Icaritin, an inhibitor of beta-site amyloid cleaving enzyme-1, inhibits secretion of amyloid precursor protein in APP-PS1-HEK293 cells by impeding the amyloidogenic pathway

Materials

APP-PS1-HEK293 cells were purchased from Shanghai Enzyme-linked Biotechnology (Shanghai, China). ICT (purity >98.3%) was obtained from Nanjing Zelang Medical Technologies (Nanjing, China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Biosharp (Hefei, China). A lactate dehydrogenase (LDH) assay kit was obtained from Beyotime (Shanghai, China).

An electrochemiluminescence (ECL) kit was purchased from Thermo Fisher (Waltham, MA, USA). A BACE1 Fluorescence Resonance Energy Transfer (FRET) Assay kit was obtained from Panvera (Madison, WI, USA). A PrimeScript™ RT Reagent kit and SYBR™ Premix Ex Taq were purchased from TaKaRa Biotechnology (Shiga, Japan). Antibodies against anti-BACE1, anti-presenilin 1, anti-AβP_1–40, anti-AβP_1–42, anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and goat anti-rabbit immunoglobulin (Ig)G H&L (DyLight 488) were obtained from Abcam (Cambridge, UK). Anti-C-terminal fragment β (CTFβ) antibody was purchased from Biolegend (San Diego, CA, USA). Anti-sAPPβ (6A1) antibody and anti-sAPPa (2B3) antibody were obtained from Immuno-Biological Laboratories (Fujioka-Shi, Japan). Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG antibody was purchased from Abmart (Shanghai, China).

BACE1 inhibition assay

The BACE1 inhibition assay was executed using a BACE1 FRET kit according to manufacturer instructions. A stock solution of ICT in dimethyl sulfoxide was prepared (20 mmol/L). The sample was diluted further in assay buffer to prepare a 3× concentration of test compounds. A total of 10 μL of BACE1 substrate (a new “red” FRET peptide substrate based on the “Swedish” mutant, 3× concentration) was added to 10 μL of 3× concentration of each test compound in separate wells of a black 96 well microplate and mixed gently. Then, 10 μL of 3× BACE1 was added to each well to start the reaction. Plates were incubated for 90 min at 25 °C in the dark. Then, 10 μL of sodium acetate was added to each well to stop the reaction. Finally, fluorescence measurements were undertaken with a multimode microplate reader (Multiskan™ GO; Thermo Scientific, Waltham, MA, USA) at an excitation wavelength of 545 nm and emission wavelength of 585 nm. The half-maximal inhibitory concentration (IC₅₀) was calculated by plotting the obtained relative fluorescence unit per hour (RFU/h) against the logarithmic of the inhibitor concentration. The measured inhibition data were analyzed in Prism 7 (GraphPad, La Jolla, CA, USA) by nonlinear regression (curve fitting).

Cell culture

APP-PS1-HEK293 cells subcultured by 1:3, generations were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose (SH30022.01B; Hyclone, Logan, MT, USA) supplemented with 10% fetal bovine serum (04-001-1A; Hyclone, Logan, MT, USA) and penicillin–streptomycin (SV30010; Hyclone, Logan, MT, USA) at 37 °C in a humidified atmosphere of 5% CO₂.

Cell viability by the MTT and LDH assays

Cell viability was determined by MTT and LDH assays. Briefly, APP-PS1-HEK293 cells cultured in 96 well plates (5,000/well) were treated with different concentrations of ICT for 16 h. The medium was removed and 50 μL of MTT reagent (0.5 mg/mL) in DMEM was added to each well. Then, cells were incubated for 3 h at 37 °C. After dissolving formazan crystals with 150 μL of dimethyl sulfoxide, absorbance at 570 nm was measured on a microplate reader (Multiskan™ GO; Thermo Scientific, Waltham, MA, USA). An LDH assay kit was used to evaluate injury/damage to cells according to manufacturer protocols, and absorbance at 490 nm was measured.

Quantitative polymerase chain reaction

Total RNA from cultured cell samples were extracted using TRIzol™ Reagent (Sigma–Aldrich, St. Louis, MD, USA) through a series of rinse, elution, and centrifugation steps. RNA samples were reverse-transcribed using the PrimeScript RT Reagent kit (RR047A; TaKaRa Biotechnology, Shiga, Japan). SYBR Premix Ex Taq (RR820A; TaKaRa Biotechnology, Shiga, Japan) was used following manufacturer guidelines for measurement of mRNA expression of BACE1, PS1, and α-Secretase. All primer sequences are summarized in Table 1. Fluorescence signals were amplified and detected using a StepOnePlus™ sequence detector (Thermo Scientific, Waltham, MA, USA) and analyzed with Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA). The cycle threshold (Ct) for each sample was averaged from triplicates. The 2^−ΔΔCt approach was used whereby fluorescence signals were normalized to the corresponding housekeeping gene GAPDH.

Western blotting

APP-PS1-HEK293 cell lysates were prepared. Briefly, after 30 min of incubation with lysis buffer at 4 °C, insoluble material was removed by centrifugation at 12,000 rpm for 30 min at 4 °C. The protein concentration of the lysate was determined using a bicinchoninic acid protein assay kit (GK5012; Generay, Shanghai, China). Equal amounts (10 μg) of each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (P0012AC; Beyotime, Shanghai, China) and transferred to polyvinylidene difluoride (PVDF) membranes (IPVH00010; Merck, Kenilworth, NJ, USA). PVDF membranes were blocked for 1 h in a 5% (w/v) fat-free milk solution in TBST (Tris buffer containing 0.5% (v/v) Tween-20). PVDF membranes were incubated overnight at 4 °C in 5% (w/v) bovine serum albumin solution in TBST with antibodies (all purchased from Abcam) against BACE1 antibody (1:1,000 dilution; ab2077), anti-PS1 (1:1,000; ab76083), anti-A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10; 1:1,000; ab1997), anti-AβP_1–40 (1:1,000; ab20068), and anti-GAPDH (1:1,000; ab8245). PVDF membranes were washed thrice (10 min each time) by TBST and incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (1:5,000; Abmart, Berkeley Heights, NJ, USA). Then, PVDF membranes were washed with TBST thrice and visualized using the ECL kit. Band intensity was quantified with Quantity One (Bio-Rad Laboratories, Hercules, CA, USA).

Immunofluorescence

After treatment with different concentrations of ICT for 16 h, cells plated on coverslips were fixed with methanol for 20 min at 4 °C. They were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 5 min at room temperature, and treated with blocking medium (5% bovine serum albumin in phosphate-buffered saline) for 30 min. Then, they were incubated with antibodies against anti-BACE1 (1:1,000 dilution), anti-PS1 (1:1,000), and anti-ADAM10 (1:1,000) at room temperature for 2 h. Immune-reacted primary antibody was detected following 1 h incubation in the dark at room temperature with a secondary antibody conjugated with Dylight 488 (1:1,000 dilution). Cells were further stained with 4′, 6-diamidino-2-phenylindole (C1002; Beyotime) for 2 min in the dark at room temperature and washed. Then, they were placed onto microscope slides in mounting medium. Observations were carried out using a fluorescence microscope (BX43F; Olympus, Tokyo, Japan).

Statistical analyses

Data are the mean ± SEM from at least three independent experiments. Comparisons of more than two groups were made with one-way ANOVA, followed by Dunnett’s post hoc test. SPSS v22.0 (IBM, Armonk, NY, USA) was employed for statistical analyses P < 0.05 was considered significant.

IC₅₀ of ICT for BACE1

ICT inhibited BACE1 activity and the IC₅₀ was 5.70 ± 1.09 μM (Fig. 2). ICT inhibition was like that seen for other flavonoids, such as quercetin and apigenin (Sathya et al., 2012).

Protective effect of ICT in APP-PS1-293 cells

Cell viability was measured by the MTT assay and cell-membrane damage was determined according to LDH leakage. These parameters were used to ascertain the neuroprotective effects of ICT in APP-PS1-293 cells. Compared with the control group, at ICT concentrations of 5 μM and 10 μM, the viability increased and LDH leakage decreased in APP-PS1-293 cells. These results suggested a neuroprotective effect of ICT in APP-PS1-293 cells at concentrations of 5 μM and 10 μM (Fig. 3).

Effect of ICT on the amyloidogenic pathway of APP in APP-PS1-HEK293 cells

To investigate whether the amyloidogenic pathway is involved in the protective effects of ICT in APP-PS1-HEK293 cells, we detected some of the mRNA and proteins involved in this pathway.

First, we focused on changes in expression of α-, β- and γ-secretase. We noted a significant increase in expression of ADAM10 in the ICT group compared with that in the control group (Fig. 4A; Fig. 5; Fig. 6A). We observed a significant decrease in BACE1 expression in the ICT group compared with that in the control group (Fig. 4B; Fig. 5; Fig. 6B). We documented a significant decrease in PS1 expression in the ICT group compared with that in the control group (Fig. 4C; Fig. 5; Fig. 6C). These results suggested that ICT could promote the non-amyloidogenic pathway, and inhibit the amyloidogenic pathway, of APP.

To verify our results, we detected some APP fragments. Compared with the control group, protein expression of sAPPβ and CTFβ was reduced significantly, whereas that of sAPPα increased. Finally, we found that protein expression of AβP_1–40 and AβP_1–42 in APP-PS1-HEK293 cells was lower in ICT-treated groups compared with that in the control group.