High Magnesium Inhibits The Calcification Of Vascular Smooth Muscle Cells Induced By High Phosphorus In Rats By Regulating BMP-2

Objective: Vascular calcification,which has been considered as a common complication of end-stage renal disease for many years,is an important cause of cardiovascular disease(CVD)in patients with end-stage renal disease.However,recent studies have shown that magnesium ion can attenuate vascular calcification,but the underlying mechanism is still unclear.Our previous study found that high magnesium can in a certain degree of inhibition of high phosphorus induced vascular smooth muscle cells(VSMCs)calcification and osteogenic transdifferentiation and its possible mechanism is by reducing the expression of VSMCs of bone related factors,research also found that vascular calcification processes associated with bone formation similar,is organized actively regulated process involving many promotion and inhibition of calcification factors.Bone morphogenetic protein(BMPs)plays an important role in vascular calcification.The BMP-2(Bone morphogenic protein-2,BMP-2)is the most widely studied,is recognized as a BMP osteogenetic activity in the family of the strongest subtypes.Therefore,in this study,the effect of high magnesium on the expression of bone morphogenetic protein 2(BMP-2)was studied by using VSMCs calcification as a model.Methods:1 Experimental methods and grouping1.1 In vitro VSMCs experiment: 4 week old healthy male SD rats(80g-100g)were selected from the animal experimental center of Hebei Medical University.The VSMCs method was used to culture SD rat thoracic aorta.The VSMCs were randomly divided into 4 groups: normal control group,high phosphorus group,high magnesium group and 2-APB intervention group.(1):normal control group with 10% fetal bovine serum DMEM culture;(2)highphosphorus group: add 10 m M beta glycerophosphate in normal medium;(3)high magnesium group joined the Magnesium Sulfate 3m M high phosphate medium(Mg SO4);(4)2-APB group: add 3m M Magnesium Sulfate and10-4m M 2-APB high phosphorus medium.1.2 Vascular ring experiment: select 1 healthy male SD rats aged 240-260weeks(from the animal experimental center of Hebei Medical University,),under sterile conditions to remove the thoracic aorta,the blood vessels into2mm-3 mm long vascular ring.Vascular rings were randomly divided into 4groups(n=5): normal control group,high phosphorus group,high magnesium group,magnesium channel inhibitor(2-APB)intervention group.Culture medium and cell culture.The remaining 2 rats were cultured in vitro according to the above method,and were repeated for 2 times.2 VSMCs calcification was detected by alizarin red staining.The results were as follows: the calcium salt deposition was orange red.Determination of cell calcium content: BCA method for the determination of cell protein content,the results of protein content of calcium content(mg/g protein).The results of ALP assay showed that the high magnesium environment inhibited the activity of ALP.3 The activity of ALP was detected by enzyme linked immunosorbent assay,and the protein was used to calibrate the ALP activity.RT-PCR and Western-blot were used to detect the expression of BMP-2 and Runx2 in each group of cells.4 Statistical analysis Data analyses were conducted using SPSS 17.0 software(SPSS Company,Chicago,IL,USA).All results were presented 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.For all the statistical tests,significance was defined as P<0.05.Result:1 Effects of high magnesium on the calcification of vascular smooth muscle cells and thoracic aorta induced by high phosphorus1.1 After 14 days of cell culture,alizarin red staining and calcium content showed that,compared with normal control group,cell calcium salt deposition was significantly increased in high phosphorus and magnesium channel inhibitor intervention group(P<0.05).And calcification in VSMCs was significantly decreased in high magnesium group(P<0.05).1.2 After 14 days of cell culture,von Kossa staining showed that,compared with normal control group,calcification in aortic rings was significantly increased in high phosphorus group and magnesium channel inhibitor intervention(P<0.05)and decreased in high magnesium group(P<0.05).2 Effect of high magnesium on expression of BMP-2,Runx2 and ALP in vascular smooth muscle cells induced by high phosphorus.The results of RT-PCR and Western-blot showed that the expression of BMP-2 and Runx2 in the high phosphorus group was significantly higher than that in the normal control group(P<0.05).Compared with the high phosphorus group,the expression of BMP-2 and Runx2 in the high magnesium group was significantly decreased(P<0.05).Compared with the high magnesium group,the expression of BMP-2 and Runx2 increased in the magnesium channel inhibitor(P<0.05).3 Effect of high magnesium on the expression of BMP-2 and Runx2 in rat thoracic aortic rings induced by high phosphorus.The expression of BMP-2and Runx2 in aortic rings was higher in high phosphorus group(P<0.05)and lower in high magnesium group(P<0.05).The expression of BMP-2 and Runx2 were increased after magnesium channel inhibitor was given(P<0.05).Conclusion:The high magnesium environment can inhibit the occurrence of vascular calcification in rat vascular rings induced by phosphorus.The mechanism may be achieved by regulating the expression of BMP-2 and its downstream Runx2,which may affect the transformation of vascular smooth muscle cells into osteogenic/ chondrogenic differentiation.