Acerogenin C was isolated from A. nikoense as previously described (10). All cell culture-associated reagents were obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Lipofectamine 2000™ was obtained from Invitrogen (Thermo Fisher Scientific, Inc.). The anti-HO-1 (catalog no. sc-10789), Nrf-2 (catalog no. sc-722), p-Akt (catalog no. sc-514032), Akt (catalog no. sc-5298), proliferating cell nuclear antigen (PCNA; catalog no. sc-56) and β-actin (catalog no. sc-1616) antibodies, and small interfering RNA (siRNA) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The HO-1 inducer cobalt protoporphyrin IX (CoPP) and all other chemical reagents were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

Mouse hippocampal HT22 cells were donated from Professor Youn-Chul Kim (Wonkwang University, Iksan, Korea). The cells (5×106 cells/dish) were cultured in Dulbecco's modified Eagle's medium containing streptomycin (100 µg/ml), 10% fetal bovine serum (FBS), and penicillin G (100 U/ml) at 37°C in an atmosphere containing 5% CO₂ and 95% air.

HT22 cells were maintained at 2×104 cells/well and treated with acerogenin C (1, 10, 20, 40 and 50 µM) for 48 h. Alternatively, HT22 cells were pretreated with acerogenin C (10, 20 and 40 µM) or trolox (50 µM) for 12 h, and then treated with glutamate (5 mM) for 12 h. In addition, HT22 cells were treated with acerogenin C (30 µM) in the presence or absence of 50 µM tin protoporphyrin XI (SnPP), (HO)-1 siRNA, or 10 µM LY294002, and then exposed to glutamate (5 mM) for 12 h. All incubations were performed at 37°C and 5% CO₂. Following incubation, the cell culture medium was removed from each well and replaced with fresh medium. Cells were incubated with 0.5 mg/ml MTT for 1 h and the formed formazan crystals were dissolved in 150 µl 99.7% dimethyl sulfoxide. Optical density was measured at a wavelength of 590 nm on a microplate reader (Bio-Rad, Hercules, CA, USA).

HT22 cells homogenized in PER-Mammalian Protein Extraction buffer (Pierce; Thermo Fisher Scientific, Inc.) supplemented with 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail I (EMD Millipore, Billerica, MA, USA). Cytoplasmic and nuclear fractions were separated using NE-PER Nuclear and Cytoplasmic Extraction reagents (Pierce; Thermo Fisher Scientific, Inc.).

HT22 cells were incubated with acerogenin C (10, 20 and 40 µM) or CoPP (20 µM) for 12 h. Alternatively, HT22 cells were treated with 30 µM of acerogenin C for 0, 30, 60 and 120 min, and transiently transfected with Nrf2 siRNA and then treated with 30 µM acerogenin C for 12 h (Fig. 4B). In addition, HT22 cells were pre-incubated with or without 10 µM LY294002 for 1 h and then incubated in the absence or presence of 30 µM of acerogein C for 60 min (p-AKT) or 12 h (HO)-1. Pelleted HT22 cells were obtained by centrifugation at 200 × g for 3 min at room temperature. Following the washing of cells with PBS, they were lysed using Tris-HCl buffer (20 mM; pH 7.4) supplemented with a protease inhibitor mixture containing chymostatin (1 mg/ml), aprotinin (5 mg/ml), pepstatin A (5 mg/ml) and phenylmethylsulfonyl fluoride (0.1 mM). Equal amounts of protein were resolved using SDS-PAGE and transferred to a Hybond-enhanced chemiluminescence nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membrane was blocked with 5% skimmed milk at room temperature for 1 h, and then incubated with anti-HO-1, anti-Nrf2, anti-p-Akt, anti-Akt, anti-PCNA, or anti-β-actin antibodies (all of which were used at a 1:1,000) at 4°C overnight. The membrane was subsequently incubated with horseradish peroxidase-conjugated anti-goat (catalog no. ap106p; 1:1,000), rabbit (catalog no. ap132p; 1:1,000) and mouse (catalog no. ap160p; 1:1,000) secondary antibodies, obtained from EMD Millipore, followed by Enhanced Chemiluminescence (GE Healthcare, Chicago, IL, USA) detection substrate. Secondary antibodies were diluted in 3% skimmed milk in TBST buffer. The bands were quantified by densitometry (ImageJ software version 1.47; National Institutes of Health, Bethesda, MD, USA). Nuclear and cytoplasmic extracts of cells were prepared using NE-PER reagents, as per the manufacturer's protocol (Thermo Fisher Scientific, Inc.).

Lipofectamine 2000™ (Invitrogen; Thermo Fisher Scientific, Inc.) containing Opti-MEM without FBS were used to temporarily transfect HT22 cells with 50 nM HO-1 siRNA (catalog no. sc-35554) and Nrf2 siRNA (catalog no. sc-37030) (both from Santa Cruz Biotechnology, Inc.) for 6 h. The cell culture medium was replaced with fresh medium with 10% FBS.

Total RNA was isolated from the cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and quantified spectrophotometrically at 260 nm (ND-1000; Thermo Fisher Scientific, Inc.), Total RNA (1 µg) was reverse-transcribed into cDNA using the High Capacity RNA kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The obtained cDNA was amplified using the SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd., Dalian, China) and a StepOnePlus Real-Time PCR (Applied Biosystems). Reaction mixture contained diethyl pyrocarbonate-treated water, primer, and SYBR-Green PCR Master Mix. The sequences of primer were designed by PrimerQuest (Integrated DNA Technologies, Inc., Coralville, IA, USA). The primer sequences are: HO-1 forward, 5′-CTCTTGGCTGGCTTCCTT-3′ and reverse, 5′-GGCTCCTTCCTCCTTTCC-3′; and GAPDH forward, 5′-ACTTTGGTATCGTGGAAGGACT-3′ and reverse, 5′-GTAGAGGCAGGGATGATGTTCT-3′. The thermal cycling conditions used were as follows: Pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 sec and annealing at 60°C for 1 min. A total of 40 cycles were performed. The data was analyzed using StepOne software (version 2.3; Applied Biosystems; Thermo Fisher Scientific, Inc.), and the cycle number at the linear amplification threshold (quantification cycle; Cq) was recorded for the endogenous control gene and the target gene. Relative gene expression (target gene expression normalized to the expression of the endogenous control gene) was calculated using the comparative Cq method (2^−ΔΔCq) (23).

All data were expressed as the mean ± standard deviation from at least three independent experiments. To compare each experimental group, one-way analysis of variance was used followed by the Newman-Keuls post hoc test. Statistical analysis of all data was conducted using GraphPad Prism software (version 3.03; GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To verify the cytotoxic effects of acerogenin C (Fig. 1A), its effect on the viability of HT22 cells was analyzed using the MTT assay. There were no significant effects at 1–50 µM acerogenin C; however, the viability notably decreased at 50 µM (Fig. 1B). In addition, the neuroprotective effect of acerogenin C on HT22 cells against glutamate-associated cytotoxicity and ROS generation was investigated. To demonstrate the neuroprotective effect of acerogenin C, HT22 cells pretreated with different concentrations of acerogenin C (10, 20 and 40 µM) for 3 h and 5 mM glutamate for 12 h were subject to an MTT assay. The results demonstrated that the viability of 20 and 40 µM acerogenin C-treated cells was significantly increased compared with untreated cells (Fig. 2). Trolox, well-known for its anti-oxidative effect, was used as a positive control and provided a notable cytoprotective effect at a concentration of 50 µM.

HO-1 expression serves a key cytoprotective role in HT22 cells. It catalyzes the oxygen-dependent degradation of heme, producing CO, free iron and bilirubin/biliverdin (7). Consequently, the HO-1 induced protective effect in HT22 cells was investigated. HO-1 mRNA expression was evaluated following treatment with acerogenin C. Cobalt protoporphyrin (CoPP) was used as a positive control. CoPP is a well-known HO-1 inducer. Treatment with cerogenin C increased HO-1 mRNA and protein expression in HT22 cells in a dose-dependent manner (Fig. 3A and B). Subsequently it was demonstrated that tin protoporphyrin XI (SnPP), a known inhibitor of HO-1 expression, and HO-1 siRNA significantly blocked the protective effect of acerogenin C (Fig. 3C). These results suggest that HO-1 contributes to the cytoprotective effect of acerogenin C in HT22 cells.

The Nrf2/HO-1 pathway is important in the prevention of oxidative stress-induced damage. Under normal conditions, Nrf2 is located within cell cytoplasm. During oxidative stress, Nrf2 is phosphorylated and translocated into the nucleus, and binds to the specific ARE sequences (5,6). Therefore, the translocation of Nrf2 to nuclei in HT22 cells was observed. Cells were treated with acerogenin C for 0, 30, 60 and 120 min, and Nrf2 protein levels were detected by western blotting. Cytosolic Nrf2 levels were decreased; however, nuclear Nrf2 levels were increased following treatment with acerogenin C (Fig. 4A). PNCA is widely used as a control in antibody validation of nuclear protein. In addition, the role of Nrf2 in HO-1 activity was analyzed using Nrf2 siRNA. Hippocampal HT22 cells were temporarily transfected with Nrf2 siRNA and were treated with acerogenin C to induce HO-1 activity. Nrf2 siRNA inhibited the nuclear translocation of Nrf2 and transient transfections with Nrf2 siRNA also eliminated the induction of HO-1 expression by acerogenin C (Fig. 4B). These results demonstrated that HO-1 induction by acerogenin C is associated with the Nrf2 nuclear translocation pathway in HT22 cells.

Multiple studies demonstrated that the PI3K/Akt pathway is associated with the expression of HO-1 (24). Therefore, it was investigated whether acerogenin C induces expression of HO-1 via the PI3K/Akt signaling pathway. Following treatment with 40 µM acerogenin C, the levels of Akt were significantly increased (Fig. 5A). In addition, treatment with LY294002, a known inhibitor of PI3K, abolishes HO-1 expression and cytoprotection by acerogenin C (Fig. 5B and C). Therefore, acerogenin C-induced HO-1 expression is associated with the PI3K/Akt signaling pathway in HT22 cells.