High glucose (HG) impairs endothelial progenitor cell (EPC) function. The activation of p38 mitogen-activated protein kinase and the inhibition of the Akt/eNOS/NO pathway serve central roles in this process. Icariin has protective effects in endothelial cells. The aim of the present study was to investigate the effects of icariin on HG-induced EPC dysfunction, including proliferation, migration and tube formation. Experiments were performed with EPCs isolated from the femurs and tibias of Sprague-Dawley rats
Endothelial progenitor cells (EPCs), derived from bone marrow or peripheral blood cells, have been shown to be incorporated into the foci of physiological and pathological neovascularization (
Icariin (C33H40O15; molecular weight, 676.66), a flavonoid extracted from several plants in the genus Epimedium, exhibits various pharmacological activities, including enhancing immune function, stimulating osteoblast proliferation, antioxidative stress, antiapoptosis, stimulation of angiogenesis and improving cardiovascular function (
Male Sprague Dawley (SD) wild-type rats (SPF grade; 180–200 g; 2–3 weeks; n=3) were obtained from Wuhan University Experiment Animal Center. These rats were allowed free access to standard rat chow and water, and were kept in an environment with controlled temperature and lighting (24°C; 12/12 h-light/dark cycle; humidity, 50–60%). Mononuclear cells were isolated from bone marrow from the femurs and tibias of SD rats, and cultured in endothelial basal medium (EBM-2 SingleQuots; Lonza Group, Ltd.) containing 5% FBS, human vascular endothelial growth factor A, human fibroblast growth factor-2, human epidermal growth factor, insulin-like growth factor-1 and ascorbic acid (EBM-2 SingleQuots; Lonza Group, Ltd.) to induce mononuclear cells differentiation into EPCs at 37°C in an atmosphere containing 95% air and 5% CO2. After 3 days in culture, the non-adherent cells were removed, and the adherent cells were maintained in new media. EPCs were characterized by FITC-Ulex europaeus agglutinin I (cat. no. L9006; Sigma-Aldrich; Merck KGaA) and DiI-acetylated low-density lipoprotein (cat. no. H7970; Beijing Solarbio Science & Technology Co., Ltd.) as previously described (
Cell proliferation was assessed using a CCK-8 kit (Dojindo Molecular Technologies, Inc.). In each well of a 96-well plate, 5,000 EPCs were seeded and cultured for 12 h at 37°C in an atmosphere containing 95% air and 5% CO2. After synchronization in EBM-2 with 0.1% FBS for 12 h, the EPCs were treated with icariin at three different concentrations (0.01, 0.1 or 1 µM) for 24 h at 37°C (
To evaluate the migratory ability of EPCs, a Transwell chamber assay (Corning, Inc.) was performed. Briefly, EPCs were seeded at a density of 5×104 cells/well in the upper chamber with serum-free EBM-2 and different stimulation conditions, and the lower chamber was filled with serum-free EBM-2 containing stromal cell-derived factor 1a (SDF-1a; 100 ng/ml). After incubation for 4 h at 37°C, the cells on the top of the filter were removed, and the migrated cells on the bottom of the filter were fixed in 95% alcohol for 30 min and stained with 0.1% crystal violet for 10 min at room temperature. Then, the cells on the filter were counted manually in at least three random selected high-power fields (magnification, ×100) in each well under a light microscope (Olympus Corporation).
A 24-well culture plate was coated with Matrigel (BD Biosciences), which was allowed to solidify for 30 min at 37°C. EPCs (5×104/well) were seeded and incubated at 37°C for 8 h. Tube formation was defined as a structure exhibiting a length four times its width. The total length of the tube formation was measured in three random fields (magnification, ×100; Olympus Corporation) per group using Adobe Photoshop CS5 software (Adobe Systems, Inc.) (
NO production was measured in culture medium with a total NO assay kit (cat. no. S0023; Beyotime Institute of Biotechnology). Briefly, EPCs (density, 1×104/ml) were plated on dishes and exposed to various treatments. Then, the supernatants were collected following centrifugation (140 × g; 10 min) at room temperature and analyzed according to the manufacturer's protocol. The total NO production of EPCs was determined by measuring the concentrations of nitrate and nitrite using the Griess method, and was normalized to standards in the total NO assay kit.
Cells were lysed in a RIPA lysis buffer according to the manufacturer's protocol (BioVision, Inc.). Protein concentrations were determined by a bicinchoninic acid protein assay (Beyotime Institute of Biotechnology). Proteins samples (4.5 µg/µl; 20 µl per lane) were separated using 10% SDS-PAGE and transferred onto PVDF membranes. For western blot analysis, the PVDF membranes were blocked at room temperature. with 5% non-fat dried milk that was dissolved in Tris-buffered saline containing 0.1% tween-20 for 90 min and probed with antibodies (1:1,000) against phosphorylated (p)-p38 (cat. no. sc-7973; Santa Cruz Biotechnology, Inc.), p38 (cat. no. ab7952; Abcam), protein-CREB (cat. no. 11273; Signalway Antibody LLC), CREB (cat. no. 9197; Cell Signaling Technology, Inc.), p-Akt (cat. no. 4060; Cell Signaling Technology, Inc.), Akt (cat. no. 21054; Signalway Antibody LLC), p-eNOS (cat. no. 9574; Cell Signaling Technology, Inc.), eNOS (cat. no. 21170; Signalway Antibody LLC) and GAPDH (cat. no. sc-365062; Santa Cruz Biotechnology, Inc.) overnight at 4°C. After washing three times, the membranes were incubated with peroxidase-conjugated secondary antibodies (Goat Anti Rabbit IgG/HRP; 1:50,000; cat. no. 31460; Pierce; Thermo Fisher Scientific, Inc.; Goat Anti Mouse IgG/HRP; 1:50,000; cat. no. 31430; Pierce; Thermo Fisher Scientific, Inc.) for 1 h at room temperature, and enhanced chemiluminescence (Thermo Fisher Scientific, Inc.) was performed. The autoradiographs were scanned using Adobe Photoshop CS5 software, and the protein ratios were calculated (
The experimental data are presented as the mean ± SD. One-way analysis of variance followed by Bonferroni post hoc test was used for comparisons between continuous variables. All statistical analyses were performed using SPSS 19.0 for Windows (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.
High glucose, which is a risk factor for CAD (
p38 and its downstream target CREB have been reported to play a critical role in EPC downregulation induced by HG (
It has been reported that the inhibitory effects of HG on Akt and eNOS phosphorylation are involved in HG-induced EPC dysfunction (
The present study demonstrated that icariin could ameliorate the inhibition of EPC proliferation, migration and tube formation induced by HG. Additionally, icariin significantly reduced the activation of the p38/CREB pathway and stimulated the Akt/eNOS/NO pathway in EPCs treated with HG. These results indicated potential mechanisms underlying the protective effects of icariin on EPCs, and suggested that icariin may be a useful agent for improving EPC function in a HG microenvironment.
Previous studies reported that icariin exerts endothelial protection effects (
Several mechanisms may be involved in the HG-induced reduction in EPC number, and impairment in EPC proliferative and migratory abilities. p38 MAPK and its downstream target CREB have been shown to decrease the number and proliferation of EPCs (
Another important mechanism involved with HG-induced impairments in EPC migration is the inhibition of PI3K/Akt/eNOS activation and NO production (
There are certain limitations to the present study. Only
Collectively, the results of the present study demonstrated that icariin can attenuate HG-induced EPC dysfunction
Not applicable.
The present study was supported by the National Natural Science Foundation of China (grant no. 81600226).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
HJ designed and directed the experiments. SC, ZW and HZ performed the experiments. SC, ZW, HB and DH collected and analyzed the experimental data. SC and HJ wrote the manuscript. HZ and HB investigated the relevant literature and revised the manuscript. All authors read and approved the final manuscript.
The present study was approved by Institutional Animal Care and Use Committee, the Animal Care and Use Committee of Wuhan University (permit no. WDRM20161204).
Not applicable.
The authors declare that they have no competing interests.
Icariin rescues the EPC dysfunction induced by HG. (A) EPCs were treated with various concentrations of icariin for 24 h in HG conditions. Cell proliferation was determined by a Cell Counting Kit-8 assay. (B) EPCs were treated with or without 1
Icariin inhibits the HG-induced phosphorylation of p38 and CREB in EPCs. (A) EPCs cultured in 5.5 (Control) or 25 mM glucose (HG) were treated with or without 1 µM icariin for 30 min. Levels of p-p38, total p38, p-CREB and total CREB were determined via western blot analysis. (B) Quantitative results of the phosphorylated levels of p38. (C) Quantitative results of the phosphorylated levels of CREB. Data are presented as the mean ± SD; n=3/group. *P<0.05 vs. Control group, #P<0.05 vs. HG group. CREB, cAMP response element binding protein; EPC, endothelial progenitor cell; HG, high glucose; p-, phosphorylated.
Icariin induces Akt and eNOS phosphorylation, and NO production in HG-treated EPCs. (A) EPCs were cultured in 5.5 (Control) or 25 mM glucose (HG) for 3 days with or without treatment with 1 µM icariin for 30 min. Levels of p- and total Akt and eNOS were determined via western blot analysis. (B) Quantitative results of the phosphorylated levels of Akt (C) Quantitative results of the phosphorylated levels of eNOS (D) EPCs were treated with or without 1 □M icariin for 3 h. The relative levels of intracellular NO were determined by the concentrations of nitrate and nitrite. Data are presented as the mean ± SD; n=3/group. *P<0.05 vs. Control group, #P<0.05 vs. HG group. eNOS, endothelial nitric oxide synthase; EPC, endothelial progenitor cell; HG, high glucose; NO, nitric oxide; p-, phosphorylated.
Schematic of the potential role and mechanisms of icariin in HG-induced EPC dysfunction. Icariin can inhibit the activation of the p38/CREB signaling pathway induced by HG in EPCs, and activate the Akt/eNOS/NO signaling pathway that is inhibited by HG in EPCs. It is proposed that via these mechanisms, icariin attenuates HG-induced EPC dysfunction. CREB, cAMP response element binding protein; eNOS, endothelial nitric oxide synthase; EPC, endothelial progenitor cell; HG, high glucose; NO, nitric oxide; p-, phosphorylated.