Fructus Corni (
Calcium (Ca2+) is a ubiquitous and essential second messenger in cells, which mediates a broad of cellular functions including secretion, excitation, contraction, metabolism, transcription, growth, proliferation, division and apoptosis (
Recently, two key factors involved in SOCE have been identified. These are the endoplasmic reticulum (ER) Ca2+ sensor protein stromal interaction molecule 1 (STIM1) and the plasma membrane Ca2+ channel protein Orai1 (
The traditional Chinese herbal medicine Fructus Corni (
In the present study, the aim was to investigate the effects of FCE on neuronal differentiation. The effects of FCE treatment on neurite outgrowth, intracellular Ca2+ and the expression of STIM1 provide useful information to the therapeutic potential of FCE in neurodegenerative diseases.
Fresh Fructus Corni (
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
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 (
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 (
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 (
Following treatment with 60 µg/ml FCE, morphological changes indicating neurite outgrowth were observed in the PC12 cells (
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 (
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 (
In the present study, it was shown that FCE promotes neurite outgrowth and differentiation of PC12 cells. Notably, the FCE-induced neuritogenesis was demonstrated to be associated with the downregulation of STIM1 expression at the mRNA and protein levels, as well as the inhibition of extracellular Ca2+ influx.
The second messenger Ca2+ plays a crucial role in regulating a number of different cellular processes by modulating Ca2+-regulated proteins and the corresponding signaling pathways. There are two main sources of intracellular free Ca2+: Ca2+ released from intracellular Ca2+ storage organelles, most notably the ER, and Ca2+ influx from sources external to the cell (
The essential roles of STIM1 and Orai1 in SOCE have been confirmed in a number of studies. STIM1 is a single-pass transmembrane protein in the ER membrane that functions as a Ca2+ sensor in the ER, while Orai1 is the pore-forming subunit of SOC channels in cell membranes (
The upstream pathway leading to SOCE in PC12 cells has not been elucidated. Ca2+ levels in the ER are mainly controlled by the phospholipase C (PLC)/IP3/IP3 receptor pathway with activation by tyrosine kinase-type or Gq-related G protein-coupled receptors (
In conclusion, the findings of the present study provide the first evidence of induction of neurite outgrowth in PC12 cells by the traditional Chinese herbal medicine FCE. Additionally, the results demonstrate that FCE-induced neuritogenesis is associated with the suppression of STIM1 expression and the inhibition of Ca2+ influx. These results provide a useful information regarding the therapeutic potential of FCE for the treatment of neurodegenerative disease.
Fructus Corni extract
nerve growth factor
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
dimethyl sulfoxide
store-operated Ca2+ entry
endoplasmic reticulum
stromal interaction molecule 1
Ca2+ release-activated Ca2+
voltage-gated Ca2+ channel
inositol 1,4,5-trisphosphate
transient receptor potential channel
Effects of FCE on PC12 cell viability. PC12 cells were seeded in a 96-well plate at an initial density of 1×104 cells/well, and were (A) incubated with various concentrations of FCE for 48 h or (B) incubated with 60 µg/ml FCE for various periods of time. Cell viability was determined by MTT assay. The data are presented as a percentage of the vehicle treated control ± standard error of the mean of three independent experiments. A Student's t-test was used for the determination of significance. *P<0.05, **P<0.01 and ***P<0.001 compared with the untreated control. FCE, Fructus Corni extract; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.
Effects of FCE on neurite outgrowth from PC12 cells. PC12 cells (1×105 cells/ml) were seeded onto 24-well plates. After 24 h of stabilization, NGF or FCE was added and the cells were cultured for an additional 48 h. STIM1 shRNA-treated PC12 cells were also observed. (A) Morphological observations of PC12 cells from the different groups (control, 50 ng/ml NGF-treated, STIM1 shRNA-treated and 60 µg/ml FCE-treated groups). Scale bar, 100 µm. (B) The percentage of neurite-bearing cells was calculated from 5 random fields per well, and the data are expressed as a mean percentage of 6 wells in a representative experiment. Data are presented as mean ± standard error of the mean (SEM). ***P<0.001 compared with the control group. (C) Neurite lengths in PC12 cells of the various groups were calculated. Data are presented as mean ± SEM. ***P<0.001 compared with the untreated control. FCE, Fructus Corni extract; NGF, nerve growth factor; STIM1, stromal interaction molecule 1; shRNA, short hairpin RNA.
FCE treatment reduces the expression of STIM1 mRNA in PC12 cells. The relative mRNA level of STIM1 expression in PC12 cells of different groups (control, 50 ng/ml NGF-treated, STIM1 shRNA-treated and 60 µg/ml FCE-treated groups) was measured by reverse transcription-quantitative polymerase chain reaction. Data are presented as mean ± standard error of the mean from three experiments. **P<0.05, comparison with the untreated control. FCE, Fructus Corni extract; NGF, nerve growth factor; STIM1, stromal interaction molecule 1; shRNA, short hairpin RNA.
FCE treatment suppresses the expression of STIM1 protein in PC12 cells. (A) Expression of STIM1 protein in the PC12 cells of different groups (control, 50 ng/ml NGF-treated, STIM1 shRNA-treated and 60 µg/ml FCE-treated groups) was observed through immunofluorescence staining. Scale bar, 100 µm. (B) PC12 cells were treated with STIM1 shRNA or control shRNA, and the expression of STIM1 protein was detected by western blotting with specific anti-STIM1 antibody. Data are expressed as mean ± standard error of the mean (SEM). ***P<0.001 compared with the control group. (C) The expression of STIM1 protein in PC12 cells of different groups was detected by western blotting, and assessed by semi-quantitative analysis. All experiments were conducted in triplicate. Data are expressed as mean ± SEM. ***P<0.001 compared with the untreated control. FCE, Fructus Corni extract; NGF, nerve growth factor; STIM1, stromal interaction molecule 1; shRNA, short hairpin RNA.
FCE-induced neurite growth in PC12 cells is associated with the inhibition of Ca2+ influx. (A) Cytosolic Ca2+ was measured in the PC12 cells of different groups (control, 50 ng/ml NGF-treated, STIM1 shRNA-treated and 60 µg/ml FCE-treated groups), and representative traces of intracellular Ca2+ changes (fluorescence emission at 505 nm with alternating excitation wavelengths of 340 and 380 nm) in PC12 cells were observed. (B) Involvement of inhibiting extracellular Ca2+ influx in FCE-induced neurite growth of PC12 cells. Fluorescence emission at 505 nm was monitored while excitation wavelengths were alternated between 340 and 380 nm at a frequency of 0.5 Hz The intracellular Ca2+ measurements were determined as a 340/380 nm ratio obtained from different groups. All data shown are from individual experiments representative of at least three independent replicates. Data are expressed as mean ± standard error of the mean. **P<0.001 compared with the untreated control. FCE, Fructus Corni extract; NGF, nerve growth factor; STIM1, stromal interaction molecule 1; shRNA, short hairpin RNA.