Effect of vitamin K2 on β-glycerophosphate-induced calcification in rat vascular smooth muscle cells and the mechanism / 中华肾脏病杂志

Objective To explore the effect of vitamin K2 on β-glycerophosphate(β-GP)-induced rat vascular smooth muscle cells (VSMCs) calcification and and the mechanism.Methods VSMCs were obtained from rat aortic,and identified by immunocytochemistry,then randomly divided into control group,high phosphorus group,vitamin K2 group (the group was settled three subgroups according to the concentration of vitamin K2 based on the high phosphorus medium,namely 10 μmol/L,25 μmol/L,50 μmol/L) and noggin (bone morphogenetic protein pathway inhibitor) group.Calcification was visualized by Alizarin red staining,calcium load in cells was quantified by o-cresolphthalein complexone method and alkaline phosphatase (ALP) activity was measured after stimulating 14 days,gene expressions of bone morphogenetic protein-2 (BMP-2),SMAD1,SMAD7 and Runx2 mRNA were detected by RT-PCR,Runx2 protein levels was detected by Western blotting after stimulating 3 days.Results Compared with the cells in control group,high phosphorus induced cell calcification,increased ALP activity,up-regulated the expression of BMP-2,SMAD1,Runx2 mRNA (P ＜ 0.05) and down-regulated the expression of SMAD7 (P ＜ 0.01),while compared with high phosphorus group,the calcium deposition,ALP activity and the expression of BMP-2,SMAD1,Runx2 mRNA were remarkably reduced in a dose-dependent manner by treatment with vitamin K2 (P ＜ 0.05) and the expression of SMAD7 was increased (P ＜ 0.01).Compared with high phosphorus group,SMAD1 and Runx2 expression in noggin group were remarkably reduced(P ＜ 0.01).Conclusion Vitamin K2 inhibits β-glycerophosphate-induced VSMCs calcification which correlates with the suppression of the expression of osteoblast markers through the down-regulation of bone morphogenetic protein pathway.

Effect and mechanism of magnesium on vascular calcification induced by hyperphosphate / 中华肾脏病杂志

Objective To explore the effect and mechanism of magnesium on calcification induced by hyperphosphate.Methods Vascular smooth muscle cells (VSMCs) were primarily cultured in vitro and induced calcification by β-glycerophosphate (β-GP).VSMCs were randomly divided into control group,high phosphorus group (10 mmol/L β-GP),magnesium intervèntion group(10 mmol/L β-GP + 3 mmol/L MgSO4) and 2-aminoethoxy-diphenylborate (2-APB,an inhibitor of magnesium transporter) intervention group(10 mmol/L β-GP+3 mmol/L MgSO4+ 10-4 mol/L 2-APB).Calcium deposition and alkaline phosphatase (ALP) activity were measured by alizarin red staining,quantification of calcium and euzyme linked immunosorbent assay.RT-PCR and Western blotting were used to observe the expression of core binding factor α-1 (Cbfα-1) mRNA and protein,respectively.In vivo,male Sprague-Dawley rats (n=24) were randomly divided into control group (methylcellulose+high phosphorous diet),vascular calcification group (adenine suspension + high phosphorous diet),high magnesium intervention group(adenine suspension+high phosphorous and magnesium diet).The aortic pulse wave velocity (PWV) was measured,and vascular calcification was determined by von Kossa stain and quantification of calcium.Cbfα-1 in aortic was measured by immunohistochemistry.Results In vitro,compared with high phosphorus group,calcification,ALP activity (P ＜ 0.05) and Cbfα-1expression in VSMCs were significantly decreased in magnesium intervention group after incubation for 14 days,but the addition of 2-APB might inhibit the protective effect of magnesium on VSMCs.Dynamic observation of Cbfα-1 showed that magnesium significantly inhibited the expression of Cbfα-1 (P ＜ 0.05) on the third day and the inhibitory role was obviously increased in a time-dependent manner.Consistent with the findings in vitro,the aortic PWV,calcification were all significantly reduced (P ＜ 0.05) in high magnesium intervention group with high serum magnesium level,when compared with vascular calcification group.Immunohistochemistry showed that hypermagnesemia downregulated obviously the expression of Cbfα-1 induced by hyperphosphatemia(P ＜ 0.05).Conclusion Magnesium protects against vascular calcification by inhibiting osteogenic differentiation of VSMCs.

Preparation of lappaconitine-loaded chitosan/sodium β-glycerophosphate injectable thermosensitive hydrogels and research on its properties / 中国药学杂志

OBJECTIVE: To prepare lappaconitine (LA)-loaded chitosan/sodium β-glycerophosphate (CS/β-GP) thermosensitive hydrogels and investigate its phase transition mechanism of gel formation process and release properties in vitro. METHODS: The injectable CS/β-GP thermosensitive hydrogels were prepared with biodegradable CS as carrier material and β-GP as coagulation accelerator.The release behavior in vitro was studied by dynamic dialysis, and the phase transition mechanism of gel formation process was further investigated by rheologieal method. RESULTS: The optimized process condition was as follows;the concentration of β-GP and CS was 560 and 22 mg·mL-1, respectively, CS was dissolved by 0.1 mol·L-1 HOAc, and the valume ratio of CS to β-GP was 8.75:1.25 (V/V), the gelation time of CS/β-GP thermosensitive hydrogels with volume ratio of 8.75:1.25 (V/V) at 37℃ was 5 min 38 s.The in vitro release study showed that these injectable CS/β-GP thermosensitive hydrogels had sustained release effect for LA, and the release behavior could be well described by the Higuchi model and Korsmeyer-Peppas model.The mechanism of LA releasing from CS/β-GP thermosensitive hydrogels was attributed to drug dissolution and diffusion.Rheologieal studies showed that the CS/β-GP thermosensitive hydrogels belonged to thixotropic system and exhibited non-Newtonian and shear-thinning fluid behavior as well as "solid-like" gelatin behavior. CONCLUSION: LA-Loaded CS/β-GP injectable thermosensitive hydrogels with good elasticity and gel strength properties are prepared successfully, and they show sustained release effect of LA in vitro.

Preparation of Wei medicine Kuijiean thermosensitive hydrogel and study on its release properties / 中草药

By developing a rapid gelation chitosan (CS) /β-glycerophosphate (β-GP) thermosensitive hydrogel containing Kuijiean, to decrease the loss of drug and confirm the capabilities of the drug delivery. Thermosensitive hydrogel was a carrier, gel time was chosen in this study as the index to investigate the effect of β-GP concentration, pH value, and temperature on the thermosensitive hydrogel by single factor experiments. The properties of the hydrogel were characterized regarding shape and surface morphology by using scaning electron microscopy (SEM); The chemical structure diversification of hydrogels upon gelation was charactered by FTIR spectrometer. By the experiments of the drug loaded thermosensitive hydrogel to deliver drug in vitro, the diversification of the capabilities of the drug delivery was evaluated. The temperature of Kuijiean thermosensitive hydrogel was (37.0 ± 4.5) ℃, by which the sol gel could transform into semi-solid gel at (6.00 ± 0.82) min, the drug release rate slowed down obviously, and the cumulative release rate was only (67.78 ± 0.35)% (n = 3) by 24 h, while the cumulative release rate of the equal quantity of raw material drug was (90.43 ± 0.62)% (n = 3) by 24 h. The release behavior was close to the Weibull model, the drug release mechanism was a double mechanism combining drug diffusion and gel erosion. It is true that achieves the transformation of Kuijiean from sol to semi-solid gel at 37.0 ℃. The CS/β-GP gel system allows the sustained release of the Kuijiean.

Effects of Magnesium Ions on the Calcification of Vascular Smooth Muscle Cells / 天津医药

Objective To explore the effects of the different concentrations of magnesium ions on vascular smooth muscle cell (VSMC) calcification in rats. Methods VSMCs were obtained from rat aortic, and were identified by immunocy-tochemistry. VSMCs were then randomly divided into control group, high phosphorus group and magnesium intervention group. VSMCs were cultured with 10%fetal bovine serum in control group. VSMCs were cultured with high phosphorus in high phosphorus group. VSMCs were cultured with different concentrations of magnesium chloride based on the high phos-phorus medium in magnesium intervention group (final concentrations of magnesium ions were 1, 2 and 3 mmol/L). The calci-um content and alkaline phosphatase(ALP)activity were measured after the stimulation for 7 days. The expression of Cbfα1 mRNA was detected by RT-PCR. Results Compared with control group, calcium deposits were found significantly higher in high phosphorus group and magnesium intervention group. The calcified nodules gradually reduced with the increased magnesium ion concentration in the intervention group. The calcium contents were significantly lower in the intervention groups (2 and 3 mmol/L) compared with those of high phosphorus group (P<0.05), but no difference was found between 1 mmol/L magnesium intervention group and high phosphorus group. There were no significant differences in the ALP activity and Cbfα1 mRNA expression between intervention groups (2 and 3 mmol/L) and control group (P<0.05). The ALP activity and the expression of Cbfα1 mRNA were gradually decreased with the increased magnesium ion concentration in the inter-vention group, and which were lower than those of high phosphorus group (P<0.05). Conclusion Magnesium can reduce calcification and osteoblastic transdifferentiation, which may be achieved by reducing the expression of Cbfα1 in VSMCs.

Preparation and properties of chitosan/β-glycerophosphate thermosensitive hydrogel containing amino-carbon nanotubes / 中国药学杂志

OBJECTIVE: To synthesis chitosan/β-glycerophosphate (CS/β-GP) thermosensitive hydrogel containing multi-walled carbon nanotubes-polyethyleneimine (MWCNTs-PEI) complexes and to lay a foundation for further research of dual slow-release delivery system. METHODS: Chitosan thermosensitive hydrogel containing MWCNTs-PEI was prepared by MWCNTs-PEI dispersed to the chitosan thermosensitive hydrogel. As the indicator of the gelling time, the experiment studied the effect of β-GP concentration, pH, temperature and MWCNTs-PEI composite quality on the thermosensitive chitosan hydrogel, and then it was charactered by using transmission electron microscopy (SEM), infrared spectrometer(IR), and initially investigated in vivo compatibility. RESULTS: The dynamic rheology method investigated the gelling temperature were about 37.0°C. Within a certain range, the gelling time of thermosensitive chitosan hydrogel was shortened with the increase of concentration of β-GP, pH, temperature, and the quality of MWCNTs-PEI complexes, and they could be transformed into the hydrogel in vivo. The addition of MWCNTs-PEI complex didn't react chemically with the thermosensitive chitosan hydrogel and significantly make the holes of the chitosan thermosensitive hydrogel smaller by SEM and FT-IR, eventually leading to the swelling rate and the corrosional ratio decrease. CONCLUSION: Chitosan/β-glycerophosphate thermosensitive hydrogel containing amino-carbon nanotubes has a rapid gelation and good temperature-sensitivity, which can serve as a good double sustained-release carrier.

A new repressible alkaline phosphatase in Neurospora crassa EM 5297a.

Neurospora crassa Em 5297a can utilize sodium β-glycerophosphate as a sole phosphorous source (in the place of KH2PO4). Under these conditions a repressible alkaline phosphatase is elaborated which has different pH optimum towards β-glycerophosphate (10.2) and pyrophosphate (9.0) as substrates. This enzyme does not require any metal ion for its activity and could be assayed in the presence of EDTA. However, under conditions of cobalt toxicity, the activity of this enzyme is high and is decreased in copper and nickel toxicities.