[Effect and mechanism of intermittent alkaline stimulation on high phosphorus induced calcification in vascular smooth muscle cells of rats].

Objective: To explore the effect and possible mechanisms of intermittent alkaline on rat vascular smooth muscle cells (VSMCs) calcification induced by high phosphorus. Methods: VSMCs were isolated from rat thoracic aorta and cultured in vitro. The fourth generation VSMCs were randomly divided into control group, high phosphorus+ pH7.4, high phosphorus+ pH7.5, high phosphorus+ pH7.6 and high phosphorus+ pH7.7 group with random number table. The control group was cultured in DMEM with 10% fetal bovine serum. Other groups were cultured in DMEM with 10 mmol/L ß-glycerophosphate and alkalized by 7.4% NaHCO(3) to adjust the pH respectively. After the intervention of 4 hours, the control group was replaced with the normal medium containing 10% fetal bovine serum, the other 4 groups were replaced with high phosphorus based on the pH value of the culture medium, and then replaced the culture medium every other day. After 4 days intervention, the mRNA and protein expression of L type calcium channel ß(3) subunit(LTCC ß(3)) and Runt related transcription factor 2 (Runx2) were detected by RT-PCR and Western blot. After 4 days intervention, the level of VSMC calcium ion was detected by Fluo-3/AM. After 14 days intervention, alkaline phosphatase (ALP) activity was measured by enzyme linked immunosorbent assay (ELISA) and the calcification was observed by measuring calcium content. Results: (1) Compared with control group, the gene and protein expressions of LTCC ß(3) were higher in high phosphorus+ pH7.4 group (0.49±0.03 vs. 0.23±0.02 and 0.45±0.03 vs. 0.26±0.02 respectively, all P<0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.86±0.05) and protein(0.62±0.04) expressions of LTCC ß(3) were higher in high phosphorus+ pH7.5 group (P<0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.99±0.05) and protein(0.80±0.03) expressions of LTCC ß(3) were higher in high phosphorus+ pH7.5 group (all P<0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.16±0.05) and protein(0.93±0.03) expressions of LTCC ß(3) were higher in high phosphorus+ pH7.7 group (all P<0.05). (2) Compared with control group, calcium ion influx were higher in high phosphorus+ pH7.4 group (124.61±6.06 vs. 75.68±7.82, P<0.05). Compared with high phosphorus+ pH7.4 group, calcium ion influx was higher in high phosphorus+ pH7.5 group(210.85±9.75, P<0.05). Compared with high phosphorus+ pH7.5 group, calcium ion influx was higher in high phosphorus+ pH7.6 group(298.44±11.42, P<0.05). Compared with high phosphorus+ pH7.6 group, calcium ion influx was higher in high phosphorus+ pH7.7 group(401.13±11.41, P<0.05). (3) Compared with control group, the mRNA and protein expressions of Runx2 and ALP were higher in high phosphorus+ pH7.4 group (0.60±0.04 vs. 0.34±0.03, 0.42±0.04 vs. 0.21±0.02, 67.2±4.3 vs. 23.2±2.3 respectively, all P<0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.76±0.05) and protein(0.68±0.03) expressions of Runx2 and ALP(102.1±5.4) were higher in high phosphorus+ pH7.5 group (all P<0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.90±0.05) and protein(0.90±0.05) expressions of Runx2 and ALP(139.3±4.9) were higher in high phosphorus+ pH7.6 group (all P<0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.11±0.05) and protein(1.08±0.06) expressions of Runx2 and ALP(197.0±6.7) were higher in high phosphorus+ pH7.7 group (all P<0.05). (4) Compared with control group, the calcium content were higher in high phosphorus+ pH7.4 group ((75.4±4.3)mg/g pro vs.(25.2±2.1)mg/g pro, P<0.05). Compared with high phosphorus+ pH7.4 group, the calcium content were higher in high phosphorus+ pH7.5 group ((100.8±5.7) mg/g pro, P<0.05). Compared with high phosphorus+ pH7.5 group, the calcium content were higher in high phosphorus+ pH7.6 group ((143.5±6.1) mg/g pro, P<0.05). Compared with high phosphorus+ pH7.6 group, the calcium content were higher in high phosphorus+ pH7.7 group ((205.1±8.2) mg/g pro, P<0.05). Conclusion: Intermittent alkaline stimulation can promote high phosphorus induced rat VSMCs calcification possibly through upregulating LTCC ß(3) subunit gene and protein expression, increasing calcium ion influx and enhancing VSMCs phenotypic transformation.

[Role of nuclear factor of activated T-cells cytoplasmic 1 on vascular calcification in rats with chronic renal failure].

Objective: To explore the role of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) on vascular calcification in chronic renal failure rats. Methods: Nineteen male Sprague-Dawley (SD) rats were randomly divided into three groups: sham-operated group (n=6), 5/6 Nephrectomy (Nx) group (n=6), 5/6 Nx+ calcitriol group (n=7). Vascular calcification was determined by von Kossa staining and orthocresolphthalein complexone (OCPC) method. Protein expressions of NFATc1 and runt-related transcription factor 2 (Runx2) in aortas were measured by immunohistochemistry.In vitro, vascular smooth muscle cells (VSMCs) were primarily cultured and calcification was induced by ß-glycerophosphate (ß-GP). These cells were then randomly divided into control group, calcification group (10 mmol/L ß-GP) and cyclosporin A (CsA) intervention group (10 mmol/L ß-GP+ 1 µg/ml CsA). Calcium deposition was measured by Alizarin red staining and OCPC method; alkaline phosphatase (ALP) activity was measured by enzyme-linked immunosorbent assay. RT-PCR and Western blotting were used to observe the mRNA and protein expression of VSMCs NFATc1 and Runx2 respectively. Results: Compared to that in sham-operated and 5/6 Nx group, the expression of NFATc1 was obviously up-regulated in 5/6 Nx+ calcitriol group (7.20±0.46 vs 1.52±0.77, 2.04±1.31, P<0.05). In vitro, VSMCs calcification was successfully induced by high phosphorus environment, and RT-PCR and Western blotting showed that the expressions of NFATc1 and Runx2 were up-regulated (P<0.05). The calcification level in CsA intervention group was lower than that in calcification group [(60.86±7.95) vs (107.20±11.07) mg/g, P<0.05], and expression of Runx2 (mRNA and protein level) and ALP activity [(48.63±3.02) vs (98.75±3.46) U/g, P<0.05] decreased as well. Conclusion: NFATc1 contributes to accelerating vascular calcification in rat with chronic renal failure, the possible mechanism of which is that NFATc1 promotes VSMCs transformation to osteogenic phenotype.

Embryotrophic factor-3 from human oviductal cells enhances proliferation, suppresses apoptosis and stimulates the expression of the beta1 subunit of sodium-potassium ATPase in mouse embryos.

BACKGROUND: Embrytrophic factor-3 (ETF-3) from human oviductal cells enhanced the development of mouse preimplantation embryos. This report studied the embryotrophic mechanisms of the molecule. METHODS AND RESULTS: Mouse embryos were incubated with ETF-3 for 24 h at different stages of development. ETF-3 treatment between 96 and 120 h post-HCG increased the cell count of blastocysts, whilst treatment between 72 and 96 h post-HCG enhanced the expansion and hatching of the blastocysts. ETF-3 increased the cell number of the embryos by suppressing apoptosis and increasing proliferation as determined by TUNEL and bromodeoxyuridine uptake assays, respectively. Real-time quantitative PCR showed that the in vivo developed and ETF-3-treated blastocysts had a significantly higher mRNA copy number of Na/K-ATPase-beta1, but not of hepsin, than that of blastocysts cultured in medium alone. The former gene was associated with cavitation of blastocysts while the latter was related to hatching of blastocyst. The beneficial effect of ETF-3 on blastocyst hatching was also seen when ETF-3-supplemented commercially available sequential culture medium for human embryo culture was used to culture mouse embryos. CONCLUSIONS: ETF-3 improves embryo development by enhancing proliferation, suppressing apoptosis and stimulating expression of genes related to blastocyst cavitation. Supplementating human embryo culture medium with ETF-3 may improve the success rate in clinical assisted reproduction.

[Anti-tumor effects of ailing-1 on human megakaryoblastic leukemia cells].

OBJECTIVE: To evaluate the anti-cancer effect of Ailing-1 on human megakaryoblastic leukemia cells in culture. METHODS: Using living cell counting, 3H-TdR incorporation and clonal proliferation assay. RESULTS: Ailing-1 has various cytotoxicity on cell level, DNA synthesis and clone proliferation and shows good does-effect relation. The ED50 of Ailing-1 is greater than that of homoharringtonine through three different methods, consistent with the clinical result. Drug resistance has not been found. CONCLUSION: Ailing-1 is a promising anti-tumor drug.

Measurements of n-p correlations in the reaction of relativistic neon with uranium.

We report a preliminary measurement of coincident neutron-proton pairs emitted at 45 degrees in the interaction of 400, 530, and 650 MeV/A neon beams incident on uranium. Charged particles were identified by time of flight and momentum, as determined in a magnetic spectrometer. Neutral particles were detected using a thick plastic scintillator, and their time of flight was measured between an entrance scintillator, triggered by a charged particle, and the neutron detector. The scatter plots and contour plots of neutron momentum vs. proton momentum appear to show a slight correlation ridge above an uncorrelated background. The projections of this plane on the n-p momentum difference axis are essentially flat, showing a one standard deviation enhancement for each of the three beams energies. At each beam energy, the calculated momentum correlation function for the neutron-proton pairs is enhanced near zero neutron-proton momentum difference by approximately one standard deviation over the expected value for no correlation. This enhancement is expected to occur as a consequence of the attractive final state interaction between the neutron and proton (i.e., virtual or "singlet" deuterons). The implications of these measurements are discussed.