酸性环境抑制高磷诱导大鼠VSMCs 钙化的机制研究

Mechanisms of acidic environment inhibiting vascular smooth muscle cell calcification induced by high phosphorus in rats

摘要:

目的 探讨酸性环境对高磷诱导的大鼠血管平滑肌细胞（VSMCs）钙化的影响及其机制。 方法 体外分离培养大鼠VSMCs，采用免疫细胞化学法鉴定。将VSMCs 按随机数字表法分为正常对照组、 高磷＋ pH 7.4 组、高磷＋ pH 7.1 组。刺激4 d 后，采用逆转录聚合酶链反应和Western blot 检测活化T 细胞 核因子c1（NFATc1）、Runt 相关转录因子2（Runx2）基因和蛋白的表达。刺激14 d 后，对各组细胞进行 钙化染色、钙含量和碱性磷酸酶（ALP）活性测定。结果 与正常对照组比较，高磷＋ pH 7.4 组的钙含量、 ALP 活性、Runx2 和NFATc1 表达升高（P <0.05）；与高磷＋ pH 7.4 组比较，高磷＋ pH 7.1 组的钙含量、 ALP 活性、Runx2 和NFATc1 表达降低（P <0.05）。相关性分析发现，NFATc1 蛋白表达水平与ALP 活性、 Runx2 蛋白表达水平呈正相关（P <0.05）。结论 酸性环境可以抑制高磷诱导的大鼠VSMCs 钙化，其机制可 能是通过降低NFATc1 表达，抑制VSMCs 表型转化来实现的。

Abstract:

Objective To explore the effect and possible mechanisms of acidification on high phosphorusinduced vascular smooth muscle cell (VSMC) calcification in rats. Methods VSMCs were isolated from rat aorta, and randomly divided into control group and high phosphorus group. The high phosphorus group was further settled into two subgroups, i.e. high phosphorus+pH 7.1 group and high phosphorus+pH 7.4 group, which were treated with β-glycerophosphate, and acidified by HCl to adjust the pH. The mRNA expressions of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1 ) and Runt-related transcription factor 2 (RUNX2 ) in VSMCs were detected by RT-PCR after stimulation for 4 days. The activity of alkaline phosphatase (ALP) and calcium deposition were tested after culture for 14 days. Results Compared to the control group, calcium depositon and ALP activity were significantly increased in the high phosphorus+pH 7.4 group (P < 0.05), and the expressions of NFATc1 and RUNX2 were upregulated as well (P < 0.05). Meanwhile, the calcification level and the expressions of RUNX2 and NFATc1 in the high phosphorus+pH 7.1 group were lower than those in the high phosphorus+pH 7.4 group (P < 0.05). NFATc1 expression level was positively correlated with ALP activity and RUNX2 expression level (P < 0.05). Conclusions Acidic environment can inhibit VSMC calcification induced by high phosphorus in rats, its mechanism is possibly by downregulation of NFATc1 expression and prevention of VSMC phenotypic transformation.