistent as possible.

2.4. Hematoxylin-Eosin (HE) Staining

The morphological changes of the CA1 area of the mouse hippocampus was observed by pathological examination. At the end of the MWM experiment, the mice were cut and their brain tissue was removed and quickly placed on the ice tray along the sagittal suture. The brain tissues were fixed in 4% formalin solution at 4°C for 8 h, taken out in a 70% ethanol solution for 5 min, then placed in 80%, 90%, 95%, and absolute ethanol for gradient dehydration for 4 h each time, respectively; finally, tissues were immersed in xylene for 30 min, and then embedded in paraffin. Continuous coronal sections at the optic chiasma area (including the hippocampus) were taken, and the slice thickness was 3 μm. Each specimen was taken for 10 consecutive slices for the HE tests.

2.5. Silver Staining

Silver nitrate staining was applied to observe neuronal tangles (NFTs) in the CA1 area of the mouse hippocampus. In brief, the paraffin section was dewaxed and placed in a 20% silver nitrate aqueous solution and immersed in a 37°C incubator for 30 min in the dark; after distilled water washing for 3 min, 10% of the formaldehyde solution was treated for several seconds until the section was yellow; after washing with distilled water for 5 min, the ammonia silver droplets were applied to dye for 40 s; 10% formaldehyde was again treated for 2 min and then 5% sodium thiosulfate solution was used to fix for 5 min. Finally, NFTs were observed under an optical microscope.

2.6. TUNEL Staining

Apoptosis of neurons in the CA1 area of the mouse hippocampus was measured by TUNEL staining (Roche, Nutley, NJ, USA). The paraffin-embedded tissue was cut into 4-5 μm thick sections. The sections were then incubated in 50 μL of the TUNEL mixture (47.5 μL of TUNEL label containing fluorescein isothiocyanate-conjugated dUTP and 2.5 μL of TUNEL enzyme) in a humidified chamber (60 min, 37°C). Control sections were incubated with 50 μL of TUNEL label solution containing no TUNEL enzyme. Sections were photographed and TUNEL-positive nuclei were detected with IP Lab Imaging Analysis Software (Fairfax, VA, USA). Apoptotic index was calculated using TUNEL-positive nuclei/total number of nuclei×100 automatically.

2.7. Western Blotting Analysis

RIPA lysate (Beyotime, Shanghai, China) was used to obtain total proteins, 100 μg of which were segregated using SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. TBST containing 5% skim milk was used for membrane incubation for 1 h. Then, the membranes experienced incubation with primary antibodies including Anti-Bax antibody (ab77566, 1 μg/mL, Abcam, Cambridge, MA, USA), anti-Bcl-2 antibody (ab196495, 1 : 500, Abcam), anti-Caspase-3 antibody (ab13847, 1 : 500, Abcam), anti-Cleaved Caspase-3 antibody (ab2302, 1 μg/mL, Abcam), anti-Nrf2 antibody (ab137550, 1 : 500, Abcam), anti-Heme Oxygenase 1 antibody (ab13243, 1 : 2000, Abcam), anti-NQO1 antibody (ab2346, 0.3 μg/mL, Abcam), anti-ROCK1 antibody (EP786Y) (ab45171, 1 : 2000, Abcam), anti-ROCK2 antibody (ab71598, 1 μg/mL, Abcam), and anti-GAPDH (ab181603, 1 : 10000, Abcam) at 4°C overnight. The membranes were washed in TBST three times and incubated with anti-rabbit IgG H&L (HRP) secondary antibody (ab6721, 1 : 2000, Abcam) at room temperature for 1.5 h. After washing using TBST thrice, the membranes were subjected to color reaction by ECL Plus from Life Technology, and GAPDH was detected as control groups.

2.8. Primary Culture of Hippocampal Neurons

C57BL/6 mice born for 0-48 h were taken out and the brain was taken aseptically after sacrifice. The hippocampus tissue was isolated, washed in a Petri dish containing D-Hank’s solution, and then 1-2 mL 0.02% EDTA and 0.25% trypsin digest were added into the dish; the hippocampus tissues were cut into small pieces, then transferred into a 10 mL centrifuge tube for digesting for 10-15 min; then, 4-6 mL DMEM/F12 medium containing 20% fetal bovine serum to terminate digestion was added to the decomposition liquor; the digestive solution was filtered with nylon sieve, centrifuged at 1000 rpm for 10 min, and cells were collected. Hippocampus cells were seeded in a 96-well culture plate at 6 cells at 200 μL per well and incubated in a 37°C, 5% CO2 incubator. After 7 days of culture, cells were used for follow-up experiments.

2.9. AD Cell Model and Drug Administration

Amyloid β-protein 25-35 (Aβ25-35, Sigma-Aldrich, St. Louis, MO, USA) was applied to induce hippocampal neuron cells into AD cells. At first, Aβ25-35 was diluted to a concentration of 5 μg/μL with sterile physiological saline and incubated at 37°C for one week to become aggregated Aβ25-35. Hippocampal neuron cells were divided into six groups: blank control group (control), model group (Aβ25-35), low-dose GLT group (3.0 μmol L-1 GLTs), middle-dose GLT group (30.0 μmol L-1 GLTs), high-dose GLT group (300.0 μmol L-1 GLTs), and vehicle group (drug control group which added an equivalent medium and 20 μmol L-1 Aβ25-35). Hippocampal nerve cells in the treatment group were added with GLTs at the above dosage, and the model group was added with the same amount of culture medium. After 24 h of culture, Aβ25-35 with a concentration of 20 μmol L-1 was added to both the treatment group and the model group, and the culture was continued for 24 h. The blank control group was given the same amount of medium.

2.10. MTT Assay

Hippocampus cells were inoculated into the 96-well plates at a density of 4 cells/well and stored in DMEM with FBS in it. At 37°C, cells were incubated for 4 h and 50 μL of MTT (0.5 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added into each well and had their growth condition observed at the 12th h. To solubilize the crystals, 150 μL dimethylsulfoxide (DMSO) was added into each well after the supernatant was subsequently removed. The optical density (OD) was measured at 590 nm by a microplate reader (Bio-Rad, Hercules, CA, USA).

2.11. Determination of SOD, MDA, and LDH Levels

After drug treatment, the levels of superoxide dismutase (SOD), malondialdehyde (MDA), and lactic dehydrogenase (LDH) in the hippocampal neuron cell were then measured using a SOD test kit, MDA test kit, and LDH test kit, which were purchased from Nanjing Jiangcheng Bioengineering Institute (Nanjing, China). The experiment was conducted in accordance with the instructions of the kit. The parallel experiment was repeated three times.

2.12. Flow Cytometry Analysis

To detect cell apoptosis, PE Annexin V (BD Biosciences, San Jose, CA, USA) was applied, keeping to the instructions of the manufacturers. An FACSCalibur FCM (BD Biosciences) was used to observe cell apoptosis. Experiments in triplicate helped to reduce errors. FACS Diva software was adopted at data analysis.

2.13. Statistical Analysis

Experimental data analyses are performed with GraphPad Prism v6.0 statistical software and presented as  (SD) of results from three or more independent repetition experiments. Student’s test was used to compare the differences between two groups. One-way ANOVA was applied to analyze three groups or above.  values < 0.05 accepted as the scale for statistical significance.