SDF-1α/CXCR4 axis promotes migration and invasion of pancreatic cancer cells through inducing epithelial-mesenchymal transition

AIM:To investigate the role of SDF-1α/CXCR4 axis in pancreatic cancer cell migration and invasion.METHODS:The mRNA expression of CXCR4 in 4 pancreatic cancer cell lines was detected by RT-qPCR. The migration and invasion abilities of PANC-1 cells with the axis activated by exogenous SDF-1α or inhibited by CXCR4 inhibitor AMD3100 were detected by Transwell assays. The cell viability was measured by MTS assay. The protein expression of the epithelial-mesenchymal transition (EMT)-related molecules in the cells treated with exogenous SDF-1α or AMD3100 was determined by Western blot.RESULTS:All of the 4 pancreatic cancer cell lines expressed CXCR4 mRNA, while the PANC-1 cell line expressed the most. Exogenous SDF-1α promoted the migration and invasion abilities of PANC-1 cells, which was inhibited by AMD3100. The PANC-1 cells treated with exogenous SDF-1α for 72 h grew faster, while SDF-1α combined with AMD3100 made little significance to the viability of PANC-1 cells. Exogenous SDF-1α induced EMT of PANC-1 cells by up-regulating the expression of SNAIL and TWIST, and AMD3100 reversed this effect.CONCLUSION:SDF-1α/CXCR4 axis enhances the migration and invasion abilities of pancreatic cancer cells through inducing EMT.     

Resveratrol modulates SDF-1/CXCR4 signaling to inhibit hypoxia-ischemia-induced apoptosis of neurons from the brain of newborn rats

AIM:To investigate the effect of resveratrol (RSV) on apoptosis and stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway in hypoxia-ischemia-induced neurons from the brain of newborn rats and its mechanism. METHODS:The cortex neurons from the brain of newborn rats were given oxygen-glucose deprivation (OGD) treatment to mimic neonatal hypoxic-ischemic encephalopathy (HIE). The cortex neurons were randomly divided into 4 groups:control group, HIE model group, HIE+RSV-low (10 μmol/L) group, and HIE+RSV-high (50 μmol/L) group. After OGD treatment for 2 h, the neurons were cultured with indicated dose of RSV for 24 h. The apoptosis was analyzed by flow cytometry. Western blot was used to determine the levels of apoptosis-related proteins, SDF-1 and CXCR4. Real-time PCR was used to detect the mRNA expression of SDF-1 and CXCR4. Additionally, to explore the effects of RSV on cell apoptosis and apoptosis-related proteins after the suppression of SDF-1/CXCR4 signaling, a CXCR4 antagonist AMD3100, and RSV were used to co-treat OGD-injured neurons for 24 h. RESULTS:RSV alleviated OGD-induced neuronal apoptosis, down-regulated cleaved caspase-3 and cytochrome C levels, and up-regulated the ratio of Bcl-2/Bax. Compared with the control group, OGD treatment increased the expression of SDF-1 and CXCR4 (P<0.05). Compared with the HIE model group, RSV further up-regulated the expression of SDF-1 and CXCR4 (P<0.05). AMD3100 reversed the effects of RSV on OGD-induced cell apoptosis. CONCLUSION:RSV suppresses hypoxia-ischemia-induced apoptosis of neurons from the brain of newborn rats via up-regulating SDF-1/CXCR4 signaling pathway.     

The SDF-1/CXCR4 axis repairs hypoxia-ischemia brain damage via-regulating oriented differentiation of mesenchymal stem cells

AIM:To explore the effect of stromal cell-derived factor-1(SDF-1)/CXC chemokine receptor 4(CXCR4)axis on the neural differentiation of rat mesenchymal stem cells(rMSCs) and recovery from hypoxia-ischemia brain damage(HIBD). METHODS:The rMSCs were isolated from the rat bone marrow, and expanded in vitro. The mRNA and protein levels of CXCR4 in rMSCs treated with SDF-1α(10 μg/L) and hypoxia for 0 h, 6 h, 12 h, 24 h, 48 h and 72 h were detected by RT-PCR, Western blotting and flow cytometry. The mRNA and protein levels of SDF-1α in the hippocampus of the rats with hypoxia-ischemia for 1 d, 3 d, 5 d, 7 d, 14 d and 21 d were also detected by the same methods. The protein levels of neuron-specific enolase(NSE) and glial fibrillary acidic protein(GFAP), as well as the positive rate of neural-induced rMSCs pretreated with AMD3100(a CXCR4 antagonist) at dose of 5 mg/L were determined by Western blotting and immunocytochemistry. RESULTS:Compared with the normal controls, both mRNA and protein levels of CXCR4 increased in rMSCs exposed to hypoxia for 6 h and 12 h, and the results were also confirmed by flow cytometry. As expected, the mRNA level of SDF-1α in the hippocampus of HIBD rats was higher than that in normal control rats(P<0.01). Moreover, the mRNA expression of CXCR4 was extremely up-regulated in rMSCs treated with SDF-1α at concentration of 10 μg/L, and the results were also confirmed by Western blotting and flow cytometry analysis(P<0.01). The protein levels and positive cell numbers of NSE and GFAP were extremely decreased in rMSCs pretreated with AMD3100 at concentration of 5 mg/L. CONCLUSION:Compared with normal rats, SDF-1α level in the hippocampus of the rats with hypoxia-ischemia is increased. Hypoxia and micro-dose of SDF-1α induce the expression of CXCR4 in rMSCs, while CXCR4 antagonist reduces the neural differentiation of rMSCs, suggesting that SDF-1/CXCR4 axis may be deeply involved in the neural differentiation of rMSCs during the process of repairing HIBD.     

Role of CXCR4 in changes of protein C system in ulcerative colitis mice

AIM: To explore the role of chemokine receptor CXCR4 in the pathogenesis of protein C system (PCS) in ulcerative colitis (UC).METHODS: In vivo, the mice were divided into control group and UC group. The macroscopic score, microscopic score and ulcer index were assessed. The mRNA levels and activity of myeloperoxidase (MPO), cyclooxygenase-2 (COX-2), stromal cell-derived factor-1α (SDF-1α) and monocyte chemotactic protein 1 (MCP-1) both in colonic tissue and plasma were determined. The expression and location of CXCR4, β-arrestin, p-JNK, endothelial cell protein C receptor (EPCR) and thrombomodulin (TM) were detected. The activity of protein C (PC) and protein S (PS) was measured in each group. In vitro, mouse colonic microvascular endothelial cells were isolated, cultured and identified. Both CXCR4-overexpressing and CXCR4-silencing colonic mucosa microvascular endothelial cells were constructed. The effects of SDF-1α on the protein levels of EPCR, TM, β-arrestin and p-JNK, and on the activity of PC, PS and activated protein C (APC) were observed.RESULTS: Compared with control group, UC mice showed increased gross score, histopathological score and ulcer index (P<0.05). The mRNA levels and activity of MPO, COX-2, SDF-1α and MCP-1 in colon and plasma were increased (P<0.01). The protein levels of CXCR4, β-arrestin and p-JNK were up-regulated, EPCR expression was down-regulated in colon, and the activity of PC and PS in plasma was decreased (P<0.05 or P<0.01). CXCR4 overexpression further aggravated SDF-1α-induced PCS inhibition in colonic mucosa microvascular endothelial cells, and further up-regulated the protein levels of β-arrestin and p-JNK (P<0.05).CONCLUSION: PCS is inhibited in UC. CXCR4 is involved in the regulation of PCS inhibition by mediating chemokines and acting on colonic mucosa microvascular endothelial cells through β-arrestin-JNK pathway.     

Effects of erythropoietin on expression of SDF-1 and its receptor in peripheral blood endothelial progenitor cells from rats with chronic renal failure

AIM:To investigate the effects of erythropoietin (EPO) on the expression of homing factors in peripheral blood endothelial progenitor cells (EPCs) from rats with chronic renal failure (CRF). METHODS:The CRF model was established by a two-stage 5/6 nephrectomy procedure in rats. Experimental rats were randomly divided into three groups: sham operation group, CRF model group and EPO treatment group. From the third week after the second stage of 5/6 nephrectomy procedure, rats in EPO treatment group received subcutaneous injection of human recombinant EPO at 50 U/kg every time and three times a week for 6 weeks, and then all the rats were sacrificed. EPCs were isolated from rat peripheral blood and primarily cultured. The mobilization, angiogenesis and functional activity of EPCs in vitro were detected. The mRNA and protein expression of EPO, EPO receptor (EPOR), stromal cell-derived factor 1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) in EPCs was also detected by the methods of real-time PCR and Western blotting. RESULTS:Compared with CRF model group, the expression of EPO and EPOR in EPCs in EPO treatment group was significantly up-regulated (P<0.05). Moreover, the expression of SDF-1 and its receptor CXCR4 in EPCs was also up-regulated by administration of EPO (P<0.05). CONCLUSION: EPO can mobilize EPCs from CRF rat peripheral blood, which may be associated with the increased expression of SDF-1 and its receptor CXCR4.     

Effect of AMD3100 on dengue virus type 2-induced apoptosis in Eahy926 cells

AIM: To investigate the role of AMD3100 (an inhibitor of CXCR4) in dengue virus type 2 (DV2)-induced apoptosis in human umbilical vein endothelial cell line Eahy926. METHODS: The expression of factor Ⅷ in Eahy926 cells was examined by immunohistochemistry staining. The cells were divided into untreated group, DV2 infection group and DV2+AMD3100 group. Flow cytometric analysis was used to detect the expression of CXCR4 in Eahy926 cells 24 h, 36 h, 48 h and 60 h after DV2 infection. In addition, the percentage of apoptotic cells was also analyzed by flow cytometry. Immunofluorescence was performed to detect the phosphatidylserine (PS) on the surface of Eahy926 cells.RESULTS: Eahy926 cells were factor Ⅷ-positive. Compared with untreated group, the expression of CXCR4 increased in DV2 infection group, most markedly 48 h after infection (66.13%±10.30%, P<0.05). The percentage of apoptotic Eahy926 cells after DV2 infection was the highest at 36 h (29.85%±15.78%, P<0.05). The percentage of DV2-induced apoptotic cells in DV2+AMD3100 group was higher than that in DV2 infection group. The green fluorescence-labeled cells in DV2 infection group and DV2+AMD3100 group were more than those in untreated group. CONCLUSION: DV2 infection induces apoptosis and increases the expression of CXCR4 in Eahy926 cells. AMD3100, the inhibitor of CXCR4, may be a promoter of apoptosis in Eahy926 cells after DV2 infection.     

Donor age affects confluent EPCs on phenotypic transition,proliferation and migration of smooth muscle cells

AIM: To explore the effects of confluent endothelial progenitor cells (EPCs) derived from young and aged rats on the phenotype conversion, proliferation and migration of vascular smooth muscle cells (SMCs). METHODS: Mononuclear cells were obtained from the bone marrow of young (1~2 month old) and aged (19 to 26 month old) Sprague-Dawley rats and cultured with medium DMEM/F12 (containing 15% fetal bovine serum, endothelial cell growth supplements (ECGs) 100 g/L, 1×10⁵ units/L of penicillin and streptomycin, respectively). EPCs were characterized as double positive for DiI-Ac-LDL uptake and lectin binding. Abdominal aorta was obtained from 1 to 2 month old Sprague-Dawley rats. Vascular SMCs were cultured by tissue explant method and identified by α-SM-actin immunofluorescence. In transwell co-culture system, the confluent EPCs located in the upper chamber and SMCs were seeded on the lower chamber. The experiments were divided into passage 3 SMCs group (P3), passage 4 SMCs group (P4), passage 4 SMCs co-culture with EPCs derived from young rats group (P4YE) and passage 4 SMCs co-culture with EPCs derived from aged rats group (P4AE). The protein expression of α-SM-actin and osteopontin was detected by Western blotting. [³H]-TdR incorporation assay was used to determine the proliferation. SMC migration was analyzed by scratch wound healing assay. RESULTS: Compared with P3 group, α-SM-actin expression in P4 group significantly decreased and osteopontin protein expression obviously increased, whereas no significant change was found in P4YE group. Compared with P4 group, confluent EPCs derived from young and aged rats both markedly increased α-SM-actin and decreased osteopontin expression in P4 SMCs. Compared with aged rat-derived EPCs, young rat-derived EPCs were more effectively to induce a delayed SMC phenotype transition (from contractile phenotype to a synthetic phenotype), and to inhibit SMC proliferation and migration. CONCLUSION: Co-culture of confluent EPC induces a delayed vascular SMC phenotype transition and inhibits SMCs proliferation and migration. Young rat derived EPCs are more effective to induce a delayed vascular SMC phenotype transition and has stronger inhibitory effects on SMCs proliferation and migration compared with that derived from aged rats.     

Inhibitory effects of triptolide on cell proliferation and metastasis in Raji cells in vitro

AIM:To investigate the inhibitory effects of triptolide on cell proliferation and metastasis in Burkitts lymphoma cell line Raji cells.METHODS:The effects of triptolide on the growth of Raji cells were studied by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium(MTT) assay. The effects of triptolide on the cell apoptosis of Raji cells were detected by using Annexin Ⅴ/PI double-labled cytometry. The effects of triptolide on CXCR4 expression on Raji cells were studied by flow cytometric analysis. Chemotaxis assays were performed to observe the effects of triptolide on migration of Raji cells towards recombinant human SDF-1α (rhSDF-1α）in vitro.RESULTS:Triptolide inhibited the proliferation of Raji cells in a dose- and time-dependent way with a 24 h IC50 value of 43.06 nmol/L and a 36 h IC50 value of 25.08 nmol/L. Following the treatment of triptolide, the cell apoptosis rate was increased as the treatment concentration increased and the culture time extended. The effects were dose- and time- dependent. Triptolide could downregulate the expression of CXCR4 on Raji cells in a dose-dependent manner. Moreover, chemotaxis assay suggested that triptolide could block the migration of Raji cells to rhSDF-1α in vitro, and the inhibition was dose-dependent.CONCLUSION:Triptolide could inhibit the cell proliferation and induce the cell apoptosis of Raji cells. Furthermore， it could block the cell metastasis of Raji cells in vitro and the underlying mechanism might be related to the inhibition of the SDF-1/CXCR4 axis.     

Effect of hypoxia-inducible factor-1α knockdown by siRNA on viability and CEACAM1 expression in oral squamous cell carcinoma

AIM: To investigate the relationship between hypoxia-inducible factor-1α (HIF-1α) and viability and apoptosis of oral squamous cell carcinoma and its mechanism. METHODS: The expression of HIF-1α and carcinoembryonic antigen-related cell adhesion molecular 1 (CEACAM1) at mRNA and protein levels in oral squamous cell carcinoma cell lines Tca8113 and CAL27 and normal epithelial cell line NOK was determined by RT-PCR and Western blot. The expression of HIF-1α in CAL27 cells was silenced by RNA interference (RNAi) technique. The cells were divided into blank control group, non-sense control group and siRNA-HIF-1α group. The viability of CAL27 cells was measured by MTT assay and the apoptotic rate was analyzed by flow cytometry. The protein levels of HIF-1α, P21, vascular endothelial growth factor (VEGF), Bcl-2 and Bax were examined by Western blot. RESULTS: The expression of HIF-1α and CEACAM1 in oral squamous cell carcinoma cells was significantly higher than that in normal cells (P<0.05), and the expression of HIF-1α and CEACAM1 was positively correlated. The protein expression of HIF-1α in siRNA-HIF-1α group was significantly lower than that in blank control group (P<0.05). Knockdown of HIF-1α significantly inhibited CAL27 cell viability (P<0.05), promoted apoptosis (P<0.05), increased the protein levels of P21 and Bax (P<0.05), and significantly decreased the levels of VEGF and Bcl-2 (P<0.05). CONCLUSION: HIF-1α is over-expressed in oral squamous cell carcinoma. Knockdown of HIF-1α significantly inhibits cell viability and promotes apoptosis possibly through regulating the expression of HIF-1α downstream target genes and tumor angiogenesis.     

Migration of enhanced green fluorescent protein labeled bone marrow after transplantation into rat cerebral infarct

AIM:To investigate the role of SDF-1α in migrating of bone marrow stromal cells to the injured areas.METHODS:Ischemic brain lesion model was created in rats by permanent middle cerebral artery occlusion (MCAO).48 SD rats were divided randomly into 2 groups.Group 1:phosphate buffered saline (PBS 1 mL) for control (n=25); Group 2:BMSCs (2×106) were injected intravenously at 24 h after MCAO (n=24).After propagated in BMSCs, Ad5/F35 GFP (green fluorescent protein) was infected to BMSCs.The expression of SDF-1α (stromal cell-derived factor-1α) mRNA in the penrumbral tissue was assayed by real-time quantitative PCR.The expression of CXCR4 on MSCs was detected by flow cytometry.Confocal microscopy was used to detect the GFP-labeled MSCs migration.RESULTS:Ad5/F35 GFP signals was observed in almost infected BMSCs.The expressions of SDF-1α mRNA in the thalamus and hippocampus of the ischemic brains were peaked at 3rd day after stroke, followed by a decrease at 14th day post-ischemia.The expression of SDF-1α mRNA in the cortex of the ischemic brains was peaked at 7th day post-ischemia, still at high level at 14th day post-ischemia.The median percentage of surface CXCR4 expression in BMSCs was 14%.GFP labeled BMSCs were detected in the origination of the middle cerebral artery (olfactory area) at 6 h, after 3 days in the prenumbra tissue such as thalamus, and in the cortex more labeled cells were found after 14 d post-ischemia.CONCLUSION:BMSCs can pass through the blood brain barrier of ischemic rats.Its mechanism might be associated with the expression of SDF-1α in the ischemic brain.     

Transforming growth factor α promotes the proliferation,migration and adhesion of human endothelial progenitor cells

AIM: To explore the effects of transforming growth factor-α (TGF-α) in the monoclonal formation, proliferation, migration and adhesiveness of human endothelial progenitor cells (EPCs). METHODS: The isolated and cultured EPCs were treated with various concentrations of TGF-α (final concentrations of 1, 5, 10 μg/L, respectively). At the same time, the PBS control and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) group (10 μg/L TGF-α plus 1: 1 000 EGFR-TKI) were set. The effects of TGF-α on monoclonal formation, proliferation, migration and adhesiveness of EPCs were determined by clone formation experiment, thiazolyl blue tetrazolium bromide (MTT), EdU, Transwell and adhesion assays, respectively. The expression of epithelial growth receptor (EGFR) and vascular endothelial growth factor (VEGF) were measured by Western blotting. RESULTS: Different concentrations of TGF-α all significantly induced the monoclonal formation, proliferation, migration and adhesiveness of EPCs (P<0.01), which were inhibited by EGFR-TKI. The results of Western blotting showed that TGF-α also induced the expression of EGFR and VEGF with a certain concentration effect (P<0.01). CONCLUSION: By combining with EGFR induced the expression of VEGF, TGF-α significantly promotes the related cell function of monoclonal formation, proliferation, migration, adhesiveness in EPCs.     

Protective effects of salidroside on endothelial progenitor cells damaged by radiation

AIM: To explore the protective effects of salidroside on endothelial progenitor cells (EPCs) damaged by radiation and its mechanisms.METHODS: EPCs from normal peripheral blood were cultured in fibronectin-coated flasks with endothelial progenitor medium. The effects of salidroside on the viability, migration, adhesion and apoptosis of radiation-damaged EPCs were detected. The viability, apoptosis and migration of the cells were assayed by CCK-8 assay, flow cytometry and Transwell chamber experiment, respectively. The cell adhesion assay was performed by re-plating the cells on fibronectin-coated dishes, and then the adherent cells were counted. The expression of Akt protein in the cells was assessed by Western blotting. RESULTS: Salidroside improved the viability, and migratory and adhesive capacities of the EPCs, and decreased the apoptosis after radiation. Salidroside also increased the protein level of phosphorylated Akt. However, the effects of salidroside on radiation-damaged EPCs were inhibited by phosphatidylinositol 3-kinase inhibitor LY294002. CONCLUSION: Salidroside protects EPCs from radiation damages and its mechanism is associated with enhancing phosphatidylinositol 3-kinase/Akt signaling pathway.     

Effect of AG490 on expression of VEGF and HIF-1α in HEL cells

AIM: To investigate the effect of AG490 on the expression of VEGF and HIF-1α, and the capacity of invasion in human erythroleukemia (HEL) cells. METHODS: The HEL cells were treated with AG490 at different concentrations. The cell viability was detected by CCK-8 assay. The apoptosis was detected by Hoechst staining. The apoptosis and the cell cycle were analyzed by flow cytometry. The capacity of migration was evaluated by Transwell assay. The mRNA expression level of JAK2 was measured by RT-PCR. The protein levels of p-JAK2, VEGF and HIF-1α were determined by Western blot. RESULTS: The HEL cell viabilities were 88%, 75%, 48%, 10% and 0.12% after treated with AG490 at 20, 40, 60, 80 and 100 μmol/L for 48 h, respectively. The results of Hoechst staining showed that brilliant blue cells in 80 μmol/L AG490 group was significantly increased compared with control group for 48 h. The apoptosis rate of 80 μmol/L AG490 group was significantly increased compared with control group at 48 h after AG490 treatment. The number of membrane-permeating HEL cells in 20 μmol/L AG490 group at 24 h after AG490 treatment was significantly lower than that in control group (P<0.05). The mRNA level of JAK2 decreased in a concentration-dependent manner after the HEL cells were treated with different concentrations of AG490 for 48 h. The protein levels of p-JAK2, VEGF and HIF-1α were lower in AG490 treatment groups than those in control group (P<0.05). CONCLUSION: AG490 inhibits the expression of VEGF and HIF-1α in HEL cells by inhibiting JAK2 pathway.     

Effects of Danggui Buxue decoction on endothelial progenitor cells,serum VEGF and SDF-1 in atherosclerotic rabbits

AIM: To investigate the effects of Danggui Buxue decoction (DBD) on serum vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1), as well as the activity of circulating endothelial progenitor cells (EPCs) in atherosclerotic rabbits. METHODS: The model of atherosclerosis was established using immune injury and fatty diet for 4 weeks in New Zealand rabbits (n=25). All modeled rabbits were randomized into 5 groups with 5 animals in each group. The rabbits in atherosclerosis group were intragastrically administered with distilled water. The rabbits in simvastatin group were treated with simvastatin at the dose of 1.7 mg/kg. The rabbits in DBD high-dose, middle-dose and low-dose treatment groups were given DBD at the doses of 6 g/kg, 3 g/kg and 1.5 g/kg, respectively. All drugs were given once a day for 2 weeks. After treatment, the levels of serum VEGF and SDF-1 were measured. The mononuclear cells isolated from the rabbit peripheral blood were cultured for 7 days in vitro, and then attached cells were cultured with both DiI-ac-LDL and FITC-UEA-1 for identification. The proliferation was detected by MTT method. The cell migration was observed using Transwell chambers. The adhesion determination and in vitro angiogenesis assay were also performed. RESULTS: Compared with atherosclerosis group, the levels of serum VEGF and SDF-1 were elevated (P<0.05), the proliferation, migration, adhesion and angiogenesis of EPCs were all improved in DBD high-dose, middle-dose treatment groups and simvastatin group (P<0.05). CONCLUSION: DBD elevates the levels of serum VEGF and SDF-1 to improve the activity of EPCs in the process of atherosclerosis.     

Expression of hepatocyte nuclear factor 4α in experimental colitis in mice

AIM: To investigate the role of hepatocyte nuclear factor 4α (HNF4α) in the pathogenesis of ulcerative colitis (UC) by measuring the expression of HNF4α in the colon tissues in experimental colitis mice. METHODS: BALB/c mice were exposed to 2% or 2.5% (W/V) dextran sulfate sodium (DSS) to induce acute colitis, and the severity of colitis was assessed by observation of disease activity index (DAI), histological injuries and inflammatory cytokines. The correlation between the expression of HNF4α and the severity of disease as well as E-cadherin (E-CAD), junctional adhesion molecule 1 (JAM-1) and desmocollin 2 (DSC-2) was analyzed. RESULTS: Compared with the normal controls, DAI, histological injuries and the mRNA expression of inflammatory cytokines in DSS-treated mice were significantly elevated (P<0.05). The expression of HNF4α at protein and mRNA levels was significantly decreased (P<0.01). The result of Pearson analysis indicated an inverse correlation between the protein expression of HNF4α and the severity of disease (P<0.01). The positive correlation between the mRNA expression of HNF4α and E-CAD/JAM-1/DSC-2 (P<0.01) was also observed. CONCLUSION: There is a close relationship between the expression of HNF4α and the severity of colitis as well as the intercellular linking proteins. The low expression of HNF4α in intestine might aggravate the function of intestinal mucosal barrier, thus promoting the development of UC.     

Proton-sensing receptor OGR1 mediates inhibitory effects of acidic environment on viability and tube formation ability of endothelial progenitor cells

AIM: To analyze the expression of ovarian cancer G-protein-coupled receptor 1 (OGR1), a proton-sensing receptor, in the endothelial progenitor cells (EPCs), and to explore the role of OGR1 in acid-regulated viability and tube formation ability of EPCs. METHODS: The method of FITC-UEA-I and DiI-Ac-LDL double staining was used to identify the EPCs. The expression of OGR1 in mouse EPCs was analyzed by real-time-PCR and Western blot. After treatment with different pH media, the OGR1 expression was analyzed in EPCs. The viability and cell cycle distribution of the EPCs were analyzed by CCK-8 and flow cytometry assay. The migration and vascularization of EPCs were measured by scratch test, Transwell migration assay and tube formation experiments after silencing OGR1 using small interfering RNA(siRNA). RESULTS: The isolated endothelial progenitor cells were well differentiated, and FITC-UEA-I and DiI-Ac-LDL staining was positive. The expression of OGR1 at mRNA and protein levels was observed in mouse EPCs. The expression of OGR1 was increased gradually with the decrease of pH value of the medium, while the expression of OGR1 was the highest in the medium of pH 6.4 (P<0.05). pH 6.4 medium inhibited the viability of the EPCs and induced cell cycle arrest at G₀/G₁ phase. Knock-down of OGR1 expression by siRNA partially reversed the effect of acidic environment on the EPCs (P<0.05). The abilities of migration and tube formation of EPCs were inhibited by pH 6.4 medium, while transfection of siRNA to silence OGR1 expression partially reversed the effect (P<0.05). CONCLUSION: The expression of OGR1 in EPCs is positive, and OGR1 mediates the effects of acid on the viability, migration and tube formation ability of EPCs.     

Effects of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells

AIM: To observe the expression of hypoxia-inducible factor-1alpha (HIF-1α) and stromal cell-derived factor 1 (SDF-1) in pulmonary artery endothelial cells (PAECs) of SD rats and to investigate the role of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells to PAECs. METHODS: Immunomagnetic beads were used to separate and purify the CD34⁺/CXCR4⁺ progenitor cells derived from the peripheral circulation of SD rats. The expression of HIF-1α and SDF-1 in PAECs exposed to hypoxia (1% O₂, 5% CO₂ and 94% N₂) was detected by immunofluorescence, Western blotting and ELISA. The migration index and adhesion rate were measured in the progenitor cells, which were subjected to the following different treatments: (1) normoxia (21% O₂); (2) hypoxia 12 h; (3) hypoxia 12 h +HIF-1α inhibitor (2ME2); (4) hypoxia 12 h+SDF-1 neutralizing antibody; (5) hypoxia 12 h+2ME2+SDF-1 neutralizing antibody.RESULTS: The expression of HIF-1α and SDF-1 in PAECs was effectively induced by the hypoxic exposure, and both of them reached the peak levels after 12 h of hypoxic treatment (P<0.01), while administration of 2ME2 decreased the hypoxia-induced SDF-1 expression (P<0.05). Treatment of the PAECs with 2ME2 or SDF-1 neutralizing antibody attenuated the migration index and adhesion rate of progenitor cells to the PAECs (P<0.05). CONCLUSION: There is a HIF-1α/SDF-1 signaling axis in hypoxia-exposed PAECs, which may play a crucial role in the migration and adhesion of progenitor cells to PAECs.     

Effect of plumbagin on levels of Nox4/ROS and α-SMA in human hepatic stellate cells

AIM: To observe the effect of plumbagin on the mRNA and protein expression of nicotinamide adenine dinucleotidephosphate oxidase 4 (Nox4), reactive oxygen species (ROS) level and protein expression of α-smooth muscle actin (α-SMA) in the HSC-LX2 cells stimulated with transforming growth factor β1 (TGF-β1) in vitro. METHODS: HSC-LX2 cells were cultured in vitro and divided into blank group, model group, high-, medium- and low-dose (2, 1.5 and 1 μmol/L) plumbagin groups. After incubated with each drug for 72 h, the mRNA expression of Nox4 was detected by RT-PCR. ROS levels were tested by in situ loading probe method. The protein contents of Nox4 and α-SMA were measured by Western blot. RESULTS: Compared with model group, after treated with plumbagin for 72 h, the mRNA expression of Nox4, ROS level and α-SMA protein were significantly decreased in high-and medium-dose plumbagin groups (P<0.01). CONCLUSION: Plumbagin inhibits the activation of HSC-LX2 cells via decreasing the expression of Nox4, thus decreasing ROS levels.     

Effects of high glucose on proliferation,migration, adhesion and secretion of rat late endothelial progenitor cells

AIM: To investigate the effects of high glucose on the proliferation, adhesion, migration and secretion potentials of late endothelial progenitor cells (EPCs) from bone marrow. METHODS: Mononuclear cells were collected from rat bone marrow by density gradient centrifugation and cultured with M199 medium. The early EPCs were identified by DiI-ac-LDL and FITC-UEA-1 double staining. The late EPCs were identified by RT-PCR to detect the expression of von Willebrand factor (vWF) and VE-cadherin. Moreover, the cells were identified by FACS to detect the expression of CD133 and vascular endothelial growth factor receptor-2(VEGFR-2). The 3rd generation of EPCs was harvested and incubated with glucose in a series of concentrations (5, 10, 20 or 40 mmol/L). The cell proliferation, adhesion, migration and the secretion of chemokines such as monocyte chemoattractant protein-1(MCP-1) and interleukin-8 (IL-8) were assayed with MTT, adhesion test, modified Boyden chamber assay and ELISA, respectively. RESULTS: Compared with normal glucose (5 mmol/L)treatment, high glucose (10, 20, 40 mmol/L) dose-dependently degraded the proliferation and migration of late EPCs (P<0.05 or P<0.01). At the same time, treatment with glucose at the concentration of 40 mmol/L decreased the adhesion of EPCs (P<0.05) and increased the release of MCP-1 and IL-8 by late EPCs. CONCLUSION: High glucose inhibits proliferation, adhesion and migration of late EPCs, and enhances the secretion of inflammatory factors, indicating that the high glucose correlates with the vascular complications of patients with diabetes.     

Effects of propofol on expression of CXCR4/CXCR7 and migration ability of human breast cancer MCF-7 cells in vitro

AIM:To investigate the effects of propofol on the expression of CXC-chemokine receptor (CXCR)4/CXCR7 and migration ability of human breast cancer MCF-7 cells in vitro. METHODS:MCF-7 cells were randomly divided into 4 groups,control group, lipid emulsion group, 3 mg/L and 8 mg/L propofol group. The cell viability was measured by MTT assay. The migration ability was detected by wound-healing assay and Transwell assay. The mRNA level of CXCR4/CXCR7 was detected by RT-qPCR. The protein expression leve of CXCR4/CXCR7 was determined by Western blot. RESULTS:Compared with control group, the scratching healing rates in 3 and 8 mg/L propofol group were decreased (P<0.05), and the chemotactic index was also decreased (P<0.05). The protein expression level of CXCR4/CXCR7 was decreased in 3 and 8 mg/L propofol group(P<0.05). However, both the mRNA level of CXCR4/CXCR7 and the viability of the MCF-7 cells kept no change. CONCLUSION:Propofol down-regulates the protein expression of CXCR4/CXCR7 and inhibits the migration ability of breast cancer MCF-7 cells in vitro.