Synthetic Aβ_1-42 (> 95% purity) was purchased from Sangon Biotech Company (Shanghai, China). AS (purity 98.86%, MW 959.133, Figure 1A) was purchased from PUSH Bio-Technology, Co., Ltd. (Chengdu, Sichuan, China). TAK-242 (resatorvid) was purchased from MedChemExpress (Monmouth Junction, NJ, United States). CCK-8 assay and Annexin V-FITC apoptosis detection kit were purchased from Dojindo Chemical Technology Co., Ltd. (Shanghai, China). All antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, United States). Hoechst 33258 kit, JC-1 kit, Dulbecco’s modified Eagle’s medium (DMEM), dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Beyotime (Haimen, Jiangsu, China).

The hBMEC line was purchased from Procell Life Science and Technology, Co., Ltd. (Wuhan, Hubei, China). The cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin in an atmosphere of 95% air, and 5% CO₂ at 37°C in a humidified incubator and renewed with new medium every 3–5 days.

Synthetic Aβ_1-42 was prepared to a final stock solution of 100 μM by dissolving in hexafluor-2-propanol (HFIP) to foster the fibrillization state. Briefly, 1 mg of Aβ_1-42 was dissolved in 1 ml HFIP solution and distributed into centrifuge tubes after 15 min ultrasonic treatment. HFIP was removed by evaporation under vacuum at room temperature for overnight and the tubes were stored at -20°C before use. For experiments, one tube containing Aβ_1-42 solution was taken out 24 h before experiment and resuspended in HFIP/water (ratio 3:7) for 15-min ultrasonic treatment and stirred for another 24 h; then, the solvent HFIP was evaporated under speedvac for 10 min to make 100 μM of fibrillar Aβ_1-42 as the final solution. TAK-242 is a specific inhibitor of TLR4 receptor and used as a positive control for comparison of the effect of AS (Li et al., 2006). TAK-242 was dissolved in DMSO to a final concentration of 100 mM and stored at -20°C for future use. The concentration (1 μM) selection of TAK-242 for the present in vitro study was based on previous reports from the literature (Raveendran et al., 2011; Hussey et al., 2013). AS was first dissolved in methanol at a concentration of 10 mM as the stock solution and further diluted with culture medium without bovine serum to appropriate concentrations.

The effects of AS and/or Aβ_1-42 on the proliferation of hBMECs were determined by CCK-8 assay. The cells (∼80–90% confluency) were seeded in 96-well plates (1.0 ml) at a density of 5 × 10⁴ cells/well and cultured for 24 h. Then, the culture medium was replaced with serum-free DMEM and the cells were treated with different concentrations (3.125, 6.25, 12.5, 25, 50, and 100 μM) of AS or TAK-242 (1 μM) for 12 h. The vehicle solution was used as control for all experiments after it was compared to 1% methanol solution by CCK-8 assay and showed no significant difference in cell growth inhibition between the both solutions (97.00 ± 2.30% vs. 96.85 ± 3.37% in cell viability). The cells were washed with phosphate buffered solution (PBS) for 15 min at room temperature after 12 h treatment and then treated with 50 μM Aβ_1-42 or serum-free DMEM (vehicle control) for another 24 h. After being cultured for 24 h, CCK-8 (10 μl/well) was added into the cells and incubated for 1 h. The absorbance was measured at 450 nm using SpectraMax M3 microplate readers (Molecular Devices Corporation, Sunnyvale, CA, United States). All experiments were performed three times in triplicate.

The changes in the mitochondrial membrane potential were determined by JC-1, a fluorescent dye with potential-dependent accumulation in mitochondrial membrane as a monomer (fluoresced green) or dimer (fluoresced red-orange) in living cells as previously described (Wu et al., 2013). Briefly, the cells were seeded at a density of 2 × 10⁵ cells/well in 6-well plates and treated with different concentrations of AS (25, 50, and 100 μM), TAK-242 (1 μM), or same volume of serum-free DMEM (vehicle control) for 12 h, then treated with Aβ_1-42 (50 μM) or medium for another 24 h at 37°C. The cells were washed with PBS twice and incubated with JC-1 (5 μM) for 20 min at 37°C in a CO₂ incubator, then washed with PBS three times for 5 min each time at 37°C in the dark. Fluorescent images were visualized and recorded under a fluorescence microscope at ×100 magnifications (AMG EVOS, Thermo Fisher Scientific, Inc., Waltham, MA, United States). All experiments were performed three times in triplicate.

Morphological assessment of apoptotic cells was processed by Hoechst 33258 staining method as described earlier (Yang et al., 2013). Briefly, hBMECs were seeded in 6-well culture plates at a density of 1 × 10⁵ cells/well and treated with various concentrations of AS (25, 50, and 100 μM), TAK-242 (1 μM), or same volume of serum-free DMEM (vehicle control) for 12 h, then treated with Aβ_1-42 (50 μM) or medium for another 24 h at 37°C. After incubating with fixative solution for 10 min, the medium was removed and washed with PBS for 15 min. Then, the cells were treated with 500 μL staining solution of Hoechst 33258 (10 μg/ml) for 5 min, followed by washing with PBS for 15 min at a dark room for reducing the background. Finally, the nuclear morphological changes of apoptotic cells were observed under a fluorescent microscope at ×400 magnifications (AMG EVOS, Thermo Fisher Scientific, Inc., Waltham, MA, United States) and the percentage of apoptotic cells was calculated according to the ratio of apoptotic cells to total cells. All experiments were performed three times in triplicate.

The cellular apoptosis of hBMECs were also determined by flow cytometry with an Annexin V-FITC apoptosis detection kit following manufacturer’s instruction. Briefly, the cells (∼80–90% confluency) were seeded in 6-well plates at a density of 2 × 10⁵ cells/well and cultured for 24 h. Then, the culture medium was replaced with serum-free DMEM and the cells were treated with AS at 25, 50, and 100 μM, TAK-242 at 1 μM, or same volume of serum-free DMEM (vehicle control) for 12 h. The cells were washed with PBS for 15 min at room temperature after 12 h treatment and treated with Aβ_1-42 at 50 μM or same volume of medium for another 24 h. The cell suspensions were centrifuged at 1,500 × g for 3 min at room temperature. After centrifugation, the apoptotic cells were resuspended in Annexin V-FITC binding buffer. A total of 5 μL Annexin V-FITC and 5 μL PI were successively added to the cells and incubated for 15 min in the dark at room temperature. The quantity of stained cells was determined by flow cytometry (FACSCalibur, BD Biosciences, San Diego, CA, United States). All experiments were performed three times in triplicate.

The effects of AS and/or Aβ_1-42 on the levels of TNF-α and IL-6 in hBMECs were determined by ELISA following manufacturer’s instruction. Briefly, the cells (∼80–90% confluency) were seeded in 96-well plates at a density of 2 × 10⁵ cells/well and cultured for 24 h. Then, the culture medium was replaced with serum-free conditioned medium and the cells were treated with AS at 25, 50, and 100 μM, TAK-242 at 1 μM, or same volume of medium (vehicle control) for 12 h. The cells were washed with PBS for 15 min at room temperature after 12 h treatment and treated with Aβ_1-42 at 50 μM or same volume of medium for another 24 h. The cell suspensions were centrifuged at 1,500 × g at room temperature for 5 min. Supernatants were analyzed for the levels of TNF-α and IL-6 by ELISA kit (R&D Systems, Minneapolis, MN, United States). The absorbance was determined at 450 nm using SpectraMax M3 microplate readers (Molecular Devices Corporation, Sunnyvale, CA, United States). All experiments were performed three times in triplicate.

The hBMECs were seeded at a density of 4.0 × 10⁵ cells/well on 2.0 ml 6-well plates and replaced the culture medium with serum-free DMEM after 24 h culture. Then, the cells were treated with medium (vehicle control), AS (25, 50, and 100 μM), or TAK-242 (1 μM) for 12 h. The cells were washed with PBS for 15 min at room temperature after 12 h treatment and treated with Aβ_1-42 at 50 μM or same volume of medium for another 24 h, then, withdrawn the culture medium, and washed with cold PBS for harvest. The cell pellets were disrupted in cell lysis buffer (RIPA buffer, 0.5% NP-40, 50 mM Tris-HCl, 120 mM NaCl, 1 mM EDTA, 0.1 mM Na₃VO₄, 1 mM NaF, 1 mM PMSF, and 1 μg/mL leupeptin, pH 7.5), and then, the lysates were centrifuged at 9600 × g for 15 min at 4°C. Equal amounts of protein samples (30 μg) were electrophoresed on 12% SDS–PAGE gel for separation, followed electrophoresis, the proteins were electrotransferred from the gels to PVDF membranes to form blots. Non-specific binds were blocked with 5% skim milk in Tween-PBS buffer for 1 h, and then the membranes were incubated with anti-TLR4, anti-MyD88, anti-TRAF6, anti-NF-κB p65, and anti-NF-κB p65 (phospho S468) primary (1:1000) and secondary (1:5000) antibodies or β-actin (13E5) rabbit monoclonal antibody (1:1000) at 4°C overnight and washed three times with Tween-PBS buffer. Then, the blots were incubated for 1 h at room temperature with a 1:1000 dilution of horseradish peroxidase-labeled anti-rabbit or anti-mouse IgG (H+L) and washed three times with Tween-PBS buffer. The membranes were then developed and visualized by incubation with ECL Western detection reagents (Thermo Fisher Scientific Inc., Waltham, MA, United States). The specific protein bands were visualized and analyzed with a ChemiDoc image analyzer (Bio-Rad, Hercules, CA, United States). All experiments were performed three times in triplicate.

The cells were seeded at a density of 1.0 × 10⁵ on glass coverslips and treated with medium (vehicle control), AS (25, 50, and 100 μM), or TAK-242 (1 μM) for 12 h. Then, the cells were washed with PBS for 15 min at room temperature after 12 h treatment and treated with Aβ_1-42 (50 μM) or same volume of medium for another 24 h. The cells were fixed in 4% paraformaldehyde for 30 min at 4°C and washed with PBS for three times with 5 min for each time, then the cells were incubated with 0.1% Triton X-100 for 10 min, and blocked with 1% bovine serum albumin (BSA) for 30 min. The cells were incubated with rabbit monoclonal primary antibody (1:500) against NF-κB p65 at 4°C overnight, followed by HRP-conjugated goat anti-rabbit anti-NF-κBp65 secondary antibody (1:2000) for 2 h and washed with PBS for 15 min. The cells were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) for 10 min and washed with PBS for 15 min. The signals were detected and assessed under an inverted phase contrast fluorescence microscope (LD laser: 405 nm, 25 mW; multi-line Ar laser: 458, 488, and 515 nm, 40 mW; HeNe green laser: 543 nm, 1 mW; and HeNe red laser: 635 nm, 20 mW) at ×400 magnifications (OLYMPUS company, model: CKX41, Shinjuku-ku, Tokyo, Japan). All experiments were performed three times in triplicate.

All experimental data were analyzed by IBM SPSS18.0 statistical software (SPSS Inc., Chicago, IL, United States) and expressed as mean ± SD. One-way or two-way univariate analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) test were performed for the analysis of variance and comparison of different groups. A difference at p < 0.05 was considered as statistical significance (marked as ^∗) and higher significance level was set at p < 0.01 (marked as ^∗∗).

First, we evaluated the effects of AS at various concentrations (3.125, 6.25, 12.5, 25, 50, and 100 μM) and TAK-242 (1 μM) alone for 12 h exposure on cell viability of hBMECs by CCK-8 assay. The results showed that AS up to 100 μM and TAK-242 at 1 μM had no significant cell growth inhibition with the cell viability over 95% (data not shown). Next, we treated the cells with three higher concentrations (25, 50, and 100 μM) of AS and 1 μM TAK-242 for 12 h, then followed by treatment of 50 μM Aβ_1-42 for additional 24 h. The results showed that Aβ_1-42 (50 μM) treatment for 24 h significantly (p < 0.01) inhibited the cell proliferation with the cell viability of 50.89 ± 5.12% compared to 96.85 ± 3.37% in control cells treated with vehicle. However, AS treatments (25, 50, and 100 μM) significantly attenuated cell growth inhibition (p < 0.01) induced by Aβ_1-42 (50 μM) in a concentration-dependent manner (Figure 1B). Pretreatment of TAK-242 (1 μM) also protected the cells from Aβ_1-42-induced cell growth inhibition closed to that of 100 μM of AS (Figure 1B). These data suggest that AS and TAK-242 were effective to attenuate cell growth inhibition induced by Aβ_1-42 in hBMECs.

Mitochondria play a critical role in cellular apoptosis and decrease of mitochondrial membrane potential is a hallmark event in the early stage of apoptosis in cells (Wlodkowic et al., 2011). Therefore, we evaluated the effects of AS (25, 50, and 100 μM) and TAK-242 (1 μM) for 12 h followed by Aβ_1-42 (50 μM) treatment for additional 24 h on mitochondrial membrane potential (ΔΨm) in hBMECs using JC-1 fluorescence and the results are illustrated in Figure 2. From the representative photographs, we can see that the fluorescence intensity of red and the ratio of red/green in hBMECs were significantly (p < 0.01) decreased (∼2.5-fold) by Aβ_1-42 (50 μM) treatment compared to that of the control cells treated with vehicle (Figures 2Aa,b,B). However, the ratio of red/green fluorescence was significantly increased (p < 0.01) in the cells pretreated with AS at 25, 50, or 100 μM in a concentration-dependent manner (Figures 2Ac–e). TAK-242 at 1 μM (Figure 2Af) also exhibited similar effect as 100 μM of AS on the ratio of red/green fluorescence. The data indicate that mitochondrial membrane potentials of hBMECs were significantly decreased by Aβ_1-42 but AS and TAK-242 restored the declined ΔΨmm induced by Aβ_1-42 (Figure 2B), therefore to inhibit Aβ_1-42-induced apoptosis via maintaining high mitochondrial membrane potential.

The crucial factor for pathogenesis of AD is neuronal cell damage and loss due to Aβ-induced apoptosis (Loo et al., 1993; Crews and Masliah, 2010). Therefore, we studied the protective effect of AS on apoptosis induced by Aβ_1-42 in hBMECs.

Hoechst 33258 staining is a classical and rapid method for the detection of apoptosis by observation of chromatin condensation under a fluorescence microscopy (Yang et al., 2013). The representative morphological photographs of hBMECs and the cell apoptotic ratio by Hoechst 33258 staining method are illustrated in Figure 3. The photograph in Figure 3Aa shows the characteristic preserved normal features of hBMECs in the control cells without Aβ_1-42 treatment, which the cell nuclei appear blue fluorescence. However, higher levels of apoptotic cells with condensation of nuclear chromatin and fragmentation stained with white color were detected in the cells treated with Aβ_1-42 (50 μM) for 24 h (Figure 3Ab). Interestingly, the numbers of apoptotic cells were markedly decreased with pretreatment of AS (25, 50, and 100 μM) or TAK-242 (1 μM) for 12 h (Figures 3Ac–f). The summarized results of apoptotic ratio of cells are shown in Figure 3B and indicate that Aβ_1-42 (50 μM) treatment significantly increased apoptosis (over 70% apoptotic cells) compared to that of control cells treated with vehicle (less than 10% apoptotic cells, p < 0.01), while pretreatment of AS significantly inhibited Aβ_1-42-induced apoptosis in a concentration-dependent manner (p < 0.01) in hBMECs. In additional TAK-242 (1 μM) exhibited inhibitory effect on Aβ_1-42-induced apoptosis as more potent than that of 50 μM but less than that of 100 μM of AS.

Annexin V is labeled with fluorescein (FITC) as fluorescent probe and one of the sensitive methods to detect the early apoptosis of cells (Wlodkowic et al., 2011). Therefore, we further investigated the quantity of apoptotic cells induced by Aβ_1-42 and the protective effects of AS (25, 50, and 100 μM) and compared to that of TAK-242 (1 μM) with Annexin V/PI double staining by flow cytometric analysis and the results are illustrated in Figure 4. The data showed that 24 h treatment of Aβ_1-42 (50 μM) significantly induced apoptosis in hBMECs to 66.40 ± 4.82% compared to 13.82 ± 2.22 % (p < 0.01) in control cells treated with vehicle (Figures 4Aa,b). Interestingly, pretreatment of AS at 25, 50, and 100 μM significantly decreased apoptotic cells (p < 0.01) induced by Aβ_1-42 (50 μM) compared to Aβ_1-42 alone to 35.20 ± 5.26%, 22.20 ± 3.66%, and 15.28% ± 2.18% in hBMECs, respectively (Figures 4Ac–e,B). Pretreatment of TAK-242 (50 μM) also significantly inhibited Aβ_1-42-induced apoptosis with 18.48 ± 2.58% in hBMECs (Figures 4Af,B). The results clearly demonstrated that AS effectively inhibited Aβ_1-42-induced apoptosis in a concentration-dependent manner in hBMECs by both methods of Hoechst 33258 Staining and Annexin V/PI Double Staining with flow cytometric analysis and indicate that AS and TAK-242 have protective effect on hBMECs cells from β_1-42-induced apoptosis.

The TNF-α is one of the most important inflammatory mediators and appears in the earliest process of apoptosis (Wang W.Y. et al., 2015). IL-6 is a key pro-inflammatory cytokine and plays an important role in inflammation and immune response of the body as well as in the regulation of metabolic, regenerative, and neural processes (Scheller et al., 2011). Therefore, we evaluated the effect of AS on the levels of TNF-α and IL-6 induced by Aβ_1-42 and compared to that of TAK-242 in hBMECs by ELISA analysis.

The expressions of TNF-α and IL-6 were significantly increased (p < 0.01) by Aβ_1-42 (50 μM) treatment compared to vehicle treatment in hBMECs (Figure 5). However, pretreatment of AS at 25, 50, and 100 μM significantly decreased the elevated expressions of TNF-α and IL-6 induced by Aβ_1-42 in hBMECs (Figure 5). TAK-242 at 1 μM had similar effect as 100 μM of AS on decreasing the elevated expressions of TNF-α and IL-6 induced by Aβ_1-42 in hBMECs (Figure 5). These results suggest that AS and TAK-242 effectively inhibited Aβ_1-42-induced high expressions of TNF-α and IL-6 and the effects of AS and TAK-242 on decrease of apoptosis induced by Aβ_1-42 may be via inhibiting the expressions of TNF-α and IL-6 in hBMECs.

The TLR4/NF-κB signaling pathway plays an important role in the regulation of cell apoptosis (Liu et al., 2013). NF-κB signaling activation involves phosphorylation and nuclear translocation of the p65 protein (Christian et al., 2016). We speculated that the anti-apoptotic effect of AS may be related to inhibition of the expressions of proteins in TLR4/NF-κB signaling pathway and translocation of NF-κB p65 in hBMECs. Therefore, we evaluated the effects of AS on the expressions of TLR4, MyD88, TRAF6, p-NF-κB p65, and total NF-κB p65 proteins and NF-κB p65 translocation. We treated hBMECs with medium (vehicle control), AS (25, 50, and 100 μM) or TAK-242 (1 μM) for 12 h followed by Aβ_1-42 (50 μM) treatment for additional 24 h and analyses by Western blotting and Immunofluorescence assay. The results showed that Aβ_1-42 treatment significantly upregulated the protein expressions of TLR4, MyD88, TRAF6, and p-NF-κB p65 (p < 0.01) but no effect on total NF-κB p65 compared to vehicle treatment in hBMECs (Figure 6). However, pretreatment of AS significantly (p < 0.01 vs. Aβ_1-42 treated group) downregulated the highly elevated levels of TLR4, MyD88, TRAF6, and p-NF-κB p65 induced by Aβ_1-42 in hBMECs (Figure 6). Pretreatment of TAK-242, a specific TLR4 inhibitor, also significantly decreased (p < 0.01) the protein expressions of TLR4, MyD88, TRAF6, and p-NF-κB p65 being upregulated by Aβ_1-42 in hBMECs (Figure 6).

Next, we determined the localization of NF-κB p65 in hBMECs. Aβ_1-42 (50 μM) treatment resulted in a significant translocation of p65 from a cytoplasmic to a nuclear localization compared to that of vehicle treatment (p65 mainly located in the cytoplasm) in hBMECs (Figures 7Aa–b,B). However, pretreatment of AS (25, 50, and 100 μM) significantly inhibited the nuclear translocation of p65 in a concentration-dependent manner compared to Aβ_1-42 treated group (p < 0.01) in hBMECs (Figures 7Ac–e,B). Similarly, pretreatment of TAK-242 (1 μM) also significantly (p < 0.01) inhibited the nuclear translocation of p65 (Figures 7Af,B). The results indicate that AS and TAK-242 effectively inhibited Aβ_1-42-induced high expressions of TLR4, MyD88, TRAF6, and p-NF-κB p65 proteins and NF-κB p65 nuclear translocation. Therefore, the effects of AS and TAK-242 on inhibition of apoptosis induced by Aβ_1-42 may be through suppressing TLR4/NF-κB signaling pathway in hBMECs.