中电导钙激活钾离子通道抑制剂对高磷诱导大鼠胸主动脉平滑肌细胞钙化的作用及机制

Effects of intermediate conductance calcium-activated potassium channel blocker TARAM-34 on β-glycerophosphate induced vascular smooth muscle cells calcification

目的探讨中电导钙激活钾离子通道（KCa3．1）抑制剂1-[（2-氯苯基）二苯甲基]-1H-吡唑（TRAM-34）对高磷诱导大鼠胸主动脉平滑肌细胞钙化的作用及其机制。方法体外培养原代大鼠主动脉平滑肌细胞和胸主动脉环，将传代培养至第4代的细胞和血管环均分为3组：对照组（加入含10％胎牛血清的DMEM培养基）、高磷组（在对照组培养基的基础上，加入10mmoL／L13-甘油磷酸）和TRAM-34干预组（在高磷组培养基的基础上，加入20nmol／LTRAM-34）。采用邻甲酚酞络合酮比色法检测细胞和血管环的钙含量；采用荧光探针法测定胸主动脉平滑肌细胞内的钙离子浓度；采用RT—PCR和Western blot检测各组细胞中调节成骨和软骨分化的Runt相关转录因子2（Runx2）的表达水平；采用免疫组织化学法检测各组胸主动脉环中Runx2的表达水平；采用磷酸苯二钠法测定胸主动脉平滑肌细胞中碱性磷酸酶（ALP）活性。结果（1）胸主动脉平滑肌细胞体外培养12d后，高磷组钙含量高于对照组[（121．67±6．17）mg／g蛋白比（84．38±8．17）mg／g蛋白，P〈0．05]，TRAM-34干预组钙含量[（93．31±11．36）mg／g蛋白]低于高磷组（P〈0．05）。胸主动脉环体外培养12d后，高磷组钙含量高于对照组[（7．17±0．57）mg／g蛋白比（1．18±0．13）mg／g蛋白，P〈0．05]，TRAM-34干预组钙含量[（4．71±0．42）mg／g蛋白]低于高磷组（P〈0．05）。（2）胸主动脉平滑肌细胞体外培养4d后，高磷组细胞内的钙离子浓度高于对照组（349．22±40．47比151．67±16．94，P〈0．05），TRAM-34干预组细胞内的钙离子浓度（194．67±22．21）低于高磷组（P〈0．05）。（3）胸主动脉平滑肌细胞体外培养4d后，lit—PCR结果显示，高磷组细胞Runx2mRNA表达水平高于对照组（0．630±0．033比0．340±0．058，P〈0．05），TRAM-34干预组细胞Runx2mRNA表达水平（0．399±0．023）低于高磷组（P〈0．05）；Westernblot结果显示，高磷组细胞Runx2蛋白表达水平高于对照组（0．865±0．031比0．414±0．011，P〈0．05），TRAM-34干预组细胞Runx2蛋白表达水平（0．575±0．014）低于高磷组（P〈0．05）。胸主动脉环体外培养4d后，免疫组织化学结果显示，高磷组的Runx2表达水平高于对照组（0．113±0．010比0．0674-0．008，P〈0．05），TRAM-34干预组的Runx2表达水平（0．069±0．006）低于高磷组（P〈0．05）。（4）胸主动脉平滑肌细胞培养12d后，高磷组ALP活性高于对照组[（96．56±9．84）U／g蛋白比（46．92±4．60）U／g蛋白，P〈0．05]，TRAM-34十预组ALP活性[（70．20±8．41）U／g蛋白]低于高磷组（P〈0．05）。结论KCa3．1抑制剂TRAM-34抑制高磷诱导大鼠胸主动脉平滑肌细胞钙化，可能是通过抑制平滑肌细胞钙离子内流，降低Runx2．的表达水平，抑制平滑肌细胞向成骨或成软骨细胞表型转化来实现。

Objective To observe the role of TRAM-34 （ 1- （ （2-chlorophenyl） diphenylmethyl） - 1H-pyrazole） , the blocker of intermediate conductance calcium-activated potassium channel （KCa3.1）, on β-glycerophosphate induced vascular calcification in vitro. Methods Vascular smooth muscle cells （VSMCs） were obtained from rat thoracic aorta, and VSMCs after the fourth passage and aortic rings were divided into control group （cultured in DMEM with 10% fetal bovine serum）, high phosphorus group （cultured in DMEM with 10% fetal bovine serum and 10% 13-glycerophosphate） and TRAM-34 group（20 nmol/L TRAM-34 was added into high phosphorus DMEM）. Calcium deposition of VSMCs and aortic rings were measured by o-cresolphthalein complexone method. Calcium influx of VSMCs was measured by immunofluorescence probe Fluo-3 AM. The expression of runt-related transcription factor 2 （ Runx2 ） was detected by RT-PCR and Western blot for cells and immunohistochemistry for aortic rings. ALP activity was measured by alkaline phosphatase activity detection kit. Results （1） Compared with control group, calcification was significantly increased in high phosphorus group （ （ 121.67 ± 6. 17） mg/g vs. （ 84. 38 ± 8. 17） mg/g,P 〈0. 05） and this effect could be attenuated by TRAM-34 （ （93.31 ± 11.36） mg/g, P 〈 0. 05 vs. high phosphorus group） after 12 days culture. Similar results were found in aortic rings cultured for 12 days--high phosphorus group：（7.17 ± 0. 57） mg/g vs. control：（ 1.18 ± 0. 13 ） mg/g （P 〈 0. 05 ） and TRAM-34： （4. 71 ±0. 42） mg/g, P 〈0. 05 vs. high phosphorus group. （2） Compared with control group, the calcium influx was higher in high phosphorus group （349.22 ±40.47 vs. 151.67 ± 16.94, P 〈0. 05） and reduced in TRAM-34 group （ 194. 67 ± 22. 21, P 〈 0. 05 vs. high phosphorus group ） in VSMCs simulated for 4 days. （ 3 ） Both mRNA and protein expressions of Runx2 in high phosphorus groups were higher than in control group （0. 630 ±0. 033 vs. 0. 340 ±0. 058 and 0. 865 ±0. 031 vs. 0. 414 ±0. 011, both P 〈 0. 05） and lower in TRAM-34 group （0. 399 ± 0. 023 and 0. 575 ± 0. 014, both P 〈 0.05 vs. high phosphorus group） in VSMCs simulated for 4 days. Besides, compared with high phosphorus group, the expression of Runx2 was decreased in control group （0. 113 ± 0. 010 vs. 0. 067 ± 0.008,P 〈 0.05） and TRAM-34 group （0. 069 ±0. 006 ,P 〈0. 05） after aortic rings were cultured for 4 days. （4） Compared with control group, the activity of ALP was significantly increased in high phosphorus group （ 96.56 ± 9. 84 vs. 46. 92±4.60,P 〈0.05） and decreased in TRAM-34 group（70.20 ± 8.41, P 〈0.05 vs. high phosphorus group） in VSMCs simulated for 12 days. Conclusion KCa3. 1 blocker TRAM-34 can inhibit β-glycerophosphate induced VSMCs and aortic ring calcification through inhibiting calcium influx, downregulating Runx2 expression and attenuating osteogenic differentiation.