17β estradiol

Budesonide (gift from AstraZeneca, Sweden), Org34517 (gift from Chiesi Farmaceutici, Parma, Italy), progesterone (57-83-0, Cayman Chemical), 17β-estradiol (50-28-2, Cayman Chemical), dexamethasone (D8893), triiodo-l-thyronine (T2877), retinoic acid (R2625), 9-cis-retinoic acid (R4643), 7β-hydroxycholesterol (H6891), and hemin (51280) (all from Sigma-Aldrich) were dissolved in dimethyl sulphoxide (DMSO) (D2650, Sigma-Aldrich) as stocks of 1 to 10 mM. Final DMSO concentrations on cells were ≤0.1%. Dihydroxy vitamin D3 (D1530, Sigma-Aldrich) was dissolved in absolute ethanol as a stock of 10 mM. Recombinant human IL1B (201-LB, R&D Systems) was dissolved in phosphate-buffered saline (PBS) (14190144, Thermo) containing 0.1% bovine serum albumin (BSA) (A3059, Sigma-Aldrich). G418 (A1720, Sigma-Aldrich) was dissolved in sterile water as stocks of 100 mg/ml.

Aβ (25–35) (Sigma Aldrich, St. Louis, MO, USA) was dissolved in sterile distilled water at a concentration of 1 mM, then incubated in a capped vial at 37 °C for 5 days to allow formation of the aggregated form. It was then stored frozen at −20 °C until use. 17β-Estradiol, S-equol, and ICI-182,780 (all from Cayman Chemical, Ann Arbor, MI, USA) were dissolved in 99.5% ethanol to make stock solutions, which were used for experiments at a final concentration of 10 nM for estradiol and 1 μM for equol and ICI-182,780 in culture medium. It should be noted that no cytotoxic effect of the vehicle (99.5% ethanol) per se on cells was observed via the analysis of cell viability in our preliminary experiments that were conducted to determine the appropriate concentrations of the aforementioned treatments for the present study.
To induce cell death, cells were incubated with (Aβ) or without (C) 1 μM Aβ (25–35) for 24 h. To study the effects of estradiol (E2) and equol (Eq), cells were preincubated with estradiol (E2 + Aβ) or equol (Eq + Aβ) for 24 h prior to Aβ (25–35) exposure. Estradiol was used as a positive control and ICI-182,780 was used as an ER antagonist. It was added 1 h before the estradiol or equol treatment.

Aβ_1–42 and Aβ_1–40 were purchased from Peptide Institute Inc. (Osaka, Japan), and SH-SY5Y cells (human neuroblastoma; EC94030304) were obtained from the European Collection of Authenticated Cell Cultures (London, UK). Dulbecco’s Modified Eagle Medium (DMEM) Ham’s/F-12 medium and all-trans retinoic acid (ATRA) were obtained from Fuji Film Wako Pure Chemical Co., Ltd. (Osaka, Japan). Penicillin G sodium, streptomycin sulfate, amphotericin B, and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific K.K. (Waltham, MA, USA). 17β-estradiol and G-15 were procured from Cayman Chemical Company (Ann Arbor, MI, USA), and raloxifene hydrochloride was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The other chemicals used in this experiment were commercially available and of the purest grade. Their structures are shown below (Figure 1). Special-grade products were used for other reagents.