镁离子对高磷诱导的大鼠胸主动脉血管环钙化的影响

The effects of magnesium ions on the calcification of aortic rings induced by high phosphorus in rats

目的探讨不同浓度镁离子对高磷诱导的大鼠胸主动脉血管环钙化的影响。方法体外分离大鼠胸主动脉血管环,将其随机分为正常对照组(含10%胎牛血清DMEM培养基)、高磷组(含10 mmol/Lβ-甘油磷酸高磷培养基)和镁干预组(高磷培养基加不同浓度硫酸镁,使镁离子终浓度分别为1、2、3 mmol/L,镁干预组Ⅰ~Ⅲ)。给予14天干预后,采用von Kossa染色及邻甲酚酞络合酮比色法检测胸主动脉血管环钙化情况;免疫组织化学方法检测胸主动脉血管环L型钙通道α1c(LTCCα1c)、LTCCβ3亚基、Runt相关转录因子2(Runx2)和平滑肌22α(SM22α)表达情况。结果与正常对照组比较,高磷组钙沉积增加,镁干预组随镁离子浓度增大棕黑色颗粒沉积逐渐减少,钙含量测定结果与之相一致(P<0.05)。Runx2、LTCCα1c、LTCCβ3亚基的表达除镁干预组Ⅲ与正常对照组差异无统计学意义外,其余各组均高于正常对照组(P<0.05);镁干预组随镁离子浓度增大,Runx2、LTCCα1c、LTCCβ3亚基的表达水平均逐渐降低,且均低于高磷组(P<0.05)。SM22α表达除镁干预组Ⅲ与正常对照组差异无统计学意义外,其余各组均低于正常对照组(P<0.05)。相关性分析显示,LTCCβ3、LTCCα1c亚基均与Runx2表达呈正相关(r=0.692,P<0.001;r=0.716,P<0.001),Runx2与SM22α表达呈负相关(r=-0.671,P=0.001)。结论镁离子在一定程度上可以抑制高磷诱导的大鼠胸主动脉血管环钙化,其可能通过下调LTCCα1c、LTCCβ3亚基表达,抑制VSMCs向成骨/成软骨表型转化,进而抑制血管环钙化的发生。

Aim To investigate the effect and possible mechanisms of the different concentrations of magnesium ions on aortic rings calcification induced by high phosphorus in rats. Methods Aortic rings were isolated from rat thoracic aorta and cultured in vitro,and randomly divided into control group,high phosphorus group and magnesium intervention group（final concentrations of magnesium ions were 1,2 and 3 mmol/L）. Aortic rings were cultured with 10% fetal bovine serum in control group. After 14 days of intervention,the expressions of L-type calcium channel（LTCC） α1 cβ3 subunit,Runt-related transcription factor 2（Runx2） and smooth muscle 22α（SM22α） were detected by Immunohistochemistry. Calcium concentration of aortic rings was measured by Von Kossa staining and quantification of calcium.Results Compared with control group,calcified nodules in high phosphorus group was increased,and calcified nodules gradually reduced with the increased magnesium ion concentration in the intervention group. The expression of LTCCα1 c、β3 subunits in high phosphorus group increased（P〈0. 05）. With the increase of magnesium ion concentration,the expression of Runx2、LTCC α1 c、β3 decreased（P〈0.05） and the expression of SM22α increased（P〈0.05） in high magnesium group. There results of Pearson correlation analysis showed that SM22α was negatively corelated with Runx2（r =-0.671,P = 0.001）,and LTCCα1 csubunit was positively corelated with Runx2（r = 0.712,P〈0.001）,LTCCβ3 subunit was positively corelated with Runx2（r = 0. 0.692,P〈0.001）. Conclusion Magnesium can promote high phosphorus induced rat aortic rings calcification,and its mechanism is possibly achieved by downregulatiing LTCC α1 cβ3 protein expression,and reducing the transformation of VSMCs into osteogenic/osteogenic phenotype.