Song X, Zhang Y, Wang H, Wen H, Zhao C, Lan Y, Pan L, Zhang C, Cheng M
School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, China.
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, China.
J Physiol Pharmacol. 2016 Jun;67(3):363-75.
The objective of this study is to investigate whether the inhibition of tradinterol (SPFF) against acetylcholine (ACh)-induced proliferation is mediated by Ca(2+) signaling in airway smooth muscle cells (ASMCs), and whether stereoselectivity of the drug exists. Guinea pig ASMCs were primarily prepared with the method described and treated with ACh combined to SPFF isomers for 24 or 48 hours, respectively. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to determine the proliferation of the guinea pig ASMCs. Ca(2+) fluorescent intensity in the guinea pig ASMCs, expressed with percentage increase in fluorescence when the intensity was determined with varioskan flash or shown with percentage increase in Geo Mean (GM) measured with flow cytometry, was recorded. Images of the intensity were obtained with fluorescent microscope. 2-APB, an (inositol 1,4,5-trisphosphate receptor) IP3R blocker, and NiCl2, a store-operated channel (SOC) inhibitor, were used to investigate the mechanism of SPFF isomers regulating intracellular Ca(2+) via IP3R on sarcoplasmic reticulum (SR) and/or SOC on plasma membrane. (-)SPFF and (±)SPFF, treated for 48 hours, showed significant inhibition against ACh-induced proliferation. The Ca(2+) elevation induced by ACh was concentration-dependently suppressed by SPFF isomers. (-)SPFF is the most effective but the potency of (±)SPFF is less than that of the former and stronger than that of (+)SPFF based on the half maximal inhibitory concentration (IC50) value. No significant additive effect was observed when (-)SPFF/(±)SPFF was used alone and combined with NiCl2/2-APB. As far as (+)SPFF is concerned, no similar phenomenon was observed. (-)SPFF and (±)SPFF but (+)SPFF showed significant inhibition against the percentage increase in fluorescence induced by CaCl2. It is likely that the influence of IP2RSOC-mediated Ca(2+) signaling in ASMCs helps (-)SPFF and (±)SPFF contribute to the suppression of ASMCs proliferation. Stereoselectivity of SPFF isomers may lead to different levels of suppression of ACh-induced intracellular Ca(2+) and ASMCs proliferation. Moreover, cell cycle analysis with flow cytometry was applied to the evaluation of the action in human ASMCs in order to further confirm the anti-proliferative effect of the drugs. It was found that (-)SPFF, (±)SPFF but (+)SPFF suppressed the elevated rate of cell population in Phase S over all the cells stimulated with ACh, when SPFF and its isomers were individually exposed to the cells for 72 hours. These results that demonstrate the different stereoselective activities of SPFF are in consistent with those obtained from the guinea pig ASMCs.
本研究的目的是探讨曲托喹酚（SPFF）对乙酰胆碱（ACh）诱导的气道平滑肌细胞（ASMCs）增殖的抑制作用是否由Ca(2+)信号介导，以及该药物是否存在立体选择性。采用所述方法原代培养豚鼠ASMCs，分别用ACh与SPFF异构体处理24或48小时。采用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-H-溴化四氮唑（MTT）法测定豚鼠ASMCs的增殖情况。记录豚鼠ASMCs中Ca(2+)荧光强度，用多功能酶标仪测定强度时以荧光增加百分比表示，或用流式细胞仪测定几何平均值（GM）增加百分比表示。用荧光显微镜获取强度图像。使用肌醇1,4,5-三磷酸受体（IP3R）阻滞剂2-APB和储存-操纵性通道（SOC）抑制剂NiCl2，研究SPFF异构体通过肌浆网（SR）上的IP3R和/或质膜上的SOC调节细胞内Ca(2+)的机制。处理48小时后，(-)SPFF和(±)SPFF对ACh诱导的增殖表现出显著抑制作用。SPFF异构体浓度依赖性地抑制ACh诱导的Ca(2+)升高。根据半数最大抑制浓度（IC50）值，(-)SPFF最有效，(±)SPFF的效力低于前者但高于(+)SPFF。单独使用(-)SPFF/(±)SPFF并与NiCl2/2-APB联合使用时，未观察到明显的相加效应。就(+)SPFF而言，未观察到类似现象。(-)SPFF和(±)SPFF但(+)SPFF对CaCl2诱导的荧光增加百分比表现出显著抑制作用。ASMCs中IP2RSOC介导的Ca(2+)信号的影响可能有助于(-)SPFF和(±)SPFF抑制ASMCs增殖。SPFF异构体的立体选择性可能导致对ACh诱导的细胞内Ca(2+)和ASMCs增殖的抑制水平不同。此外，应用流式细胞术进行细胞周期分析以评估对人ASMCs的作用，以进一步证实药物的抗增殖作用。当SPFF及其异构体分别作用于细胞72小时时，发现(-)SPFF、(±)SPFF但(+)SPFF抑制了所有受ACh刺激的细胞中S期细胞群体的升高率。这些证明SPFF不同立体选择性活性的结果与在豚鼠ASMCs中获得的结果一致。
