Icariin (ICA), which is an essential bioactive component extracted from the herb
Neural stem cells (NSCs) are multipotent stem cells, which are derived from neural tissues, either from the central or peripheral nervous systems. NSCs are self-renewing and can give rise to various cell types of the nervous system, including neurons, astrocytes and oligodendrocytes, through asymmetric cell division (
ICA (purity >98%, high-performance liquid chromatography grade) with the molecular formula C33H42O15 and a molecular weight of 676.65 was purchased from Shaanxi Scidoor Hi-tech Biology Co., Ltd. (Xi'an, China). Hank's Balanced Salt Solution (HBSS), Dulbecco's modified Eagle's medium (DMEM)/F12 medium, fetal bovine serum (FBS), B27, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), Glutmax and poly-L-lysine-coated plates were purchased from Thermo Fisher Scientific, Inc. (Beijing, China). Mouse anti-nestin was obtained from BD Pharmingen (cat. no. 560341; San Jose, CA, USA), mouse anti-β-III-tubulin (cat. no. T8578), mouse anti-glial fibrillary acidic protein (GFAP; cat. no. G3893) and rabbit anti-galactocerebroside (GalC; cat. no. G9152) were obtained from Sigma Aldrich (Shanghai) Trading. Co., Ltd.(Shanghai, China). Cy2- or Cy3-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). RNeasy kit and AMV reverse transcriptase were obtained from Qiagen, Inc. (Valencia, CA, USA). Cy3-dCTP and Cy5-dCTP were purchased from CapitalBio, Inc. (Beijing, China). SYBR Premix Ex Taq for quantitative polymerase chain reaction (qPCR) was obtained from Sangon Biotech Co., Ltd. (Shanghai, China).
The use of human fetuses obtained following spontaneous abortion was approved by the Ethic Committee of Xuanwu Hospital of Capital Medical University (Beijing, China), and written informed consent was obtained from the study participants. The corpus striatum of 16–20-week fetuses were obtained from 5 fetuses following spontaneous abortion, and were dissected and chopped in cooled HBSS using a tissue chopper. The tissue was further triturated by pipetting up and down 10 times to dissociate cells. After filtering through a 300 micron stainless steel sieve, in order to remove the large pieces of tissue, the dissociated cells were pelleted by centrifugation at 200 × g for 5 min at room temperature, washed once with HBSS, and were re-suspended at a density of 2×106 cells/ml in DMEM/F12 medium containing 2% B27, 20 ng/ml EGF, 20 ng/ml bFGF and 1% Glutmax (DMEM/F12 complete medium). Cells were seeded in 100 mm dishes and cultured at 37°C in a humidified atmosphere containing 5% CO2. Cell medium was changed every 3 days. After an initial culture for 7–10 days typical NSC clusters appeared, which were pelleted by centrifugation at 200 × g for 5 min at room temperature and were treated with accutase. Accutase-digested cells were washed once with HBSS, re-suspended in DMEM/F12 complete medium and used for all subsequent assays. For NSC characterization, the cells were seeded onto poly-L-lysine-coated chamber slides (1×104/cm2). Following a 12 h culture, the cells were subjected to immunostaining for the analysis of nestin expression.
Growth factor-free DMEM/F12 medium supplemented with 1% FBS was used to induce cell differentiation. Cell clusters were digested with accutase as aforementioned, were washed once with HBSS, re-suspended in differentiation medium, and were seeded onto poly-L-lysine-coated chamber slides (1×104/cm2). Cells were cultured for 7 days, and were subsequently subjected to fixation for immunostaining, in order to determine the expression of β-III-tubulin, GFAP and GalC.
Cells were fixed in ice-cold 4% paraformaldehyde for 15 min, followed by three washes with phosphate-buffered saline (PBS; pH 7.4). After blocking with 10% goat serum (Jackson ImmunoResearch, Inc., West Grove, PA, USA) in PBS containing 0.3% Triton X-100 at 37°C for 1 h, the cells were incubated with various primary antibodies at 4°C overnight. The antibodies used were as follows: Mouse anti-nestin (1:1,000), mouse anti-β-III-tubulin (1:1,000), mouse anti-GFAP (1:500) and rabbit anti-GalC (1:100). After three washes with PBS, Cy2- or Cy3-conjugated secondary antibodies (1:500) were added to the cells and were incubated at 37°C for 30 min, followed by three washes with PBS. Cells were then mounted with VectaShield mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA) and 4′,6-diamidino-2-phenylindole was used for nuclear counter-staining. Fluorescent signals were visualized using a confocal laser microscope system (MRC1024; Bio-Rad Laboratories, Inc., Hercules, CA, USA).
To study the effects of ICA on NSC proliferation, accutase-dissociated single cells were suspended in DMEM/F12 complete medium at a density of 2×105 cells/ml. A 100
cDNA microarray analysis was performed for gene expression profiling, and the results were compared between the cells treated with ICA and the untreated control cells. Cell treatment with 10
qPCR was performed to verify the array results. Primers (
All results are expressed as the mean ± standard error of the mean, and were analyzed using one-way analysis of variance followed by Tukey's post-hoc test using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.
NSCs were isolated from the corpus striatum of 16–20-week human fetuses obtained following spontaneous abortion. After culturing in DMEM/F12 complete medium for 7–10 days, typical NSC clusters appeared in the culture. Immunostaining analysis revealed that neurosphere-forming cells expressed nestin, a marker of NSCs (
Using a CCK-8 cell proliferation assay kit, the effects of ICA on NSC proliferation were determined. As shown in
Gene expression in 10
The mRNA expression levels of fibroblast growth factor receptor 1 (FGFR1), and four genes of the Wnt signaling pathway: Frizzled class receptor 7 (FZD7), dishevelled segment polarity protein 3 (DVL3), catenin beta-1 (CTNNB1) and glycogen synthase kinase-3β (GSK-3β), were further determined by qPCR. The results indicated that the mRNA expression levels of FZD7 (
NSCs possess the characteristics of self-renewal and multipotency (
In our previous study,
In order to provide information regarding the mechanisms underlying ICA actions, the present study analyzed ICA-regulated gene expression in NSCs. A cDNA microarray analysis, which is an important tool used to uncover the molecular basis of several biological processes (
In conclusion, NSCs derived from the corpus striatum of 16–20-week human fetuses obtained following spontaneous abortion were successfully isolated. The NSCs expressed nestin, an NSC marker, and could differentiate into various types of cells of the nervous system. Treatment with ICA promoted the proliferation and modulated gene expression in NSCs, thus suggesting that ICA may exert its neuroprotective effects by regulating NSC activity. Taken together, the findings suggest that ICA may be potentially applied for the treatment of neurodegenerative disorders.
The present study was supported by the National Natural Science Foundation of China (grant nos. 81273498 and 81274120), the Beijing Natural Science Foundation (grant nos. 7112061 and 7132110), the Beijing Science and Technology Program (grant no. Z131102002813066), the Capital Health Development Scientific Grant (grant no. 2011-1001-05), and the Beijing Health and Technical High-level Personnel Plan (grant nos. 2011-1-7 and 2009-3-66).
Isolation and characterization of neural stem cells (NSCs). Cells isolated from the corpus striatum were cultured in Dulbecco's modified Eagle's medium/F12 complete medium and were characterized. (A) Representative image of nestin-positive cells (green) in a neurosphere. As shown by immunostaining, when NSCs were cultured in differentiation medium they expressed (B) β-III-tubulin, a neuronal marker [red, β-III-tubulin-positive; blue, 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining], and (C) glial fibrillary acidic protein (GFAP), an astrocyte marker (red, GFAP-positive; blue: DAPI nuclear staining). Scale bar, 100
Icariin (ICA) promoted neural stem cell (NSC) proliferation. NSCs were treated with various concentrations of ICA, as indicated, and cell proliferation was assessed using the Cell Counting kit-8 cell proliferation assay kit. (A) Cells incubated with 10
Icariin (ICA) regulated gene expression in neural stem cells. cDNA microarray analysis identified genes whose expression was altered following ICA treatment (
Quantitative polymerase chain reaction primer sequences.
Genes | Reference number | Sequence | Product size |
---|---|---|---|
FZD7 | NM_003507 | F: 5′-GGCGCCTCTGTTCGTCTAC-3′ | 106 bp |
R: 5′-GGTCTTGGTGCCGTCGTGT-3′ | |||
CTNNB1 | NM_001904 | F: 5′-CACGTGCAATCCCTGAACTGA-3′ | 121 bp |
R: 5′-CGCATGATAGCGTGTCTGGAA-3′ | |||
DVL3 | NM_004423 | F: 5′-GGTACTGCGGGAGATTGTG-3′ | 168 bp |
R: 5′-GGAAGGTGCCGGTCAT-3′ | |||
GSK-3β | NM_002093 | F: 5′-GTCCGATTGCGTTATT-3′ | 176 bp |
R: 5′-TTCGGAACAGCTGATACAT-3′ | |||
FGFR1 | NM_023105 | F: 5′-AAGGCATCATTTGGTGAACAGAAC-3′ | 133 bp |
R: 5′-TCACACAACATTGCTTCAAGGTAGG-3′ | |||
GAPDH | NM_002046 | F: 5′-ATGACATCAAGAAGGTGGTG-3′ | 177 bp |
R: 5′-CATACCAGGAAATGAGCTTG-3′ |
F, forward primer; R, reverse primer; bp, base pair; FZD7, frizzled class receptor 7; CTNNB1, catenin beta-1; DVL3, dishevelled segment polarity protein 3; GSK-3β, glycogen synthase kinase-3β; FGRF1, fibroblast growth factor receptor 1; GAPDH, glyceraldehyde 3-phosphate dehyrdrogenase.
Differences in gene expression in the top 10 pathways analyzed using Molecular Annotation System software.
Pathway | Total genes | P-value | Genes |
---|---|---|---|
Regulation of actin cytoskeleton | 19 | 2.0E-8 | ITGA7 |
Focal adhesion | 22 | 2.3E-7 | CAST |
MAPK pathway | 20 | 5.2E-7 | PRKX↓, MEF2C↓, PDGFRA |
ECM-receptor interaction | 12 | 8.5E-7 | ITGA7 |
Axon guidance | 12 | 2.0E-6 | EPHA3↓, NCK2↓, EPHA2 |
Cell communication | 11 | 7.0E-6 | INA↓, LAMC1 |
TGF-beta pathway | 9 | 1.2E-5 | ACVR1↓, RPS6KB2↓, MYC |
Wnt pathway | 10 | 3.8E-5 | WNT7B |
Gap junction | 9 | 5.2E-5 | TUBB3↓, PRKX↓, PDGFRA |
Glutathione metabolism | 6 | 5.9E-5 | GSS |
↑Indicates gene upregulation and ↓indicates gene downregulation by icariin. MAPK, mitogen-activated protein kinase; ECM, extracellular matrix; TGF, transforming growth factor.
Differentially expressed genes in the Wnt signaling pathway.
Gene | Fold change (ICA/control) | Molecular functions |
---|---|---|
WNT7B | 2.482 | Wnt ligand |
FZD7 | 2.850 | Non-G-protein coupled receptor |
DVL3 | 2.191 | Catalytic activity |
GSK-3β | 0.468 | Kinase and inactivating agent |
MYC | 4.390 | Transcription regulator |
CCND1 | 11.677 | Mitotic and cell cycle regulation |
CTNNB1 | 2.500 | Coactivator of transcription |
TCF7 | 2.804 | RNA polymerase II transcription |
LEF1 | 10.663 | Transcription regulator |
JUN | 2.086 | Transcription cofactor |
ICA, icariin.