OBJECTIVE: To investigate the effectiveness and preliminary mechanisms of icariin (ICA) in enhancing the reparative effects of adipose-derived stem cells (ADSCs) on skin radiation damagies in rats. METHODS: Twelve SPF-grade Sprague Dawley rats [body weight (220±10) g] were subjected to a single dose of 10 Gy X-ray irradiation on a 1.5 cm×1.5 cm area of their dorsal skin, with a dose rate of 200 cGy/min to make skin radiation damage model. After successful modelling, the rats were randomly divided into 4 groups ( n=3), and on day 2, the corresponding cells were injected subcutaneously into the irradiated wounds: group A received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL), group B received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL)+1 μmol/L ICA (0.1 mL), group C received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL) pretreated with a hypoxia-inducible factor 2α (HIF-2α) inhibitor+1 μmol/L ICA (0.1 mL), and group D received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL) pretreated with a Notch1 inhibitor+1 μmol/L ICA (0.1 mL). All treatments were administered as single doses. The skin injury in the irradiated areas of the rats was observed continuously from day 1 to day 7 after modelling. On day 28, the rats were sacrificed, and skin tissues from the irradiated areas were harvested for histological examination (HE staining and Masson staining) to assess the repair status and for quantitative collagen content detection. Immunohistochemical staining was performed to detect CD31 expression, while Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to measure the protein and mRNA relative expression levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB), fibroblast growth factor 2 (FGF-2), interleukin 10 (IL-10), transforming growth factor β (TGF-β), HIF-2α, and Notch1, 2, and 3. RESULTS: All groups exhibited skin ulcers and redness after irradiation. On day 3, exudation of tissue fluid was observed in all groups. On day 7, group B showed significantly smaller skin injury areas compared to the other 3 groups. On day 28, histological examination revealed that the epidermis was thickened and the dermal fibers were slightly disordered with occasional inflammatory cell aggregation in group A. In group B, the epidermis appeared more normal, the dermal fibers were more orderly, and there was an increase in new blood vessels without significant inflammatory cell aggregation. In contrast, groups C and D showed significantly increased epidermal thickness, disordered and disrupted dermal fibers. Group B had higher collagen fiber content than the other 3 groups, and group D had lower content than group A, with significant differences ( P<0.05). Immunohistochemical staining showed that group B had significantly higher CD31 expression than the other 3 groups, while groups C and D had lower expression than group A, with significant differences ( P<0.05). Western blot and qRT-PCR results indicated that group B had significantly higher relative expression levels of VEGF, PDGF-BB, FGF-2, IL-10, TGF-β, HIF-2α, and Notch1, 2, and 3 proteins and mRNAs compared to the other 3 groups ( P<0.05). CONCLUSION ICA may enhance the reparative effects of ADSCs on rat skin radiation damage by promoting angiogenesis and reducing inflammatory responses through the HIF-2α-VEGF-Notch signaling pathway.
Animals ; Rats, Sprague-Dawley ; Skin/pathology* ; Rats ; Vascular Endothelial Growth Factor A/genetics* ; Basic Helix-Loop-Helix Transcription Factors/genetics* ; Signal Transduction ; Flavonoids/pharmacology* ; Adipose Tissue/cytology* ; Stem Cells/cytology* ; Receptors, Notch/metabolism* ; Radiation Injuries, Experimental/metabolism* ; Wound Healing/drug effects* ; Male

To evaluate radiosensitivity and the effects of radiation on the expression of vascular endothelial growth factor (VEGF) and VEGF receptors in the canine oral melanoma cell line, TLM 1, cells were irradiated with doses of 0, 2, 4, 6, 8 and 10 Gray (Gy). Survival rates were then determined by a MTT assay, while vascular endothelial growth factor receptor (VEGFR)-1 and -2 expression was measured by flow cytometry and apoptotic cell death rates were investigated using an Annexin assay. Additionally, a commercially available canine VEGF ELISA kit was used to measure VEGF. Radiosensitivity was detected in TLM 1 cells, and mitotic and apoptotic cell death was found to occur in a radiation dose dependent manner. VEGF was secreted constitutively and significant up-regulation was observed in the 8 and 10 Gy irradiated cells. In addition, a minor portion of TLM 1 cells expressed vascular endothelial growth factor receptor (VEGFR)-1 intracellularly. VEGFR-2 was detected in the cytoplasm and was down-regulated following radiation with increasing dosages. In TLM 1 cells, apoptosis plays an important role in radiation induced cell death. It has also been suggested that the significantly higher VEGF production in the 8 and 10 Gy group could lead to tumour resistance.
Animals ; Apoptosis/*radiation effects ; Cell Line, Tumor/radiation effects ; Dogs ; Dose-Response Relationship, Radiation ; Enzyme-Linked Immunosorbent Assay/veterinary ; Melanoma/genetics/metabolism ; Mouth Neoplasms/genetics/metabolism ; Radiation Tolerance ; Tetrazolium Salts/metabolism ; Thiazoles/metabolism ; Up-Regulation/*radiation effects ; Vascular Endothelial Growth Factor A/genetics/metabolism/*radiation effects ; Vascular Endothelial Growth Factor Receptor-1/genetics/metabolism/*radiation effects ; Vascular Endothelial Growth Factor Receptor-2/genetics/metabolism/*radiation effects

BACKGROUNDOsteopontin (OPN) is a secreted phosphoglycoprotein (SSP) that is overexpressed in a variety of tumors and was regarded as a molecular marker of tumors. In this study, we intended to demonstrate the role of OPN in human breast cancer cell line MDA-MB-231.

METHODSRecombinant plasmid expressing small interfering RNA (siRNA) specific to OPN mRNA was transfected into MDA-MB-231 cells to generate the stable transfected cell line MDA-MB-343, and the empty plasmid tansfected cells (MDA-MB-neg) or wildtype MDA-MB-231 cells were used as control cells respectively. Expression of OPN, hypoxia inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) proteins was analyzed by Western blotting analysis. The radiosensitivity of cells was determined by detecting cell apoptosis, cell proliferation and cell senescence.

RESULTSHIF-1 and VEGF proteins in MDA-MB-343 cells were significantly downregulated upon the efficient knockdown of OPN expression under either hypoxia or normoxia environment. Moreover, expression of OPN protein was upregualted upon hypoxic culture. Stable OPN-silencing also decreased cell invasion, increased cell apoptosis and cell senescence, as well as reduced clonogenic survival, resulting in increase radiation tolerance.

CONCLUSIONSSuppression of OPN gene expression can enhance radiosensitivity and affect cell apoptosis in breast cancer cells. OPN seems to be an attractive target for the improvement of radiotherapy.

Breast Neoplasms ; genetics ; metabolism ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; drug effects ; genetics ; Humans ; Hypoxia-Inducible Factor 1 ; genetics ; metabolism ; Osteopontin ; genetics ; metabolism ; RNA, Small Interfering ; Radiation Tolerance ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVETo investigate the effect of static magnetic field on deep wound healing of SD rats and VEGF during the wound healing and different strength static magnetic field on deep wound healing of SD rats.

METHODSDivided forty-eight SD rats into three groups: 0.16 T magnetic disk treatment (0.16 T group), 0.32 T magnetic disk treatment (0.32 T group), control group. General wounds healing situation was observated on the 3, 6, 9, 12 day. The area of every wound was calculated. The tissue of granulation was dyeing by immune tissue chemical decoration method, in which VEGF protein content with its range in tissue was measured.

RESULTSThe healing index of 0.16 T magnetic group wounds were larger than that of control group on 6th and 9th day, there were statistical difference. The healing index of 0.32 T magnetic group wounds were larger than that of control group on 3rd, 6th, 9th and 12th day, there were statistical difference. The healing index of 0.32 T group wounds contrasted to that of 0.16 T group wounds had no statistical significance. Observation of VEGF at the course of wound healing:the expressing of VEGF in magnetic group wounds on 3rd and 6th was stronger than in control group wounds, there were statistical difference. While there were no obvious difference between them on 9th and 12th day (P>0.05). But the contrast between that in 0.32 T group and in 0.16 T group had no statistical difference. The expressing strength of VEGF in magnetic group reached the peak amplitude on the 6th day, and that in control group reached peak amplitude on 9th day. And the peak amplitude of magnetic group was stronger than that of control group.

CONCLUSIONStatic magnetic disc of 0.16T and 0.32 T can promote deep wound of SD rats heal. The mechanism of static magnetic field promoting wound heal may be relative to the expressing highly of VEGF during early and middle time.

Animals ; Magnetic Field Therapy ; Male ; Rats ; Rats, Sprague-Dawley ; Regeneration ; physiology ; radiation effects ; Skin ; radiation effects ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; Wound Healing ; radiation effects

BACKGROUNDLow-power helium-neon (He-Ne) lasers have been increasingly widely applied in the treatment of cardiovascular diseases, and its vasodilation effect has been proven. The aim of this study was to determine the effects of low-power He-Ne laser irradiation directed at the precardial region of Wistar rats on capillary permeability in the myocardium and the expression of myocardial vascular endothelial growth factor (VEGF).

METHODSSixteen rats were divided randomly into control and irradiated groups (n = 8, each). A He-Ne laser (632.8 nm) was applied to the irradiated group with a dose of 60.5 J/cm(2). Ferritin was perfused into the left femoral vein and capillary permeability was examined under an electron microscope. VEGF expression in the myocardium was investigated by immunohistochemical methods, RT-PCR, and image analysis.

RESULTSThe ultrastructures of the myocardial capillaries were examined. Compared to the control group, more high-density granules (ferritin), which were present within the capillary endothelium and the mitochondrions of myocardial cells in the internal layer of the myocardium, were observed in the irradiated group. VEGF staining of the myocardium was stronger in the irradiated group than that in the control group. The optic density of the irradiated group (0.246 +/- 0.015) was significantly higher than that of the control group (0.218 +/- 0.012, P < 0.05). Finally, the levels of RT-PCR products of VEGF165 mRNA were 2.79 times higher in irradiated rats than in the control rats.

CONCLUSIONSOur study demonstrates that He-Ne laser irradiation (in doses of 60.5 J/cm(2)) increases myocardial capillary permeability and the production of VEGF in myocardial microvessels and in myocardium. Our study provides experimental morphological evidence that myocardial microcirculation can be improved using He-Ne laser irradiation.

Animals ; Capillary Permeability ; radiation effects ; Female ; Heart ; radiation effects ; Immunohistochemistry ; Lasers ; Microscopy, Electron ; Myocardium ; metabolism ; ultrastructure ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Vascular Endothelial Growth Factor A ; analysis ; genetics