Pharmacological data on Combination of Astragaloside Ⅳ with Tanshinone ⅡA inhibits the Angiotensin II-stimulated proliferation and migration of vascular smooth muscle cells via the STIM1, TRPC1/6, and NFATc3 signaling pathway

Objective: Astragaloside Ⅳ and Tanshinone ⅡA combined application is widely used to clinically treat coronary heart disease and atherosclerosis in China. However, it is not clear if a combined application is better than a single application of either drug alone. This study aimed to evaluate the role of TRPC/NFATc-mediated calcium signaling in the angiotensin II-induced migration and proliferation of vascular smooth muscle cells and the inhibitory effect of astragaloside Ⅳ combined with tanshinone ⅡA that occurs via the STIM1, TRPC1/ 6, and NFAT3 signaling pathway. Materials and methods: Primary VSMCs were obtained from the abdominal aortas of SD rats, cultured by tissue block adherence and identified by immunocytochemistry. VSMC migration and proliferation were investigated by transwell migration and MTT assays, respectively. Intracellular Fluo-3/AM [Ca2+]i fluorescence was detected using a laser confocal microscope. The protein expression of STIM1, TRPC1, TRPC6, calcineurin and NFATc3 was determined using a Western blot. Results: Astragaloside Ⅳ combined with tanshinone ⅡA could significantly inhibit angiotensin II-induced VSMC proliferation, migration and [Ca2+]i increases, and this combination was superior to either drug alone and was not significantly different from the TRPC blocker SKF96365. Western blotting revealed that Ang II induced obvious increases in VSMC STIM1, TRPC1, TRPC6, CaN and NFATc3 protein expression and that astragaloside Ⅳ combined with tanshinone ⅡA and SKF96365 significantly inhibited the STIM1, TRPC1, TRPC6, CaN and NFATc3 protein expression induced by angiotensin II, indicating that astragaloside Ⅳ combined with tanshinone ⅡA has the same blocking effect on TRPC-NFATc calcium signaling as SKF96365 and is better than the application of a single drug alone. Conclusions: Astragaloside Ⅳ combined with tanshinone ⅡA can inhibit the proliferation and migration of angiotensin II-induced VSMCs and is superior to the single application of either astragaloside Ⅳ or tanshinone ⅡA, and the mechanism is related to the inhibition of the STIM1, TRPC1/6, CaN, and NFATc3 calcium signaling pathway.

研究目的：黄芪甲苷Ⅳ（Astragaloside Ⅳ）与丹参酮ⅡA（Tanshinone ⅡA）联合疗法在我国临床中被广泛用于治疗冠心病（coronary heart disease）及动脉粥样硬化（atherosclerosis），但目前尚不明确联合用药是否优于单一药物治疗。本研究旨在评估TRPC/NFATc介导的钙信号通路在血管紧张素Ⅱ（angiotensin Ⅱ）诱导的血管平滑肌细胞（vascular smooth muscle cells, VSMCs）增殖与迁移中的作用，以及黄芪甲苷Ⅳ联合丹参酮ⅡA通过STIM1、TRPC1/6及NFAT3信号通路发挥的抑制效应。
材料与方法：采用组织块贴壁法培养SD大鼠腹主动脉来源的原代VSMCs，并通过免疫细胞化学法完成细胞鉴定。分别采用Transwell迁移实验与MTT比色法检测VSMC的迁移能力与增殖活性。使用激光共聚焦显微镜检测细胞内Fluo-3/AM标记的游离钙离子浓度（[Ca²+]i）荧光信号。采用蛋白质印迹法（Western blot）检测STIM1、TRPC1、TRPC6、钙调神经磷酸酶（calcineurin, CaN）及NFATc3的蛋白表达水平。
结果：黄芪甲苷Ⅳ联合丹参酮ⅡA可显著抑制血管紧张素Ⅱ诱导的VSMC增殖、迁移及细胞内[Ca²+]i升高，且该联合用药效果优于单一药物治疗，与TRPC通道阻滞剂SKF96365的作用无显著差异。蛋白质印迹结果显示，血管紧张素Ⅱ可显著上调VSMC中STIM1、TRPC1、TRPC6、CaN及NFATc3的蛋白表达水平；而黄芪甲苷Ⅳ联合丹参酮ⅡA与SKF96365均可显著抑制血管紧张素Ⅱ诱导的上述蛋白表达升高，表明黄芪甲苷Ⅳ联合丹参酮ⅡA对TRPC-NFATc钙信号通路的阻断效果与SKF96365一致，且优于单一药物治疗。
结论：黄芪甲苷Ⅳ联合丹参酮ⅡA可抑制血管紧张素Ⅱ诱导的VSMC增殖与迁移，其效果优于单一使用黄芪甲苷Ⅳ或丹参酮ⅡA，其作用机制与抑制STIM1、TRPC1/6、CaN及NFATc3钙信号通路相关。