Fresh Fructus Corni (C. officinalis Sieb. et Zucc.) was purchased from Tong Ren Tang (Beijing, China). The Fructus Corni (100 g) was extracted by heating in 1,000 ml distilled water at 80°C for 2 h, and then concentrated using a rotary evaporator (Rotary evaporator R-480; Büchi Labortechnik AG, Flawil, Switzerland). The concentrated extracts were lyophilized using a freeze dryer (FDU-540; Elaya, Tokyo, Japan). Following the lyophilization, a powder was obtained (yield, 25.82 g) and was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) to provide a stock solution of concentration 100 mg/ml and stored at 4°C. The stock solution of FCE was then diluted with medium to the desired concentration prior to use.

Rat β-nerve growth factor (NGF) was purchased from R&D Systems (Minneapolis, MN, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) was obtained from Sigma-Aldrich. Rabbit polyclonal anti-STIM1 (S6072) and mouse monoclonal anti-β-actin (A5316) antibodies were purchased from Sigma-Aldrich.

PC12 (adrenal gland pheochromocytoma) cells (that were a gift from Professor Yanhong Liao of Huazhong University of Science and Technology, Wuhan, China) were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone™, GE Healthcare, Logan, UT, USA) supplemented with 10% heat-inactivated horse serum (Gibco, Grand Island, NY, USA), 5% heat-inactivated fetal bovine serum (Gibco), and antibiotics (100 µg/ml streptomycin and 100 U/ml penicillin; Gibco) at 37°C in a 5% CO₂ incubator. Cells were supplied with fresh medium three times per week, and were split at a 1:3 ratio twice per week.

PC12 cells were seeded into 96-well plates (200 µl/well) at a density of 5×10⁴ cells/ml. They were treated with various concentrations (0, 10, 20, 40, 60, 80 and 100 µg/ml) of FCE for 48 h. At the end of the drug incubation period, MTT (Sigma-Aldrich) working solution (0.5 mg/ml) was added to each well, and the plates incubated for an additional 4 h at 37°C. Following centrifugation at 350 × g for 5 min, the medium was replaced with DMSO. The absorbance of reduced MTT at 570 nm was measured with a plate reader (Bio-Rad Laboratories, Hercules, CA, USA). The rate of inhibition of cell proliferation was calculated from the optical density (OD) values as follows: Inhibitory rate of PC12 cell proliferation (%) = (OD value control group - OD value test group) × 100/OD value control group. Based on the IC₅₀ of FCE in PC12 cells, a single concentration of 60 µg/ml was determined according to the results and it was selected for evaluation in the following tests. The MTT assay was also conducted using 60 µg/ml FCE with various incubation times (0, 1, 6, 12, 18, 24, 48 and 72h) to evaluate whether the effect of FCE was time-dependent. Based on the results a 48-h culture time was used for the PC12 cells treated in different groups.

Lentiviral plasmids carrying STIM1 short hairpin RNA (shRNA) were purchased from Sigma-Aldrich. The lentivirus was produced by the Hope Center Viral Vectors Core of Washington University (St. Louis, MO, USA). For control infection, non-targeted shRNA was used.

STIM1 shRNA and non-targeted shRNA were transduced into PC12 cells using lentivirus (Hope Center Viral Vectors Core of Washington University) according to the instructions provided by the manufacturer. Briefly, PC12 cells were infected with lentivirus at day 8 in vitro, and selected with puromycin dihydrochloride (Santa Cruz Biotechnology, Inc.) for 1 week. At 3 days after infection, western blotting or immunofluorescence staining was performed.

PC12 cells (1×10⁵ cells/ml) were seeded onto 24-well plates and cultured for 1 day, after which time 60 µg/ml FCE or 50 ng/ml NGF was added and the cells were cultured for an additional 2 days. The cells were then fixed with 4% paraformaldehyde (Sigma-Aldrich) in phosphate-buffered saline (PBS), and cell morphology was assessed under a phase-contrast microscope (IBE2003; Chongqing COIC Industrial Co., Ltd, Chongqing, China). STIM1 shRNA-treated cells were also cultured and assessed, without 60 µg/ml FCE or 50 ng/ml NGF addition. Neurite extension from the PC12 cells was regarded as an index of neuronal differentiation. Processes with a length equivalent to ≥1 diameters of the cell body were regarded as neurites. The differentiation of PC12 cells was evaluated by examining the proportion of neurite-bearing cells to total cells in randomly selected fields. The mean differentiation score was obtained for >100 PC12 cells in each well. In certain experiments, images of the cells were captured, and the total length of the neurite extension per positive cell and average length of neurites in positive cells were determined in randomly chosen fields using Motic Images Plus software (version 2.0S; Motic Instruments Inc., Richmond, Canada).

Cells were blocked with 0.1% Tween-20 and 5% goat serum in PBS for 30 min and then stained for 2 h at room temperature with monoclonal mouse anti-Myc-Cy3 (1:200, cat. SAB4700448, Sigma-Aldrich) and polyclonal rabbit anti-GAP-43 (1:50, cat. BA0878, Boster, Pleasanton, CA, USA) antibodies. Primary antibody was then visualized using ECL™ donkey monoclonal anti-rabbit secondary antibody (1:1,000, cat. A-21206, Thermo Fisher Scientific Inc., Waltham, MA, USA). Fluorescent images were captured with a confocal microscope (Olympus Fluoview 500; Olympus, Tokyo, Japan).

Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and reverse transcribed using M-MLV-Reverse Transcriptase (Promega Corporation, Madison, WI, USA), according to the manufacturer's instructions. qPCR was performed using the SYBR-Green Master PCR mix (Applied Biosystems, Foster City, CA, USA) on the TP800 Real Time PCR System (Takara Bio, Otsu, Japan). qPCR of cDNA was performed using the following forward (F) and reverse (R) primer sequences: STIM1: F, 5′-GGACGATGATGCCAATGGTGATGT-3′; R, 5′-TTCCACAGGTCCTCCACGCTGAT-3′; β-actin: F, 5′-TGGACATCCGCAAAGAC-3′; R, 5′-GAAAGGGTGTAACGCAACTA-3′. The amplification conditions were: 5 min at 94°C; 35 cycles of 45 sec at 94°C, 1 min at 56°C and 1 min at 72°C; followed by 10 min at 72°C. All quantification was normalized to an endogenous gene, β-actin. For relative quantification, 2^−(Ct-Cc) where Ct and Cc are the mean threshold cycle differences after normalizing to β-actin, was calculated and used as an indication of the relative expression levels (15).

PC12 cells were collected, washed once with ice-cold PBS, and lysed with a lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.2% deoxycholic acid and 1:100 protease inhibitor cocktail). Lysates were centrifuged for 10 min at 12,000 × g and supernatants were analyzed for protein concentration using a BCA Protein Assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Equal amounts of proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies against STIM1 (1:1,000) and β-actin (1:1,000), respectively. Immunoreactive bands were visualized by enhanced chemiluminescence. Images of the bands were captured using a scanner (HP Scanjet 7400C; Hewlett-Packard, Palo Alto, CA, USA), and the intensities of the were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

Ratiometric imaging of intracellular Ca²⁺ using cells loaded with fura-2 was measured as previously described (16). All cells for these experiments were grown in round coverslips (30 mm) under normal tissue culture conditions, except where specified. Coverslips with cells were placed in a cation-safe solution composed of (in mM): 107 NaCl, 7.2 KCl, 1.2 MgCl₂, 11.5 glucose and 20 HEPES-NaOH pH 7.3, and loaded with fura-2-acetoxymethyl ester (2 µM final concentration) for 30 min at 37°C. Cells were washed, and de-esterification was allowed for a minimum of 15 min. Ca²⁺ measurements were made using a Leica DMI6000 B fluorescence microscope (Leica Microsystems, Wetzlar, Germany) controlled by SlideBook software (Intelligent Imaging Innovations, Denver, CO, USA). Fluorescence emission at 505 nm was monitored while excitation wavelengths were alternated between 340 and 380 nm at a frequency of 0.5 Hz; intracellular Ca²⁺ measurements were determined as a 340/380 nm ratio obtained from groups (n=15-25) of single cells. External solutions were (in mM): 135 NaCl, 5.4 KCl, 10 HEPES, 0.02 NaH₂PO₄, 2 Mg²⁺ and 10 glucose pH 7.4. Measurements shown are representative of a minimum of three independent experiments.

All results are expressed as the means ± standard error of the mean of data obtained from triplicate experiments. Data in two groups were analyzed by Student's t-test. Multiple comparisons of the data were performed by analysis of variance followed by Tukey's test. All the analyses were performed using the SPSS software package, version 17.0 (SPSS, Inc., Chicago, IL, USA). Differences with P<0.05 were considered statistically significant.

To examine the effects of FCE treatment on cell proliferation, MTT assays were used to detect the viability of PC12 cells. When the PC12 cells were treated with various concentrations of FCE for 48 h, the proliferation of PC12 cells was inhibited in a concentration-dependent manner (Fig. 1A). According to the inhibitory rates of FCE at different concentrations on PC12 cells (data not shown), the IC₅₀ was 58.248 µg/ml. A concentration of 60 µg/ml FCE was selected for use in this study. Furthermore, a time-dependent reduction in cell viability was observed (Fig. 1B). The PC12 cells were incubated for 48 h with different treatments prior to the following experiments.

Following treatment with 60 µg/ml FCE, morphological changes indicating neurite outgrowth were observed in the PC12 cells (Fig. 2A). NGF, a prominent neurotrophic factor, was used to induce neurite outgrowth in PC12 cells as a positive control. In addition, the morphological changes induced by the knockdown of STIM1 expression with specifically targeted shRNA were observed to elucidate the effect of STIM1 on neurite outgrowth from PC12 cells. The results demonstrated that FCE treatment stimulated neurite extension in PC12 cells in a similar manner to NGF. In addition, neurite outgrowth from the PC12 cells also was observed in the STIM1 shRNA-treated group, suggesting that STIM1 may be involved in the process of neural differentiation (Fig. 2A). Furthermore, FCE significantly increased the percentage of neurite-bearing cells and the length of the branches (Fig. 2B and C).

SOCCs, including STIM1, play a important role in cellular proliferation and differentiation. In this study, the knockdown of STIM1 expression with specifically targeted shRNA was carried out, and it effectively reduced the expression of STIM1 mRNA (Fig. 3) and protein (Fig. 4B). Treatment with FCE also reduced the mRNA expression of STIM1 in the PC12 cells (Fig. 3), concurrently with its inducing effect on neurite outgrowth. Immunofluorescence staining confirmed that FCE treatment suppressed the expression of STIM1 protein in PC12 cells (Fig. 4A). Consistent with these results, the expression of STIM1 protein was also found to be significantly decreased in the FCE treatment group compared with the control group by western blot assay (Fig. 4C).

To examine whether sustained Ca²⁺ influx is critical for neurite growth in PC12 cells with FCE treatment, cytosolic Ca²⁺ was measured by the fura 2 assay (Fig. 5). FCE treatment inhibited Ca²⁺ influx in a time-dependent manner in PC12 cells, which differed from the NGF and STIM1 shRNA groups. These results suggest that FCE-induced neurite growth may involve a signaling pathway and mechanism different from that of NGF.