| Objective: Patients with ESRD has a vastly increased risk of cardiovascular disease,at present has become a worldwide problem.Studies show that cardiovascular disease is the first cause of death for patients with chronic kidney disease(CKD).And vascular calcification is independent risk factors of cardiovascular disease.Membrane in vascular calcification is the main characteristic of vascular calcification in patients with chronic kidney disease(CKD).Vascular smooth muscle cells is an important component of the blood vessels in the membrane.Magnesium can inhibit vascular calcification,The pathogenesis of vascular calcification is a complex mechanism.In this study,we Explore magnesium effects on vascular calcification in chronic renal failure rats and the possible mechanism.Method:1 Primary culture of rat VSMCs.VSMCs were obtained from rat aortic,then randomly divided into control group,high phosphorus group(10mmol/L ?-GP)and magnesium intervention group(10mmol/L ?-GP+3mm ol/L Mg SO4,)2-APB(an inhibitor of magnesium transporter)intervention group.2 The preparation of the aortic rings.Aortic rings were obtained from rat aortic,then randomly divided into control group,high phosphorus group(10mmol/L ?-GP)and magnesium intervention group(10mmol/L ?-GP+3mmo l/L Mg SO4,2-APB(an inhibitor of magnesium transporter)intervention group.3 Calcification of VSMCs and aortic rings.Calcium deposition of VSMCs was measured by Alizarin red staining,quantification of calcium and enzyme linked immunosorbent assay.Calcium deposition of aortic rings was measured by Von Kossa staining and quantification of calcium.4 Alkaline phosphatase(ALP)activity.Cell culture for 7 days,PBS wash three times,0.1%Triton X-100 4? stay the night,ALP)activity was measured by enzyme linked immunosorbent assay.5 The expression of Runx2 was detected by RT-PCR and Western Blot after cells stimulated for 7 days.6 The expression of Runx2 was detected by Immunohistochemistry after aortic rings were cultured for 7 days.7 The expression of LTCC ?1C and ?3 was detected by RT-PCR and Western Blot after cells stimulated for 7 days.8 Deteation of intracelluar calcium ion level of VSMCs by fluorescence probeDeteation of intracelluar calcium ion level of VSMCs by fluorescence probe after cells were cultured for 7 days.9 Statistic methodStatistical analysis Data analyses were conducted using SPSS 17.0 software.All results were expressed as mean ± standard deviation.Differences among groups were determined by analysis of variance(ANOVA),and the Student-Newman-Keuls method was determined by post-hoc testing.P<0.05 denoted a statistically significant difference.Results:1 Magnesium can inhibit calcification of VSMCs and aortic rings.After cells stimulated for 7 days,compared with control group,Alizarin red staining and Von Kossa staining obviously shown that calcification in VSMCs and aortic rings were significantly increased in high phosphorus group(P<0.05)and decreased in 2-APB intervention group(P<0.05).2 Compared with control group,the activity of ALP in high phosphorus groups were increased(P<0.05).3 The expression of Runx2 was detected by RT-PCR and Western Blot after cells cultured for 7 days show that compared with control group,calcification in aortic rings was significantly increased in high phosphorus group(P<0.05)and decreased in magnesium intervention group(P<0.05).4 The expression of Runx2 in aortic rings was downregulated as the magnesium with incubated by ?-GP(P<0.05).5 Compared with high phosphorus group and 2-APB intervention group,the expression of LTCC ?1C ?3 subunit decreased(P<0.05)in high high magnesium group.6 The result of intracelluar calcium ion level of VSMCs show that compared with high phosphorus group,intracelluar calcium ion was downregulated as the magnesium with incubated by ?-GP.Conclusions: Magnesium may downregulate LTCC gene expression,prevent calcium influx and then inhibite osteogenic differentiation so as to reduce ?-glycerophosphate induced VSMCs calcification. |
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