Daphnetin (purity >98%) was obtained from Sigma-Aldrich (St. Louis, MO, USA), and the ERK inhibitor U0126 was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). H₂O₂ (30%) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Rabbit monoclonal antibodies against β-actin (catalog no., 4970S), Akt, phospho (p)-Akt (Ser 473; catalog no., 9272S), p38 mitogen-activated protein kinase (MAPK; catalog no., 8690S), p-p38 MAPK (Thr180/Tyr182; catalog no., 4511S), ERK (catalog no., 4695S), p-ERK (Thr202/Tyr204; catalog no., 4376S), JNK/stress-activated protein kinase (SAPK; catalog no., 9258S), p-JNK/SAPK (Thr183/Tyr185; catalog no., 4668S), poly ADP-ribose polymerase (PARP; catalog no., 9532S), cleaved-caspase 3 (catalog no., 9664S; 1:500), pro-caspase 3 (catalog no., 9665S) and HSP70 (catalog no., 4872S) were all purchased from Cell Signaling Technology, Inc and used at 1:1,000 dilution, unless otherwise specified. Rabbit polyclonal antibody against glyceraldehyde 3-phosphate dehydrogenase (catalog no., AP0063; 1:1,000) was purchased from Bioworld Technology, Inc. (St. Louis Park, MN, USA). Secondary antibodies coupled to IRDye800 fluorophore (catalog no., 926-32211; dilution, 1:5,000) for use with the Odyssey^® Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) were purchased from LI-COR Biosciences.

Rat pheochromocytoma PC12 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (Invitrogen™; Thermo Fisher, Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/v) heat-inactivated horse serum, 5% fetal bovine serum and 1% antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) (Hyclone™; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in 5% CO₂/95% air humidified atmosphere. Culture medium was changed every 2–3 days.

Cell viability was detected using Cell Counting Kit-8 (CCK-8; KeyGen Biotech Corp., Ltd., Nanjing, China). Briefly, PC12 cells were plated onto 96-well plates at a density of 2×10⁴ cells per well 24 h prior to treatment. Cells were treated with various concentrations of daphnetin (0, 2.5, 5.0, 10.0 and 20.0 µM) for 2 h, then stimulated with H₂O₂ (200 µΜ) for 24 h, followed by incubation with 10 µl CCK-8 working solution at 37% for 4 h. The absorbance of each well at 450 nm was measured using a Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific, Inc.). Three repeats were performed for each of the different treatments.

The apoptotic ratio was analyzed using Annexin-V/PI Double Staining Assay (KeyGen Biotech Corp., Ltd.), according to the manufacturer's protocol. Briefly, the cells were washed, trypsinized and resuspended with 500 µl binding buffer, then stained with Annexin V-FITC (5 µl) and PI (5 µl). The stained cells were visualized directly using a Guava EasyCyte™ System (EMD Millipore, Billerica, MA, USA), and the data were analyzed using Guava TUNEL ExpressPro version 8.0 software (EMD Millipore).

Cells were rinsed twice with ice-cold phosphate-buffered saline (PBS) and lysed on ice in a lysis buffer containing 20 mM Tris (pH 7.5), 2 mM EDTA, 135 mM NaCl, 2 mM DTT, 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 10% glycerol, 1% Triton X-100, 1 mM Na₃VO₄, 10 mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 1 mM PMSF, supplemented with a complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) for 30 min. Lysates were centrifuged at 12,500 × g for 15 min at 4°C. Equal amounts of proteins were subjected to 12% SDS-PAGE, and then transferred onto nitrocellulose membranes (GE Healthcare Life Sciences, Chalfont, UK). The membrane was blocked with 5% skimmed milk for 1 h at room temperature, washed with Tris-buffered saline with Tween 20 three times, then incubated with the indicated primary antibodies at 4°C overnight. Subsequently, the membranes were incubated with secondary antibodies for 1 h at room temperature. The antibody-antigen complexes were visualized by the Odyssey Infrared Imaging System using IRDye800 fluorophore-conjugated antibodies (LI-CORBiosciences).

PC12 cells were pretreated with various concentrations of daphnetin (5, 10 and 20 µM) for 2 h, then stimulated with H₂O₂ (200 µΜ) for 24 h. Subsequently, cell morphology was observed using an inverted microscope (DP72; Olympus Corporation, Tokyo, Japan).

Cells were pre-incubated with daphnetin for 2 h, then stimulated with H₂O₂ for 24 h. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized in 0.2% Triton X-100 and then incubated with DAPI (1 µg/ml) for 5 min in the dark. Subsequent to washing with PBS, nuclear morphology was observed and photographed using a fluorescence microscope (DP72; Olympus Corporation).

Data are expressed as the mean ± standard deviation. One-way analysis of variance was used to determine the significance of the difference between two groups. P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using SPSS version 17.0 software (SPSS, Inc., Chicago, IL, USA).

PC12 cells were treated with various concentrations of H₂O₂ (50, 100, 200, 400 and 600 µM) for 24 h, and subsequently cell viability was determined using CCK-8. As shown in Fig. 1A, H₂O₂ reduced cell viability in a dose-dependent manner (P<0.05 vs. control). For the cells treated with 200 µM H₂O₂, the cell viability was 48.75±1.63%. Since, the cell viability was reduced by ~50% with 200 µM H₂O₂ at 24 h, in subsequent experiments these variables were selected as the standard dose and time point for the induction of PC12 cell apoptosis. In PC12 cells treated with various doses of daphnetin and then stimulated with 200 µM H₂O₂, cell viability was increased in a daphnetin dose-dependent manner (P<0.05 vs. H₂O₂-treated group; Fig. 1B).

Observation of cell morphology revealed that untreated PC12 cells exhibited typical long fusiform-like morphology. By contrast, H₂O₂-treated (200 µM; 24 h) PC12 cells were shrunken, scattered and floating. Pretreating cells with daphnetin significantly reversed the apoptotic morphological alterations observed with H₂O₂-treated cells (Fig. 2A). To further determine the role of daphnetin in suppressing H₂O₂-induced PC12 cell apoptosis, the apoptotic ratio was determined by Annexin V-FITC/PI assay. As shown in Fig. 2B, cells exposed to H₂O₂ for 24 h had an apoptotic ratio of 55.6%. However, the number of apoptotic cells, when cells were pretreated with various concentrations of daphnetin, was significantly reduced; the apoptotic ratio was 34.6, 31.3, 26.6%, for 5, 10 and 20 µM daphnetin, respectively.

The activation of apoptosis-associated proteins was detected using western blotting to confirm the occurrence of apoptosis. Daphnetin pretreated cells clearly attenuated the H₂O₂-induced cleavage of PARP and caspase 3, and enhanced the level of pro-caspase 3 (Fig. 2C) compared with cells not pretreated with daphnetin. DAPI staining was used to determine the apoptotic status of the PC12 cells. Staining with DAPI revealed that the round nuclei of normal cells was homogeneous, and when exposed to 200 µM H₂O₂ for 24 h cells underwent nuclear condensation and fragmentation. However, these alterations in nuclear morphology were significantly attenuated by pretreatment with daphnetin (Fig. 2D). Overall, these results suggest that daphnetin has a protective effect in H₂O₂-induced PC12 cell apoptosis.

To determine the mechanism by which daphnetin inhibits H₂O₂-induced apoptosis, the phosphorylation levels of MAPKs in PC12 cells were investigated. PC12 cells were stimulated with 200 µM H₂O₂ for various times, and the phosphorylation levels of p38, ERK and JNK were detected by western blotting. The results showed that stimulation of PC12 cells with H₂O₂ resulted in an increase in phosphorylation of JNK, p38 and ERK (Fig. 3A). The enhanced phosphorylation of JNK and p38 was significantly attenuated by daphnetin pretreatment in a dose-dependent manner (Fig. 3B). However in the presence of daphnetin, increased ERK phosphorylation observed with H₂O₂ stimulation was not attenuated. In addition, with daphnetin stimulation, phosphorylation of ERK was clearly elevated, but ERK activation had no additive effect on daphnetin and H₂O₂ stimulation (Fig. 3B).

In order to determine other mediators in daphnetin neuroprotection, HSP70, a well-known chaperone with cytoprotective effects, was investigated. PC12 cells were stimulated with daphnetin, and total cellular protein was extracted and subjected to western blotting with an anti-HSP70 antibody. The present results revealed that the level of HSP70 was increased upon daphnetin stimulation in a dose- and time-dependent manner (Fig. 4A and B). The level of HSP70 peaked at 12 h.

The phosphorylation of upstream kinases was examined to investigate the possible signal transduction pathways involved in daphnetin-induced HSP70 expression. PC12 cells were treated with daphnetin (20 µM) for various times and the phosphorylation level of ERK and Akt was detected by western blotting. As shown in Fig. 5A, the activation of ERK was enhanced in a time-dependent manner following daphnetin stimulation. ERK activation peaked at 12 h, which is consistent with the expression of HSP70. By contrast, Akt phosphorylation was not significantly different compared with unstimulated PC12 cells. Overall, these observations suggested that ERK signaling activation may be involved in daphnetin-induced HSP70 expression.

To test this hypothesis, U0126, a specific inhibitor of ERK, was used to verify the activation of ERK in daphnetin-induced HSP70 expression. PC12 cells were pretreated with 20 µM U0126 for 2 h, and then exposed to 20 µM daphnetin for a further 12 h, and the expression of HSP70 and phosphorylated ERK were detected using western blotting. As shown in Fig. 5B, attenuation of ERK activation by U0126 clearly reduced the protein expression level of HSP70. Therefore, the present data suggest that HSP70 is involved in daphnetin-mediated cytoprotection via ERK signaling.