【摘要】 目的：探讨Ca²⁺释放激活钙通道蛋白1（Orai1）在大鼠主动脉钙化中的作用机制。方法：取30只8周龄的雄性SD大鼠（200～250 g），随机分为正常生长培养液（GM）组、高钙磷钙化培养液（CM）组、溶剂对照二甲基亚砜组、Orai1抑制剂3,5-双（三氟甲基）吡唑衍生物（BTP2）处理组及内质网应激（ERS）抑制剂4-苯基丁酸（4-PBA）处理组，每组6只。制备离体主动脉环，给予10 mmol/L β-甘油磷酸和3 mmol/L CaCl₂刺激7 d构建主动脉钙化模型，造模同时给予BTP2或4-PBA干预。采用茜素红和冯·科萨染色评估主动脉钙化的严重程度；Western blot检测Orai1、成骨分化相关指标[Runt相关转录因子2（RUNX2）和骨形态发生蛋白2（BMP2）]、收缩表型指标[平滑肌肌球蛋白重链（SMMHC）和平滑肌蛋白22α（SM22α）]和ERS信号通路相关蛋白[磷酸化真核生物翻译起始因子2α（peIF2α）和活化转录因子4（ATF4）]的表达水平；基于Fluo-4 AM负载的细胞内Ca²⁺成像技术，利用激光共聚焦显微镜测量血管平滑肌细胞内游离Ca²⁺浓度([Ca²⁺]_i)的变化，观察Orai1抑制剂BTP2对钙池操纵性钙（SOC）通道介导的钙内流的影响；血管张力实验观察BTP2对SOC通道介导的血管收缩的影响。结果：茜素红和冯·科萨染色结果显示，与GM组相比，CM组血管钙化程度显著增强（P<0.01）;Western blot结果显示，与GM组相比，CM组RUNX2和BMP2蛋白表达显著上调（P<0.05或P<0.01）,Orai1蛋白表达显著上调（P<0.05），同时p-eIF2α和ATF4蛋白表达亦显著上调（P<0.01或P<0.05）。与CM组相比，给予10??mol/L BTP2干预后，血管钙化程度显著减轻（P<0.05）；且逆转了RUNX2和BMP2蛋白表达（P<0.01），同时下调p-eIF2α和ATF4蛋白表达（P<0.01）；此外，与CM组相比，给予5 mmol/L 4-PBA干预逆转了RUNX2和BMP2蛋白表达（P<0.05或P<0.01）；激光共聚焦显微镜检测血管平滑肌细胞[Ca²⁺]_i结果表明，BTP2显著抑制了SOC通道介导的钙内流（P<0.05）；血管张力测定结果显示，BTP2显著抑制了SOC通道介导的血管收缩（P<0.05）。结论：在大鼠主动脉钙化模型中Orai1表达上调，Orai1抑制剂BTP2可抑制SOC通道介导的钙内流和血管收缩，同时减轻主动脉环成骨样表型转化，其机制可能与ERS有关。更多还原

【Abstract】 AIM: To investigate the mechanism of Ca²⁺ release-activated calcium channel protein 1（Orai1） in rat aortic calcification. METHODS: Thirty 8-week-old male SD rats（200 to 250 g） were randomized into 5 groups: growth medium（GM） group, calcification medium（CM） group, dimethyl sulfoxide group, Orai1 inhibitor 3,5-bis（trifluoromethyl）pyrazole derivative（BTP2） group, and endoplasmic reticulum stress（ERS） inhibitor 4-phenylbutyrate（4-PBA） group. Then, isolated aortic rings were prepared and stimulated with 10 mmol/L β-glycerophosphate and 3 mmol/L CaCl₂ for 7 d to establish a calcification model. Concurrently, BTP2 or 4-PBA intervention was performed during modeling. The severity of calcification was evaluated via alizarin red and von Kossa staining. Additionally, the expression levels of Orai1, osteogenic differentiation markers [Runt-related transcription factor 2（RUNX2） and bone morphogenetic protein 2（BMP2）], contractile markers [smooth muscle myosin heavy chain（SMMHC） and smooth muscle protein 22α（SM22α）], and key molecules involved in the ERS signaling pathway, especially phosphorylated eukaryotic translation initiation factor 2α（p-eIF2α） and activating transcription factor 4（ATF4）, were assessed by Western blot. By utilizing laser confocal microscopy, intracellular free Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells（VSMCs） was monitored using Fluo-4 AM fluorescent probes. The effect of BTP2 on store-operated calcium（SOC） channel-mediated vasoconstriction was further evaluated using an in vitro vascular tension assay. RESULTS: The results of alizarin red and von Kossa staining revealed a marked increase in aortic ring calcification following stimulation with high levels of calcium and phosphate（P<0. 01）. In an in vitro rat aortic ring calcification model, there was significant up-regulation of Orai1 protein expression（P<0. 05）, accompanied by elevated levels of osteogenic differentiation-related markers RUNX2 and BMP2（P<0. 05 or P<0. 01）. In addition, the expression of ERS pathway markers was markedly elevated, especially peIF2α and ATF4（P<0. 01 or P<0. 05）. Treatment with BTP2 resulted in significant attenuation of aortic ring calcification（P<0. 05） and reversal of osteogenic differentiation markers, including RUNX2 and BMP2 protein expression（P<0. 01）, while concurrently down-regulating the ERS pathway proteins p-eIF2α and ATF4（P<0. 01）. Furthermore, compared with the CM group, treatment with 4-PBA reversed the expression levels of RUNX2 and BMP2（P<0. 05 or P<0. 01）. The assessment of [Ca²⁺]_i in VSMCs indicated that BTP2 significantly inhibited SOC channel-mediated calcium influx（P<0. 05）. In vitro vascular tension assays demonstrated that BTP2 significantly inhibited SOC channel-mediated vasoconstriction（P<0. 05）. CONCLUSION: An aortic ring calcification model is successfully developed, demonstrating up-regulation of Orai1 expression. Treatment with Orai1 inhibitor BTP2 effectively suppresses SOC channel-mediated calcium influx and vasoconstriction, consequently inhibiting aortic calcification. This mechanism may be associated with ERS.更多还原