间歇性碱刺激对高磷诱导的大鼠血管环钙化的影响及可能机制

Effect of intermittent alkaline stimulation on aortic ring calcification in rat induced by high phosphorus and its mechanism

目的探讨间歇性碱刺激对高磷诱导的大鼠胸主动脉血管环钙化的影响及可能机制。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、高磷组(含10 mmol/Lβ-甘油磷酸的培养基)和间歇性碱刺激组(在高磷培养基的基础上调整pH值为7.7)。干预14天后,采用免疫组织化学方法检测L型钙通道(LTCC)β3亚基、Runt相关转录因子2(Runx2)和平滑肌蛋白22α(SM22α)的表达情况;采用硝酸银染色法和钙含量测定法检测血管环钙化程度。结果与正常对照组相比,高磷组的钙含量、Runx2和LTCCβ3亚基表达水平均增加(P<0.001);与高磷组相比,间歇性碱刺激组的钙含量、Runx2和LTCCβ3亚基表达水平均有显著增加(P<0.001)。同时,高磷组棕黑色钙化结节较正常对照组增多,间歇性碱刺激组棕黑色钙化结节较高磷组增多。与正常对照组相比,高磷组的SM22α表达水平下降(P<0.001);与高磷组相比,间歇性碱刺激组的SM22α表达水平显著下降(P<0.001)。相关性分析显示,LTCCβ3亚基与Runx2蛋白表达呈正相关(r=0.740,P=0.002),与SM22α蛋白表达呈负相关(r=-0.670,P=0.006)。结论间歇性碱刺激可以促进高磷诱导的大鼠胸主动脉血管环钙化,其可能通过上调LTCCβ3亚基表达,促进血管平滑肌细胞向成骨/成软骨表型转化,进而促进血管环钙化的发生。

Aim To explore the effects of intermittent alkaline stimulation on vascular ring calcification from the thoracic aorta of rats induced by high phosphorus and the possible mechanism. Methods Aortic rings were isolated from rat thoracic aorta and cultured in vitro,and randomly divided into three groups： control group,high phosphorus group（ containing 10 mmol/L β-glycerol phosphate medium） and intermittent alkaline stimulation group（ adjusting pH to 7.7 on the basis of high phosphorus medium）. After 14 days of intervention,the expressions of L-type calcium channels（ LTCC）β3subunit,runt-related transcription factor 2（ Runx2） and smooth muscle protein 22α（ SM22α） were detected by immunohistochemical method. The degree of vascular ring calcification was detected by silver nitrate staining and calcium content test. Results Compared with control group,calcium content,the expressions of Runx2 and LTCCβ3were significantly increased in high phosphorus group（ P0.001）. Compared with high phosphorus group,calcium content,the expressions of Runx2 and LTCCβ3were significantly increased in intermittent alkaline stimulation group（ P0.001）. At the same time,the number of brown and black calcified nodules in high phosphorus group was higher than that in control group,and the number of brown and black calcified nodules in intermittent alkaline stimulation group was higher than that in high phosphorus group. Compared with control group,the expression of SM22α was significantly decreased in high phosphorus group（ P〈0. 001）. Compared with high phosphorus group,the expression of SM22α was significantly decreased in intermittent alkaline stimulation group（ P0.001）. Correlation analysis showed that the expression of LTCCβ3was positively correlated with Runx2 protein expression（ r = 0. 704,P = 0. 002） and negatively correlated with SM22αprotein expression（ r =-0. 670,P = 0. 006）. Conclusion Intermittent alkaline stimulation can promote high phosphorus induced vascular ring calcification from the thoracic aorta of rats. Its mechanism may be upregulation of LTCCβ3protein expression,enhancing the transformation of vascular smooth muscle cells into osteogenesis/chondrogenesis phenotype,and then promoting the occurrence of vascular ring calcification.