Research Of Magnesium Ion On The Calcification Induced By High Phosphorus In Vascular Smooth Muscle Cells

Objective：High phosphorus is an independent risk factor for vascular mediacalcification in chronic kidney disease (CKD), and can lead to severecomplications such as cardiovascular events, the mechanism of highphosphorus induced calcification may be by vascular smooth muscle cells(VSMC) membrane type Ⅲ sodium phosphate transporter (Pit-1) into cells,and cause the VSMC phenotypic transformation. Further researches haveshown that magnesium ions can not only restrain activity of Pit-1channel toreduce the transfer of phosphorus into the cells, and but also can be acompetitive antagonist of phosphorus in cells, and play a role in adjusting thelevels of phosphorus in cells, but the relationship between magnesium ionsand vascular smooth muscle cells calcification is unclear. This study aims tothrough the VSMCs induced by high phosphorus calcification model toexplore the relationship between the magnesium ions and VSMCscalcification, in order to make clear the role of magnesium ions in VSMCscalcification.Methods:1Primary culture of VSMCsPrimary culture to get vascular smooth muscle cells (VSMCs), andidentify the cells by morphology and immune method, and then depict normalgrowth curve of smooth muscle cells.2Experimental and Intervention groupsVSMCs were randomly divided into negative control group, group ofhigh phosphorus and magnesium intervention groups, negative control groupusing low glucose DMEM medium containing10%fetal bovine serum, highphosphorus group on the basis of normal medium added10mmol/L β-glycero- phosphate for high phosphorus medium. Magnesium intervention groups wereadded magnesium chloride in the medium on the basis of high phosphorus, thefinal concentration of magnesium ions were1mmol/L (magnesiumintervention group1),2mmol/L (magnesium intervention group2) and3mmol/L (magnesium intervention group3), the stimulating time is7days.3The influence of different magnesium ion concentration of VSMCscalcificationThe calcification of VSMCs detected by Alizarin Red calcificationstaining and colorimetric method to determine the intracellular calciumcontent.4Different magnesium ion concentrations on the ALP activity of VSMCscalcificationTake3to5cells seeded in6-well plates, the density of cells is2×105/pore, intervention7days, refused the medium, and then cells werewashed two to three times with PBS, and added500μl0.1%Triton X-100toeach hole at4℃environment, about12～24h, cells were lysed and thenrepeated pipetting into centrifuge tubes centrifuged by chemiluminescencedetection ALP activity.5Different magnesium ion concentrations on the VSMCs Cbfα1mRNAexpression of calcified VSMCsBy RT-PCR method to study the effect on the VSMCs Cbfα1mRNAexpression levels of different magnesium ion concentrations.6Statistical methodsStatistical analysis of data using SPSS13.0software for analysis. Theexperimental data were described by mean±standard deviation(x s).Compared differences between two groups by t test, differences amongmulti-groups by one-way analysis of variance (ANOVA) and means of twosubgroups comparison in multi-groups by Student-Newman-Keuls (SNK).With P<0.05for the difference was statistically significant.Results:1Primary culture of VSMCs Use organization block adherent method to obtain the original generationof rat VSMCs, usually about3~4d a few cells can climb out from stick wallof artery tissue block, for long fusiform, scattered arrangement, the cellnumber increased significantly and confluent over time, about6~7d cellscovered most of the bottle bottom, when the number of cells accounts for the80~90%of the total bottom area can be represented. After represented cellswere round or oval at first, stick a wall after can be gradually extend intospindle, abundant cytoplasm, nuclear lobes ovoid, crisscross overlap multiplecells growth, is a typical "peak to valley" performance. Detection of smoothmuscle cells by immunocytochemistry method specific antibodies-alphasmooth muscle actin (α-SMA actin), that the cell cytoplasm strong expressionis positive results, immune reaction products was brown.2The influence of different magnesium ion concentration of VSMCscalcificationAlizarin Red calcification staining results showed: While compared withthe negative control group, VSMCs calcification dyeing were increased insome extent degrees in the other groups, and magnesium intervention groupscompared with high phosphorus group, the calcified nodules of VSMCs byAlizarin Red staining gradually decreased regularly, when the magnesium ionsconcentration was2mmol/L, the calcified nodules began to markly shrink,showed a concentration dependence, calcified nodule reached to least whenthe concentration was3mmol/L.Calcium content by colorimetric method results showed: Compared withthe negative control group, the levels of calcium content were significantlyelevated in other groups, while compared with high phosphorus group, thelevels of calcium content gradually die down along with the magnesium ionsconcentration in magnesium intervention groups, the result showed a negativecorrelation, the magnesium ion concentration, the greater the calcium contentis lower, and when the concentration of magnesium ion is2mmol/L, calciumcontent begin to reduce, when the concentration of magnesium ion is3mmol/L, Calcium content test results also corresponding to the minimum, the comparative differences between groups with statistical significance (P <0.05).3Different magnesium ion concentrations on the ALP activity of VSMCscalcificationALP activity assay results: Compared with the negative control group,the level of ALP in VSMCs rised in other groups in some degree, while incontrast to the high phosphate and magnesium intervention groups, the levelsof ALP also gradually reduced along with the rising of the magnesium ionconcentration, the change trend of ALP activity are similar with calciumcontent results, also showed a concentration dependence, when magnesiumions concentration was3mmol/L, ALP activity reached to minimum.4Different magnesium ion concentrations on the VSMCs Cbfα1mRNAexpression of calcified VSMCsResults of RT-PCR to detect the expression of each group VSMCs inCbfα1mRNA display: Compared with the control group, the level of Cbfα1mRNA in VSMCs increased respectively in high phosphate and magnesiumintervention groups, while the Cbfα1mRNA levels gradually reduced in theintervention groups in contrast to the high phosphate group, the changetendency is associated with magnesium ion concentrations, Cbfα1mRNAlevels reached to least when the concentration was3mmol/L.Conclusion:1Magnesium ions can inhibit VSMCs induced by high phosphoruscalcification, and showed a concentration dependence.2The mechanism of Magnesium ions suppression the calcification ofVSMCs induced by high phosphate may occur through inhibition oftransdifferentiation to achieve synergy.