A combination of lycopene and human amniotic epithelial cells can ameliorate cognitive deficits and suppress neuroinflammatory signaling by choroid plexus in Alzheimer's disease rat

 Neuroinflammation characterized by glial activation and release of proinflammatory mediators is considered to be correlated with cognitive deficits in Alzheimer's disease (AD).

• The choroid plexus (CP), an epithelial layer that forms the blood-cerebrospinal fluid barrier, is able to modulate the cognitive function, through changes in the neuroinflammatory response and in brain immune surveillance.
• Previously, some studies have demonstrated that lycopene (LYCO) or human amniotic epithelial cells (HAECs) could attenuate inflammation in AD
• However, it is unclear if LYCO can interact with HAECs to improve neuroinflammation at the CP
• Thus, this study chose the region of interest, considered the feasibility, and tested the therapeutic agent for immunomodulatory effects in an acutely induced AD rat model.

Alzheimer’s disease (AD)

• The exact mechanism of neurodegenerative changes in AD has not been completely elucidated
• Recent indications highlight neuroinflammation as a key player in neuropathological feature of AD
• Lycopene (LYCO), an aliphatic hydrocarbon carotenoid, present in the ripened tomatoes, has been identified as a potential useful agent in the management of neoplastic diseases
• This study aimed to explore the effect of lycopene and HAECs in combination with their combination on the CP in the AD rat model
• Currently, human amniotic epithelial cells (HAECs) derived from the amnion exhibit considerable advantages over other stem cells and have drawn much attention from researchers

Human amniotic epithelial cells isolation and in vitro culture

• Complete isolation and culture procedures were carried out within a biosecurity cabinet under sterile conditions.
• Briefly, the amnion was manually stripped from the chorion and enzymatically removed by TrypZean (Sigma-Aldrich, St Lois, MO, USA) and collected by centrifugation (500 × g, 10 min, each time for 5 min).
• HAECs were placed in cell culture bottle with D-MEM/F12 medium containing 1 g/L glucose, 10 ng/mL EGF, and 5% fetal bovine serum for 7 days under incubating condition at 37°C with 5% CO2, changing medium every 3 days.

Flow cytometry and immunofluorescent staining

• To identify the phenotypic characteristics of freshly isolated HAECs and detect stem cell-related cell surface markers, primary cells were labeled with the following specific primary antibodies for 30 min at 4°C
• Where appropriate cells were fixed and permeabilized for intracellular antibody staining according to the manufacturer's instructions
• All primary antibodies were purchased from BD Bioscience (San Jose, CA, USA).
• The relevant isotype control antibodies were used as negative controls
• Cells were washed with fluorescence-activated cell sorting buffer, and then centrifugated at 300 × g for 5 minutes
• Each sample was tested for 20,000 cells

Preparation of Aβ, lycopene, and surgery

• Aβ1-42 (3 nmol/3 mL) was prepared in artificial cerebrospinal fluid (ACSF)
• The solution was incubated at 37°C for 3 days to form the aggregated Aβ and stored at −70°C.
• Lycopene was dissolved in 0.5% Carboxymethylcellulose-Na (CMC-Na) and administered at a dose of 5 mg/kg body weight for 35 days.

Animal and treatments

• Seventy two adult male Wistar rats (SPF class, weighing 250-300 g) were provided by the Chinese Academy of Sciences and housed in a room maintained under controlled temperature and on a 12 h/12 h light/dark cycle prior to experimentation.
• All rats were randomly divided into six groups (1-Alzheimer's disease [AD group]: AD-like pathology rat model was induced continuously by bilateral intraventricular injection [ICV] 10 µL/d of aggregated Aβ for 14 days [AD+PS group]].
• 2- Alzheimer’s disease+Physiological salt solution: AD rats were treated continuously by intragastric gavage of equal-dose physiological salt solution daily for 35 days;
• Alzheimer's disease+lycopene: AD+LYCO group]
• Beckman–Laurel neuropathy (AD+PBS group): AD rats received by ICV injection 10 µl of PBS
• Alzheimer’'s disease +HAECs: AD group
• A schematic diagram of drug treatment schedule and protocol design.

Assessment of behavioral parameters

• Forty-two days later, the learning and memory abilities of rats (six rats each group) were evaluated by the Morris water maze
• The apparatus consisted of a circular water tank (180 cm in diameter and 60 cm high) with a platform 2 cm below the water level inside the tank with water maintained at 28.5±2°C at a height of 40 cm.
• Each rat was trained for four consecutive days to find the hidden platform. For each trial, these animals were released from four randomly assigned start positions respectively.

Tissue sample collection and storage

• After behavior assessment, all other rats were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg)
• The top of the cranium was removed from the skull and the two lateral ventricle CPs were dissected out using a Dumont No. 5 forceps
• For protein levels analysis and detection of gene expression by real-time quantitative PCR, the CP samples from each brain were rapidly stored at −80°C

Cerebrospinal fluid isolation and ELISA assay

• Usually, 14 h after animal management in the last time, CSF was collected and evaluated by the cisterna magna puncture technique
• In brief, rats were anesthetized and placed on a stereotactic instrument so that the head formed a 135° angle with the body
• A needle attached to a 1mL syringe was carefully inserted to the dura, from the caudal end of the incision
• Collection of CSF by pulling back the plunger
• Approximately 10 µL CSF could be aspirated from each rat
• Brain Aβ1-42 was detected using commercially enzyme-linked immunosorbent assay (ELISA) kit
• The levels of cytokines in the brain and CSF were measured by immunoassay using a commercially available ELISA kit

Detection of gene expression by real-time quantitative PCR (RT-qPCR)

• Total RNA was purified by using Trizol
• Real-time PCR of cDNA was performed using forward and reverse primer sequences
• The mRNA expressions of TLR4, p65 subunit of NF-κB, and β-actin in CP were measured using the comparative critical threshold (Ct) method
• Data were analyzed using FluorChem FC2 software

Western blot analysis.

• The CP tissues were harvested and washed with phosphate buffered saline, and homogenized in ice-cold RIPA lysing buffer for 30 min, and then centrifuged at 12,000 × g for 10 min at 4°C until the supernatant was separated and used as a Western blot for nucleoprotein analysis.

Statistical Analysis

• GraphPad Prism 7.0 was used to analyze data. Normal distribution of data was first tested by Shapiro-Wilk test, P>.05 was accepted.
• Statistical analysis was performed using analysis of variance (ANOVA), followed by post hoc Duncan’s multiple range test. Significance levels were indicated *.01≤P<.05; ⁎⁎0.001 ≤ P<.001.

Morphology of HAECs and expression of cell markers

• The cells adhered to the wall and grew in 2-3 days after inoculation, showing round and strong refraction.
• When hematoxylin eosin staining, the cells P0 generation were uniform in size, blue in nucleus, red in cytoplasm, and rich in fluid (Fig. 2A).
• Cells also showed the positive expression of stem cell-related cell surface marker CD73 (75.83%), weak positive expression, of CD90 (34.74%), and the low expression of CD105 (8.95%).

Effect of combination of LYCO and HAECs on escape latency of AD rats

• The mean escape latency did not differ between any of the groups on the first day of testing in Morris water maze, but from the second day onward, there was a significant difference in transfer latency.
• Rats injected with ICV Aβ1-42 showed a lower ability to find the platform and learn its location in the fifth day of training, whereas those injected with LHYCO showed significantly lower latency.

Effect of combination of LYCO and HAECs on time in target quadrant of AD rats.

• Compared with AD or AD+PS group, the treatment group with the HAEC showed a significant increase in the average time spent in the quadrant (P<.01), which could improve Aβ-induced memory loss
• The rats treated with HAEC also experienced a longer time span than those treated with AD+sham, suggesting that the therapy also increased behavioral performance.

Effect of combination of LYCO and HAECs on total distance traveled to reach the hidden platform (path length) of AD rats

• After training, a postinjection day test was conducted to assess the distance traveled. There was a significant difference in path length of rats injected with ICV Aβ1-42 as compared to those who were not given the drug (P<.001), suggesting improvement in memory.
• The total distance to arrive the platform also showed significant amelioration of memory deficits.

Effects of combination of LYCO and HAECs on the levels of Aβ1-42 and cytokines in AD rats

• Results showed that there were significant differences in Aβ levels in the hippocampus of AD or AD+PS group rats compared to controls (P<.01)
• The levels of IL-10 and TGF-β1 in the cerebrospinal fluid (CSF) were also detected by ELISA and showed that TNF-α and IL-1β levels were significantly increased in the combined group than in the control group
• In addition, HAEC transplantation also decreased the Aβ level significantly compared to control group rats, suggesting that the protective effect of the combination on hippocampus cell injury was associated with anti-inflammatory mechanism

Effects of combination of LYCO and HAECs on the expressions of TLR4, NF-κB p65 mRNA, and protein at the CP

• Compared to the AD+PS group, lYCO significantly down-regulated the expressions OFG mRNA and protein (P<.05) and protein expressions, respectively.
• Treatment of HAEC stem cells resulted in significantly decreased LYR4 mRNA levels and protein levels, whereas treatment of TTRs resulted in significant decreases in both levels.

Discussion Alzheimer's disease (AD) is an irreversible, progressive neurodegenerative disease overlaid with neuroinflammation and cognitive decline

• No cure yet exists for AD, despite many years and humongous efforts to find efficacious pharmacological treatments
• In recent years, HAECs and alternative sources of adult stem cells have been gaining interest in regenerative medicine for the treatment of neurodegenative diseases
• Plant extracts and their bioactive compounds have received considerable attention because of their distinct pharmacology profiles, such as the rapid onset of action, less side effect profile, potential drug synergies, and most importantly, their ability to improve proliferation, differentiation, and therapeutic efficacy of stem cells
• The CP as the gatekeeper of the brain represents a suitable target for therapeutic intervention, which has potential to be manipulated in AD
• LYCO also improves cognition and memory ability of rodents in different pathological conditions
• It is a potentially protective molecule for a variety of neurologic lesions related to β-amyloid peptide
• Recent study aimed to explore its health beneficial potentials and possible mechanism

Reference

• 10.1016/j.jnutbio.2020.108558