Objective To explore the effect and possible mechanisms of acidification on high phosphorus induced vascular smooth muscle cells (VSMCs) calcification in rats. Methods VSMCs were isolated from rat aorta, identified by immunocytochemistry, and randomly divided into control group and high phosphorus group according to the pH, the high phosphorus group was further settled into three subgroups, high phosphorus + pH6. 8, + pH7. 1 and + pH7. 4, which were treated with β-glycerophosphate, and acidified by HCL to adjust the pH respectively. The VSMC mRNA expressions of L-type calcium channel (LTCC) subunitsα1C , β2 , β3 , Runt-related transcription factor 2 ( Runx2) and Smad1 mRNA were detected by RT-PCR after stimulated for 4 days; meanwhile, the VSMCs were loaded with calcium probe Fluo-3 / AM, and the concentration of calcium was measured by Fluo-3 / AM among different groups. The activity of alkaline phosphatase and calcium deposition were tested after culturing 14 days. Results Compared with control group, calcium concentration and the activity of ALP in VSMCs were significantly increased in high phosphorus + pH7. 4 group after incubation for 14 days (88. 26 ± 6. 43 vs. 22. 39 ± 3. 19, 94. 33 ± 3. 08 vs. 20. 39 ± 1. 18, both P ﹤ 0. 05), the expression of Smad1, Runx2 and LTCC β3 subunit mRNA in high phosphorus + pH7. 4 group was increased as well (0. 65 ± 0. 05 vs. 0. 07 ± 0. 01, 0. 37 ± 0. 02 vs. 0. 01 ± 0. 00, 0. 80 ± 0. 01 vs. 0. 34 ± 0. 13, all P ﹤ 0. 05). Besides, Smad1, Runx2 and LTCCβ3 subunit mRNA were decreased in high phosphorus + pH7. 1 group and high phosphorus + pH6. 8 group (0. 25 ± 0. 02 vs. 0. 37 ± 0. 02, 0. 09 ± 0. 01 vs. 0. 37 ± 0. 02, 0. 44 ± 0. 04 vs. 0. 65 ± 0. 05, 0. 23 ± 0. 01 vs. 0. 65 ± 0. 05, 0. 64 ± 0. 11 vs. 0. 80 ± 0. 01, 0. 43 ± 0. 01 vs. 0. 80 ± 0. 01, all P ﹤ 0. 01), compared with those in high phosphorus + pH7. 4 group as the pH decreased. There was no significant difference in LTCC α1C and β2 subunits mRNA among each groups (P = 0. 08, P = 0. 74). Calcium influx in VSMCs was partially blocked as pH was changed ( P ﹤ 0. 01) . Conclusions Acidic environment can inhibit high phosphorus induced rat VSMCs calcification, its mechanism is possibly by downregulation of LTCC β3 subunit gene expression, decreasing calcium ion influx and preventing VSMC phenotypic transformation to achieve.%目的：探讨酸性环境对高磷诱导的大鼠血管平滑肌细胞(VSMCs)钙化的影响及可能机制。方法体外分离培养大鼠 VSMCs,采用免疫细胞化学方法鉴定。将 VSMCs 按随机数字表法分为正常对照组、高磷+ pH7.4、高磷+ pH7.1和高磷+ pH6.8(在高磷培养基的基础上调整 pH 值为6.8、7.1和7.4三个亚组)。刺激4 d 后,采用 RT-PCR 检测 L 型钙通道(LTCC)α1C、β2和β3亚基, Runt 相关转录因子2(Runx2)及 Smad1基因的表达；应用钙离子探针 Fluo-3/ AM 检测 VSMCs 胞外钙离子内流的效应变化。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+ pH7.4组钙含量、ALP 活性、Runx2和 Smad1mRNA 的表达明显增高(88.26±6.43比22.39±3.19、94.33±3.08比20.39±1.18、0.37±0.02比0.01±0.00、0.65±0.05比0.07±0.01,均为 P ﹤0.05)；与高磷+ pH7.4组比较,高磷+ pH7.1组和高磷+ pH6.8组的钙含量、ALP 活性、Runx2和 Smad1mRNA 的表达均降低(69.95±1.72和50.74±3.29、51.11±2.05和34.62±1.13、0.25±0.02和0.09±0.01、0.44±0.04和0.23±0.01,均为 P ﹤0.05)。与正常对照组比较,高磷+ pH7.4组的 LTCCβ3亚基 mRNA 表达水平增加(0.80±0.01比0.34±0.13, P ﹤0.01),高磷+ pH7.1组和高磷+ pH6.8组的 LTCCβ3亚基 mRNA 表达水平均下降(0.64±0.11和0.43±0.01,均为 P ﹤0.01)。各组之间 LTCCα1C 和β2亚基 mRNA 的表达差异无统计学意义(P =0.08和0.74)。与正常对照组比较,高磷+ pH7.4组的 VSMCs 胞内钙离子浓度增加(438.33±7.50比149.54±20.89,P ﹤0.05)；高磷+ pH7.1组和高磷+ pH6.8组的 VSMCs 胞内钙离子浓度均降低(329.66±16.64和234.00±17.43,均为 P ﹤0.01)。结论酸性环境可以抑制高磷诱导的大鼠VSMCs 钙化,其可能是通过抑制 LTCC β3亚基表达,降低 VSMCs 钙离子内流,阻滞 VSMCs 发生表型转化来实现的。
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