Microarray analysis of verbenalin-treated human amniotic epithelial cells reveals therapeutic potential for Alzheimer’s Disease

Characteristics of differentially expressed genes (DEGs)

In the present study, control hAEC spheroids were maintained in the placental basal medium, and the treatment hAEC spheroids were treated with 20 μM of verbenalin for seven days. The effective concentration of verbenalin on hAEC was determined using the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Supplementary Figure 1). Microarray analysis was conducted on three biological replicates of day 7 (d7) control and treatment samples, and two biological replicates of day 0 (d0) control samples. Genes satisfy both p-value <0.05 (one-way between-subjects ANOVA) and fold-change (in linear space) > 1.1 criteria simultaneously were considered as differentially expressed genes (DEGs) and were included for gene ontology (GO) analysis.

We found a total of 383 unique genes were consistently differentially expressed in all three replicates of verbenalin-treated cells and were considered as DEGs for further analysis. Among the DEGs, 137 genes were upregulated, and 246 genes were downregulated. Figure 1A shows a volcano plot displaying the DEGs. The red dots represent the significantly upregulated genes, and the green dots represent the significantly downregulated genes. There were several genes which showed large relative differences, or fold changes, however, the fold changes were not consistent among the replicates, thus, were not included for further analysis (Figure 1A, grey dots represent the non-significant genes).

Figure 1. (A) Volcano plot displaying DEGs between verbenalin-treated and untreated-control hAECs on day 7 (performed in Transcriptome Analysis Console version 4 software). The vertical axis (y-axis) corresponds to -log10 p-value of the ANOVA p-values, and the horizontal axis (x-axis) displays linear fold change. The red dots represent the up-regulated genes; the green dots represent the downregulated genes. (B) Distribution of fold changes in mRNA expression levels in verbenalin-treated hAECs (C) Pie chart showing the enriched (p < 0.05) tissue expressions by the DEGs between verbenalin-treated and untreated-control hAECs on day 7 (analyzed by DAVID online tool).

Figure 1B shows the distribution of fold changes of the DEGs. Most of the DEGs (80%) showed fold change <1.4, probably because we did not add any supplement other than verbenalin in the treated cells. For the GO analysis, we have included all the genes with fold change >1.1 (and, p<0.05) to explore the molecular changes which might be small in magnitude but are consistent. Tissue expression analysis by the ‘functional annotation’ tool of DAVID (Database for Annotation, Visualization and Integrated Discovery) software revealed that 49% of DEGs were brain-specific (Figure 1C). Further analysis of brain-specific DEGs showed that 34% genes were previously reported to be expressed in pons (n= 131), 23% in medulla oblongata (n= 90), 21% in parietal lobe (n= 82), and 20% in subthalamic nucleus (n= 75). Gene family analysis of the DEGs revealed 25 genes were transcription factors (TFs), 11 were protein kinases, and seven were growth factors. Top 20 significantly upregulated and downregulated genes and their related GO have been listed in Supplementary Tables 1 and 2, respectively.

Significantly enriched cellular components and biological process

Figure 2 shows significantly enriched cellular components (Figure 2A) and biological processes (Figure 2B) by DEGs in verbenalin-treated hAECs according to false discovery rate (FDR) q-value and p-value, respectively. Significantly enriched top cellular components include, but not limited to, cell projection (GO: 0042995), cytoskeleton (GO: 0005856), cell junction (GO: 0030054), neuron part (GO: 0097458), dendrite (GO: 0030425), and synapse (GO: 0045202). Significantly enriched top biological processes are positive regulation of dendrite development (GO: 1900006), negative regulation of type 2 immune response (GO: 0002829), guanosine triphosphatase (GTPase) activity (GO: 0043547), vasoconstriction (GO: 0045907), protein ubiquitination (GO: 0031398), regulation of GTPase mediated signal transduction (GO: 0051056), cell motility (GO: 20000145), and microtubule cytoskeleton organization (GO: 0000226).

Figure 2. (A) Significantly enriched cellular components for DEGs. (B) Top biological processes as per p-value (modified Fisher’s exact) by DEGs. (C) Significantly enriched KEGG pathways by DEGs (p < 0.05; modified Fisher’s exact test). All the gene ontology enrichment analyses were performed using DAVID online tool.

Significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways

Figure 2C shows the significantly enriched (p < 0.05; modified Fisher’s exact test) KEGG pathways by the DEGs. Several signaling pathways, namely ErbB, mitogen-activated protein kinase (MAPK), Gonadotropin-releasing hormone (GnRH), and calcium signaling pathways, as well as pathways in cancer, were significantly overrepresented. Other significantly enriched signaling pathways include regulation of actin cytoskeleton, adherens junction, axon guidance, and dorsoventral axis.

Gene set enrichment analysis (GSEA) reveals regulation of AD-related gene sets

Table 1 listed important gene sets that were significantly enriched by the DEGs between d7 verbenalin-treated and control hAECs. Gene sets were identified using the Molecular Signatures Database (MSigDB) of GSEA. Interestingly, we found that 44 DEGs were overlapped with the gene set that is upregulated in the brain from patients with AD (p=1.31 e^-12). We also found that 29 DEGs were overlapped with the gene set that is downregulated in the brain from AD patients (p=1.02 e^-7) [25]. Neurogenesis-, neuron differentiation-, and nervous system development- associated gene sets were also highly enriched.

Verbenalin treatment regulated AD-associated genes in hAECs

We found that a total of 73 AD-associated genes were regulated in the verbenalin-treated hAECs, among which 44 were reported to be upregulated (termed as ‘group A’ genes), whereas 29 genes were reported to be downregulated (termed as ‘group B’ genes) in the brain from patients with AD [25]. In the verbenalin-treated hAECs, 32 ‘group A’ genes were downregulated, and 12 were upregulated. Figure 3A shows the heatmap of the relative gene intensity of the downregulated AD-associated genes. Seven genes of this ‘group A’ genes were previously reported to be upregulated at the incipient stage of AD, namely TAO Kinase 2 (TAOK2), Carnitine Palmitoyl Transferase 2 (CPT2), Tumor Protein D52 (TPD52), Interferon, Alpha 5 (IFNA5), Transmembrane Protein 8A (TMEM8A), Family with Sequence Similarity 3, Member A (FAM3A), and Rho GTPase Activating Protein (ARHGAP) 17 (ARHGAP 17), all of which were downregulated in the treatment cells. Several transcription factors were among the ‘group A’ genes, such as Cut-Like Homeobox 1 (CUX1), Nuclear Receptor Subfamily 2, Group F, Member 6 (NR2F6), SERTA Domain Containing 2 (SERTAD2), TATA-Box Binding Protein Associated Factor (TAF) 11 (TAF11), TAF15, Transcription Factor 7-Like 1 (TCF7L1), Trichorhinophalangeal Syndrome I (TRPS1), and Upstream Transcription Factor 2 (USF2). Among the ‘group B’ genes, 25 were downregulated, and four were upregulated in the verbenalin-treated cells. Further analysis of these 25 genes showed overlap with the genes associated with ‘cell death’ (n=6), ‘genes whose expression significantly and positively correlated with the density of calbindin-containing gamma-Aminobutyric acid-ergic (GABAergic) neurons from prefrontal cortex (Brodmann area 9 (BA9) brain region) across all subjects with psychiatric disorders, such as bipolar disorder, depression, and schizophrenia’ (n=10) [26], and ‘genes whose expression significantly and positively correlated with oligodendrocyte density in layer VI (n=7), and layer III (n=5) of the BA9 brain region in patients with bipolar disorder’ [26]. We also found 11 genes of ‘group B’ were reported to be strongly associated with late-onset of AD [27] (Figure 3B), and five genes were associated with neurodegeneration (Figure 3C). Figure 3D shows the boxplots for the relative ratios of gene intensity (genes presented in the heat maps) compared between d7 control vs. d0 control and verbenalin-treated hAECs vs. d0 control. When compared with d0 control, d7 verbenalin-treated hAECs showed a better effect on AD-associated genes than d7 untreated control hAECS. Several genes (n = 22) associated with interactions of pathological hallmark proteins tubulin polymerization promoting protein/P25, β-amyloid, and α-synuclein [28] were also found to be regulated in the treatment cells. List of AD-associated genes and their fold changes compared with d0 and d7 controls is given in the Supplementary Table 3.

Figure 3. Heat maps showing relative expression intensity of genes reported to be (A) upregulated in AD human brain, (B) strongly associated with late-onset of AD, (C) associated with neurodegenerative diseases in untreated control hAECs on day 0 and day 7, and in verbenalin-treated hAECs on day 7. (D) Boxplots for the relative ratios of gene intensity (genes presented in the heat maps) in day 7 control (Control_D7) and verbenalin-treated hAECs compared with day 0 control. Box ranges from 25^th to 75^th percentile, the line in the middle represents the median value, the whiskers represent the min, max, and mean values, and the error bar represents the SD. Significance was computed by One-way ANOVA for linear distribution and Mann-Whitney U test for nonlinear distribution. Heat maps were generated using Morpheus online tool.

Verbenalin treatment regulated the gene expression of the receptors of the ErbB pathway

The dysregulation of ErbB signaling in humans is associated with the development of AD. In the microarray analysis, we found that verbenalin treatment significantly downregulated the expression of epidermal growth factor (EGF) receptor (EGFR) (fold change -1.41), and vascular endothelial growth factor B (VEGFB) (fold change -1.89). On the other hand, the expression of neuregulin 1 (NRG1) was significantly upregulated (fold change 1.34). A similar gene expression result was found in the real-time PCR (RT-PCR) analysis (Figure 4A). Although not significant, EGFR and NRG1 showed similar protein expression patterns as gene expression (Figure 4B). However, VEGF showed a slight upregulation in protein expression (Figure 4B). NRG1 is the first discovered member of the NRG family that contains the EGF-like domain. NRGs and related EGF-domain containing proteins interact with different receptor tyrosine kinases of the ERBB family (ERBB1- 4) and initiate intracellular signaling pathways in a specific way. NRG1 is the direct ligand for ERBB3 and ERBB4 tyrosine kinase receptors, and concomitantly recruits ERBB1 and ERBB2 coreceptors, resulting in ligand-stimulated tyrosine phosphorylation and activation of the ERBB receptors. Adenomatous polyposis coli (APC) that acts as a mediator of ERBB2-dependent stabilization of microtubules at the cell cortex was downregulated (fold change -1.13).

Figure 4. Effect of verbenalin treatment on the expressions of EGF, VEGF, and NRG1. The hAECs were treated with 20 μM of verbenalin (Ver) for 7 days, while the control cells were maintained in the placental basal medium. (A) Gene expressions were evaluated by real-time PCR. Each value represents the mean ± SD (n = 3). Asterisks refer to statistical significance (*p < 0.05, **p < 0.01) by One-way ANOVA as compared with control (Ctrl). (B) Boxplots of protein concentration (ng/ml) obtained by ELISA (n = 4). Box ranges from 25^th to 75^th percentile, the line in the middle represents the median value, the whiskers represent the min, max, and mean values, and the error bar represents the SD. The difference in protein concentration between treatment and control group was measured using One-way ANOVA for linear distribution.

Verbenalin treatment regulated expression of genes of Rho family GTPases

The Rho GTPases belong to the Ras superfamily of small (molecular weight ~21kDa) guanine nucleotide-binding proteins (G-proteins). The most extensively studied members of the Rho family are Ras Homolog Family Member A (RHOA), Ras-related C3 botulinum toxin substrate 1 (RAC1), and cell division cycle 42 (CDC42). We found that verbenalin treatment significantly downregulated the expression of several genes of ARHGAP, such as ARHGAP21 (fold change -2.00), ARHGAP17 (fold change -1.38), ARHGAP4 (fold change -1.37), ARHGAP19 (fold change -1.11). ARHGAP21 functions as the GTPase activator for RHOA and CDC42, ARHGAP17 for CDC42, and ARHGAP19 for RHOA. We also found the downregulation of family with sequence similarity 65, member B (FAM65B), an inhibitor of the small GTPase RhoA (fold change - 1.93). Additionally, pleckstrin homology domain-containing family G member 6 (PLEKHG6), a guanine nucleotide exchange factor activating the small GTPase RhoA, was also downregulated (fold change -1.38).

Verbenalin treatment regulated genes associated with circadian entrainment

Circadian entrainment is the biological process by which endogenous oscillations are synchronized with external cues, such as daily light and temperature cycles, within a period of ~24 h. The circadian clock coordinates the daily molecular, hormonal, physiological, and behavioral rhythms. We found several genes associated with circadian rhythm were downregulated in the verbenalin-treated hAECs, such as TFs NK2 homeobox 1 (NKX2-1; fold change -1.13) and retinoic acid-induced protein 1 (RAI1; fold change -1.13), imprinted gene GNAS complex locus (GNAS; fold change -1.27), and G protein beta polypeptide 1 (GNB1; fold change -1.19). Although not specific, there was also the downregulation of voltage-gated Ca²⁺ channel CACNA1C (fold change -1.13) and Ca²⁺/calmodulin-dependent kinase (CAMK) II gamma (CAMK2G; fold change -2.07) in the verbenalin-treated cells.

Verbenalin treatment showed neuroprotective effects against Aβ-induced cytotoxicity in human neuroblastoma SH-SY5Y cells

As unexpectedly, our study results showed that verbenalin treatment could significantly regulate AD-associated genes in hAECs, we investigated its neuroprotective effects against Aβ-induced cytotoxicity in human neuroblastoma-derived SH-SY5Y cells. The human SH-SY5Y cell line is a widely-used cellular model to examine the toxic effects of amyloid peptides. We found that verbenalin was nontoxic up to the concentration of 20 μM (Figure 5A). Therefore, 20 μM of verbenalin was used for determining its effects on Aβ-induced neuronal cell damage. When SH-SY5Y cells were exposed to 5 μM of Aβ for 72 h, there was significant cell death compared with untreated controls (Figure 5B). However, in cultures pre-treated with 20 μM of verbenalin for 24 h (Figure 5B), the Aβ-induced cell death was significantly reduced compared to only Aβ-treated conditions, suggesting the neuroprotective effect of verbenalin against Aβ-induced cytotoxicity in SH-SY5Y cells.

Figure 5. Neuroprotective effects of verbenalin (Ver) on amyloid beta (Aβ)-induced toxicity in human neuroblastoma SH-SY5Y cells. (A) Cells were exposed to verbenalin at concentrations of 1, 5, 10, 20, and 40 μM for 72 h. The control cells were not treated. Cell viability was measured by the MTT assay and was calculated as a percentage of that in the control group (100%). The results are expressed as the means ± standard error of the mean (SEM) of independent experiments (n = 6, 96-well plate). ***p < 0.001 as compared to control. Cells were pre-treated with 20 μM verbenalin for 24 h and then exposed to 5 μM Aβ for 72 h. The results are expressed as the means ± standard error of the mean (SEM) of independent experiments (n = 6, 96-well plate). ^†p < 0.1, *p < 0.05, **p < 0.01 compared with the group exposed to Aβ only (ANOVA followed by Dunnett’s multiple comparisons test). (B) Cell viability was measured by the MTT assay and was calculated as a percentage of that in the control group (100%). (C) A bioluminescence assay was used to measure cellular ATP levels, and the results are shown as relative intracellular ATP levels. (D) Levels of intracellular reactive oxygen species (ROS) were measured using a fluorescence cell-based assay, and results are shown as relative intracellular ROS (n=4).

Verbenalin treatment significantly ameliorated the Aβ-induced decline of ATP levels in SH-SY5Y cells

Figure 5C shows the effect of verbenalin treatment on Aβ-induced ATP decline. Exposure to 5 μM of Aβ for 24 h resulted in a significant decrease in ATP production compared to control cells. However, pretreatment with verbenalin for 24 h could rescue the reduction of ATP production in Aβ-treated SH-SY5Y cells (p < 0.01).

Verbenalin treatment attenuated Aβ-induced reactive oxygen species (ROS) generation in SH-SY5Y cells

The effect of verbenalin treatment on oxidative stress was detected by testing the level of intracellular ROS. Figure 5D shows that after exposure to 5 μM of Aβ for 24 h, ROS production was increased compared to untreated cells (p = 0.22). When the cells were pretreated with 20 μM of verbenalin for 24 h, ROS production was decreased compared to only Aβ-treated SH-SY5Y Cells (p = 0.44), suggesting the preventive effect of verbenalin against Aβ-induced oxidative stress. However, the changes did not achieve statistical significance.

Verbenalin treatment regulated the expressions of EGFR, VEGF, and NRG1 in Aβ-induced SH-SY5Y cells

We investigated how verbenalin treatment could modulate the expressions of EGFR, VEGF, and NRG1 in Aβ-induced neurotoxic condition. Figure 6A shows that verbenalin could inhibit Aβ-induced EGFR activation, and upregulated the expressions of VEGF and NRG1. EGFR and NRG1 showed similar protein expression patterns as gene expression (Figure 6B).

Figure 6. Effect of verbenalin treatment on the expressions of EGF, VEGF, and NRG1 in Aβ-induced human neuroblastoma SH-SY5Y cells. (A) Gene expressions were evaluated by real-time PCR. Each value represents the mean ± SD (n = 4). (B) Boxplots of protein concentration (ng/ml) obtained by ELISA (n = 4). Box ranges from 25^th to 75^th percentile; the line in the middle represents the median value; the error bar represents the SD. Asterisks refer to statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001) by One-way ANOVA followed by Dunnett’s multiple comparisons test (for linear distribution) as compared with only Aβ-treated group.

Amnion epithelial cells extraction and culture

The detailed methodology has been explained elsewhere [64]. Briefly, AECs were isolated from the delivered term placenta of the mothers who underwent cesarean delivery. The amnion was separated from the chorion manually and was washed with 200 mL of Hank’s Basic Salt Solution – Calcium and Magnesium Free (CMF-HBSS) and then was cut into smaller pieces using surgical scissors. AECs were maintained in Placenta Epithelial Cell Basal Medium (PromoCell, Cat. # C-26140). The medium was changed every 2-4 days. To subculture AECs, the plates were first washed twice with 10 mL of PBS, and then 3 mL of pre-digestion buffer (pre-warmed to 37°C) was added to the plate. After incubation at 37°C for 5 minutes, five mL of 0.05% (w/v) trypsin-EDTA (pre-warmed to 37°C) was added to the plate and incubated at 37°C for 10 minutes. Five mL of Dulbecco’s Modified Eagle Medium (DMEM) was added to stop the reaction. The cell suspension was then centrifuged at 200 RPM for 4 minutes at 4°C twice. After centrifuge, the supernatant was discarded each time, and the cells were suspended in the placental basal medium.

Preparation of 3D culture plates, spheroid formation, and compound supplement

We used a 3D culture plate (ElplasiaTM, Kuraray Co., Ltd., Cat # RB 500 400 NA 24) for the study. Lipidure ^TM (NOF Corporation, Cat. # CMS206; 400 μL) solution was placed into each well of the 3D plate at the concentration of 50 mg in 10 mL absolute ethanol. After two minutes, the Lipidure ^TM solution was aspirated out, and the plate was dried for 3 hours. Then 400 μL of PBS was placed in each well. The plate was centrifuged at 2000 g for 15 minutes at room temperature. The PBS was then discarded, and the wells were washed twice with 400 μL of PBS. The plates were then stored in the cell culture incubator until use.

Spheroids were formed by seeding 1 × 10⁶ AECs in Placenta Basal Epithelial Cell Medium into each well of the 24-well plate. The initial culture was maintained for 24 hours. Control samples for d 0 were collected before adding the treatment. For the treatment samples, the medium was changed with 20 μM of verbenalin (Sigma-Aldrich, Japan) every 48 hours for three times. Control samples were maintained in the Placental medium, which was also changed in every 48 hours. Finally, the treatment and control samples were collected from a one-week culture.

RNA extraction and quantification

RNA was extracted using Isogen (Nippon Gene Co. Ltd., Toyama, Japan) kit following the manufacturer’s guide. NanoDrop 2000 spectrophotometer (ThermoScientific, Wilmington, DE, USA) was used to quantify the integrity of RNA.

Affymetrix microarray gene expression

We conducted Affymetrix microarray gene expression profiling using GeneChip® 3’ Expression Arrays and 3’ IVT PLUS Reagent Kit (Affymetrix Inc., Santa Clara, CA, USA). We used 250 ng of total RNA from each sample to generate amplified and biotinylated complementary RNA (cRNA) from poly (A) RNA in a total RNA sample following the users’ manual. Human Genome U219 array strips (HG-U219) were hybridized for 16 hours in a 45°C incubator, washed and stained. Imaging was conducted in the GeneAtlas Fluidics and Imaging Station. Each HG-U219 array strip is comprised of more than 530,000 probes, which cover approximately 36,000 transcripts and variants and represent more than 20,000 unique genes.

Microarray data processing and analysis

Expression Console Software (provided by the Affymetrix) was used to normalize the raw data following the robust multichip average (RMA) algorithm (http://www.affymetrix.com). Subsequent analysis of the gene expression data was carried out in the freely available Transcriptome Analysis Console (TAC) version 4 (Thermofisher Inc.). In this present study, we have considered both fold changes and variability of gene expression for the identification of DEGs. Raw fold change between two experimental conditions does not take the variance of gene expression among the replicates into account. Therefore, it does not provide statistical confidence that the genes will show similar fold-change threshold in future experiments. Therefore, we defined DEGs as the genes that satisfy both p-value <0.05 (one-way between-subjects ANOVA) and fold-change (in linear space) > 1.1 criteria simultaneously. MSigDB of GSEA was used to determine whether a priori-defined set of genes shows statistically significant and concordant differences between two biological states, i.e., verbenalin-treated versus non-treated control (https://software.broadinstitute.org/gsea/index.jsp) [65, 66]. MSigDB emphasizes a genomic, unbiased approach to define the gene sets, which are curated from published expression profiles and allows researchers to evaluate microarray data at the level of gene sets, which tend to be more reproducible and more interpretable. Gene annotation, tissue expression, and pathway analysis for the DEGs were conducted using an online data mining tool DAVID ver. 6.8 [67, 68]. Heat maps were generated using visualization software Morpheus (https://software.broadinstitute.org/morpheus). All data generated or analyzed during this study are included in this published article and its supplementary files. Microarray data are deposited in the Gene Expression Omnibus (GEO) under Accession Number: GSE137061 (https://www.ncbi.nlm.nih.gov/geo/info/linking.html).

Real-time PCR

To synthesize cDNA from total RNA, the SuperScript III reverse transcriptase kit (Invitrogen, Carlsbad, CA, USA) was used. Primers and probes for human vascular endothelial growth factor B (VEGFB) (Hs00173634_m1), human epidermal growth factor receptor (EGFR) (Hs01076090_m1), human neuregulin 1 (NRG1) (Hs01101538_m1) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Hs02786624_g1) were purchased from Applied Biosystems (Foster City, CA, USA). The gene expression was normalized to GAPDH expression. The real-time PCR amplification and product detection were performed with a 7500 Fast Real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (Applied Biosystems). Each reaction was run in triplicate, and data were analyzed using the ΔΔCt method.

Cell cytotoxicity test

The SH-SY5Y cells (American Type Culture Collection (ATCC), Manassas, VA, USA) and hAECs were seeded in 96-well plates at a density of 2.0 × 10⁵ cells/ml and 1.0 × 10⁵ cells/ml respectively and were incubated for 24 h. The following day, the cells were treated with various concentrations (1, 5, 10, 20, and 40 μM) of verbenalin for 72 h. After the treatment, the MTT (Dojindo Laboratories, Kumamoto, Japan) solution was added to each well (10 μl/well) and was incubated at 37°C for 24 h. Then, the generated formazan crystal was dissolved with 100 μl of 10% Sodium Dodecyl Sulfate (SDS) (Nippon Gene, Tokyo, Japan) in each well, and was incubated overnight at 37°C. After that, the absorbance was measured at 570 nm using a microplate reader (Power Scan HT, BioTEK Japan Inc.). The values were normalized to the value of the medium and were calculated as the percentage (%) of control.

In vitro neuroprotection assay

The human neuroblastoma SH-SY5Y cell line was obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). The cells were maintained in a mixture of 1:1 (v/v) of Dulbecco’s modified eagle medium (DMEM), and nutrient mixture F-12 (Ham) supplemented with 15% heat-inactivated FBS (Gibco, Japan), 1% MEM non-essential amino acids solution (Biological Industries, Beit Haemek, Israel) and 1% penicillin (5000 μg/mL)− streptomycin (5000 IU/mL) solution (Lonza, Basel, Switzerland) on 100 mm culture dish (Falcon, Corning, NY, USA) or 96-well culture plates (Falcon) at 37 °C in a 95% humidified air−5% CO2 incubator. A serum-free Eagle’s minimum essential medium (Opti-MEM) (Gibco, Japan) was used to culture the cells for the neuroprotection assay.

The neuroprotective activity was determined by the MTT reduction assay. SH-SY5Y cells were seeded at a density of 2.0 × 10⁴ cells/well in the 96-well culture plates and incubated for 24 h. The cells were pre-incubated with 20 μM verbenalin in Opti-MEM for 24 h and then were subjected to treatment with 5 μM Aβ and the sample for 24 h. The treated medium was replaced with 100 μL of Opti-MEM in the absence of verbenalin and Aβ. Subsequently, 10 μL of MTT (Dojindo Laboratories, Kumamoto, Japan) dissolved in PBS (−) at 5 mg/mL was added into the medium. After overnight incubation at 37 °C, 100 μL of 10% SDS (w/v) was added and incubated until the formazan product dissolved. The absorbance was measured at the wavelength of 570 nm with a Varioskan Lux multimode microplate reader (Thermo Fisher, Waltham, MA, USA). The proliferation of SH-SY5Y cells was shown by the percentage of the Aβ-treated group.

ATP assay

SH-SY5Y cells were seeded at a density of 2.0 × 104 cells/well in the 96-well culture plates and incubated for 24 h. The cells were then pre-incubated with 20 μM verbenalin in culture medium for 24 h. After 24 h incubation, the medium was replaced with 100 μl of culture medium containing 5 μM Aβ and 20 μM verbenalin. After 24 h, 100 μl of Cellular ATP measurement reagent (CA2-100, TOYO B-NET, Tokyo, Japan) was added and stirred for 1 min on a plate shaker. Then 150 μl of the suspension was transferred to a white plate and was let stand for 10 min in a luminometer set at 23 °C. The luminescence was measured with a Varioskan Lux multimode microplate reader.

Cellular ROS assay

Cellular ROS production was measured using OxiSelect Intracellular ROS Assay Kit (Green Fluorescence, STA-342, Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, SH-SY5Y cells were seeded at a density of 2.0 × 10⁴ cells/well in the 96-well culture plates and were incubated for 24 h. The cells were then pre-incubated with 20 μM verbenalin in culture medium for 24 h. The culture medium was later replaced with 100 μl of 0.1x DCFH-DA and the cells were incubated for 1 h at 37 °C. Then 0.1x DCFH-DA was replaced with 100 μl of Opti-MEM containing 5 μM of Aβ and 20 of μM verbenalin. The cells were incubated at 37 °C for 24 h. The sample-containing medium was replaced with 200 μl of 1 × Cell Lysis Buffer, shaken for 1 min on a plate shaker, and incubated for 5 min at room temperature (24 °C). Finally, 150 μl of cell lysate was transferred to a black plate. Fluorescence wavelength was detected at 480nm excitation / 530nm emission with a Varioskan Lux multimode microplate reader.

Protein isolation and detection

SH-SY5Y cells were seeded at a density of 6.0 × 105 cells / 3ml / well in a 6-well plate. After 24 h of incubation at 37 °C, the cells were pre-incubated in 3 ml of Opti-MEM medium containing 20 μM Verbenalin for another 24 h. The medium was then replaced with 3 ml of Opti-MEM medium containing 5 μM Aβ and 20 μM verbenalin. The medium was collected after 72 h. commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure VEGF (ab100662), NRG1 (ab100614), and EGFR (ab100505) according to the manufacturer’s instructions (Abcam, Cambridge, UK). The absorbance was measured at 450 nm on a Varioskan Lux multimode microplate reader.

Statistical analysis

Data were analyzed using GraphPad Prism (version 8.0, GraphPad Software Inc., San Diego, CA) and SPSS ver.26 (Armonk, NY: IBM Corp). Data were expressed as the means ± standard error of the mean (SEM) unless otherwise mentioned. One-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test for linear distribution and Mann-Whitney U test for nonlinear distribution was carried out for the comparisons between treatment groups. Differences were considered statistically significant at the value of P < 0.05. Graphs were prepared using OriginPro software (OriginPro 2020, OriginLab Corporation, Northampton, MA, USA).

Ethics approval

The protocol was reviewed and approved by the Ethical Review Committee of the University of Tsukuba. Informed written consent was obtained from the mothers who donated the placenta after delivery.