| Objective: Vascular calcification, a common phenomenon in patients with chronic kidney disease(CKD), is highly correlated with cardiovascular disease(CVD) mortality. Prior studies have demonstrated that, in addition to traditional risk factors, nontraditional risk factors, including uremic toxin and a disturbed bone and mineral metabolism(in particular hyperphosphatemia) are considered to be associated with the high prevalence of vascular calcification in patients with CKD. Exposure of vascular smooth muscle cells(VSMCs) to high phosphate condition results in obvious mineralization with various putative processes, such as apoptosis, osteogenic differentiation of VSMCs, imbalance between expression of calcification inducers(core binding factor α-1) and inhibitors(matrix Gla protein and osteopontin). Since the mechanism underlying vascular calcification is very complicated, there is still no effective treatment for vascular calcification. Physiologically, magnesium is required for numerous fundamental functions in humans, including maintaining vascular tone and heart rhythm. In this regard, magnesium deserves clinical attention due to its pleiotropic potentials for interfering with vascular calcification. Recently, it has been showed that magnesium carbonate may inhibit the progression of coronary artery calcification. However, the concrete relation between magnesium and vascular calcification still remains unknown and requires further study. Thus, in this paper we investigated the effect and mechanism of magnesium on vascular calcification induced by β-glycerophosphate(β-GP) in rat VSMCs.Methods:1 Primary culture of VSMCs:VSMCs were obtained from tunica media of adult male Sprague Dawley rat thoracic aorta using the explant culture method, and identified by morphology and immune methods.2 Experimental and Intervention groups:VSMCs were divided into control group(low glucose DMEM medium containing 10% fetal bovine serum), high phosphorus group(normal medium added 10mmol/L β-GP),magnesium intervention group(high phosphorus medium added 3mmol/L Mg SO4) and 2-APB(an inhibitor of magnesium transporter) intervention group(high phosphorus medium added 3mmol/L Mg SO4 and 10-4mol /L 2-APB) randomly. After incubation for different days,0 day,3 days,6 days,10 days,14 days, the cells were analyzed.3 Detection the calcification in different groups after incubation for 14 days: Calcium deposition was measured by Alizarin red staining, and the calcium content in the supernatant was measured with the o-cresolphthalein complexone method using a Calcium Assay kit according to the manufacturer’s instructions after VSMCs were incubated for 14 days.4 Determination of alkaline phosphatase activity after incubation for 14 days: The cells were cultured for 14 days after intervention. Alkaline phosphatase(ALP) activity was measured using an Alkaline Phosphatase Activity Detection kit in accordance with the manufacturer’s protocol.5 Expression of Cbfα1 after incubation for different time periods:RT-PCR was used to observe the expressive of VSMCs Cbfα1 m RNA after VSMCs were incubated for 0 day,3 days,6 days,10 days and 14 days, respectively.6 Expression of MGP and OPN after incubation for different time periods:RT-PCR was used to observe the expressive of VSMCs MGP and OPN m RNA after VSMCs were incubated for 0 day,3 days,6 days,10 days and 14 days, respectively.7 Statistical methods:Data analyses were conducted using SPSS 19.0 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 used for post-hoc testing. P<0.05 denoted a statistically significant difference.Results:1 Primary VSMCs are cultured successfully: The original generation of VSMCs were obtained using the explant culture method as previously described, and usually after about 3- 4 days a few cells climbed out from stick wall of artery tissue block with long fusiform and scattered arrangement. After 6-7 days the cells increased significantly and confluent for passage. When cells passaged, cells were round or oval at first, then gradually extend into spindle with abundant cytoplasm and nuclear lobes ovoid. Finally multiple cells were crisscrossed and presented a typical "peak to valley" performance. VSMCs were identified by a positive staining of α-Smooth muscle actin in the cell cytoplasm where the reaction product was brown.2 Magnesium attenuates β-GP-induced calcification and ALP activity : VSMCs incubated with calcifying medium(β-GP) for 14 days exhibited evident calcification observed by Alizarin red staining, when compared with the control cells. However, these changes were clearly reversed in the cells maintained in the high-magnesium medium. In agreement, quantitative analysis indicated that calcium content in the VSMCs was significantly reduced under high-magnesium conditions compared with calcifying conditions. Furthermore, ALP activity, a vital marker of calcification, was markedly enhanced following β-GP treatment, while inhibited in the presence of magnesium. VSMCs treated with 2-APB, a specific inhibitor of TRPM7, exhibited no magnesium-induced reduction in calcification, and recovered the magnesium-inhibited decrease of ALP activity on the 14 th day.3 Magnesium inhibits β-GP induced osteoblastic factor Cbfα1 expression of VSMCs in a time-dependent manner: Compared with high phosphorus group and 2-APB intervention group, Cbfα1 expression in VSMCs was significantly decreased in magnesium intervention group after VSMCs were incubated for 14 days. Dynamic observation shown that on the third day the level of Cbfα1 m RNA began to increase significantly and a notable time-dependent increase in the Cbfα1 expression level was observed in the VSMCs incubated with calcifying medium. Short-term(3-day) incubation in the high-concentration magnesium medium markedly reduced β-GP-induced Cbfα1 expression, with the effect remaining enhancement throughout the culture period.4 Magnesium regulates the secretion of calcification inhibitors--MGP and OPN in a time-dependent manner: Compared with high phosphorus group and 2-APB intervention group, MGP and OPN expression in VSMCs was significantly increased in magnesium intervention group after VSMCs were incubated for 14 days. RT-PCR indicated that β-GP treatment led to a significant reduction in the levels of MGP and OPN by day 3 of incubation, and that this reduction was time-dependent. Incubation for 3 days in β-GP with magnesium inhibited the β-GP-induced reduction in the levels of MGP and OPN. Furthermore, long-term(14-day) exposure to the high-magnesium environment restored, and also gradually upregulated, the expression levels of MGP and OPN.Conclusion:1 Magnesium is able to efficiently reduce β-GP-induced calcification in rat VSMCs.2 The mechanism underlying this magnesium-induced reduction in calcification is inhibiting the transdifferentiation of VSMCs by reducing the expression of Cbfα1, while upregulating the expression of calcification inhibitors--MGP and OPN-- in a time-dependent manner. |
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